Glioblastoma multiforme (GBM) has a poor prognosis—despite aggressive primary treatment composed of surgery, radiotherapy and chemotherapy, median survival is still around 15 months. It starts to grow again after a year of treatment and eventually nothing is effective at this stage. Recurrent GBM is one of the most disappointing fields for researchers in which their efforts have gained no benefit for patients. They were directed for a long time towards understanding the molecular basis that leads to the development of GBM. It is now known that GBM is a heterogeneous disease and resistance comes mainly from the regrowth of malignant cells after eradicating specific clones by targeted treatment. Epidermal growth factor receptor, platelet derived growth factor receptor, vascular endothelial growth factor receptor are known to be highly active in primary and recurrent GBM through different underlying pathways, despite this bevacizumab is the only Food and Drug Administration (FDA) approved drug for recurrent GBM. Immunotherapy is another important promising modality of treatment of GBM, after proper understanding of the microenvironment of the tumour and overcoming the reasons that historically stigmatise GBM as an ‘immunologically cold tumour’. Radiotherapy can augment the effect of immunotherapy by different mechanisms. Also, dual immunotherapy which targets immune pathways at different stages and through different receptors further enhances immune stimulation against GBM. Delivery of pro-drugs to be activated at the tumour site and suicidal genes by gene therapy using different vectors shows promising results. Despite using neurotropic viral vectors specifically targeting glial cells (which are the cells of origin of GBM), no significant improvement of overall-survival has been seen as yet. Non-viral vectors ‘polymeric and non-polymeric’ show significant tumour shrinkage in pre-clinical trials and now at early-stage clinical trials. To this end, in this review, we aim to study the possible role of different molecular pathways that are involved in GBM’s recurrence, we will also review the most relevant and recent clinical experience with targeted treatments and immunotherapies. We will discuss trials utilised tyrosine receptor kinase inhibitors, immunotherapy and gene therapy in recurrent GBM pointing to the causes of potential disappointing preliminary results of some of them. Additionally, we are suggesting a possible future treatment based on recent successful clinical data that could alter the outcome for GBM patients. A group of malignant brain tumours called ‘Gliomas’ arises from the supporting neuroglial cells . Although current WHO classification includes epigenetic, genetic and clinicopathological features of brain tumours, most clinicians still divide brain tumours into low-grade glioma including  and high-grade glioma (HGG)  . GlioblaAs suggested by the term ‘multiforme’, GBM is characterised by intra-tumour heterogeneity not only on cellular but also on molecular levels . This heTill now, no clinical reflection of the four subgroups with only a slight survival advantage to the pro-neural subtype , 14.This review article considers the major therapeutic strategies currently being investigated in the field of recurrent GBM, focusing on approaches with not only pre-clinical but also clinical data. We aim to discuss novel and experimental tyrosine kinase receptor inhibitors, immunotherapy and gene therapy pointing to the underlying pathways that lead to their promising role in recurrent GBM. We also added a final section on the most important future direction that scientists are trying to apply to treat recurrent GBM based on pre-clinical data to improve the outcome of these patients.We searched the MEDLINE, PubMed database for high impact factor journals at least 1.8 with high citations. We also searched clinical trials.gov for phase I, II trials with reported PFS, OS published on tyrosine receptor kinase inhibitors, immunotherapy and gene therapy in recurrent GBM and discussed them. Ongoing trials and pre-clinical relevant up-to-date studies related to this subject have also been discussed. We used in our search the keywords glioblastoma, GBM, recurrent GBM and recurrent high-grade glioma.The RTK inhibitors are one of the most extensively studied drugs in oncology, we will discuss in the following section four of the main growth factor receptors in GBM and targeted treatments against them focusing on the applied clinical experience from clinical trials.Human EGFR  is over-expressed in 40%–60% of primary GBM tumours and mostly occurs in the classical subtype  15], bu, bu15], EGFR receptor is activated by two mechanisms as mentioned above, either by receptor over-expression or multiple ligand-independent and ligand-dependent pathways which will lead to stimulation of subsequent downstream mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase (PI3K) and Src kinase pathway besides signal transducers and activators of transcription (STAT) transcription factor activation . These eSometimes phosphatase and TENsin homolog (PTEN) activity is lost which mainly acts as a tumour suppressor protein that inhibits the PI3K pathway, in this situation, there will be resistance to EGFR kinase inhibitors . PatientEGFR TKIs are classified into three main categories: first-generation inhibitors that target EGFR and its co-receptor HER2 and bind to it reversibly like ; second-generation irreversible inhibitors  and third-generation TKIs.Almost all trials for recurrent GBM patients ‘based on a high percentage of them expressing EGFR (40%–60%)’ evaluating EGFR TKIs utilised a continuous daily dosing schedule but also included unenriched participants . Some stGefitinib was evaluated in phase II single-arm trial that included 57 patients during the first recurrence and found that no radiological response was found in them, PFS-6 (PFS at 6 months) was 13% and OS was 39.4 weeks. There was no correlation between EGFRvIII mutation’s presence or absence of EGFR over-expression with the outcome . AnotherErlotinib was tested on 44 recurrent GBM patients and again the radiological response was not observed and PFS-6 was 3% . Later sResulting from the hypothesis of thinking of the possibility that stimulation of downstream pathways or activation of other survival pathways may cause EGFR resistance, subsequent studies evaluated the combination of EGFR TKI with agents that inhibit these signalling pathways . Many stSome of the finished and ongoing trials of anti-EGFR in rGBM are demonstrated in Depending on the pre-clinical findings that VEGF signal activation acts as a mediator of EGFR resistance, a combination of EGFR inhibitor and VEGF2R2 inhibitors was introduced to clinical trials to be tested , 39. Pha2/day for TMZ when combined. And the phase II results of the same trial found that the PFS-6 for the afatinib monotherapy arm, afatinib-protracted TMZ arm and combination arm was 3%, 23% and 10%, respectively. Different biomarkers were evaluated like EGFR, EGFRvIII, PTEN, pAKT and O6-methylguanine-DNAmethyltransferase (MGMT) but none of them correlated with the outcome although a non-significant association with EGFRvIII expression and better outcome were observed in patients treated with afatinib.As regards trials that evaluated second-generation EGFR, in phase I/II study of afatinib with or without protracted TMZ in patients with GBM after recurrence, provided that all patients received standard chemo-radiotherapy with TMZ at presentation . The phaDacomitinib single agent was evaluated in two phases II studies , 44. Thein vivo and in vitro and some of these inhibitors are approved for clinical trials. Imatinib (Gleevec) is one of these drugs which has an inhibitory effect on PDGFR. Although imatinib has activity in other malignancies, it did not show significant activity in HGG especially GBM in the recurrent settings. The tumour growth and OS remained unchanged whether it was used as a single agent or in combination with hydroxyurea [in vitro studies on GB cells found that imatinib increases the migration and invasion of GB cells, a fact that explains the previous failures of the drug [PDGFR is another member of the TKI family and is overexpressed in HGGs, especially in GBs . Plateleroxyurea , 49. Recthe drug .in vitro HGG cell lines [Tandutinib is another platelet derived growth factor receptor B (PDGFRB) inhibitor, which was evaluated in clinical trials in recurrent GBM and was found to have a little effect . AG1433 ll lines . In 2019ll lines   and their differentiation into endothelial cells, that is why it has a role in angiogenesis . It was in vitro more than TMZ [Although FGFR mutations are not frequent in GBM, several studies suggest that modification of the FGFR signalling pathway stimulates GBM progression and patient survival . Small mthan TMZ . In Decethan TMZ .The multi-targeted approaches may represent a good choice for effective selection of resistant tumour subtypes. Vandetanib is one of these multitargeted TKI  that was evaluated in clinical trials for patients with recurrent GBM. The drug was safe and tolerable but its antitumour effects were limited .in vitro and clinical trials, and PD173074 had good results in vitro. Sunitinib is a multi-kinase inhibitor of VEGFR, PDGFR, fms-related receptor tyrosine kinase 1 (FLT1), FLT1/kinase insert domain receptor (KDR), FLT3 and Ret Proto-Oncogene (RET) kinases with no encouraging results in patients with GBM [Another phase I clinical trial proved the safety of administrating vandetanib in combination with sirolimus in patients with rGBM . Two othwith GBM .Immunotherapy stimulates the immune system to recognise, target and get rid of tumour cells. Many trials have focused on developing immunomodulating therapies to restore the functional ability of different immune cells against neoplastic cells and with promising results in several tumour types including melanoma, lung cancer, urothelial tumours and colon cancer . Recent To leverage the natural immune response and restore its elimination ability of glioblastoma malignant cells, crucial understanding of the BBB and the tumour microenvironment and its complex interaction with the immune system is required In GBM, the BBB integrity is changed due to endothelial tight junctions damage reﬂecting molecular composition changes . The BBBIt is also believed that simultaneous ionising radiations’ exposure augments the strength of immunotherapy by: 1) a direct and indirect destruction to tumour cells causing cell deaths, 2) an alteration of the cancer stromal microenvironment and 3) activation of CD8 + T cells. Radiation induces activation of sequential biological mechanisms and biochemical events, including stimulation of interferon genes pathway and up-regulation of transforming growth factor β, leading to initiation of immune responses . TherefoCytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and PD-1 pathways operate at distinct stages of an immune-response, resulting in negative effects on T-cell activity . CTLA-4 Generally, anti-PD-1 drugs have a well-tolerated adverse event profile rather than anti-CTLA-4. It is suggested that a greater toxicity could be related to a better responses. Additional results are needed to decide whether this hypothesis is validated. Despite