In multivariate regression analysis, the stent placement method (CIU-SP vs. C-SP) was independently associated with the I-PSS daytime frequency on day 14 (P = 0.017). This study suggests that CIU-SP significantly improved stent-related daytime frequency compared with C-SP, and it may benefit especially those patients who have mild urinary symptoms before the placement of ureteral stents.A previous randomized, controlled trial had demonstrated that complete intraureteral stent placement (CIU-SP) was superior to conventional stent placement (C-SP) in terms of improvement of stent-related urinary symptoms. However, it is unclear as to which subdomain symptom and cohort could benefit the most from CIU-SP compared to C-SP in urinary symptoms while considering the baseline urinary status. To determine this, a post-hoc analysis was performed using data from a previous study . We assessed the mean changes in the International Prostate Symptom Score (I-PSS) and the Overactive Bladder Symptom Score (OABSS) from baseline to day 14. Statistical comparison between the two groups was performed using analysis of covariance with adjustment of baseline urinary status as a covariate. Among 80 patients, the total I-PSS was significantly lower in the CIU-SP group than in the C-SP group in the cohort with mild urinary symptoms (P = 0.005), but not in those with moderate/severe symptoms (P = 0.521). The CIU-SP group showed significantly improved I-PSS and OABSS daytime frequencies, with the highest  These stents are also useful for preserving patency in ureteral injury during the wound healing process2. In the case of ureteroscopic lithotripsy (URS), ureteral stents are commonly placed with the intention of preventing urinary obstruction due to ureteral edema at the surgical site. This leads to a reduction in the risk of postoperative urinary infection3. However, despite such advantages, this process causes ureteral stent discomfort, such as body pain and impairment of urinary symptoms, and affects the patient’s quality of life (QoL)4. Most of these symptoms have been considered to be attributed to ureteral spasm or contact of the distal end of the stent on the bladder wall5. However, there is no uniform consensus on how to improve stent-related urinary discomfort.Ureteral stents are an effective medical device for improving urinary passage obstruction mainly due to ureteral stricture and stone impaction6. The main concept of CIU-SP was omitting the distal end of ureteral stents to reduce irritation on the trigonal area of the bladder. We found that CIU-SP significantly reduced stent-related pain (as a primary outcome measure) and lower urinary tract symptoms (LUTS) (as secondary outcome measures) compared with C-SP on postoperative days 3 and 14. With regard to urinary symptoms, these should be affected not only by URS or the ureteral stent placement procedure, but are also largely affected by baseline LUTS status9. To appropriately assess the efficacy of CIU-SP in stent-related urinary symptoms To appropriately assess the efficacy of CIU-SP in ameliorating stent-related urinary symptoms and identify the subgroup that could benefit from CIU-SP compared to C-SP, we performed a post-hoc analysis to compare the mean changes in urinary symptom scores from baseline to day 14 between the two stent placement groups, with adjustment of the baseline urinary status as a covariable10. Furthermore, we examined the most relevant subdomain in urinary symptom scores that is associated with improvement by CIU-SP vs. C-SP.Recently, our research group conducted a prospective, randomized, controlled trial to evaluate the efficacy of complete intra-ureteral stent placement (CIU-SP) vs. conventional stent placement (C-SP) in patients with an indwelling loop type of ureteral stent after URS11) and 3.11 ± 2.31 (categorized as mild symptoms12), respectively. Of 80 patients, 41  and 39  had mild and moderate/severe I-PSS, respectively; and 67  had mild symptoms, and 13  had mild and moderate/severe OABSS, respectively. Baseline characteristics between the two groups were well balanced, except for OABSS urgency  and Overactive Bladder Symptom Score (OABSS) were 9.57 ± 7.83 , I-PSS daytime frequency , and OABSS daytime frequency  were significantly correlated with the stent placement method  , simple linear regression analysis was performed. The I-PSS incomplete emptying  and stent placement method  were significantly associated with the I-PSS daytime frequency   was an independent clinical factor for predicting improvement of the I-PSS daytime frequency.6, this post-hoc analysis had several strengths for accurately evaluating stent-related LUTS for the following reasons: (1) measuring the mean change in urinary symptoms from baseline to day 14; (2) applying analysis of covariance for adjusting baseline covariates; (3) selecting day 14 as the final evaluation date for minimizing the confounding effect of discomfort of the URS procedure13; and (4) examining details regarding stent-related LUTS using the validated LUTS-specific indicators12. Therefore, our results have added new evidence on management of improving stent-related urinary discomfort and QoL.In contrast to our previous report17. Charles et al. found that nocturia and daytime frequency were the primary and secondary chief complaints, respectively, based on the American Urological Association Symptom Index in 1240 men with BPH15. McVary et al. suggested that the symptom of bother and QoL were affected two-fold by storage I-PSS questions  vs. questions on voiding symptoms 16. OAB, namely a storage symptom disorder, affected patients’ mental health, work productivity, and health-related QoL, regardless of sex17.In the field of LUTS research, storage-related symptoms are the most clinically bothersome and affect patients’ QoL in those with benign prostatic hyperplasia (BPH)/LUTS or overactive bladder (OAB)18. According to such underlying mechanism(s), CIU-SP may be a reasonable placement method because of not presenting the distal end of the stent. This can prevent iatrogenic physical irritation on the bladder wall. Notably, in our study, CIU-SP significantly reduced daytime frequency among urinary symptom subdomains compared with C-SP, whereas the rate of nocturia was equal in both groups. Based on the mechanism(s) mentioned above, these findings are easy to understand because the absence of a distal tip of the stent might reduce physical stimulus on the bladder during daytime activities.In the present study, we found that patients’ QoL was affected approximately three-fold by a storage symptom compared with a voiding symptom, despite the fact that both symptoms equally occurred because of stent placement. Similar to the approach for BPH/LUTS or OAB, management of storage symptoms is important in controlling ureteral stent-related urinary discomfort. Physical or chemical stimulation on the bladder wall causes release of chemicals, including ATP, acetylcholine, prostaglandins, and nitric oxide, which modulate the activity of either afferent nerves or muscular components of the bladder wall6 could be attributed to the inclusion of more than half of the patients with favorable urinary symptoms before the URS surgery in the study. In other words, patients who have moderate or more urinary symptoms could tolerate even conventional stent placement. This information can be applied in daily clinical practice as well as in clinical trial protocols associated with ureteral stent placement.Furthermore, we also found a negative correlation between the degree of the urinary status at baseline and the change in urinary symptoms after ureteral stent placement. Indeed, patients who had mild urinary symptoms could benefit from CIU-SP, whereas those with moderate/severe symptoms showed little impact, regardless of placement techniques. Thus, the clinical significance of CIU-SP demonstrated in our previous study22. These results regarding improvement of urinary symptoms are conflicting, and negative results have been found in several randomized, controlled trials22. We believe that trials using medication should consider not only improvement of stent-related irritation, but also urinary symptoms that the patient has23. Nevertheless, most ureteral stent studies did not take into account the change from baseline or baseline LUTS status22. Therefore, actual results of previous studies may change if considering these factors for statistical analysis. Unlike trials of medications, we focused on examining the pure effect of stent placement methods on urinary symptoms, and found that CIU-SP was a better placement technique than C-SP. Unfortunately, even when using CIU-SP, some patients still suffer from stent-related urinary symptoms. Therefore, further investigations are required to determine the mechanism(s) that are associated with these symptoms , for providing a more comfortable method for patients who require ureteral stent placement.To date, many studies have attempted to alleviate stent-related discomfort by agents, such as alpha-1 blockers, anticholinergics, and beta-3 adrenergic receptor agonists6, we did not assess the safety of CIU-SP in patients with distal ureteral stones. Therefore, further studies including patients with distal ureteral stones are required in the future.We acknowledge several limitations that should be interpreted with caution. First, this post-hoc study was not intended to be designed when performing the primary study. Therefore, there was a lack of calculation of required sample size for this study. Second, we used only the loop type of stents instead of double j stents, which are commonly used. Although this study could not determine the best management for using double j stents, we believe that our findings could be useful for developing a new stent design that can reduce irritation of the bladder wall. Finally, as we previously mentionedThis study highlights the asymmetrical relationship between ureteral stent-related storage and voiding LUTS for patients’ urinary QoL. CIU-SP may be a more beneficial strategy for patients who have mild urinary symptoms at baseline than for those who have moderate/severe symptoms. Furthermore, CIU-SP might help reduce ureteral stent-related storage symptoms compared with C-SP, especially in terms of improving daytime frequency.6. This study was approved by the ethics board of Kansai Medical University (IRB No. 2016503), and all patients provided written informed consent. This trial strictly followed the 2010 Consolidated Standards of Reporting Trials (CONSORT) statement guidelines24. Details of this trial, and inclusion and exclusion criteria were described previously6. Briefly, patients who were aged > 20 years who underwent unilateral URS with planned ureteral stent insertion were included. However, patients who had concomitant use of alpha-1 blockers, anticholinergics, corticosteroids, calcium channel blockers, and analgesics, distal ureteral stones, and preoperative ureteral stenting were excluded. Patients were equally randomized in a 1:1 ratio into the CIU-SP and C-SP groups. The modified intention-to-treat population, except for those without follow-up, sufficient clinical data, or those who withdrew consent after randomization, was used for analysis 70676. Th11 and the OABSS 12 at baseline and day 14 were obtained  with a string was inserted by the two placement methods  according to the actual ureteral length. Finally, the stent string was cut approximately 10 cm from the tip of the urethra after insertion of the urethral catheter. For discharge medication, only oral diclofenac sodium 25 mg was allowed to be used during the study period . On day 14, the ureteral stent was removed at the outpatient clinic with the extraction string.For all patients, the I-PSS 6.The primary endpoint was the mean change in the total I-PSS and OABSS from baseline to day 14. The secondary endpoints were the mean change in the I-PSS and OABSS subscores from baseline to day 14. These analyses were not part of the protocol-specified primary or secondary endpoints of the previous studyt test was used to compare continuous variables. The correlations between data were evaluated using Spearman’s rank correlation test. For analysis of endpoints, ANCOVA was applied for comparison between the two groups (the placement group as a factor and baseline as a covariate). Simple or multiple linear regression analysis was applied to assess the association between the urinary symptom scores and potential factors, with 15 subjects per variable as the minimum required sample size25. The t statistic in linear regression analysis, which was calculated as the ratio of an estimated coefficient (β) to its standard error, was used to test the hypothesis that a coefficient is equal to zero. All statistical analyses were performed using EZR version 1.37 26. A two-sided P value of < 0.05 was considered statistically significant.Continuous data are expressed as mean ± standard deviation. The Chi-square test was used to compare nominal variables, and the paired or non-paired Supplementary Figure 1.