a different spectrum of adverse events, a safe and effective management of checkpoint inhibitors’ toxicity is mainly based on early sign and symptom recognition.Overall, objective responses to ICI in rGBM were seen in the following trials shown in In one retrospective analysis which investigated the effect of ipilimumab 3 mg/kg every 3 weeks with bevacizumab found an over-all response rate (ORR) of 31% in patients with rGBM . A phaseOngoing, three important phase III studies investigating the role of nivolumab in newly-diagnosed and recurrent glioblastoma are awaiting the final analysis publication, but the initial results are disappointing, checkmate-143 was the only one included patients with recurrent GBM, the other two trials included primary GBM only. The first trial was a randomised, open-label, phase III CheckMate-143 trial (NCT02017717), nivolumab monotherapy did not show significant change of OS in comparison to bevacizumab in rGBM patients . The secLastly unlike adult brain GBM, paediatric GBM is linked to genetic syndromes like Li-Fraumeni (Tp53) syndrome and bi-allelic mismatch repair genes syndrome (bMMRD), it is associated with increased incidence of malignancies in the first years of life; most common malignancies include glioblastoma, haematological malignancies and gastrointestinal tract cancers. BMMRD results from homologous germline mutations in one of the following genes . ‘DNA mismatch repair deficient (DMMRD) GBM’ has the highest mutational load in all cancers. Knowing that non-DMMRD cancers with high mutational load like melanoma, lung and bladder malignancies have high response to ICI lead to potential therapeutic opportunity to this subset of patients with GBM. One study did exome sequencing and neoantigen prediction on 37 dMMRD cancers (hyper-mutated) and compared them to adult and paediatric GBM patients, bMMRD GBM had high mutational load compared to sporadic paediatric and adult GBM. Based on this data, two siblings with dMMRD who had recurrent multifocal GBM treated with nivolumab and this treatment resulted in strong radiological response and significant prolongation of clinical response .DC is a highly specialised antigen processing and presenting cell which plays a vital role in initiating immune response and useful in immunotherapy as providing a way for cytotoxic T lymphocytes, natural killer cells and cytokines to kill tumour cell directly or indirectly . Severalp = 0.03) compared to the control group and a trend towards better PFS was also detected .Patients who received DC vaccine had significantly prolonged OS : 0.49 to 0.97, p = 0.14; I2= 0%) [Moreover, the incidence of side effects was comparable between patients treated with dendritic cell vaccine and control  .Some trials reported improvement from 13 month OS in the control group to 15.7–35.9 months OS in those patients who received DCs . So dataGene therapy simply is delivering either tumour suppressor genes to the tumour to regulate its growth by inhibiting oncogenes, or delivering an inactive pro-drug to be activated at the site of the tumour to a lethal compound. GBM gene therapy until now has used different delivery vectors like viral vectors, non-polymeric NPs and polymeric NPs. We will discuss briefly these vectors in the following section .It is the first and commonly used vectors in GBM like neurotropic retroviruses and adenovirus that possess a specific ability to infect neuron and glial cells like, herpes simplex virus1 (HSV-1) , 118. FiToca 511 is another retroviral vector which delivers tumour suppressor gene, cytosine deaminase (CD) and oral pro-drug Toca FC, CD enzyme converts 5-fluorocytosine to active 5-fluorouracil . A phaseAs regards adenoviral vectors, a phase I trial evaluated adenoviral vector with wild P53 (Ad-p53) transfected into tumour cells showed minimal toxicity but limited ability to penetrate tumour tissue , 124. AnDespite many studies of viral vectors in GBM, it resulted only in small survival advantage and the main challenge facing it is the ability to penetrate tumour tissue.Including polymeric and non-polymeric systems. Few non-polymeric vectors have been studied in GBM including liposomes, gold nanoparticles (NPs) and RNA NP. A transferrin receptor-targeted liposome vector SG-53 encapsulates P53 wild type plasmid DNA can cross BBB targeting glioblastoma cells leading to reduction in MGMT and can induce apoptosis in xenograft mice . So, SGTNu129, a spherical nucleic acid gold NP, contains siRNA targeting B-cell lymphoma 2 (BCL-2) like protein 12 which participates in tumour progression and resistance to apoptosis [RNA NPs, for example, tested to deliver anti-miR-21 locked nucleic acid sequences to inhibit miR21 in xenograft GBM models in mice, with significant tumour regression . RNA NP Based on current evidence and preliminary results of clinical trials assessing new treatment strategies, clustering patients with recurrent GBM according to their molecular profile involved in their disease development will defiantly help optimising decisions in clinical scenarios. This fact can be attributed to the widely varying molecular nature of this disease and its unique micro-environment. The awaited clinical trials results mentioned in this review  and 2 anAlso, there is clearly much work to be done to identify novel therapeutic targets and to develop strategies for treating advanced thyroid cancer. Pre-clinical data suggests a number of areas that could be developed in the coming years. For example, GSCs is believed by scientists to be the main cause of relapse as it causes re-growth of the tumour after eradicating the main bulk of it by surgery and chemo radiotherapy  Figure . It is iNotch pathway is important for determination of cell fate, proliferation, maintenance of cell quiescence, migration and regulate neural stem cell differentiation . It starCD 133-positive GSCs over-express genes like ID4 and FABP7 which are notch-pathway activator that leads to enhance infiltration ability of GBM . Due to This pathway is important during CNS development and plays a role in self-renewal, differentiation and neural stem cell development . HoweverRecurrent GBM is a fatal disease despite progression in understanding its molecular pathways and trying to target them. EGFR, PDGFR, FGFR and VEGFR inhibitors all showed few advantages although they are amplified or mutated in rGBM. YKL-40 targeting and multi-target of tyrosine receptor kinases may be beneficial. Immunotherapy has many limitations but it is still too early to decide that it is useless, especially DC vaccination which achieved significant advantage in some trials. A future direction towards targeting cancer stem cells pathways like notch and Wnt pathways could be an excellent solution for this disease.The authors have no conflicts of interest.The authors have not received any funding for this work.


Recent advances in epigenetic research have enabled the development of epigenetic clocks, which have greatly enhanced our ability to investigate molecular processes that contribute to aging and age-related disease. These biomarkers offer the potential to measure the effect of environmental exposures linked to dynamic changes in DNA methylation, including nutrients, as factors in age-related disease. They also offer a compelling insight into how imbalances in the supply of nutrients, particularly B-vitamins, or polymorphisms in regulatory enzymes involved in 1-carbon metabolism, the key pathway that supplies methyl groups for epigenetic reactions, may influence epigenetic age and interindividual disease susceptibility. Evidence from recent studies is critically reviewed, focusing on the significant contribution of the epigenetic clock to nutritional epigenomics and its impact on health outcomes and age-related disease. Further longitudinal studies and randomized nutritional interventions are required to advance the field. Epigenetic regulation has been identified as a key factor in aging  and is lMaintaining optimal nutritional status will have an important contribution to improving health outcomes with respect to age-related disease and healthspan. Several dietary factors are emerging as key modifiers of biological age and epigenetic clock models are helping to unravel the complex interplay of diet and age-related disease. Folate and related B-vitamins, essential cofactors in 1-carbon metabolism, the main metabolic pathway for generating methyl groups for DNA methylation , are emen = 5290; ≥50 y) from the Irish Longitudinal Study on Ageing (TILDA) (Wave 1) and (n = 5186) from the Trinity Ulster Department of Agriculture (TUDA) study reported the prevalence of deficient or low vitamin B-12 status (<185 pmol/L) as 12% and 11.8%, respectively, whereas the prevalence of deficient/low folate status was ≤15% , indicating that B-vitamins may play protective roles in age-related disease –10. Highwas ≤15% .Application of epigenetic clock models to epigenomic data from dietary interventions or longitudinal studies of dietary intake offer immense potential for elucidating how nutrition can modulate age-related disease processes and improve health outcomes. As the volume of studies investigating the effect of nutrients, in particular B-vitamins, on DNA methylation in health and disease begin to increase, understanding the essential role of these nutrients in modulating DNA methylation age and age acceleration are critical.The aim of this literature review was to address this gap by providing a critical overview of recent studies using the epigenetic clock to predict biological age and age-related disease and the application of nutrition in modifying these parameters. Further longitudinal studies and randomized nutritional interventions are required. Additionally, challenges with methodology are highlighted and opportunities presented for researchers to consider for advancement of the field of nutritional epigenomics and age-related disease.The literature search for this review was conducted by searching the Medline (via OvidSP) database and PubMed for articles published in English only and limited to human studies. Both medical subject headings and keywords were used in the search to identify articles with relevant information on aging, DNA methylation clock, diet, and vitamins. This was subsequently followed by forward citation searching or “snowballing” whereby relevant references were identified from key articles, followed up, and repeating the process with each article used to obtain more literature.Medical subject headlines included: exp DNA Methylation/, exp Dietary Supplements/, exp Micronutrients/, Vitamins/, Vitamin B Complex/, Food, Fortified/, genome-wide methylation.mp. or Methylation/, Aging/or Biological Clocks/or Epigenetic clock.mp. or DNA Methylation/. The keywords used were:  or (“methylation clock” or 450K or Methyl450 or Methylation450 or beadchip or “bead chip” or 800k or epic or EWAS or genome-wide or genomewide or epigenome-wide or epigenomewide). Finally, only those articles with emphasis on vitamins, diet, micronutrients, and methylation clocks were selected, and the relevant data was extracted for the review.DNA methylation is widely regarded as the most stable epigenetic mark involved in establishing patterns of gene expression and phenotype . It usuaDNA methylation has differing functions, depending on its location within the genome. It is usually associated with transcriptional gene repression at CpG-rich promoters; however, a mechanistic link between gene body methylation and active transcription is also suggested by enrichment of 5mC within gene bodies of transcribed genes . CpG sitDNA methylation modifications are dynamic, extensively reprogrammed in early development , 28, andOne-carbon metabolism provides a direct link between nutrients, mainly folate and related B-vitamins, and DNA methylation  and therMTHFR 677TT genotype (Methionine regenerated from homocysteine serves as a precursor for SAM and is then converted to S-adenosylhomocysteine (SAH) during the methyl transfer . The celgenotype , 36. It genotype , 38. FurPerturbations in 1-carbon metabolism may occur through low intake of nutrients involved in 1-carbon metabolism , malabsoMTHFR C677T polymorphism results in reduced MTHFR enzyme activity in individuals with the 677TT genotype which encodes a thermolabile enzyme  found strong associations of MTHFR c665C > T polymorphism with blood concentrations of total plasma homocysteine and serum folate  to regulate the methylation levels of their own gene promoter or trans-regulatory elements (trans-meQTL) regulating methylation of other genes. For example, 57 CpGs were differentially methylated depending on the genotype of 6 1-carbon metabolism genes . The MTHFR rs1801133 single nucleotide polymorphisms (SNPs) (responsible for the C677T polymorphism) was shown to act as a trans-meQTL regulatory element in breast tissue associated with lower methylation of 5 CpGs   involved in 1-carbon metabolism and homocysteine metabolic pathways could mediate the CVD risk conferred by low dietary intake of B-vitamins. Furthermore, using highly robust and comprehensive microarray methods, several large EWAS have shown that supplementation with B-vitamins, predominantly folate and vitamin B-12, or dietary intake of these nutrients modulate DNA methylation at the genome-wide level in older adults  in a study involving participants from the Melbourne Collaborative Cohort Study (MCCS)  . Additioy (MCCS) . For exaStudies of monozygotic (MZ) twins have shown that although twins are epigenetically indistinguishable during the early years of life, older MZ twins exhibited remarkable differences in their epigenome, indicating that patterns of epigenetic modifications in MZ twin pairs diverge as they become older . Entropi. Each clock is derived by a linear regression algorithm that trains against the chronological age of sample donors and selects a set of CpGs, determining the weighted contribution of each CpG in the set to produce a DNA methylation age (DNAm Age) that correlates accurately with chronological age. The first of these to have a major impact was the Horvath clock  exceeds chronological age, age acceleration (AgeAccel) is said to be experienced and this measure is perhaps of most interest to scientists and clinicians studying aging and disease. AgeAccel is defined as the residual from regressing DNAm Age on chronological age, where a positive value indicates that epigenetic age is greater than expected. Horvath further characterized epigenetic age acceleration as either intrinsic (IEAA) or extrinsic (EEAA) epigenetic age acceleration. IEAA is a measure of age acceleration that is independent of age-related changes in the cellular composition of blood, whereas EEAA captures the age-related functional decline of the immune system and accounts for changes in blood cell composition such as the decrease of naïve CD8+ T cells and the increase in memory or exhausted CD8+ T cells .To investigate biological age more extensively and discriminate morbidity and mortality more accurately among individuals of the same chronological age, recently developed clocks have been trained on age-related and disease phenotypes in combination with chronological age. Two of the most robust are the DNAm Phenotypic Age predictor (DNAm PhenoAge)  and the The CpGs which are included in the clock algorithms are widely distributed across the genome and do not appear to be clustered in or near any particular genomic feature or any particular regulatory region. The methylation clocks and associated challenges have been extensively reviewed recently , 82. It Epigenetic clocks are not linear across the lifespan. Many of the current epigenetic clock studies have been conducted in adults, and as a result, many show impressive accuracy across most tissues during middle age . In lateP = 3.35 x 10-11) and both higher EEAA  and AgeAccelPheno  are associated with elevated systolic blood pressure (P = 6.2 x 10-24) and time-to-cancer  and also demonstrates a strong association with hypertension   .The influence of diet in the etiology of many age-related diseases is well established and the advent of epigenetic clocks has brought a novel approach to confirm diet as an important health factor . EpigeneP = 0.012) than in females . Furthermore, epigenome-wide methylation results from the B-Vitamins for the PRevention Of Osteoporotic Fractures (B-PROOF) study, intervening with daily folic acid and vitamin B-12 supplements in a robust 2-y RCT  and, crucially, did not include a control arm, suggestions of biological age reversal were found in all 4 robust methylation clocks available, and in each individual. This study was the first to indicate that potential regression of multiple aspects and biomarkers of aging, including immune function, was possible in humans  trial used a cocktail of drugs comprising recombinant human growth hormone (rhGH) to prevent or reverse signs of immunosenescence in a 1-y pilot trial of healthy men aged 51–65 y showing a regression of epigenetic age of −2.5 y on average . Althougn humans .Although itself not a dietary factor, it is interesting to note that growth hormone, the supplement chosen in the aforementioned epigenetic age reversal trial, has been noted to perturb mRNA and protein concentrations of DNMT1  and it hDespite their obvious strengths, DNA methylation-based clocks are unlikely to replace existing clinical biomarkers and measurements such as blood pressure, walking speed, and grip strength, which are cost-effective and easy to perform. The cost of measuring DNA methylation age prevents the standard adoption of this method, at least until it becomes more affordable. In fact, GrimAge is 61% more accurate than chronological age and 46% more accurate than previously reported epigenetic clocks in predicting time to coronary heart disease. However, despite this significant advancement, neither chronological nor GrimAge are entirely accurate estimators of coronary heart disease and further work is required to determine their role as predictors of cardiovascular and other disease outcomes.Despite the growing interest in the role of diet in influencing DNA methylation and age-related disease, most previous studies in humans were not designed with DNA methylation as the primary outcome, resulting in limited data to provide concrete evidence linking the diet to DNA methylation. The methodological aspects of appropriate study design for the investigation of diet and DNA methylation will be discussed further.The study design utilized as well as dietary or biochemical data collected are critical when investigating the link between nutrient intake or status and DNA methylation. The majority of studies so far are observational and have provided inconsistent evidence for the role of dietary factors, especially B-vitamins, in modulating DNA methylation, perhaps owing to inconsistencies in study design and choice of assay . AlthougMethods to examine DNA methylation have evolved over the years and have become more sophisticated. Although commonly used methods including HPLC-UV, LC-MS/MS, methyl acceptance assay, and pyrosequencing are still useful in analyses of DNA methylation, novel technologies such as the Infinium HumanMethylation450K BeadChip array (450K) or the Infinium MethylationEPIC BeadChip (850K) microarray provide higher resolution for analyzing DNA methylation on a genome-wide scale , 105. Alminfi (https://github.com/sjthursby/CandiMeth) are also making it easier for those with little bioinformatics experience to look at methylation across the genome in samples for which array data is available.Advantages of using these approaches include the production of large datasets that can be analyzed by streamlined analytical pipelines, providing important information on the epigenome-wide landscape. Several sophisticated computational tools and software are available for the analysis and interpretation of large EWAS datasets. The relevant concepts, computational methods, and software for the analysis and interpretation of large DNA methylation data as well as statistical considerations have been thoroughly reviewed by Bock and colleagues , 108. Thminfi , RnBeadsminfi , The Chiminfi , and metminfi . Furtherminfi , DMRcateminfi , and IMAminfi  are avaiNutritional epigenomics has highlighted diet as a critical factor with the potential to influence both healthspan and lifespan. Novel insights into how perturbations in 1-carbon metabolism influence DNA methylation and data from epigenome-wide studies of nutrition interventions offer promising insights to understanding how diet impacts the methylome during healthy aging and disease. Epigenetic clocks provide an exciting additional insight into how preventive and treatment strategies may increase the healthspan of an aging global population. Despite the heightened research interests in nutritional epigenomics, the field is still beset with several methodological challenges, which greatly impact the quality of evidence currently available. The population under study must be extensively characterized to identify and exclude possible confounding factors. Robust study designs, which utilize randomization and measure appropriate biomarkers, are required to clarify the factors underlying epigenetic aging. Replication and validation of findings in multiple independent cohorts are essential to reduce reporting of false positive findings. Epigenetic clocks described here have sampled individuals from a wide spectrum of ages. A DNA methylation clock which focuses on older people or those with specific diseases could help to more accurately predict age-related disease and help to identify factors which delay or prevent this progression. Improvements in estimating time to disease have been made in the latest GrimAge clock, which is significantly more predictive than chronological age in estimating time to various diseases; however, much additional research is required to advance our knowledge and understanding in relation to coronary heart disease. Longitudinal studies offer the important advantage of tracking individuals over extended periods to enable the identification of factors which influence the diagnosis and treatment of disease, making these studies particularly valuable for clarifying whether observed changes in DNA methylation are a result of disease or have a causal role. A better understanding of the DNA methylome during aging will offer the opportunity to promote healthy aging and identify nutritional interventions which delay or prevent age-related disease in order to influence public health outcomes and policies.