Impressive therapeutic advances have been possible through the advent of zinc-finger nucleases and transcription activator-like effector nucleases. However, discovery of the more efficient and highly tailorable clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas9) has provided unprecedented gene-editing capabilities for treatment of various inherited and acquired diseases. Despite recent clinical trials, a major barrier for therapeutic gene editing is the absence of safe and effective methods for local and systemic delivery of gene-editing reagents. In this review, we elaborate on the challenges and provide practical considerations for improving gene editing. Specifically, we highlight issues associated with delivery of gene-editing tools into clinically relevant cells. Gene-editing technologies have unprecedented potential to cure, treat, or prevent a wide array of inherited and acquired diseases. These technologies provide flexibility to modify the genome at precise locations for knockdown or restoration of gene expression, insertion of a therapeutic transgene, or correction of mutations associated with genetic diseases. Gene-editing-based clinical trials have advanced for multiple diseases.+ hematopoietic stem/progenitor cells (HSPCs) [Initiated in 2009, the first-in-human use of gene editing involved application of zinc-finger nucleases (ZFNs) to disrupt the HIV-1 major coreceptor, CCR5, in human CD4 T cells isolated from patients with chronic aviremic HIV infection NCT00842634). To make the autologous CD4 cells resistant to HIV infection, 11% to 28% of alleles are modified with ZFNs. When infused back into the patients, the cells engrafted and showed long-term persistence. In one patient from the trial, HIV RNA was undetectable [4. To mak+ HSPCs modified with CRISPR-Cas9 to upregulate the expression of fetal hemoglobin as a treatment for sickle cell disease and Clustered regularly interspaced short palindromic repeats (CRISPR) and the associated protein (Cas9) nucleases represent the platform of choice for therapeutic applications. Cas9 nucleases have a simpler design, a wider targeting range, and provide higher levels of nuclease activity compared to the early generation of protein-based gene-editing tools. The first CRISPR clinical trial in the US was initiated in 2018 and involved use of autologous CD34Despite the recent clinical trials, a major barrier for therapeutic gene editing is the absence of safe and effective methods for local and systemic delivery of gene-editing reagents. In many diseases, such as Duchene muscular dystrophy, diseased tissue is widely distributed, necessitating in vivo delivery of gene-editing reagents. Further, because many diseases affect multiple body systems, a curative therapy must enable systemic delivery of the gene-editing reagents into multiple target cell types. This review aims to provide an overview of gene-editing tools and viral and nonviral delivery systems for therapeutic gene-editing applications. We elaborate on the strategies associated with each approach, the importance of delivery for effective therapies, challenges, and advances in the field. The discussed delivery systems with their advantages and disadvantages are summarized in DNA double-strand breaks (DSBs) activated at precise locations in the genome are the basis for gene editing using site-specific nucleases. DSBs are the most biologically damaging lesions; the absence of their repair can result in apoptosis, loss of genetic material, cell death, and permanent cell cycle arrest . In someIn 1994, it was discovered that DSB repair mechanisms could be leveraged for genome editing . By creaThe NHEJ pathway is the most active DNA repair mechanism in mammalian cells. It repairs DSBs without a repair template . The NHEHDR is the more accurate DSB repair mechanism: It produces high fidelity repairs using a donor template DNA . The HDRDuring the HDR pathway, a donor DNA template can be used for targeted gene editing. Using the donor template, a sequence from the donor DNA is incorporated into the genome, resulting in edits ranging from single-nucleotide changes to insertions of multiple genes . This meSaccharomyces cerevisiae [In early studies, I-SceI, a homing nuclease isolated from revisiae , and a hrevisiae  and mousrevisiae . The I-Srevisiae .While threvisiae .Site-specific nucleases have unprecedented potential to treat a wide array of inherited and acquired diseases. The objective for gene-editing-based gene therapy is the transformation of a native DNA sequence with a desired modified sequence within the genome of target cells for correction of a mutation, disruption of gene expression, deletion to restore the reading frame, or insertion of a new gene sequence to treat a disease. To elicit a therapeutic effect, heterozygous gene modification may be sufficient to ameliorate symptoms and quality of life in patients suffering from genetic or acquired diseases. The first generation of engineered nucleases are protein-based tools, ZFNs and TALENs, used for the recognition of relatively long unique sequences in the genome .FokI endonuclease cleavage domain from the Flavobacterium okeanokoites tethered to a tandem array of Cys2His2 type zinc-finger protein DNA-binding domains from a class of eukaryotic transcription factors connected using a short intervening amino acid linker [3-Cys-X2-4-Cys-X12-His-X3-4-His-X4 (X is any amino acid) folded into a FokI dimerization requirement to form a DSB, a pair of ZFNs is designed to bind to adjacent sites, 18 to 36 bp DNA sequence, on each strand in an inverted orientation and separated by a 5 to 6 bp spacer [+ HSPCs [The ZFN architecture consists of the d linker . The zinleotides . Zinc-fileotides . As a cop spacer . Generatp spacer ,23. Co-d4+ HSPCs .FokI cleavage domains engineered to preferentially heterodimerize. However, the drawback of the obligate heterodimers is that they dimerize less efficiently than wild-type FokI cleavage domains, thus reducing cleavage activity [One major drawback of ZFN technology is the potential for unwanted gene editing at off-target sites that have mismatches to the target sequence that lead to toxicity in the form of cell death and apoptosis ,26. The activity . Despiteactivity : the tarXanthomonas spp. proteobacteria tethered to the FokI catalytic domain as found in ZFNs [FokI dimerization [TALENs consist of the TALE DNA-binding domain from the  in ZFNs . The TAL in ZFNs . The forrization . An advarization ,31. In arization . The unwrization . The advrization . FurtherStreptococcus pyogenes  uses an sgRNA to bind to sites upstream of a 5′-NNGRRT PAM sequence. The SaCas9 has been employed in preclinical studies using animal models of human diseases that demonstrated successful therapeutic application [A second major challenge associated with the CRISPR-Cas9 system is its large cargo size (~4.3 kb), which affects its delivery as a viral vector for gene therapy applications. This challenge is the impetus that led to characterization of Cas homologs from other species of bacteria. The CRISPR-Cas9 from lication ,56. In alication . The dralication . The SaCBase editors are catalytically dead or impaired CRISPR-Cas nucleases capable of introducing point mutations within a narrow region of a target site B withoutN-glycosylase (UNG) activates the repair mechanism and converts the U•G mismatch back to C•G. The authors suggested that inhibition of UNG could repress BER of U•G mismatches. The second generation of base editors (BE2) was designed to overcome the BER pathway by the addition of a uracil DNA glycosylase inhibitor (UGI) to the C-terminus of the base editor [In early studies, base editors showed low on-target gene-editing rates due to disruptions by native cellular repair mechanisms in cells. The creation of a mismatched base pair in the intermediate product could potentially activate the base excision repair (BER) mechanism. The BER pathway recognizes mismatches and performs a “base-flip” to correct the mismatch . The fire editor . BE2 proDmd and Tyr. While the most common substitutions observed in blastocysts grown from the treated embryos were the intended C-to-T, a sizeable number of blastocysts (20–36%) were found to also have unwanted C-to-A and C-to-G conversions [A major limitation of base editors is their high level of unintended nucleotide alterations, including indels or base pair conversions at the target site. To improve the activity of BE2, the dCas9 was replaced with a Cas9 nickase to generate BE3 . The BE3versions . Effortsversions . A separversions .Off-target editing is a second major limitation of base editor technologies ,70,71,72Viral delivery vectors have been used for the delivery of nucleic acids into cells and are the most-used delivery method for CRISPR-Cas9 due to the availability of well-established protocols and high transduction efficiencies. A major advantage of viral delivery systems is that having mechanisms for introducing genetic material into cells systemically, they facilitate in vivo delivery of gene-editing tools but have been engineered for safety by removal of the genes required for viral replication. Although there are numerous types of viral delivery systems, we will focus our discussion on adenovirus (AdV), adeno-associated virus (AAV), and lentivirus (LV)-mediated delivery of gene-editing tools in the proceeding paragraphs.Pten, a negative regulator of the PI3K-AKT pathway involved in nonalcoholic steatohepatitis, in mice over 4 months. However, AdV-associated immunotoxicity in the liver was observed in treated mice [Rosa26 safe harbor site that resulted in long-term phenotypic correction of Hemophilia B bleeding diathesis. However, adaptive immune responses against the AdV were detected [Acidaminococcus sp., to provide resistance to HIV-1 infection in CD4+ T cells [AdV is a nonenveloped, linear double-stranded DNA virus associated with cold-like symptoms and several other diseases. The AdV remains episomal and is suitable for transduction in both quiescent and dividing cell types . As a deted mice . Similardetected . The rec T cells . The adv T cells . HoweverAAVS1 on chromosome 19 with approximately 40% to 70% frequency [Adeno-associated virus (AAV) is a nonenveloped, single-stranded DNA virus in the parvovirus family. It is a small virus with an ~4.8 kb genome consisting of rep and cap open reading frames flanked by two inverted terminal repeats. AAVs require another virus such as AdVs or herpes simplex virus to enter the lytic cycle for replication. In the absence of a helper virus, the AAV enters the lysogenic stage by integrating into a specific region on the human chromosome where it remains in latency. AAV vectors are advantageous because they can be used for transduction in many cell types. Serological studies have revealed different serotypes of AAVs with different traits and various tropisms; the serotypes require appropriate selection of AAV vectors for specific target tissues. The most established serotype, AAV2, has a broad tropism for different tissues, including liver, muscle, brain, and retina ,88. AAV2requency . Althougrequency . Additiorequency . Removinrequency . In divirequency .i.Packaging CRISPR-Cas Components into AAV VectorsStreptococcus thermophilus has been considered for this purpose although its PAM sequence narrows the range of possible targets [Campylobacter jejuni (CjCas9). The ortholog has a 2.95 kb size for potential delivery of reporter genes and donor templates and multiple sgRNAs for multiplex editing from a single vector. The study by Kim et al. on treatment of age-related macular degeneration by targeting the Hif1a reported high specificity and comparable editing efficiencies for CjCas9 and SaCas9 when delivered in a single AAV vector in vivo [Neisseria meningitidis (3.16 kb) was used to disrupt Pcsk9 in vivo, leading to lower cholesterol levels in mice. Results demonstrated successful gene modification with an efficiency of 35% at two weeks after injection [One major limitation of the AAV vector is its small packaging capacity. SpCas9 is 4.2 kb, and the AAV system provides only 0.3 kb to package the sgRNA and other gene-editing elements. Senis et al. designed a single AAV vector using CMV, with a short polyadenylation signal, and H1 promoters for expressing Cas9 and sgRNA . However targets . SaCas9  targets . The sma in vivo . A 1-yea in vivo . In a senjection . Not allnjection . The duaii.AAV-Mediated Delivery for Therapeutic ApplicationsCEP290 that causes Leber congenital amaurosis-10 was removed in a large deletion [Rpe65 [Many preclinical studies have reported successful gene editing using AAV-mediated delivery of CRISPR-Cas9 for treatment of a wide array of diseases, including hemophilia B , phenylkdeletion , demonstn [Rpe65 . Furthern [Rpe65 .AAV CRISPR editing has been used to treat muscle disorders, such as Duchenne muscular dystrophy (DMD) ,112,113.