Hyperdynamic therapy, also called triple-H therapy, is the standard treatment and prophylaxis for aneurysmal-associated vasospasm. In patients who are able to tolerate cardiopulmonary stressors induced by this therapy, it is of benefit as a modality for prevention and treatment of delayed ischemic neurologic deficit. However, it can be a cause of significant cardiopulmonary or neurologic sequelae. In rare cases, it can be associated with posterior reversible encephalopathy syndrome (PRES), secondary to prolonged vasopressor and hypertensive therapies.We present the case of a patient with right-sided aneurysmal-associated vasospasm who, after 10 days of triple-H therapy, experienced a seizure and was found to have left-sided PRES. Right-sided vasospasm served as a protective mechanism from triple-H therapy-associated PRES. It presented a treatment conundrum due to contradictory perfusion requirements. Hypertensive therapy was curtailed and in efforts to preserve local cerebral perfusion and vasodilation, local therapy with intrathecal nicardipine was initiated. We present our case, a review of the literature, and management considerations.Therapies that have conventionally functioned as second line treatments for aneurysmal subarachnoid hemorrhage  may be beneficial as earlier treatments in the setting of vasospasm given the systemic difficulties and complications associated with HHH therapy in patients with PRES. Hyperdynamic therapy is a conventional treatment for aneurysmal subarachnoid hemorrhage-associated cerebral vasospasm. Hypertension, hypervolemia, and hemodilution are used to ensure adequate cerebral perfusion in patients with severely constricted cerebral vasculature and abnormal autoregulation. Implementation of hyperdynamic therapy however does come with risks. Pulmonary edema, myocardial ischemia, hyponatremia, indwelling venous catheter-related complications, cerebral hemorrhage, and cerebral edema are all known sequelae . A less A 33-year-old previously healthy woman presented after a sudden loss of consciousness and reported seizure. Upon arrival to the hospital, she complained of a moderate headache with severe neck pain with a Hunt Hess Score of 2. CT imaging demonstrated subarachnoid hemorrhage Fig.  from a rHer hospital course was complicated by severe vasospasm of the right anterior and middle cerebral artery. On post-bleed day 5, she began to experience visual auras and complain of blurry vision. Transcranial Doppler velocities were notably increased. She underwent endovascular intervention and administration of intra-arterial (IA) verapamil resulting in improved neurologic function. Triple-H therapy was initiated and her symptoms improved despite persistently elevated right transcranial doppler (TCD) velocities. On post bleed day 15, after 10 days of blood pressure augmentation to goal systolic blood pressures of 180 mmHg, she had an acute change in neurologic exam with a mixed aphasia and disorientation. Blood pressure was increased to a goal of 200 mmHg with no significant improvement. That same day, she experienced a complex partial seizure that progressed to a generalized tonic-clonic seizure. A repeat CTA demonstrated persistent right MCA spasm. An angiogram was performed  to prevent DIND. PRES is an uncommon but clinically significant syndrome that has been described in numerous case reports. The current hypothesis for a mechanism of PRES involves capillary leak in the setting of damaged endothelium. At high pressures, the intrinsic cerebral autoregulatory system fails and vessels are forcibly dilated leading to arteriolar distention and rupture of vascular endothelial cell connections. Extravasation of plasma proteins induces a vasogenic edema –5. EtiolManagement of PRES and cerebral vasospasm together presents a treatment challenge. The treatment for each is in direct conflict with the pathophysiology of each other. We presented a patient with unilateral vasospasm of the right ACA and MCA, with complications of vasogenic edema arising in the contralateral hemisphere. While most reports of PRES demonstrate bilateral posterior occipito-parietal hemispheric involvement, a few case reports offer similar reports of unilateral vasospasm and contralateral vasogenic edema , 8. As DA literature review was performed of hyperdynamic therapy-associated PRES in patients with aSAH. The relative paucity of literature is likely secondary to the lack of standard MRI imaging performed in aneurysm patients. Awori et al. attempted to extrapolate common risk factors for patients with aSAH and triple-H-associated PRES. However, upon an extensive literature review, the cases are too few in number to establish significant risk factors . As demoDhar et al. present a very nearly identical case of a patient with right sided vasospasm receiving triple-H therapy . Workup Voetsch et al. present a similar case of a patient with right MCA spasm and DIND requiring hyperdynamic therapy and intra-arterial nicardipine . AugmentA review of the literature was performed to synthesize a possible treatment paradigm , 10–14. Dhar et al. address the important concept that perhaps more focal treatments in the setting of PRES and significant vasospasm may be more beneficial than the conventional systemic treatments of HHH therapy . Our patA review of the English literature found eight studies that reported on the use of intraventricular or cisternal administration of nicardipine for the treatment of aneurysmal SAH-associated vasospasm Table . Few of Currently other agents including intravenous endothelin-1 receptor antagonists and intrathecal nimodipine, and milrinone are being studied as agents for the treatment of vasospasm. Endothelin receptor antagonists demonstrated potential in animal studies with reduction rates of vasospasm and increase in average vessel diameter; however, an updated meta-analysis has found that it does not demonstrate overall improved outcomes . LevosimThis case report and literature review suggest that the management paradigm of aSAH in the context of PRES should shift towards more focal modalities including intra-arterial vasodilators and intrathecal agents before reliance on HHH therapies due to the possibility of cardiopulmonary and neurologic complications. Early diagnosis of PRES in neurosurgical patients is essential for therapy modification to ensure better outcomes.


Breast cancer is the most commonly diagnosed cancer among women worldwide, and early detection remains a principal factor for improved patient outcomes and reduced mortality. Clinically, magnetic resonance imaging (MRI) techniques are routinely used in determining benign and malignant tumor phenotypes and for monitoring treatment outcomes. Static MRI techniques enable superior structural contrast between adipose and fibroglandular tissues, while dynamic MRI techniques can elucidate functional characteristics of malignant tumors. The preferred clinical procedure—dynamic contrast-enhanced MRI—illuminates the hypervascularity of breast tumors through a gadolinium-based contrast agent; however, accumulation of the potentially toxic contrast agent remains a major limitation of the technique, propelling MRI research toward finding an alternative, noninvasive method. Three such techniques are magnetic resonance spectroscopy, chemical exchange saturation transfer, and non-contrast diffusion weighted imaging. These methods shed light on underlying chemical composition, provide snapshots of tissue metabolism, and more pronouncedly characterize microstructural heterogeneity. This review article outlines the present state of clinical MRI for breast cancer and examines several research techniques that demonstrate capacity for clinical translation. Ultimately, multi-parametric MRI—incorporating one or more of these emerging methods—presently holds the best potential to afford improved specificity and deliver excellent accuracy to clinics for the prediction, detection, and monitoring of breast cancer. The American Cancer Society has estimated that within the United States in 2020, a total of 276,480 females will be diagnosed with breast cancer and 42,170 are likely to die from the disease  have enabled clinicians to detect numerous biomarkers of breast cancer and to monitor the patient's response to chemotherapy. Studies have shown a correlation between these MR-based biomarkers and histopathological features of tumors. This linkage could provide a powerful technique for monitoring the progression of the disease and the patient's response to chemotherapy –21.1 and T2 are common MRI sequences exploiting the differences in the relaxation times of protons within the tissue under examination. T1 provides longitudinal relaxation time while T2 provides transverse relaxation time for a set of protons. By exploiting the distinct T1 and T2 relaxation properties of various tissues, static MRI provides superior structural contrast between adipose and fibroglandular tissues and remains a mainstay for risk analysis, tumor detection, and treatment monitoring. Dynamic MRI techniques go one step further, elucidating functional characteristics of malignant tumors. Dynamic contrast enhanced (DCE) MRI detects T1 changes in tissues over time immediately following bolus administration of a gadolinium-based contrast agent; the hypervascularity of breast tumors results in altered uptake and washout rates, and the unique time-intensity curve can distinguish malignant from benign tumors. Recent concerns regarding lasting gadolinium accumulation and toxicity, however, have impacted patient's assent to undergo techniques requiring gadolinium-based contrast agent, including DCE MRI, and research efforts have renewed to design alternative, noninvasive methods. One leading contender is diffusion weighted imaging (DWI), which already has proven valuable as an adjunct to DCE by improving combined sensitivity. DWI can elucidate tissue properties based on the Brownian motion of water. Since diffusivity differs inside and outside cells, the pattern of tissue morphology can be established based on the restriction of motion of water molecules in densely packed cells  can be enhanced utilizing the background parenchymal enhancement (BPE) technique. This technique can identify specific regions of differences within normal mammary tissues over others which facilitates a wider prediction of the tumor microenvironment and its possible changes. These features augment the specificity and sensitivity of MRI and is advantageous in reducing false positive results. BPE is assessed by four qualitative BI-RADS categories: minimal (<25% of glandular tissue demonstrating enhancement), mild (25-50% enhancement), moderate (50-75% enhancement), or marked (> 75% enhancement). In 2011, King et al. concluded that increased BPE is strongly predictive of breast cancer odds , however1 field. Given the off-center positioning of the breasts within the body coil, and the asymmetric loading presented by the torso, transmit B1 inhomogeneity is prone to worsen at higher magnetic fields. At 3T, the body coil has been reported to produce up to 50% error in tip angle  and 3T scanners typically include a built-in body coil for transmitting radiofrequency (RF) pulses, i.e., the Bip angle , which sip angle  and T1 m mapping . These i mapping .Irrespective of the scanner's magnetic field strength, receive array coils improve signal-to-noise ratio (SNR) throughout the breast compared to utilizing the body coil to receive the RF signal . A varie2. DWI encodes water diffusion in one to three orthogonal directions (each direction corresponding to a gradient direction) and assumes unrestricted isotropic diffusion. The resulting apparent diffusion coefficient (ADC) quantifies the mean bulk diffusion per pixel and is an established quantitative surrogate for tissue cellularity. While the cell membranes and vascularity within tumors preclude unrestricted water motion, the simple DWI model accurately represents voxels (single data-specific locations on a 3D tissue construct) with high water content and low cell density and the resulting hypo intensity within breast tumors remains informative. This effect is illustrated in As a noninvasive MRI technique, diffusion weighted imaging (DWI) detects the bulk diffusion of water within tissue and offers substantial advantages in visualizing and differentiating tumors based on their vascularization patterns. The amount of diffusion weighting applied to the MRI signal is set by the operator-defined b-value, with zero indicating no diffusion weighting  and commTraditional spin-echo DWI relies on a conventional single-shot echo planar imaging readout prone to produce ghosting artifacts that hinder image quality. Other readouts such as spatio-temporal encoding mitigate ghosting artifacts at the expense of added noise . UltimatHigher-resolution DWI may be attained by reducing the field of view, which focuses on a target region within the breast. With this technique, 0.8-mm in-plane resolution can be resolved at 3T, and the resulting ADC maps provide greater detail facilitating the assessment of tumor morphology . ImagingObtaining consistently high-quality breast DWI is one of the challenges that current studies are targeting to overcome. The American College of Radiology Imaging Network (ACRIN) 6698 clinical trial has shown that ADC can be measured with excellent repeatability and reproducibility in a multi-institution setting using a standardized protocol and QA procedure . An MRI Diffusion tensor imaging (DTI) builds on the DWI technique by increasing the number of diffusion-encoding directions, thus enabling the calculation of anisotropic diffusion. While DWI characterizes isotropic diffusion within each voxel as a sphere, DTI employs at least six gradient directions and geometrically represents anisotropic diffusion within each voxel as an ellipsoid. The diffusion tensor, a matrix of directional diffusion coefficients, is established for each voxel based on the diffusion rates detected concurrent with each gradient configuration. Given the directionality of resulting diffusion information, DTI can provide additional insight into tissue microstructure through mean diffusivity—the DTI analogue to the ADC in DWI—and various anisotropy measures which provide critical information such as a tissue's vascularity, density, and cellular features. Such anisotropic features include fractional anisotropy, radial anisotropy, the individual diffusion coefficients, and the maximal anisotropy index. A mean diffusivity map is shown in While there is a consensus across studies that mean diffusivity is significantly lower in malignant tumors compared to benign lesions, there are conflicting results regarding the diagnostic utility of the anisotropy indices . Some reRemarkably, DTI metrics have been shown to have distinctive correlations with breast cancer subtypes. Onaygil et al. found statistical significance between several anisotropy indices in estrogen receptor positive and negative (ER+ and ER-) breast cancers, and separate correlations with the levels of Ki-67, a biomarker for cellular proliferation, while Ozal et al. reported identifying distinct correlations between various DTI metrics and levels of breast cancer prognostic factors: ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), Ki-67, and lymphatic invasion in invasive tumors , 55.The challenge of achieving excellent repeatability and reproducibility across sites remains ongoing with breast DTI. Studies indicate the ADC can be reproduced with more accuracy compared to DTI anisotropy metrics such as fractional anisotropy , 57.Notably, the technical development that drove substantial improvements into the DTI technique was largely motivated by the quest to map neuronal tracks of white matter in the brain. Preliminary studies reconsidering the utility of DTI for breast cancer have investigated utilizing DTI for breast tractography . Given t2, and at least 15 diffusion gradient directions, the diffusion kurtosis imaging technique can map multiple structures within a single voxel, e.g., crossing white matter fibers in the brain. In the context of breast imaging, diffusion kurtosis imaging is sensitive to intracellular structures such as membranes and organelles , on the order of b = 1000–3000 s/mmganelles  and, in ganelles . Importaganelles . A mean While technically also a perfusion imaging method, the intravoxel incoherent motion model adds additional quantitative terms to account for microvascularity. Accordingly, intravoxel incoherent motion has the potential to discern both tissue diffusivity and microcapillary perfusion without the need for contrast agents . Additio2, presenting a challenge given the lower SNR inherent with high b-value acquisition.Many other advanced diffusion methods have been proposed with the goal of probing intravoxel heterogeneity and cellularity; a review of several such methods and their suitability for cancer imaging was recently published by Tang and Zhou . General1H) MRS offers the greatest sensitivity and simplest data acquisition. Elevated levels of choline-containing compounds indicate cell membrane turnover and are a biomarker for malignant breast tumors  suggests this biomarker offers 73% sensitivity and 88% specificity (1H MRS have been associated with increased resistance of cancer cells to radiation-induced cell death (Magnetic resonance spectroscopy (MRS) provides a localized snapshot of the biochemical makeup of tissue . Proton t tumors . All chocificity . MoreoveThe recent ACRIN 6657 MRS clinical trial aimed to predict response to NAC with tCho single-voxel MRS; the results were inclusive, with only 29/119 subjects providing useable data . A prima1H MRS could be an effective addition to a mpMRI protocol  MRS separates distinct choline compounds, specifically phosphorylcholine and glycerophosphocholine, otherwise overlapped as tCho on the 1H spectrum. The role of phosphocholines in breast cancer metabolism is of broad interest  MRS can provide additional information such as the composition of breast fat and correlations that may predispose to cancer. Performing in vivo13C MRS is difficult for many reasons, including low natural abundance, low (in comparison to 1H) sensitivity, J-coupling bonds between 1H and 13C atoms that obfuscate spectral peaks, and unique hardware instrumentation requirements. The preferred 13C MRS experiment, applying broadband proton decoupling, requires RF coils operating at both the 1H and 13C frequencies; the 1H channel is used for scout imaging as well as to transmit proton-decoupling pulses across the J-coupled chemical shift band  was first introduced by Wolff and Balaban ; the MT 23Na) is abundant in the body and, unlike other non-proton nuclei that yield spectra for chemical quantification, sodium has no chemical shift dispersion and instead produces images  is frequently missed by DCE MRI and has been shown to associate with certain patterns of breast calcifications  images a low-frequency acoustic wave as it propagates throughout tissue. By calculating the local complex sheer modulus, MRE can characterize biomechanical properties of breast tissue including differences in stiffness. The initial aim of employing MRE for breast cancer was to differentiate benign lesions from malignant tumors; the more liquid-like behavior of malignant tumors provided sufficient MRE contrast to achieve this aim . More re1 and T2 in a bilateral configuration  to reconstruct the conductivity and electrical permittivity of tissue  into output categories that share similar contents . On the other hand, supervised learning aims to classify data that have been assigned with ground truth labels ; example models include regression methods and support-vector machines (SVM).As an artificial intelligence tool, machine learning may best be introduced to the clinic through structured use cases; in the case of breast cancer, these may include the application of artificial intelligence to identify suspicious microcalcifications  and, givNeural networks are machine learning models that consist of multiple interconnected layers. The study of neural networks is termed deep learning. Lately, deep learning has surpassed traditional image processing models in the segmentation and detection of novel imaging biomarkers . Convolu2-weighted breast MRI predicted NAC response with high accuracy  were insufficient to properly evaluate the fractional order calculus model (1H MRS, current issues surrounding inter-site reproducibility of single-voxel MRS may be mitigated through automated voxel placement or full 3D magnetic resonance spectroscopic imaging (Both DWI and  in vivo . Advance in vivo  can derius model . Regardi imaging , particu imaging . Continu imaging , but mor imaging .Finally, the rapidly advancing field of machine learning will facilitate more impactful applications for breast cancer detection and management, likely improving specificity, positive predictive value, and differentiation of tumor subtypes through MRI. Moreover, simultaneous assessments of biomarkers and their genomics data through radiogenomics is likely to prove instrumental in the future as we advance toward precision health or personalized medicine and simultaneously decrease the MRI associated false positive rates.AC, XL, and JR drafted the manuscript. XL generated new figures. All authors contributed to manuscript revision 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.


Esophagectomy is the mainstay of curative treatment for most patients with a diagnosis of esophageal cancer. This procedure needs to be optimized to secure the best possible chance of cure for these patients. Research comparing various surgical approaches of esophagectomy generally has failed to identify any major differences in long-term prognosis. Comparisons between minimally invasive and open esophagectomy, transthoracic and transhiatal approaches, radical and moderate lymphadenectomy, and high and moderate hospital volume generally have provided only moderate alterations in long-term survival rates after adjustment for established prognostic factors. In contrast, some direct surgeon-related factors, which remain independent of known prognostic factors, seem to influence the long-term survival more strongly in esophageal cancer. Annual surgeon volume is strongly prognostic, and recent studies have suggested the existence of long surgeon proficiency gain curves for achievement of stable 5-year survival rates and possibly also a prognostic influence of surgeon age and weekday of surgery. The available literature indicates a potentially more critical role of the individual surgeon’s skills than that of variations in surgical approach for optimizing the long-term survival after esophagectomy for esophageal cancer. This finding points to the value of paying more attention to how the skills of the individual esophageal cancer surgeon can best be achieved and maintained. Careful selection and evaluation of the most suitable candidates, appropriate and structured training programs, and regular peer-review assessments of experienced surgeons may be helpful in this respect. Although it is well-established that neoadjuvant chemotherapy or chemoradiotherapy can improve survival,1This review evaluates the existing literature that examines how variations in surgical and surgeon-related factors may influence the long-term survival of patients who undergo surgery for esophageal cancer.Efforts to improve the surgery aimed at offering better long-term survival in esophageal cancer have been devoted mainly to the role of various surgical approaches and techniques. The optimal surgical approach in the treatment of esophageal cancer is a matter of much debate. This review evaluates the existing literature addressing three key questions in relation to long-term survival: (1) Is minimally invasive esophagectomy better than open surgery? (2) Is the transthoracic approach better than the transhiatal approach? (3) Is extensive lymphadenectomy better than moderate lymphadenectomy?Only two European randomized controlled trials have compared long-term survival after minimally invasive and open surgery for esophageal cancer. The Dutch TIME-trial showed the 3-year overall survival in the totally minimally invasive esophagectomy group (59 patients) to be 40.4% versus 50.5% in the open esophagectomy group 56 patients), but this indication of a difference failed to reach statistical significance in the multivariable regression model . patients–10A recent meta-analysis including 55 studies with 14,592 patients showed an 18% reduction in 5-year all-cause mortality after minimally invasive versus open esophagectomy.8n = 106) with transthoracic esophagectomy patients (n = 114) showed no difference in overall or disease-free survival.,A Dutch RCT comparing transhiatal patients .16A recent meta-analysis of 26 studies and 48,612 patients showed that a higher lymph node yield was associated with decreased overall mortality among all esophagectomy patients  and in a subset analysis of patients receiving neoadjuvant therapy ,Comparisons of minimally invasive surgery with open surgery, transthoracic with transhiatal approach, and extensive with moderate lymphadenectomy generally have provided minor alterations in long-term survival after adjustment for prognostic factors. Although research efforts aimed at improving the overall long-term survival after esophageal cancer surgery have focused mainly on comparing various surgical approaches, these comparisons generally have resulted in minor or no differences.Compared with assessments of surgical approach, the influence of direct surgeon-related factors in relation to survival after esophageal cancer surgery has been studied less often. Four surgeon-related questions may be of special relevance for the chance of long-term survival after