+ T cell response and pre-existing immunity was reported in a clinical gene therapy trial for severe hemophilia B involving rAAV delivery of FIX. At 8 weeks after injection, all patients had therapeutic FIX levels, transient increase in liver transaminase levels, and elevated neutralizing antibody titer accompanied by subsequent decrease in transgene expression to baseline [Despite systemic AAV delivery’s safety profile and track record in clinical trials, associated immunogenicity risks remain to be resolved. One major concern stems from pre-existing immunity against AAV capsids due to prior exposure to the wild-type virus. The first evidence of a CD8baseline . In a stbaseline , suggestbaseline ,118,119 baseline ,121,122 baseline . Notablybaseline  and the baseline , excludibaseline . Howeverbaseline , and it baseline .Rian locus after systemic administration of AAV vectors in neonatal mice caused hepatocellular carcinoma [Rian locus is not conserved in humans, suggesting that mutagenesis is species-specific. However, findings of clonal integration sequences from AAV2 at known cancer driver genes, including CCNA2, CCNE1, KMT2B, and TERT in liver samples further raised safety concerns [The integration frequency of rAAV is very low; nevertheless, the genotoxicity risks still constitute valid safety concerns in the context of therapeutics, since 0.1% integration translates to a considerable number of integration events. The random integration of AAVs has the potential to result in mutagenesis due to incorporation of the vector into or near cancer-driver genes. In fact, evidence from two studies revealed that insertional mutagenesis to arcinoma ,128. Thearcinoma . Moreoveconcerns . A recenconcerns . The accHBB gene in CD34+ HSPCs to correct the mutation causing sickle cell disease [Fah that causes hereditary tyrosinemia-1 ex vivo in hepatocytes [Although most of the AAV-based CRISPR applications focus on in vivo genome editing, ex vivo editing provides an alternative approach for gene editing that avoids the safety concerns associated with systemic delivery of AAVs. Dever et al. reported successful ex vivo gene editing using the AAV-based CRISPR system targeting the  disease . Recentlatocytes . The stuRetroviridae family, which carries genomic material as RNA and has the capacity to retrotranscribe RNA into DNA using reverse transcription. After the virion binds to its receptor on a target cell, it enters the cell by endocytosis or fusion and releases its core, including reverse transcriptase and integrase. With the help of host nucleotides, reverse transcriptase synthesizes cDNA from the viral RNA and subsequently transports the cDNA to the nucleus where it integrates into the host genome [Lentiviruses (LV) belong to the t genome . Althougt genome . LV vectt genome . LVs alst genome ,136,137.Because of the life cycle of retroviruses, LVs integrate into the host genome. Viral integration may be desirable for applications where long-term transgene expression is required, such as when making gene libraries or model organisms. However, for therapeutic approaches, LVs carry safety risks for insertional mutagenesis as well as persistent expression of site-specific nucleases leading to off-target mutations . The firi.LV-Mediated Delivery for Therapeutic ApplicationsCCR5 disruption without any off-target mutations, and the transduced cells exhibited resistance to infection by R5-tropic HIV-1 and a selective advantage over cells carrying wild-type CCR5 [CXCR4 using LVs containing CRISPR-Cas9 in human CD4+ T cells [CXCR4 and CCR5 using an LV containing Cas9 and sgRNAs to obtain resistance to both R5- and X4-tropic HIV-1 infection in susceptible cell lines without any off-target mutations. Nonetheless, the rate of edited cells homozygous for both genes was relatively low, at ~10% [ICP0 to prevent Herpes simplex virus type 1 (HSV-1) infection. The study showed successful abrogation of HSV-1 infection in uninfected cells by blocking viral replication and a drastic reduction of HSV-1 replication in the infected cells [There have been many studies that use LVs for the delivery of CRISPR components for therapeutic applications and show promising results. The study by Wang et al. used the CRISPR-Cas9 system to disrupt the CCR5 gene to make cells resistant to HIV-1 infection using LV-mediated delivery. One round of co-transduction was sufficient to obtain a high frequency of ype CCR5 . A simil T cells . The gen T cells . The stu at ~10% . Roehm eed cells .ORF5 and ORF7 through LV transduction and obtained an almost complete loss of genomic RNA when targeting simultaneously [Vegfa, the gene whose dysfunction is associated with age-related macular degeneration. The CRISPR-Cas9 mediated knockdown of genomic Vegfa was observed after subretinal injection in mice caused editing efficiencies up to 93% and 84% in vitro and in vivo, respectively [Studies have explored the use of CRISPR-Cas13, which targets RNA, to combat retroviral infections. Cui et al. adapted CRISPR-Cas13b to directly disrupt the viral RNA of porcine reproductive and respiratory syndrome virus (PRRSV) in eukaryotic cells. They delivered Cas13b and crRNAs targeting the PRRSV essential genes aneously . More reaneously . LV was ectively .SNCA intron 1 to obtain an indirect downregulation of SNCA expression through DNA methylation [SNCA locus leading to downregulation of expression, causing the rescue of disease-related phenotypic perturbations, including cell viability, mitochondrial function, and oxidative stress. Importantly, significant off-target DNA methylation was observed in the control group harboring no gRNA, indicating a need to improve the specificity and the safety of the system.An all-in-one LV vector was used to deliver gRNA-dCas9-DMNT3A (DNA methyltransferase-3) targeting CpG islands in hylation . The objii.LV-Mediated Delivery for Genomic ScreeningsLVs are widely used for the creation of gene libraries and genomic screening. Shang et al. used LVs to perform a genome-wide CRISPR screen in search of genes that regulate T cell activation and successfully identified a previously uncharacterized regulator, FAM49B . Genome-Electroporation is a physical transfection method that applies high-voltage currents to cells to permeabilize the membranes to nucleic acids, chemicals, or proteins. After the application of an electrical pulse, small gaps open up in the cell membranes, allowing the entry of substances; these gaps subsequently reseal. Electroporation-mediated gene therapy was first demonstrated in 1982 in delivery of plasmid DNA into mouse L cells deficient in thymidine kinase . Initial+ HSPCs for treatment of genetic and acquired diseases [+ HSPCs from patients, gene modifying the cells ex vivo, and infusing them back into the patient for the treatment of HIV, sickle cell anemia, and β-thalassemia. Strategies for therapeutic gene editing to treat HIV typically attempt to disrupt the HIV co-receptor CCR5 [CCR5 by electroporation of Cas9 and single sgRNA into cell lines [CCR5 [+ HSPCs; these were then implanted into immunodeficient mice [CCR5 were transplanted into a patient positive for HIV and in remission for acute lymphoblastic leukemia [CCR5-deficient cells was <10% and insufficient to confer HIV resistance to the patient. The low levels of gene modified HSPCs engrafted into the bone marrow are likely the result of poor cell functionality and viability after electroporation.Electroporation has been extensively used for delivery of Cas9 into CD34diseases ,162,163.tor CCR5 . Early ill lines ,158. Howent mice . In trealeukemia . AlthougBC11A gene [BC11A to increase production of γ-globin has been proposed for sickle cell anemia and β-thalassemia [BC11A with Cas9 significantly increased fetal hemoglobin levels in vitro [BC11A by electroporation-mediated delivery of Cas9 into HSPCs for the treatment of β-thalassemia and sickle cell disease  [Therapeutic gene-editing strategies for sickle cell anemia and β-thalassemia either correct the mutated β-globin  or disru11A gene ,166. In 11A gene ,165 or l11A gene . Treatedlassemia . One stuin vitro . Current3745287) .Electroporation-mediated therapeutic gene editing has been applied for T cell cancer immunotherapies. In vitro studies demonstrated successful gene editing in T cells electroporated with CRISPR-Cas9 for recognition of cancerous cells ,172. In 2+ ions to the buffer can improve viability although the transfection of DNA can be impaired at excess concentrations [Despite success in preclinical trials, advancements are needed to further enhance electroporation-mediated delivery of Cas9 for clinical trials. Low levels of gene knockout or knockin require screening of ex vivo treated cells to enrich for successfully gene edited cells prior to transplantation. In contrast, screening is unnecessary when there are high levels of transfection efficiency. Studies have found decreased viability in cells following electroporation . Optimiztrations ,179. FurLipid mediated delivery of gene products into cells was first demonstrated in 1987 . CationiPcsk9 [Ttr in mice. The group also compared Cas9 activity in vivo with unmodified sgRNAs and chemically modified sgRNAs. Lower levels of Cas9 editing were observed in vivo using unmodified sgRNA, indicating that chemically modified sgRNAs with enhanced stability are essential for in vivo editing.Early experiments for lipid-mediated delivery of Cas9 often used Lipofectamine to deliver plasmid DNA expressing Cas9 and a targeting sgRNA into cells ,193,194.Pcsk9 . The firPcsk9 . The autBecause the Cas9 protein has a positive net charge of +22, it does not readily bind and assemble into LNPs . HoweverDelivery of Cas9 to specific tissues remains an ongoing challenge for LNP-mediated CRISPR-Cas9 delivery. In a study by Wei et al., a wide array of LNPs was shown to be tunable for delivery of Cas9 into targeted tissues, including w dendrimer lipid nanoparticles, stable nucleic acid lipid particles, and lipid-like nanoparticles . InjectiHydrodynamic delivery (HD) is a physical delivery technique originally developed for in vivo introduction of naked DNA in 1999 ,202. SinMechanistically, upon tail vein injection, the solution enters circulation through the inferior vena cava and flows through the heart, but due to the excessive load, retrograde flow occurs into the hepatic vein, where the solution is forced out into tissues . As a rei.HD for Therapeutic ApplicationsFah, the gene encoding fumarylacetoacetate hydrolase, in a mouse model of hereditary tyrosinemia by HD of CRISPR-Cas9 components along with a donor template DNA. The overall editing efficiency of 4% of the hepatocytes was sufficient to correct the disease because the edited hepatocytes had a selective advantage over the unmodified cells for repopulating the liver [Hpd, the gene encoding hydroxyphenylpyruvate dioxygenase, with indels within the range of 35–60% to prevent the accumulation of toxic metabolites in the liver [Fah-aiming sgRNA to correct an A-G splice-site mutation that caused hereditary tyrosinemia type I in the liver of Fah-deficient mice hydrodynamically injected via the tail vein [Fas, encoding for a death receptor associated with apoptosis, was disrupted using CRISPR-Cas9 to rescue Concanavalin-A-induced fulminant hepatic failure in a mouse model. Results demonstrated successful downregulation of Fas