esophagectomy: (1) What is the role of annual hospital volume versus surgeon volume? (2) How does the learning period influence the prognosis? (3) Does the age of the surgeon influence the long-term outcomes for patients? (4) Does timing of the surgery during the working week or holiday periods influence the long-term survival after surgery for esophageal cancer?Hospital Versus Surgeon Volume. Studies have consistently identified the benefits of high hospital and surgeon volumes in reducing short-term mortality after esophageal cancer surgery.22,27Proficiency Gain Curves. In more recent years, increasing attention has been paid to adverse patient outcomes during the time when a surgeon gains proficiency as an independent surgeon in undertaking a complex cancer surgery, which can be modeled as a proficiency gain curve (“learning curve”). Multi-center and national cohort studies have identified highly variable lengths in the period of proficiency gain as surgeons perform open and minimally invasive esophagectomy for short-term outcomes Table .23,24 A 23Surgeon Age. To our knowledge, only one study to date, a nationwide Swedish cohort study of 139 surgeons, has evaluated surgeon age in relation to long-term survival after esophagectomy for cancer. Younger and older surgeons tended to have worse long-term outcomes than those with ages in-between, indicating a short “surgeon age peak performance” between 52 and 56 years, which remained after adjustment for established prognostic factors .28,33Timing of Surgery. A population-based study from Sweden (1748 patients) indicated that surgery on a later weekday reduces long-term survival after surgery for esophageal cancer after adjustment for relevant confounders.32Summary of Surgeon-Related Factors. Surgeon volume seems to have a substantial influence on the chance of long-term survival after esophagectomy for esophageal cancer. Proficiency gain curves may be long before surgeons achieve stable long-term survival for their patients, possibly longer if the training period is less intensive and if the surgeon is older. Surgeon age and weekday of surgery also might be prognostic factors, although evidence is too limited for any strong conclusions to be drawn.The existing literature has generally shown only small or no differences in long-term survival depending on variations in the surgical approach for esophageal cancer, whereas some direct surgeon-related factors may have a greater impact on the life expectancy for these patients.Current evidence indicates a critical role for the skills of the individual esophageal surgeon, probably more than for the surgical approach when it comes to survival in esophageal cancer. From an esophageal cancer patient point of view, undergoing surgery by a highly qualified and experienced high-volume surgeon optimizes the chance of cure independently, it seems, of whichever surgical approach the surgeon prefers. These findings indicate a need to pay more attention to how the skills of the individual esophageal cancer surgeon are achieved and maintained. These challenges are not specific to esophageal cancer surgery but are more widespread with the introduction of advanced minimal access techniques, increasing the complexity and potential patient harm during the surgical learning curve.Careful selection of the most suitable candidates seems justified. Appropriate and structured training programs at specialized and large centers for conducting these demanding procedures are warranted. Introduction of objective assessments with feedback of anonymous peers  may help in assessing and maintaining the required surgical standards of the individual esophageal cancer surgeon.To reduce patient harm during the period of proficiency gain for surgeons, well-structured training programs before independent practice of esophagectomy are urgently needed. Future surgical training programs may be based on competency-based assessments before initiation of independent practice to replace current training systems largely based on the number of procedures, years of training, and subjective trainers’ opinions. This type of competency-based training curriculum should be followed by a period of mentorship and continual technical proficiency assessment to support esophageal surgery specialists during the initial phases of their independent practice.,Centralization of esophagectomy for cancer to high-volume surgeons has clearly been shown to improve survival.19,36Several ongoing esophageal cancer trials are focused on optimizing neoadjuvant oncological therapy.34The importance of controlling surgical quality in RCTs also has been demonstrated, with influence on mortality and locoregional recurrence.Esophagectomy is the mainstay of curative treatment for most patients with a diagnosis of esophageal cancer. Generally, RCTs comparing various surgical approaches of esophagectomy have failed to identify any major differences in long-term prognoses. Recent large cohort studies have identified some direct surgeon-related factors that seem to strongly influence the long-term survival in esophageal cancer, including surgeon volume and the proficiency gain period, and possibly also surgeon age and timing of surgery. Future research is necessary to evaluate modification of these factors and to determine how this in turn may improve long-term survival in esophageal cancer both inside and outside a clinical trial setting.


The ubiquitin proteasome system (UPS) is the main cellular degradation machinery designed for controlling turnover of critical proteins involved in cancer pathogenesis, including hematological malignancies. UPS plays a functional role in regulating turnover of key proteins involved in cell cycle arrest, apoptosis and terminal differentiation. When deregulated, it leads to several disorders, including cancer. Several studies indicate that, in some subtypes of human hematological neoplasms such as multiple myeloma and Burkitt’s lymphoma, abnormalities in the UPS made it an attractive therapeutic target due to pro-cancer activity. In this review, we discuss the aberrant role of UPS evaluating its impact in hematological malignancies. Finally, we also review the most promising therapeutic approaches to target UPS as powerful strategies to improve treatment of blood cancers. Post-translational modifications are an intriguing mechanism through which cells regulate protein homeostasis. Protein ubiquitination is the major post-translational modification that is responsible for either protein biological function alteration or specific protein degradation by the 26S proteasome . A multiSeveral studies have described the higher activity of proteasome and its aberrant role in hematological malignancies making leukemic cells particularly sensitive to proteasome inhibitors (PIs). The approval of bortezomib (Velcade) (BTZ), the first best-in-class PI for the treatment of multiple myeloma (MM), clearly opened up new perspective for the treatment of other malignancies such as mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). Therefore, research and development of new PIs, as well as the studies on PIs treatments combined with other agents used in the therapeutic setting of leukemia and lymphoma patients, represent an active and promising research area.In this review, we focus on how deregulation of the UPS components contributes to tumorigenesis and discuss the status quo of the different therapeutic molecules targeting UPS in hematological malignancies.The 26S proteasome is a macromolecular complex consisting of a catalytic 20S and regulatory 19S subunits responsible for the degradation of about 80% of the targeted intracellular proteins by an elegantly controlled mechanism. Cellular proteins undergo ubiquitin proteasome-mediated degradation fundamentally through two sequential steps. First, proteins are tagged with monomers or polymers of the ubiquitin chain; next, they are degraded by the 20S proteasome subunit . T. T134]. Despite their success, the first generation of PROTACs suffered from low potency, mainly due to their poor permeability and high protease susceptibly commonly associated with peptide-based therapeutics. However, the conception of specific ligands for E3 ligases has highly improved this technology, providing more drug-like molecules. The second generation of PROTAC is currently a very active research field, and many compounds have been synthesized so far; thus, it is impossible to cover all efforts for describing all PROTAC compounds. In this review, we will focus on some most promising examples listed below applied to leukemia therapy.Tyrosine kinase inhibitors (TKIs) are currently used to treat BCR-ABL-driven chronic myeloid leukemia (CML). However, point mutations in the tyrosine kinase domain of BCR-ABL have often occurred with consequent drug resistance insurgence. For creating BCR-ABL degrader compounds, researchers conjugated BCR-ABL TKIs dasatinib, which binds the c-ABL kinase domain to either the von Hippel–Lindau (VHL) or CRBN E3 ubiquitin ligase ligands. The resulting bifunctional compound was able to bind BCR-ABL via the TKI moiety and either VHL or CRBN via its recruiting ligand . SubsequPROTAC technology has also been applied for degrading the oncogenic protein bromodomain-containing protein 4 (BRD4). BRD4 has been reported as a required target for leukemia progression by promoting a cancer-associated transcriptional program . PROTAC Bromodomain-containing protein 9 (BRD9), a component of SWItch/Sucrose Non-Fermentable (SWI/SNF) remodeling complex, was found implicated in leukemia development and progression by regulating expression of cancer-related genes; thus, targeting BRD9 is considered a promising therapeutic strategy for leukemia . Given tSignal transducer and activator of transcription 3 (STAT3) is constitutively activated in different human cancers and it regulates a set of genes implicated in cancer cell survival, proliferation, metastasis and drug resistance, hence, it is a powerful cancer therapeutic target. Although STAT3 inhibitors have reached the clinical development stage, they demonstrated very limited clinical activity . RecentlAlthough PROTACs is a promising technology for drug discovery, especially for targeting the “undruggable” proteome, it faces important challenges for the future. For example, it is essential for identifying suitable ligands to successful design PROTACs, particularly for E3 Ub-ligase–oncoprotein interactions. Furthermore, the pharmacokinetic and pharmacodynamic characteristics of PROTACs are not very clear in clinical practice; it is likely that they require further improvement. Additionally, another possible challenge for PROTAC development is the drug resistance in treated cells due to genomic alterations of the core component of E3 ligase complexes .Protein stability deregulation of oncogenes and/or oncosuppressors is critical for cancer development and progression. Thus, our understanding of the ubiquitin code may contribute to the development of promising therapeutic agents. To date, there is enormous therapeutic potential in targeting UPS system. The exciting results reached with the use of the PIs, in some types of blood cancers, have contributed to the development of additional drugs targeting other components of UPS, although targeting UPS effectors suffers of target specificity since they can affect thousands of proteins. Furthermore, UPS components such as E3 enzymes lack a well-defined activity pocket; thus, the development of specific inhibitors remains challenging even though new advances in drug discovery, as well as development of new screening technologies, are surprisingly improving our abilities to drug demanding targets.Controlling the function of a certain protein by controlling its protein levels represents a promising new therapeutic approach in modern drug discovery. Enhancing protein degraders can extend horizons of druggable targets to those proteins that are still considered undruggable and are currently limiting medicinal research. Therefore, it will be interesting to see whether attractive oncogenic protein drivers such as c-Myc can eventually become part of a druggable proteome. PROTAC offers an excellent opportunity to target and degrade many difficult oncoproteins, suggesting that PROTAC could be developed as a new technology platform for drug discovery. The major challenge will be identifying suitable ligands for binding undruggable proteins as well as optimizing the linker molecules that connect the two chemical (PROTAC) moieties.Targeting UPS by either inhibiting or enhancing UPS effectors has proved to be one promising approach for the treatments of hematological malignancies. However, a better understanding of the mechanisms underlying E3 regulation and function in leukemia and an increase efficacy of drug discovery technologies are expected to open and expand the generation of new blood tumor therapies.


This review aims to assess and recommend approaches for targeted and agnostic High Throughput Sequencing of RNA viruses in a variety of sample matrices. HTS also referred to as deep sequencing, next generation sequencing and third generation sequencing; has much to offer to the field of environmental virology as its increased sequencing depth circumvents issues with cloning environmental isolates for Sanger sequencing. That said however, it is important to consider the challenges and biases that method choice can impart to sequencing results. Here, methodology choices from RNA extraction, reverse transcription to library preparation are compared based on their impact on the detection or characterization of RNA viruses. RNA viruses are of a global health concern from a One Health perspective, which is the intersection of human, animal and environmental health. Environmental transmission of these viruses, whether it be through food, water or recreational activities poses a risk for humans, plants and animals. It is important to adopt One Health principles for the surveillance of RNA viruses as environmental samples can (a) indicate hot spots for viral recombination, (b) serve as an important source of virus transmission, and (c) sequencing these samples allows us to pre-empt new RNA viruses and their variants of potential clinical concern. Viral persistence in the environment increases the opportunity for inter and intra-viral family recombination and increases virus-host exposure, factors that all contribute to the emergence of new viruses; that have the potential to cause large scale outbreaks. Non-enveloped viruses demonstrate remarkable persistence in the environment. Trans-kingdom virus interactions are thought to aid viral persistence in environmental settings, though this has been difficult to investigate, due to a lack of suitable cell culture systems. Furthermore, RNA viruses have high mutation rates as, unlike their DNA counterparts, most do not have a proofreading polymerase, though there are notable exceptions  have been widely applied in clinical settings but have had limited success for viral surveillance and aside from Flaviviruses . There have been important contributions regarding RNA virology from environmental HTS applications  their genetic diversity, (b) lack of conserved regions across the genome of viruses and (c) short genome lengths.resolution. In addition, the quantity of input RNA/HTS), either a targeted or agnostic approach can be taken. Targeted sequencing infers that some level of knowledge is available with respect to the target in question and that the experimental design incorporates this prior knowledge, either through amplicon-based sequencing or probe capture hybridization. An overview of how the different approaches work can be seen in In High Throughput Sequencing (ITS) between them. Unlike the 16S rRNA of bacteria, viruses lack universally conserved markers and genome plasticity. In particular, with respect to RNA viruses, this further contributes to the associated challenge, requiring a Family specific priming PCR approach. Amplicon based sequencing approaches, (particularly tiling or “jackhammer”) have been widely applied for the detection of RNA viruses with varying levels of success . Often in this case, common PCR-based approaches target highly conserved rRNA genes, such as those encoding the 16S/18S and 28S subunits or the Internal transcribed spacer (RT-PCR) amplification, primers can be designed to anneal to the most conserved sequences of the RNA virus genome(s) in question. In this case, a certain degree of validation is required. Confirmation of the PCR products via Sanger sequencing should be implemented for new primer sets to ensure specificity. There is a high likelihood that degenerate primer sets are required in order to account for virus divergence  requires prior knowledge of the specific sequence variants to be detected. Most capture-based methods use a tiling array approach, where 80 to 120-mer DNA or RNA probes are used to cover the length of the target genome/genomes. The probes typically have 10–50 bp regions between them, adopting a similar approach to the overlapping/jackhammer amplicon approach described above. Target enrichment is based on the biotinylated (or otherwise labeled) probe annealing to complementary sequences in the sample(s). The probes attach to previously fragmented genomic CBPH was initially implemented to detect Single Nucleotide Variants (SNV) in human genomic exon studies. Various studies have applied this methodology to virus specific studies using widely available commercial kits with custom design options such as SureSelect XT Target Enrichment system, Illumina TruSeq RNA Access, or SeqCap Ez probe design with separate library preparation kit. More recently, the VirCapSeq-VERT and CATCH custom virus oligonucleotide panel have become available as a general tiling array for vertebrate viruses , matrices and target viruses. Studies attempting to capture a very wide viral diversity have used panels ranging from 300,000 to 2.1 million probes per assay  though hopefully an understanding of the matrix and expected virome in question. For example, if the objective is to characterize human viruses of clinical concern in sewage, concentration and extraction methods must consider that sewage as a matrix will contain PCR inhibitors and that concentration methods may not concentrate both enveloped and non-enveloped RNA viruses of interest. Method validation and the inclusion of appropriate controls is necessary for interpretation of results and for setting quality control thresholds. As agnostic sequencing is not targeted, non-viral RNA can be captured during the library preparation and this can cause downstream issues. Indeed, obtaining sufficient genome coverage of virus RNA genomes against a background of host rRNA and SISPA) is a random priming method developed by Reyes and Kim  to a target population of blunt ended DNA molecules. The common end sequence allows one strand of the double-stranded primer to be used in repeated rounds of annealing, extension and denaturation in the presence of a high-fidelity polymerase. SISPA has been used for the discovery of new viral agents, particularly in the veterinary field  tail enrichment using TruSeq RNA kit and Circular resequencing (CirSeq). Parras-Moltó et al.  compared to CirSeq or Illumina TruSeq RNA Access kit and SISPA-generated sequences demonstrated an increased number of transition events. The accumulation of these errors could falsely indicate sub-clonal diversity or veil true diversity. Parras-Moltó et al.  is an isothermal enzymatic process where a short DNA or RNA primer is amplified to form a long single stranded DNA or RNA using a circular DNA template and specific DNA or RNA polymerases, as can be seen in Rolling Circle Amplification , was the least error prone   and Not so random (NSR) sequencing . For a detailed summary of enrichment methods, refer to Ribosomal RNA (rRNA) is the most abundant species of RNA in most cells. For agnostic RNA virus sequencing of complex samples, its presence is problematic as a large number of non-viral reads can be generated, thereby greatly limiting the number of relevant, virus-related, sequences. To increase the number of reads mapping to viral RNA, several methods have been employed to either enrich non-ribosomal RNA or remove unwanted rRNA sequences. Enrichment methods include poly-A selection [TruSeq mRNA (Illumina)], Single Primer Isothermal Amplificaiton  primers attached to magnetic beads to isolate RNA. Non-polyadenylated RNA, such as rRNA, are not captured. This approach does result in a strong bias toward the 3' end of RNA targets, though this bias is alleviated by the reduced sequencing depth required to obtain high quality viral reads   . Several product . SPIA rePDD). An overview of the rRNA depletion methods can be viewed in As an alternative to enrichment methods, rRNA can be removed using subtractive hybridization [Ribo-Zero (Illumina)], exonuclease digestion [MICROBExpress (Ambion)], endonuclease digestion (RNase H), or duplex specific nuclease (DSN)/Probe directed degradation  probes and depleted with streptavidin beads , Silica membrane based spin column technology (SMSC), and silica coated Magnetic beads (MB). Due to the physio-chemical differences between these three extraction methods, the yield, purity, and specificity of the RNA obtained varies and this can have downstream impacts.There are three common methods used for RNA extraction: organic extraction using Acid guanidinium thiocyanate phenol chloroform  or specific capture probes. Silica coated MB non-selectively bind nucleic acids in the presence of chaotropic salts via electrostatic interactions. The silica-coated beads are most suitable for applications that require nucleic acids other than mRNA while the oligo (dT) beads are best-suited for mRNA targets that are polyadenylated. Specific capture-based systems are best suited for applications that do not tolerate high concentration of non-target nucleic acids  concentration (ii) Nucleospin RNA XS generated the highest read count for RNA viruses  and (iii) viral richness is strongly impacted by extraction method. In a similar fashion, Strubbia et al.  values and higher concentrations of RNA using organic extractions is mixed, with no clear trend in either direction.a et al.  found thSNV. To date only two studies have investigated reverse transcriptase (RT) fidelity impact on next generation sequencing results  applications (see Both the efficiency of reverse transcription and fidelity is important for the detection of virus quasispecies present at low abundances and the identification of ions see . Whilst SISPA (2.2.1). As noted above, they are often six nucleotides long and are usually referred to as random hexamers, N6, or dN6. Due to their random binding, they can potentially anneal to any RNA species in the sample. Therefore, these primers may be considered for reverse transcription of RNAs without poly(A) tails, degraded RNA and RNA with known secondary structures. Some random primer sets have been constructed with viral genomes in mind, preferentially priming viral RNA over ribosomal RNA  priming has also been applied as a viral RNA enrichment method as outlined earlier 2.2.3.Oligo(dT) primers consist of a stretch of 12–18 deoxythymidines that anneal to poly(A) tails of eukaryotic mRNAs, which make up only 1–5% of total RNA. Oligo(dT) primers target polyadenylated RNAs, whereas random sequence primers target all RNAs including the abundant rRNA fraction. Mixtures of random hexamers with oligo(dT) are predominantly used in Gene-specific primers offer the most specific priming in RT  priming strategies were popular. As most publications have not assessed the yield/fidelity post cDNA synthesis, it is not possible to compare these publications based on the RT experimental design. Strubbia et al.  for GC-rich templates or betaine for AT-rich templates can reduce amplification bias for such targets. Betaine may help to keep a GC-rich template single-stranded, but it may also cause premature dissociation of the newly synthesized strand from an AT-rich template, introducing knock on effects for virome analysis  should be optimized and validated internally. “Jackhammer” PCR allows greater room for error in this aspect, as the primers are targeted across the genome, increasing the probability of successful amplification. That said however, viral RNA is a moving target and “jackhammer” approaches require up to date sequence data to perform consistently. Aside from primer design and method validation, additional considerations given to amplicon generation in HTS platforms  + selection or rRNA depletion, RNA samples are fragmentated to a certain size range, owing to the limitations in the read length of many cDNA synthesis as determined by AzNTPs:dNTPs. Following chain termination, single-stranded cDNA fragments are generated with an azido-group at their 3' ends. 3'-azido-blocked cDNA molecules can be purified and “click-ligated” to 5' alkyne-modified DNA adaptors via copper-catalysed azide-alkyne cycloaddition (CuAAC). The products of the ClickSeq reaction can be amplified using PCR to generate a cDNA sequencing library. Viral RNAs and mRNA using ClickSeq produced unbiased HTS libraries with low error-rates compared to standard methods  or enzymatic digestion. Nebulization involves directing compressed nitrogen or air forces into a DNA sample repeatedly through a small hole, producing mechanically sheared random fragments, leading to a heterogeneous mix of double-stranded DNA molecules containing 3'- or 5' overhangs as well as blunt ends  guidelines, HTS/RNA-Seq has been to slow to include extensive controls, as per other molecular methods  and comparing controls to biological samples post sequencing, cross-contamination can be identified, thereby aiding the interpretation of sequencing results. Strand specific sequencing can be used to identify the source of contamination during subtractive hybridization or viral genome vs. complementary RNA intermediates , Spike in RNA variants (SIRVs) and External RNA Control Consortium (ERCC). RNA oligonucleotides, SIRVs and ERCC samples can be applied as internal controls, spiked into each sample, including the whole process negative control. RNA oligonucleotides and ERCC can be used to assess sample inhibition, which is important to consider in complex matrices, as well as confirm method specificity , common environmental taxa introduced through cross-contamination  viruses, makes it difficult to design reasonably sized CBPH panels that capture species diversity, while also being affordable and technically feasible  of the RNA extracts may be required as SMSC/MB can carry through genomic DNA.Virus-specific approaches increase the chance to detect less abundant species through cDNA, but outcomes are dependent on the target, background RNA, reagent concentrations and priming strategy. Few studies have compared priming strategy during cDNA synthesis, and even fewer have looked at the impact of primer choice on HTS output. Random hexamers tend to produce more variable yields and should be applied at high concentrations  richness of the transcript in question  the target(s) in question and (ii) the bias introduced though polymerase choice and cycling conditions. Overall trends from the relevant studies demonstrate that thermostable, high fidelity polymerases outperform the robust alternatives. AmpliTaq Gold has been commonly applied in molecular virology and yet performed poorly in all studies, regardless of target or matrix .qRT-PCR measurements, rather than the detection of viral quasispecies. Bias observed is dependent on when and what type of fragmentation was applied. For fragmentation of RNA, RNase III-based fragmentation demonstrates a preference for double-stranded RNA sequences. This can result in uneven fragmentation of RNA leading to differential representation of specific regions of RNA  in question.While it is challenging to obtain high quality sequences from environmental samples, the information that could be gleaned is essential for maintaining public health. From developing new PCR/qPCR assays based on recent sequencing data, to monitoring antigenic drift and recombination, identifying new transmission pathways, hosts and viruses or pre-empting RNA viruses and variants of clinical concern in a One Health paradigm, the list of potential benefits goes on. AF reviewed the literature and compiled the review. AR revised the initial drafts. SK, HO'S, PC, and FC reviewed the final draft. 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.