expression, leading to the protection of hepatocytes from Fas-mediated apoptosis.HD has been applied for delivery of CRISPR-Cas for in vivo genome editing in models of human inherited metabolic diseases affecting the liver. Yin et al. achieved successful in vivo gene correction of the mutation in he liver . The Fahhe liver . More reail vein . Althougail vein . In the FVIII (BDDF8) into the Alb locus along with CRISPR components via hydrodynamic delivery and observed 0.1% and 2% knockin efficiencies with circular and linearized plasmid donors, respectively. Moreover, the linear donor design led to successful precise integration of BDDF8 in 1–2% of liver cells and corrected hemophilia A in most of the affected mice.In a separate study by Zhang et al., CRISPR-Cas9 introduced using HD was used to correct mouse models of Hemophilia A . The autCRISPR-Cas9 delivered via HD injection has been demonstrated for treatment of infectious diseases in mouse models. The study by Lin et al. was the first to demonstrate the efficacy of harnessing CRISPR-Cas to target the virus causing Hepatitis B (HBV) in vivo .Using hyii.HD for Generation of Animal Models of Human DiseaseDnajb1–Prkaca fusion mutation, which has been identified in 80–100% of patients with fibrolamellar hepatocellular carcinoma (FL-HCC), a rare form of liver cancer whose molecular basis is not established [Dnajb1–Prkaca fusion mutation as the cause of FL-HCC in wild-type mice. The study not only brings to light the molecular basis of the disease by identifying causal mutations, it provides a potential therapeutic target while establishing a mouse model of FL-HCC.HD has also been utilized as a tool to study the molecular basis of diseases and create animal models. Engelholm et al. used HD of CRISPR-Cas9 designed to delete the syntenic region on chromosome 8 in mice. Successful deletion was observed in 80% of the mice and resulted in a ablished . The mutNras and the Pten-aiming CRISPR-Cas9 through hydrodynamic injection. The results showed that only the combination of Pten disruption and Nras overexpression  was sufficient to induce HCC development in mice. By revealing the crosstalk between these two genes, the findings provide insight into HCC development, which may have important implications in liver cancer research.In a separate study, Gao and Liu investigated the role of cross-talk between Pten loss and Nras activation in driving liver cancer development in mice . Using iAlthough HD has proven to be useful in small animal models, major safety concerns prevent its translation to the clinic. One major hurdle is that the injected gene solution comprises roughly 10% of bodyweight , which iAnother major drawback of HD is the adverse effect of the injection. Upon injecting 9% of body weight into mice, a sharp increase in intravascular pressure in the liver and hepatocellular vascularity along with morphological changes were observed ,226. TheA potential tool for direct delivery of Cas9 into cells is to fuse the nuclease to a cell-penetrating peptide (CPP) that carries cargo across a cellular membrane. CPPs were first described in 1988 when researchers observed that the transcription transactivating protein isolated from the HIV virus could cross cellular membranes . For delThe development of gene-editing technologies has occurred at a rapid pace with new tools or variants of existing tools emerging almost every year. In light of gene-editing-based gene therapy trials well underway in the US, Europe, and China, modern medicine has embarked on a quest in which most monogenetic and acquired diseases have the potential to be treated or cured at the genomic level by precisely altering sequences at specific loci in human cells. As the most used and characterized gene-editing tool, CRISPR-Cas9 nuclease is currently the platform of choice for emerging translational applications of gene editing. A major barrier to clinical advancement of Cas9-mediated gene-editing therapies is the absence of safe and effective methods for delivering the CRISPR components into target cells. AAVs are a practical delivery approach for introducing CRISPR-Cas9 reagents, particularly the SaCas9, a small ortholog amenable to packaging into a single AAV vector for in vivo gene editing. However, AAVs have the potential to randomly insert into the genome, and they increase risk of hepatocellular carcinoma in neonatal mice ,128,220.Non-viral methods have the potential to address the challenges of AAV-mediated delivery for introducing gene-editing reagents into target cells. LNP-mediated delivery of transient Cas9 mRNA or RNPs is a potential solution to pre-existing immunity. However, because Cas9 mRNA and RNPs can provide peak gene editing for up to 48 h after their introduction in cells ,42, therConsidering the multitude of challenges, it is essential that the field of genome editing continues to make progress in developing novel approaches to deliver gene-editing reagents into target cells with high efficiency to further enhance the safety of gene editing and editing capabilities to unleash the full therapeutic potential of gene editing.


Purpose: To conduct a population-based study to determine whether the use of GnRH agonist and antiandrogens are associated with an increased risk of cardio-cerebrovascular disease (CCVD) in Asian patients with prostate cancer using the National Health Insurance Service-Elderly Cohort Database (NHIS-ECD).Materials and Methods: We included a total of 2,413 men aged 60 years or older with prostate cancer between January 2003 and December 2008. Outcomes of interest included the first occurrence of cardiovascular events  and cerebrovascular events .Results: The 5-year AMI-free rates of patients diagnosed with prostate cancer and treated with GnRH agonists, antiandrogens alone, or androgen deprivation therapy (ADT)-naïve interventions were 97.0%, 96.5%, and 98.3%, respectively, while the 5-year IHD-free rates were 93.2%, 92.3%, and 94.5%, respectively. Exposure to GnRH agonists or antiandrogen regimens did not significantly increase the risk of AMI or IHD compared to ADT-naïve treatment in multivariate Cox proportional-hazards models after adjusting for other covariates. Five-year IS-free rates of patients exposed to GnRH agonists, antiandrogens alone, and those with ADT-naïve prostate cancer were 94.8%, 94.7%, and 95.5%, respectively, while the five-year CVD-free rates were 92.9%, 93.3%, and 94.6%, respectively. Cox proportional-hazards models also failed to show that men who received GnRH agonist or antiandrogen treatment alone carried a significantly increased risk for IS or CVD compared to ADT-naïve patients.Conclusions: The current study based on Asian population suggests that treatment with neither GnRH agonist nor antiandrogens increases the risk of cardio-cerebrovascular disease compared to patients with ADT-naïve prostate cancer. Androgen deprivation therapy (ADT) is routinely used to treat patients with advanced prostate cancer (PCa)However, these theories remain inconclusive. Further studies are needed to confirm the association between ADT and CCVD. In particular, physiological, genetic, and ethnic differences might contribute to metabolic changes associated with ADT. However, most studies investigating the association between ADT and CCVD involved the Western population. Only a few studies involved Asian populations, including a small number of patients in a single institution, which does not represent the whole population.Therefore, this population-based study used the National Health Insurance Service-Elderly Cohort Database (NHIS-ECD). To determine whether the use of ADT was associated with an increased risk of CCVD in patients with PCa.This study used claims data acquired from NHIS-ECD released by the Korean National Health Insurance Service (KNHIS). The KNHIS provides data sharing service to support policy and academic investigations using claims data collected under the national health insurance program. The cohort members of NHIS-ECD were selected by a simple random sampling method from patients aged 60 years or older who qualified for the health insurance program in 2012. These cohort members were followed up for 12 years until 2013 unless the individual was disqualified due to death or emigration. The NHIS-ECD contains information regarding 558,147 elderly patients, accounting for approximately 10% of the entire South Korean patients aged 60 years or older. The following variables were included in the NHIS-ECD data: socio-economic status (including death and disability), principal diagnosis or comorbidities based on the International Classification of Disease, 10th Revision (ICD-10), surgical procedures, and drug prescription . Because patient data in NHIS-ECD were fully de-identified, the requirement for Institutional Review Board approval was waived.Male patients aged 60 years or older with a first diagnosis of PCa between January 2003 and December 2008, and prostatic biopsy within 30 days from the date of the first diagnosis were included in this study Figure .Patients with prevalent CCVD who had diagnosis of either condition prior to cohort entry or 6 months after cohort entry were excluded. Study subjects were classified according to treatment type. Patients treated with gonadotropin-releasing hormone [GnRH] agonists within 6 months of PCa diagnosis were defined as GnRH agonist users. Patients treated with antiandrogens alone within six months of diagnosis were defined as antiandrogen users. We included patients who were treated for at least 6 months (GnRH agonists or antiandrogens only). Patients who received neither type of treatment were defined as ADT-naïve.CCVD events included cardiovascular and cerebrovascular events. Cardiovascular events were classified into acute myocardial infarction (AMI) and ischemic heart disease (IHD) while cerebrovascular events were classified into ischemic stroke (IS) and cerebrovascular disease (CVD). Outcomes of interest included the first occurrence of AMI, IHD, IS, and CVD. Incident AMI was defined as a hospitalization or emergency room (ER) visit with an ICD-10 code of I21-I23 or I25.2 in the primary or secondary diagnosis code. Similarly, IHD, IS, or CVD was defined as a hospitalization or ER visit with an ICD-10 code of I20-I25 for IHD, I63 for IS, and I60-I69 for CVD in the primary or secondary diagnosis code.Baseline covariates were derived from medical claims during 12 months preceding the cohort entry. The variables included age, year of diagnosis of PCa, diabetes mellitus (ICD-10 code of E10-14), hypertension , and Charlson comorbidity index (CCI). CCI was calculated from medical claims one year before PCa diagnosis using the method proposed by Quan et al Each patient was followed up from the cohort entry date until the diagnosis of the first CCVD event of interest, the start of another ADT, death, or the end of study period (31 December 2013). Analysis of variance (ANOVA) was used to compare means while Chi-square test was used to compare the proportions of study patients according to the different treatment regimens. We calculated the incidence rates of AMI, IHD, IS, and CVD during treatment with GnRH agonists, antiandrogens alone, or ADT-naïve interventions. Survival analyses were performed using Cox proportional-hazards regression. All covariates in Table The current study cohort comprising a total of 2,413 prostate cancer patients included 738 who received GnRH agonist, 173 treated with antiandrogen alone, and 1502 exposed to neither type of treatment. The development of IHD, AMI, IS, and CVD in the study cohort was followed for a median of 5.79 years , 5.84 years , 5.62 years , and 5.50 years  following PCa diagnosis, respectively.The overall patient characteristics in the prostate cancer study cohort are summarized in Table Overall, the 5-year AMI-free and IHD-free rates were 97.8% and 94.0%, respectively, in the current cohort. The AMI-free rates of patients treated with GnRH agonists, antiandrogens, and those who were ADT-naïve were 97.0%, 96.5%, and 98.3%, respectively, while the IHD-free rates were 93.2%, 92.3%, and 94.5%, respectively  compared to ADT-naïve patients . Conversely, treatment with antiandrogen alone was not associated with a significantly increased risk of AMI . In multivariate Cox proportional-hazards models, the use of GnRH agonists  or antiandrogen only  in PCa patients did not significantly increase the risk of AMI after adjusting for other covariates compared to ADT-naïve patients.In crude Cox proportional-hazards models, the use of GnRH agonist in patients resulted in a significantly increased risk of AMI  or multivariate analysis . The use of antiandrogen alone did not show a significantly increased risk of IHD compared to ADT-naïve patients in crude analysis  or multivariate analysis . These results were consistent with Kaplan-Meier survival curves, showing no significant difference in the survival of patients exposed to GnRH agonist or antiandrogen alone compared to ADT-naïve group in each event category .Overall, the 5-year IS-free and CVD-free rates were 95.3% and 94.1%, respectively. The IS-free rates for PCa patients exposed to GnRH agonist, antiandrogens, and those who were ADT-naïve were 94.8%, 94.7%, and 95.5%, respectively, while the CVD-free rates were 92.9%, 93.3%, and 94.6%, respectively .The results of Cox proportional-hazards analysis stratified into IS and CVD in the current cohort are shown in Table The key findings of this Asian population-based study are as follows. First, prostate cancer patients who received GnRH agonist or antiandrogens only did not show an increased risk of cardiovascular disease compared to ADT-naïve patients. Second, the use of GnRH agonist or antiandrogens was not a significant risk factor for developing cerebrovascular disease in Asian men with prostate cancer.Despite ongoing review, the FDA drug safety communication has warned that ADT increases the risk of certain cardiovascular diseases, and physicians should monitor their development However, the relationship between ADT and CCVD remains controversial. In addition, most studies investigating the effect of ADT on CCVD have been based on western populations including Caucasians. As mentioned above, the pathophysiological events underlying changes in body composition, especially fat mass, play a key role in the development of CCVD. Significant differences in body composition and fat mass have been found between western population and Asians. In general, the prevalence of obesity is significantly higher in western population than in Asian population Physiological and genetic differences according to ethnicity also affect the relationship between ADT and CCVD. Differences in androgen receptor (AR) CAG repeat length polymorphism Our results are in agreement with previous Asian population-based studies, including the Japanese population It is worth noting that this is the first population-based observational study to investigate the relationship between ADT and the risk of CCVD among Koreans, a population with homogeneous ethnicity. To the best of our knowledge, the sample size and observation period in this study are larger than those of previous studies involving Asian population. In addition, we carefully defined the potential confounders at baseline. Male patients with a history of any cardiovascular disease  and renal failure were excluded from the analysis, which was thus more reliable by controlling potential confounders. In addition, those who were aged above 40 years in South Korea were advised to undergo health examination including electrocardiography and lipid profile. Any abnormal findings were covered by NHIS for further medical evaluation. Therefore, the analysis entailing frequent and widespread medical evaluation has considerable strength and power.Our study has some limitations due to potential confounding factors associated with lifestyle. Smoking status and obesity are known risk factors for CCVD. However, they were not evaluated in the current study. Another limitation was that patients were not categorized by cancer status such as stage, metastasis, and Gleason score in the current study because cancer profile was unavailable in the current database. However, the extent or staging of prostate cancer might not modify CCVD risk. Lastly, the potential for disease misclassification based on claim database was another limitation. The Korean NHISS covers almost all medical costs of patients with major diseases such as malignancy, coronary heart disease, and stroke. Patients only need to pay 5% of the total medical cost. However, the registration and certification criteria for patients with major diseases are stringent, which ensures sufficient reliability and trustworthiness of medical claims.The current study based on nation-wide population in Korea suggests that the use of ADT in prostate cancer patients does not increase the risk of CCVD compared to ADT-naïve patients. Although it is not sufficient to establish the association between ADT and CCVD, the Asian population differs from western population. Therefore, further well-designed studies including different regions and populations are needed to elucidate the increased risk of CCVD following ADT.


MicroRNAs (miRNAs) are non-coding RNAs that regulate multiple cellular processes during cancer progression, identified to be involved in tumorgenesis of several cancers including cancers of digestive system. However its role in gallbladder inflammatory disease (GID) and gallbladder cancer (GBC) has not been well documented. The present study was aimed to investigate the clinical significance of hsa-miRNA-335-5p (miR-335) in GBC and GID. This prospective case control study, conducted from July 1, 2014 to December 1, 2017 in Era’s Lucknow Medical College & Hospital, India, evaluated miR-335 expression by real-time polymerase chain reaction. Hundred tissue samples GID  and GBC  were studied. Relative quantification of target miR-335 expression was examined using the comparative cycle threshold method. Their expression was correlated with different clinicopathological parameters. Fishers’ exact test, Student’s t-test, and Chi-square test were used as appropriate for data analysis. Kaplan-Meier methods were used to calculate overall and disease-free survival rate. Two sided P<0.05 was considered as significant. miR-335 expression was found to be significantly low in GBC lesions when compared with GID lesions (P<0.001). The low expression level of miR-335 was correlated with histological grade (P=0.007), clinical stage (P<0.001), lymph node metastasis (P<0.001) and liver metastasis (P=0.016). Reduced expression of miRNA-335 was associated with a shorter median overall survival (7 months vs. 25 months) in GBC patients (P<0.001). Down regulation of miR-335 is associated with the severity of the disease and thus indicate that miR-335 expression may serve as prognostic marker for GBC. Gallbladder cancer (GBC) is highly aggressive and incurable disease representing the most common malignancy of the biliary tract with three-fold higher incidence in females . GBC shows striking geographical predictions in its incidence with the highest figures reported in India and Chile and relatively low levels in many western countries . The overall 5 years survival of less than 5% has been reported . The poor outcome for GBC is ascribed to late presentation, absence of specific symptoms, rapid and silent progression, ineffective therapy and limited knowledge of its etiology . To date, GBC continues to be a challenge with regards to its early stage diagnosis and optimal management of advance disease, and requires evaluation of all factors including molecular markers associated with the disease. There are many reports focused on the genetic and epigenetic alterations in GBC, which involve modifications in the expression of tumor suppressor genes and oncogenes. However, there are very few studies focused on miRNA-based epigenetic modifications involved in gallbladder carcinogenesis.MicroRNAs (miRNAs) are small, non-coding ribonucleic acids (RNAs). MiRNAs play a role on various biological processes such as differentiation, proliferation and apoptosis and control multiple gene involved in cancer. Accumulating studies indicate that miRNAs display aberrant expression patterns and functional abnormalities in many types of cancers. They act either as tumor suppressors or oncogenes according to the roles of their target genes . Changes in their expression can be used as robust and important biomarkers for cancer risk, diagnosis, prognosis, and can even be used as miRNA-based therapeutic targets. MiR-335 has been reported to differentially express in benign and malignant tumors . It is known to be associated with increased risk and prognosis of various cancers . MiR-335 has been found deregulated in breast cancer cells, gastric cancer cells, and ovarian cancer cells . But its association with aggressive behavior of GBC and gallbladder inflammatory disease (GID) has not been well evaluated. It was hypothesized that miR-335 expression would be highly down regulated in advanced stage of GBC, when compared primary stage of GBC, thus it might work as molecular marker for disease progression. Therefore we investigated the role of miR-335 expression in tissue sample of patients with different stage of GBC and compared with tissue sample of patients with GID.PatientsThis observational prospective case control study includes 50 cases of GBC who, were operated in the Department of Surgical Oncology, King George’s Medical University, Lucknow and Department of Surgery, Era’s Lucknow Medical College and Hospital, Lucknow, from July 2014 to December 2017. All patients suspected of having GBC based on clinical evaluation (Ultrasonography and computed tomography), who underwent for exploration for primary gallbladder cancer followed by either radial surgery or biopsy of metastatic lesion found during exploration. The control group comprised 50 patients of GID cases of chronic calculus cholecystitis and adenomatous hyperplasia without any gallbladder polyp or adenoma disease based on clinical evaluation and confirmed by ultrasound examination and underwent laparoscopic or open Cholecystectomy were included in the study.Ethical ComplianceProtocol and procedures employed were reviewed and approved by the Institutional Ethics Committee of Era’s Lucknow Medical College and Hospital, Lucknow India, and was in accordance with Helsinki declaration . The patients enrolled in the study provided a written informed consent for participation in this research as well as for presentation of data. The exclusion criteria were immunodeficiency disorder, patients with other cancer or who received prior chemo-radiotherapy or operated earlier. th edition 2010 . The other portion was collected in RNA later (Sigma-Aldrich ), kept at -80°C till further miR-335 processing. Tissue samples were taken into two portions; one portion of the tissue was collected in 10% buffered formalin and embedded in paraffin for histopathological examination and pathological staging was done according to American Joint Committee on cancer (AJCC) tumor node metastasis (TNM) staging 7Methods-ΔΔCt), where ΔCt =Ct miR-335-Ct RNU6B. The correlation was done in between Ct of target miRNA with respect to RNU6B. Each sample was examined in triplicate. Total ribonucleic acid (RNA) was extracted from tissue by using the mirVANA miRNA isolation Kit according to the manufacturer’s protocol. The RNA concentration was determined by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) and sample was stored at -20°C. The miR-335 and RNU6B -specific complementary de-oxyribonucleic acid (cDNA) were reverse-transcribed from two micrograms total RNA using TaqMan MicroRNA Reverse Transcription kit, Applied Biosystems, (USA). TaqMan Universal PCR Master Mix, No AmpErase (Uracil-N glycoslyase). Individual RT-PCR assays were performed in a 20μL reaction volume on a RT- PCR system (Applied Biosystems StepOnePlus™) to detect miR-335 expression in tissue. The thermal cycling conditions consists of optional AmpErase UNG activity hold at 50°C for 2 minutes, enzyme activation hold at 95°C for 10 minutes, further 40 PCR cycles of denaturation at 95°C for 15 seconds and annealing and extension step at 60°C for 60 seconds. The cycle threshold (Ct) value was recorded for both miR-335 and RNU6B, and the relative quantification of target miRNA expression was expressed as  (Reference percentage reduced expression of miR-335) final size of the sample is 50 in each group.The sample size was calculated using the formula n= Zα2) test was used to test for a statistically significant relationship between two categorical study variables. The association between miR-335 expressions and clinopathological characteristic of gallbladder cancer patients was investigated by using Chi-square test or Fisher exact test. The cut-off values of miR-335 was set by the median value of the expression levels, and were divided into two groups according to high and low expression levels of miR-335. Correlation analysis was performed using Pearson correlation method. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. The Cox proportional hazards model was used to analyze the significance of various parameters for survival. Two tailed P value <0.05 was considered statistically significant. All the clinical details and the experimental data were analyzed using GraphPad Prism version 5.0 by GraphPad Software, Inc. California, USA. Descriptive statistics of study subjects are either represented as mean with standard deviation or frequency with percentages. Normality of the data distribution was determined by Smirnov- Kolmogorov test. The chi-square ) were evaluated for the expression of miRNA - 335. Study subjects were predominantly female. Average aged subjects 50.52±1.43 in GBC and 40.14±1.35 in GID cases; with high prevalence of patients with gallstones in both case and control group . QuantitIn this study the median expression level of miR-335 (0.223) was used as a cutoff point to divide all 50 GBC patients into two groups. GBC patients who expressed miR-335 with values less than the median expression level were assigned at low expression group , and those with expression above the cutoff value were assigned to the high expression group .Significant negative correlation was found between the low expression of miR-335 with advanced clinical stage (P<0.001), tumor differentiation (P=0.007), lymph node metastasis (P<0.001) and liver metastasis (P=0.016). However miR-335 expression was not significantly related to sex, age, presence or absence of gallstone, or pain in abdomen (P>0.05) .The association between median overall survivals of GBC patients was investigated by Kaplan-Meier method and log-rank test . GBC patIn this study, we observed that down expression of miR-335 was significantly correlated with higher stage of disease. Also decreased expression of miR-335 was significantly associated with tumor differentiation, lymph node metastasis, liver metastasis and correlates with shorter survival. Down-regulated expression of miR-335 was found higher in patients with stage (III-IV) of disease. In recent years, miRNAs have been found to play a role in multiple human diseases including cancers, because they have great power to impact gene expression through posttranscriptional way. Various kinds of miRNAs have been found to correlate with tumorigenesis or tumor progression. For example, miRNA 21 was up-regulated in glioma compared with normal brain and related to cell apoptosis, tumor proliferation, invasiveness, and migration . Furthermore, many miRNAs have been found to correlate to tumor prognosis in GBC such as miRNA 34, miRNA 145, miRNA 146b, and miRNA 218 . As a potential tumor related miRNA, the gene locus of miR-335 is on chromosome 7q32.2 . It has been demonstrated to function as an oncogenic or a tumor-suppressor miRNA in various human malignancies. MiR-335 is shown to be upregulated in myeloma, gliomas, and meningiomas,  but downregulated in pancreatic adenocarcinoma, breast cancer and hepatocellular carcinoma . There have been controversial reports on the expression patterns of miR-335 even in the same cancer type. For example, Yan et al., (2012) reported that high frequency of recurrence and poor survival was associated with upregulated expression of miR-335 in gastric cancer. In contrast, Xu et al., (2012) found down regulation of miR-335 in gastric cancer, they reported that lymph node metastasis, poor pT stage, poor pN stage, and invasion of lymphatic vessels, was significantly associated with low expression of miR-335, suggesting a metastasis suppressor function of miR-335 in this disease. miR-335 has been demonstrated to play important roles gastrointestinal tract cancer including GBC . However there has been scarcity of studies on GBC showing the prognostic and therapeutic importance of miR-335. In our study we observed lower expression of miR-335 in malignant lesions as compared to benign lesions of gallbladder. Similar observation was made by  in gliomas, colorectal cancer and ovarian cancer. Peng et al., (2013), reported down regulation of miR-335 in GBC and associated with aggressive tumor behavior. In line with these finding we also observed lower expression of miR-335 in GBC and found to be significantly associated with progression of the disease. Showing that down- regulation of miR-335 might play a crucial role in gallbladder cancer progression. In the present study, we found that miR-335 is an independent prognostic factor for patients with GBC, and patients with low miR-335 expression have poorer prognosis than those with high miR-335 expression. In fact, a recent study by Peng et al., (2013) on GBC had similar findings. Indicating that miR-335 expression may be a useful marker to predict patient survival in GBC [38]However in contrast, Jiang et al., (2012) found that glioma patients with high miR-335 expression tumors had significantly shorter survival times than those with low miR-335 expression tumors. Although confirmation requires larger and more long-term studies, the variable interpretations of the significance of miR-335 expression between GBC and other types of cancers suggest that the actions and roles of miR-335 may depend on the cell type of tumor origin. Further investigations with target genes involved in miR-335 expression would help us better understand the clinical importance of miR-335 in cancer progression. Moreover it may also help us in discovering some novel therapeutic and prognostic possibilities for treating this dreaded disease.Our study indicated that miR-335 expression was decreased in GBC and was associated with tumor progression. Furthermore, miR-335 expression was found to be a marker for predicting the clinical outcome of the GBC patients. Indicating that loss of miR-335 expression may be a useful prognostic marker and a therapeutic target for GBC.The authors report no declaration of interest.


Are interstitial lung abnormalities, which are minor interstitial shadows on lung computed tomography, associated with immune checkpoint inhibitor induced–interstitial lung disease in patients with nonlung cancers?In this cohort study of 199 patients with advanced nonlung cancers treated with anti–programmed cell death 1 antibody monotherapy, patients with preexisting interstitial lung abnormalities had significantly increased risk of immune checkpoint inhibitor induced–interstitial lung disease.These findings suggest that greater attention should be accorded to the development of immune checkpoint inhibitor induced–interstitial lung disease in patients with nonlung cancers and interstitial lung abnormalities. This cohort study evaluates whether interstitial lung abnormalities are associated with immune checkpoint inhibitor–induced interstitial lung disease in patients with nonlung cancers. Immune checkpoint inhibitor–induced interstitial lung disease (ICI-ILD) is clinically serious and life-threatening. Preexisting interstitial lung abnormalities have been shown to be risk factors for ICI-ILD in patients with lung cancer.To evaluate whether interstitial lung abnormalities are associated with ICI-ILD in patients with nonlung cancers.This cohort study was conducted between December 2015 and May 2019 at Hiroshima University Hospital. A total of 199 consecutive patients with head and neck cancer, malignant melanoma, oral cavity cancer, urological cancer, and gastrointestinal cancer who received anti–programmed cell death 1 (PD-1) antibody monotherapy were included. Data analysis was conducted from December 2015 to May 2019.The associations between potential risk factors and the development of ICI-ILD were examined. Information on patient characteristics before antibody administration, including chest computed tomography findings, was obtained. The diagnosis of ICI-ILD was defined as abnormal computed tomography shadows occurring during treatment with anti-PD-1 antibodies.P < .001), and ground glass attenuation in interstitial lung abnormalities was an independently associated risk factor .A total of 199 patients were enrolled in the study. The median (range) age was 66 (20-93) years, and most patients (133 [66.8%]) were men. Nineteen patients (9.5%) developed ICI-ILD. There was no significant difference in the baseline characteristics between patients with and without ICI-ILD. The logistic regression analyses revealed that interstitial lung abnormalities were associated with increased risk of ICI-ILD (odds ratio, 6.29; 95% CI, 2.34-16.92; In this cohort study, preexisting interstitial lung abnormalities, including ground glass attenuation, were risk factors associated with ICI-ILD in patients with nonlung cancers. This observation is consistent with previously reported findings in patients with lung cancer. Therefore, we should pay more attention to the development of ICI-ILD in patients with interstitial lung abnormalities, regardless of cancer type. Among them, ICI-induced interstitial lung disease (ICI-ILD) is a clinically serious and life-threatening toxic effect.7 The reported prevalence of ICI-ILD is 4.1% in patients with lung cancer, 4.1% in patients with kidney cell carcinoma, and 1.6% in patients with malignant melanoma.8 Therefore, it is important to determine the risk factors for ICI-ILD to pay more attention to the development of ICI-ILD and initiate prompt treatment in patients with risk factors.Anti–programmed cell death 1 (PD-1) antibodies, an immune checkpoint inhibitor (ICI), exert antitumor effects by suppressing the immune tolerance to cancer cells. Recent clinical trials have demonstrated such antitumor effects, and these antibodies are approved for use for the treatment of several advanced cancers.10 In addition, we previously reported that interstitial lung abnormalities (ILAs) are a risk factor for ICI-ILD in patients with NSCLC.11 ILAs are generated by aging or smoking,14 among other factors, and minor interstitial shadows on lung computed tomography (CT) in individuals with no known ILD are present in 14% of treatment-naive patients with NSCLC.15 However, ILAs in patients with nonlung cancers would not be recognized before the administration of anti-PD-1 antibodies by physicians. In addition, there have been no reports on the risk factors for ICI-ILD in patients with nonlung cancers. Therefore, we aimed to investigate whether any patient characteristics, including ILAs, were associated with increased risk of ICI-ILD in patients with nonlung cancers.Preexisting ILD has been reported as a risk factor for the development of ICI-ILD in patients with non–small cell lung carcinoma (NSCLC).International Statistical Classification of Diseases and Related Health Problems, Tenth Revision) who received anti-PD-1 antibodies (nivolumab or pembrolizumab) at Hiroshima University Hospital between December 2015 and May 2019 were retrospectively enrolled. Information on patient characteristics before anti-PD-1 antibody administration, including chest CT findings, was obtained. Tumor responses were evaluated using the Response Evaluation Criteria for Solid Tumors version 1.1. To obtain patient consent, this study applied the opt-out method. We posted information about this study on the home page of our institution and guaranteed that the enrolled patients had an opportunity