CFTR) gene, and their applications for the development of experimental models valuable for the advancement of CF therapies.Since the early days of its conceptualization and application, human gene transfer held the promise of a permanent solution to genetic diseases including cystic fibrosis (CF). This field went through alternated periods of enthusiasm and distrust. The development of refined technologies allowing site specific modification with programmable nucleases highly revived the gene therapy field. CRISPR nucleases and derived technologies tremendously facilitate genome manipulation offering diversified strategies to reverse mutations. Here we discuss the advancement of gene therapy, from therapeutic nucleic acids to genome editing techniques, designed to reverse genetic defects in CF. We provide a roadmap through technologies and strategies tailored to correct different types of mutations in the cystic fibrosis transmembrane regulator ( CFTR) gene. CFTR encodes for a cAMP regulated chloride channel located in the apical membrane of epithelial cells that catalyze the passage of small ions through the membrane. Dysregulation of this mechanism causes an impairment of salt and fluid homeostasis that results in multiorgan dysfunctions and ultimately mortality from respiratory failure. For more than seven decades, therapies for CF were limited to the treatment of the symptoms rather than addressing the origin of the disease, the altered CFTR gene. The mutations causing CF are 352 distributed along the CFTR gene, including exonic and intronic regions [Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane regulator  at the target sites ,16. As aA turning point in the history of genome editing is represented by rare-cutting meganucleases  and their use to enhance gene substitution with donor sequences ,23, leadCFTR locus  or by the turnover of the airway epithelium. A stable integration of the CFTR transgene can be obtained through retroviral vectors and may potentially produce continuous expression by targeting staminal cells in the airway epithelium. Nevertheless, beyond difficulties in reaching the proper target cells in the epithelium, the use of retroviral vectors has been limited by the concern raised by insertional mutagenesis occasionally reported in gene therapy clinical trials [The monogenic and recessive nature of CF makes this disease an optimal target for gene complementation strategies . The delutations . Severalet cells ,10,11,12l trials .CFTR locus thus permitting physiologic expression of the ion channel. Programmable nucleases offer the opportunity to precisely target integration of DNA sequences at their cleavage sites [CFTR cDNA within the endogenous locus even though it has not been experimentally validated yet [CFTR super-exon (exons 11-23) targeting via ZFN the 5′ end of exon 11 in the CFTR locus in epithelial cell lines. While this strategy could potentially correct all the mutations located after exon 10, including the F508del, the complete phenotypic reversion was obtained only after antibiotic selection and in a small percentage of clones [With the recent development of powerful tools to manipulate the genome, new perspectives are opened in gene therapy to restore the correct CFTR expression by engineering the ge sites ,41. Thisge sites ,42,43. Hated yet . An altef clones .6 events [CFTR was a ZFN [Gene substitution is a very attractive procedure in gene therapy to replace a mutated sequence with a correct copy by exploiting the cellular HDR DNA repair pathway. HDR is very infrequent (1 out of 106 events ) and thu6 events ,23. Sequ6 events ,23. Thes6 events . Even thas a ZFN  applied as a ZFN . This apas a ZFN , TALEN [as a ZFN ,51 or CRas a ZFN .CRISPR-Cas9 was proven valid to correct ΔF508 in advanced cellular models, such as intestinal organoids derived from patients, where CFTR channel function could be restored . To compClinical and preclinical works are testing programmable nucleases as DNA cleaving tools to inactivate genes involved in infectious diseases ,28 or inCRISPR-Cas mediated sequence cleavage was successfully used in CF to repair splicing mutations, accounting for about 10% of CF cases  where thDSB dependent editing by programmable nucleases activates DNA damage signaling pathway and may results in unpredicted rearrangements and translocations ,71. To aThis technology was applied in CF by Clevers group to correct nonsense mutations in patients’ derived organoids carrying the R785X, W1282X or R553X mutations. Selected organoids showed A to G conversion in the expected positions with notably minimal efficiency (not more than 8% for any of the three tested mutations) .Prime editing is a CRISPR-based technology composed of a Cas9 nickase fused to an engineered reverse transcriptase (RT) and complexed with a prime editing guide RNA (pegRNA) . The pegAntisense oligonucleotides (ASOs) have been widely explored to therapeutically modulate gene expression by degradation of the target mRNA or by modulation of splicing elements for exon retention or skipping . ASOs weAn alternative use of ASOs consisted in inserting the missing bases in 508 CFTR at the level of RNA transcripts in a ΔF508 cellular model. Even if a phenotypic reversion was measured, correction of the mRNA does not represent a stable modification and the mechanism of correction is not fully understood .CFTR, ASOs were used to block the epithelial sodium channel, ENaC, in mice models with CF-like symptoms [+ absorption [Instead of targeting symptoms . The ratsorption . Indeed,sorption . The aersorption .The ASO therapy is limited by the life-long dependency on repetitive treatments and adverse effects that still need to be addressed .Another RNA-based approach to restore the proper amounts of CFTR transcripts was tested with the delivery of a lipid-based nanoparticle of chemically modified mRNA in patient-derived bronchial epithelial cells, showing increased membrane-localized CFTR . The nasThe Spliceosome mediated trans-splicing (SMaRT) technique was developed to repair the nascent mRNA by replacing part of the altered transcript with a correct exogenous mRNA . This apPhenotypic reversal using methods modulating CFTR mRNA by delivery or modification using ASOs or SMaRT offers the advantage of preventing potential genotoxicity generated by manipulation of the genome. However, RNA-based therapies transiently restore the function of CFTR, but are limited by requiring continuous lifetime treatments.CFTR variants associated with CF, it is very difficult to cover the entire mutation repertoire in homozygous cellular models. To overcome this limitation, isogenic models for different mutations were created using CRISPR-Cas9 mediated HDR in immortalized bronchial epithelial cells providing valuable experimental tools for CFTR functional assay [Experimental models resembling the CF phenotype, pathogenesis and symptoms are crucial for setup and evaluation of therapeutic strategies. Genome manipulation tools and the advancements of CRISPR-Cas technology gave a major impulse to the advancement of gene therapy solutions. These tools are also fundamental to advance gene therapy and drug development more in general by simplifying the production of new cellular and animal CF models ,94. Patial assay .CFTR gene with CRISPR-Cas9 mediated NHEJ. CFTR-/- sheep developed a severe disease similar to CF including pancreatic fibrosis, intestinal obstruction and the absence of vas deferens [CRISPR-Cas technology has been also applied to generate new CF animal models. A knockout sheep was generated by the disruption of the deferens . To specdeferens .CFTR locus was generated by CRISPR nucleases showing common manifestations of CF determined by the absence of CFTR ion channel, such as intestinal obstruction and reduced growth [A mouse carrying the G542X nonsense mutation in the d growth .In conclusion, CRISPR genome editing is a very promising technology to generate new therapeutic strategies, as well as new valuable experimental tools to test therapies for a wide variety of CF causing mutations .CFTR mutations. Genome editing is at the forefront not just by offering novel therapeutic solutions but also for the development of experimental models, cellular and animal, essential in translational medicine. Although there is much to be done in particular in terms of tools for in-vivo delivery of nucleic acids and genome editing molecules, hurdles and limitations seem well-defined and manageable.We have presented here emerging technologies and derived strategies as a path forward from conventional gene therapy based on delivery of therapeutic nucleic acids toward gene correction of