to refuse to participate in this study. In addition, we clarified the period for accepting the intention of refusal. This study was approved by the Hiroshima University institutional review board and conducted in accordance with the ethical standards established by the Declaration of Helsinki.16 This report followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.Consecutive patients with head and neck cancer, malignant melanoma, oral cavity cancer, urological cancer, gastrointestinal cancer, or malignant lymphoma .17 Using a sequential reading method, CT images were evaluated by 3 pulmonologists  who were unaware of other clinical findings or patient outcomes.17 In brief, reader 1 evaluated all CT images, and reader 2 reviewed ILA images and 20% of the non-ILA images that were evaluated by reader 1. Images with discordant evaluations were further reviewed by reader 3, and final decisions were agreed by consensus among the 3 readers. The correlation between reader 1 and reader 2 was determined by evaluating the Spearman rank correlation coefficient.CT data were obtained at the end-inspiration phase in the supine position using a CT scanner. We investigated the presence of abnormal findings, including preexisting ILAs on CT. The presence of ILAs was determined by observations such as ground glass attenuation (GGA), reticulation, honeycombing or traction bronchiectasis, diffuse centrilobular nodularity, and nonemphysematous cysts on CT imagesstration . These f18: (1) usual interstitial pneumonia pattern, (2) nonspecific interstitial pneumonia (NSIP) pattern, (3) organizing pneumonia (OP) pattern, (4) diffuse alveolar damage (DAD) pattern, or (5) not applicable. This classification was evaluated by 2 pulmonologists (K.S. and T.M.), who did not have information on the presence or absence of ILA or characteristics for each patient. Final decisions were agreed on by consensus between both pulmonologists. The time frame for chest CT was determined by clinicians based on the patient’s symptoms, blood tests, and chest radiographic findings. ILD was evaluated based on the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0.The diagnosis of ICI-ILD was defined as follows: (1) new abnormal shadows occurring on chest CT during treatment with anti-PD-1 antibodies .Results are expressed as medians (range) or means (SD). Comparisons between 2 groups were performed using Pearson χA total of 199 patients were enrolled in the study. The clinical characteristics of these patients are provided in Of 199 patients, 19 (9.5%) developed ICI-ILD . Among tP = .03). In contrast, the proportion of patients with ILAs was significantly higher among patients with ICI-ILD than among those without  . However, the proportions of patients with abnormal shadows, including emphysema, metastatic lung tumors, and radiation pneumonitis, did not differ significantly between the 2 groups. In urological or gastrointestinal cancers, the proportion of patients with ILAs was significantly higher in patients with ICI-ILD than those without, although the number of patients was small (eTable 6 in the The baseline characteristics of patients with and without ICI-ILD did not differ significantly . The pro < .001) . FurtherP < .001), GGA , and reticulation  were identified as significant risk factors associated with ICI-ILD in the univariate analysis  (P = .01) in ILAs to be an independent risk factor associated with ICI-ILD.Preexisting ILAs  . In mode20 However, to our knowledge, risk factors for ICI-ILD in nonlung cancers have not been previously investigated. Therefore, to our knowledge, this is the first study to identify preexisting ILAs as risk factors associated with ICI-ILD in nonlung cancers. We previously reported that preexisting ILAs are risk factors for ICI-ILD in patients with NSCLC patients.11 Therefore, greater attention should be paid to the development of ICI-ILD in patients with ILAs regardless of cancer type.This study found that preexisting ILAs were risk factors associated with ICI-ILD in patients with nonlung cancers, and GGA in ILAs was an independent risk factor. ICI-ILD is a life-threatening toxic effect, and it was observed in several clinical trials of nonlung cancers.21 ICIs exert antitumor effects via the promotion of lymphocyte activity. This could explain why the presence of ILAs, including reticulation and GGA, was associated with ICI-ILD in this study.ILAs as risk factors for ICI-ILD depends on the pathological feature of the ILAs. The present study revealed that reticulation and GGA but not honeycombing or traction bronchiectasis were significant risk factors in the univariate analysis, and only GGA was independently associated with ICI-ILD. Reticulation pathologically reflects slight fibrosis and inflammation by lymphocytes in the interstitium of the lung. GGA also involves infiltration of lymphocytes into the interstitium, reflecting active inflammation. However, honeycombing and traction bronchiectasis reflect the destruction of lung alveolar structures due to fibrosis.14 The definition of ILAs varies, and there are narrow and broad definitions. In a narrow definition, ILA is referred to as dirty lung or preclinical ILD and refers to early or minor ILD in undiagnosed cases. In contrast, ILAs in the broad definition cover early to advanced ILD.17 In this study, minimal interstitial change was narrowly defined as ILA, referring to previous reports.17 The significance of narrowly defined ILAs has been recently reported. Several studies revealed that ILAs are associated with a future decline in respiratory function, the development of interstitial changes,22 or the occurrence of lung cancer.23It has been reported that ILAs are generated by aging and smoking.5 The incidence of ICI-ILD in this study was higher than those previously reported. We believe that this may be attributable to the fact that the number of patients enrolled in this study was lower than that in clinical trials, and the participants in this study were all Japanese, who are reported to have a higher incidence of drug-induced ILD than White patients and patients from other Asian countries.24Previous systematic reviews have found that the incidence of ICI-ILD was 4.1% in patients with NSCLC, 1.6% in patients with malignant melanoma, 4.1% in patients with kidney renal cell carcinoma, and approximately 1% to 4% in patients with other cancers.7 The duration from the initiation of ICI to the development of ICI-ILD varied considerably, with a median of 87.5 days , which tended to be longer in patients with nonlung cancers than those with NSCLC, according to a previous study. These results suggest that attention should be paid to the development of ICI-ILD regardless of the line and duration of anti-PD-1 antibody administration.In this study, ICI-ILD was observed in various lines of ICI, and this result is consistent with those of previous studies.This study has limitations. First, it was retrospective and performed at a single institution. Therefore, a prospective multicenter study is warranted to verify our findings. Second, ICI-ILD was not pathologically diagnosed in this study. Therefore, the ICI-ILD findings on CT could reflect non-ICI-ILD.In this cohort study, preexisting ILAs, including GGA, were significant risk factors associated with ICI-ILD in patients with nonlung cancer. This observation is consistent with those previously reported in patients with NSCLC. Therefore, we should focus greater attention on the development of ICI-ILD in patients with ILAs, regardless of the cancer type.


We trained ML models  and determined 5-year ASCVD risk prediction, including with and without incorporation of additional EHR variables, and in Asian and Hispanic subgroups. A total of 4309 patients had ASCVD events, with 2077 in PCE-ineligible patients. GBM performance in the full cohort, including PCE-ineligible patients (area under receiver-operating characteristic curve (AUC) 0.835, 95% confidence interval (CI): 0.825–0.846), was significantly better than that of the PCE in the PCE-eligible cohort . Among patients aged 40–79, GBM performed similarly before  and after  incorporating additional EHR data. Overall, ML models achieved comparable or improved performance compared to the PCE while allowing risk discrimination in a larger group of patients including PCE-ineligible patients. EHR-trained ML models may help bridge important gaps in ASCVD risk prediction.The pooled cohort equations (PCE) predict atherosclerotic cardiovascular disease (ASCVD) risk in patients with characteristics within prespecified ranges and has uncertain performance among Asians or Hispanics. It is unknown if machine learning (ML) models can improve ASCVD risk prediction across broader diverse, real-world populations. We developed ML models for ASCVD risk prediction for multi-ethnic patients using an electronic health record (EHR) database from Northern California. Our cohort included patients aged 18 years or older with no prior CVD and not on statins at baseline ( Developed using Cox proportional hazards modeling, the PCE represent a widely used guideline-endorsed calculator to assess 10-year ASCVD event risk for individuals without a prior history of ASCVD, and are a central part of ASCVD risk reduction approaches in clinical settings2. For calculating an individualized 10-year risk of ASCVD, the PCE are applicable to patients with characteristics within prespecified ranges—including age, race, cholesterol levels, and systolic blood pressure. PCE use is consequently limited for patients with values outside the prespecified PCE ranges as well as patients without all variables available, preventing accurate guideline-based risk estimation to guide ASCVD risk reduction strategies. The PCEs were derived primarily from non-Hispanic Whites (NHWs) and African-American (AA) populations, and it is not clear whether they adequately identify risk in diverse populations based on recent analyses demonstrating inconsistent results3. Indeed, the ACC/AHA Work Group that developed the PCE noted that the risk estimator may not accurately predict risk in other racial/ethnic groups4.Risk assessment algorithms have a well-established role in guiding atherosclerotic cardiovascular disease (ASCVD) management. The 2018 update to the 2013 ACC/AHA prevention guidelines highlighted the use of the revised pooled cohort equations (PCE) to determine ASCVD risk, which is used to guide crucial management decisions, particularly the initiation of moderate to high-intensity statin therapy for long-term risk reduction5. Predictive ML algorithms have been applied broadly in various disciplines6, with burgeoning evidence of their potential utility in medicine10.Machine learning (ML) algorithms present an opportunity to develop improved and more generalizable risk prediction models. By leveraging large-scale data—from electronic health records (EHRs), for instance—such algorithms can determine combinations of variables that reliably predict an outcome. ML may incorporate variables that may not be otherwise considered in traditional predictive algorithms and thus demonstrate favorable performance compared to traditional methods2 penalty (LRL2), and with an L1 lasso penalty (LRLasso). We hypothesized that ML methods would allow the development of ASCVD risk prediction algorithms that perform favorably compared to PCE and allow risk estimation for patients to whom the PCE do not apply.The applicability of the PCE to patients with missing or out-of-range PCE variables or in racial/ethnic subgroups other than NHW or AAs is limited. The aim of this study was to develop EHR-trained ML-based ASCVD risk prediction algorithms that demonstrate broad real-world applicability, including random forests (RF), gradient boosted machines (GBM), extreme gradient boosted models (XGBoost), and logistic regression with the standard LOf 797,505 patients, 518,114 patients using statins or with prior CVD at baseline, or with <5 years of ASCVD event-free follow-up after their index visit, were excluded. The final study population (the “full cohort”) included 262,923 patients with 4309 ASCVD-related events during 5-year follow-up. Within the full cohort, 105,692 patients were not aged 40–79 years on their baseline date and 25,510 patients’ cholesterol, blood pressure, or smoking status were unknown or out of range. This left 131,721 PCE-eligible patients, and 2232 with an ASCVD event during follow-up  algorithm may be less applicable in the United States given their derivation cohorts13. This study did not address patients with missing variables or racial/ethnic groups other than NHW and AA patients. A study that recalibrated PCE using modern cohort data with improved risk prediction did not address effects on diverse racial ethnic/racial subgroups2. To our knowledge, our study is the first to explore ML-derived models for ASCVD risk prediction in patients with missing PCE variables and diverse racial/ethnic subgroups.Several groups have reported PCE recalibration to optimize risk prediction. A study from the Women’s Health Initiative demonstrated that a statistical model incorporating events from the Centers for Medicare and Medicaid Services allowed improved risk discrimination14. Our algorithm was more broadly applicable compared to PCE, including for individuals with missing variables or values outside prespecified ranges—which may be important in practice. In our study, 48% of ASCVD events occurred in patients ineligible for PCE application. In a study of Northern California patients, out of 941,546 patients with available lipid levels, ~25%  had missing or out-of-range PCE values and were excluded from traditional PCE application11. ML-based models may help bridge ASCVD risk prediction gaps with such patients to allow guideline-directed management based on risk assessment. In addition, we found that GBM performance in the full cohort had favorable sensitivity compared to that of the PCE in PCE-eligible patients when assuming intermediate prevalence of high-risk patients of 5, 10, or 25%, which further supports the clinical utility of using ML models. For an initial screening test for ASCVD risk, sensitivity  is a crucial performance metric in order to minimize misclassifying high-risk patients as low risk, and maximize opportunities to initiate potentially life-saving management.Our results have implications for ASCVD risk prediction. As a tree-based ML method, GBM is able to incorporate complex variable interactions, which may explain its favorable performance compared to non-tree-based models, such as the PCE, as has been demonstrated in other contemporary ML studies addressing cardiovascular disease15. This analysis did not assess variables beyond lipid levels. Our results raise the hypothesis that efforts at improving ASCVD risk prediction by capturing additional EHR variables may be limited. A previously reported ML-based risk calculator derived from the Multi-Ethnic Study of Atherosclerosis used the same variables as PCE16. These findings suggest that the development of improved risk prediction may require richer data or incorporation of novel variables, such as genetic information across larger patient groups, although incorporation of genetic information may be susceptible to additional bias if not carefully performed18.Consistent with other studies, we found that incorporating structured EHR data beyond traditional PCE variables in ML models did not substantially improve risk prediction. While we did not check inter-correlation of EHR variables, it is likely that we used correlated variables, which we believe may be helpful for ML methods when variables are missing for a patient. The top 20 predictive variables employed by ML models included traditional variables, such as lipids along with socioeconomic factors, including education level. A study from the UK Biobank found that the addition of apolipoproteins, direct, or calculated low-density lipoprotein (LDL) cholesterol did not improve ASCVD risk prediction3. We found that ML-based risk prediction was comparable to PCE in Asians and Hispanics, and that incorporation of additional EHR variables did not substantially improve risk discrimination. Novel ASCVD risk estimation models aimed at improved performance in racial/ethnic subgroups may require consideration of variables that are not adequately captured in current structured EHRs.PCE performance and prevalence of ASCVD risk factors vary across racial/ethnic subgroups11. We report the effect of oversampling on ML model performance , who determined that the research does not involve human subjects and granted a waiver of consent based on the nature of the project, including the use of previously collected, de-identified data. The study sample was selected from EHR data of adults >18 years of age in a community-based outpatient healthcare system in Northern California between January 1, 2009 and December 31, 2018 with at least two outpatient visits that were at least 1 year apart. The index date was defined as the first outpatient visit, which was 1 year after the patient’s first clinic visit. If there were no cholesterol lab results before a patient’s index date, the index date was shifted to the date of the first cholesterol lab result. Patients with pre-existing ASCVD, atrial fibrillation, heart failure, or other cardiovascular disease identified by the International Classification of Diseases, 9th and 10th revision (ICD-9-CM/ICD-10-CM) coding scheme were excluded  cholesterol, systolic blood pressure, blood pressure treatment status, smoking status, and diabetes status; we refer to these as the PCE variables. For each patient, for total cholesterol, HDL cholesterol, systolic blood pressure, and smoking status, the most recent value on or before the index date was used. On the date of the patient’s blood pressure measurement, the use of antihypertensive medications  was assessed. Diabetes status was identified by either a diagnosis of diabetes  or a diabetes medication (GPI2: 27) prescribed on or prior to index date. Patients were included in the PCE-eligible cohort if all PCE variables were present and within specified ranges: age 40–79 years, total cholesterol 130–320 mg/dL, HDL-C 20–100 mg/dL, and systolic blood pressure 90–200 mmHg. Race/ethnicity was self-reported. Some patients’ races were inferred based on the Social Security Record database as previously describedWe assessed the value of including additional EHR variables beyond those from the PCE. These variables were selected a priori based on EHR availability, relevance to ASCVD risk, and predictive potential. Variables were grouped into socioeconomic, clinical, and healthcare utilization categories and extracted 1 year prior to the index date.Socioeconomic variables were based on addresses and included census block group level indicators of educational attainment and median household income.21. Self-reported family (parents or sibling) medical histories were extracted. The existence of each CCS and family history was coded as a binary variable. The most recent height, weight, and diastolic blood pressure measurements before each patient’s index date were included.Patient medical problems were extracted from the EHR problem list, which were coded in ICD-9-CM/ICD-10-CM, and grouped into 283 categories using the Clinical Classification Software (CCS)Medication prescription information was incorporated by considering the first four digits of prescriptions’ GPI codes (GPI4). An indicator was created for each GPI4 denoting whether any medication from that GPI4 was prescribed. The total number of medication prescriptions in the prior year was included as a variable.Two indicator variables were included for each lab test: whether the test was ordered and returned a “normal” result, and whether it was ordered and returned an “abnormal” result. The total number of laboratory tests ordered and the total number which returned “abnormal” results were also included. LDL cholesterol was included if available.Indicators of healthcare utilization included the number of primary care, urgent care, specialty, and other  services care visits in the previous year.4. Acute myocardial infarction was defined by ICD-9-CM codes 410.* and ICD-10-CM codes I21.*, I22.*, I23.3, I24.0, I24.9, I25.9, or I51.313. Stroke events were defined based on ICD-9-CM codes 430.*, 431.*, 432.*, 433.*1, 434.*1, or 436.0 and ICD-10-CM codes G46.*, I63.*, I67.85, I69.30, I77.89, P91.0, or Z86.7314. Fatal coronary artery disease was defined by the presence of an ICD-9-CM code 411.*, 413.*, or 414.* or an ICD-10-CM code I20.*, I23.7, I24.*, I25.*, or T82.85 code followed by death within a year. Death information was retrieved from EHRs and Social Security records.In accordance with ACC/AHA Work Group guidelines, an ASCVD event was defined as the first acute myocardial infarction, stroke, or fatal coronary artery disease22. Parameters developed for NHWs were used to estimate values for Asian, Hispanic, and other non-AA populations.The 5-year ASCVD predicted risk was calculated for the PCE-eligible cohort using published parameter estimates from the PCE23.In order to determine ML risk prediction performance, the PCE-eligible patient cohort was split into an 80% training set and a 20% test set stratified by outcome. Several ML algorithms were trained on the training set, including RF, GBM, XGBoost, and LRL2 and with an LRLasso. These were the only algorithms used. They were chosen for their ability to effectively incorporate many variables into the models returned by the algorithm. The tree-based models  can model complex, high-order interactions between the input variables. Analysis was performed in Python 3.7 using the scikit-learn and xgboost packages, versions 0.21.2 and 0.90, respectivelyC-statistic, across the 5-folds. AUC CIs were calculated24. Once hyperparameters were selected and the best-performing cross-validated model was retrained on the entire training set, the final metrics of the best-performing ML models as well as the metrics of the PCE, including AUC, sensitivity, specificity, precision, and F1-score were reported and evaluated on the 20% held-out test set.First, the ML algorithms were given the PCE variables as inputs and tasked with predicting whether a patient would have an ASCVD event in the next 5 years. For each ML algorithm, 5-fold cross-validation was used on the training set to tune hyperparameters, such as tree depth, learning rate, and number of trees. Hyperparameters used and values for those hyperparameters are shown in Supplementary Table Next, ML algorithms were given access to additional variables extracted from the EHR alongside the PCE variables. As before, hyperparameters were tuned on the training set using 5-fold cross-validation. Since some EHR variables were not expected to have robust predictive power, a composite score incorporating the feature importance metrics returned by the tree-based methods as well as nonzero coefficients of LRLasso was used to prune variables  were compared26. These techniques were chosen to represent the two ends of the spectrum of imputation techniques, with mean-value imputation being the least algorithmically and computationally intensive technique, and iterative imputation being one of the most algorithmically and computationally intensive techniques. Using the full patient cohort, first with PCE variables as inputs and then with PCE+ variables as inputs, imputation and hyperparameter search was carried out using 5-fold cross-validation, and ML model performance was assessed. Supplementary Figure Third, ML algorithm performance for ASCVD risk prediction was assessed in the full patient cohort, which included patients for which the PCE were not designed . First, the hyperparameter grid search was run and ML models were trained on Asian and Hispanic subgroups, and ML performance, using only the PCE variables and using the PCE+ variables, was compared to that of the PCE. Because the number of patients in these subgroups was lower, the LRLasso model was compared to the PCE on the test set, as it is robust to overfitting when working with smaller datasetsFurther information on experimental design is available in the Supplementary InformationReporting Summary Checklist