The relationship between coronavirus disease 2019 (Covid-19) and ischemic stroke is poorly defined. We aimed to leverage genetic data to investigate reported associations.Genetic association estimates for liability to Covid-19 and cardiovascular traits were obtained from large-scale consortia. Analyses primarily focused on critical Covid-19, defined as hospitalization with Covid-19 requiring respiratory support or resulting in death. Cross-trait linkage disequilibrium score regression was used to estimate genetic correlations of critical Covid-19 with ischemic stroke, other related cardiovascular outcomes, and risk factors common to both Covid-19 and cardiovascular disease . Mendelian randomization analysis was performed to investigate whether liability to critical Covid-19 was associated with increased risk of any of the cardiovascular outcomes for which genetic correlation was identified.g=0.29, FDR p-value=4.65×10−3), body mass index  and C-reactive protein , but none of the other considered traits. In Mendelian randomization analysis, liability to critical Covid-19 was associated with increased risk of ischemic stroke . Similar estimates were obtained when considering ischemic stroke subtypes. Consistent estimates were also obtained when performing statistical sensitivity analyses more robust to the inclusion of pleiotropic variants, including multivariable Mendelian randomization analyses adjusting for potential genetic confounding through body mass index, smoking and chronic inflammation. There was no evidence to suggest that genetic liability to ischemic stroke increased the risk of critical Covid-19.There was evidence of genetic correlation between critical Covid-19 and ischemic stroke (rThese data support that liability to critical Covid-19 is associated with an increased risk of ischemic stroke. The host response predisposing to severe Covid-19 is likely to increase the risk of ischemic stroke, independent of other potentially mitigating risk factors. Similarly, patients with acute stroke have a dampened immune response and may be more susceptible to severe Covid-19.13Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the cause of the coronavirus disease 2019 (Covid-19) pandemic that has resulted in a health crisis of unprecedented magnitude.14 There are numerous plausible mechanisms by which Covid-19 may be increasing ischemic stroke risk. Covid-19 can trigger a cytokine storm with upregulation of pro-inflammatory signaling and endothelial dysfunction that predisposes to a hypercoagulable state and can lead to thromboembolic events.15 Indeed, Covid-19 also appears to promote the development of other cardiovascular disorders including myocardial injury, myocardial ischemia, arrhythmias, heart failure and venous thromboembolism.15 Furthermore, pre-existing cardiovascular disease (CVD) is associated with high mortality in people with Covid-19, which has raised the possibility of a bidirectional interaction between Covid-19 and the cardiovascular system.15 MR analyses can also allow the exploration of such bidirectional relationships.Leverage of genetic data can help overcome some of these issues. Cross-trait linkage disequilibrium score regression (LDSC) can be used to estimate the genetic correlation between traits. Mendelian randomization (MR) can be employed to investigate whether genetic variants predicting an exposure (such as Covid-19) also associate with risk of an outcome (such as ischemic stroke).Elucidating the relationship between Covid-19 and risk of ischemic stroke could prove important for optimizing prevention and treatment strategies. With this in mind, we performed cross-trait LDSC to investigate whether there is a genetic correlation between Covid-19 and ischemic stroke, and followed this up with MR analyses to investigate whether any such statistically significant correlation might be explained by liability to Covid-19 being associated with increased risk of ischemic stroke.https://github.com/verena-zuber/covid19_and_stroke).All genetic association data used in this work are publicly accessible. Appropriate patient consent and ethical approval had been obtained in the original studies from which they were obtained . StatistFirst, we performed cross-trait LDSC to estimate genetic correlations for Covid-19 with ischemic stroke, other related CVDs, and risk factors common to both Covid-19 and CVD. Second, for CVD outcomes that showed evidence of genetic correlation with Covid-19, MR analysis was performed to investigate whether liability to Covid-19 was also associated with these outcomes. Finally, bidirectional MR was carried out to investigate potential reverse associations, i.e. whether genetic liability to the CVD outcome was also associated with increased risk of Covid-19.16 In our analysis we focused on the most severe definition of Covid-19 available, where a critical case is defined as an individual who was hospitalized with laboratory confirmed SARS-CoV-2 infection and required respiratory support or died. Genetic associations were derived from 5,101 cases and 1,383,241 controls from the general population. Hospital admission and requiring respiratory support or death is a proxy for disease severity and is preferred here over other case definitions which are solely based on a positive Covid-19 test result. Previous studies have shown that bias may impact analyses identifying cases based on likelihood of testing for SARS-CoV-2 infection, because participants being tested for SARS-CoV-2 infection are selected for a wide range of genetic, behavioral and demographic traits.9Genetic association estimates for Covid-19 were obtained from release 5 of the Covid-19 host genetics consortium.Results based on other Covid-19 definitions from the Covid-19 host genetics consortium were performed as further sensitivity analysis. Firstly, we compared individuals with laboratory confirmed SARS-CoV-2 infection who had been hospitalized (cases) versus individuals with laboratory confirmed SARS-CoV-2 infection who did not require hospitalization . Secondly, we compared individuals with laboratory confirmed SARS-CoV-2 infection who had been hospitalized (cases) versus the general population . The final definition was based on individuals with reported Covid-19  versus controls from the general population .17 The common control pool included 406,111 individuals. Genetic association data were derived from the MEGASTROKE consortium.18The primary outcome was any ischemic stroke . In secondary, hypothesis-generating analyses, stroke subtypes were further explored as large artery stroke , cardioembolic stroke  and small vessel stroke .19 for heart failure were measured on 47,309 cases and 930,014 controls and taken from the HEart failuRe Molecular Epidemiology for therapeutic targetS (HERMES) consortium20 and for atrial fibrillation were measured on 65,446 cases and 522,744 controls and taken from a transethnic meta-analysis.21We considered other CVD outcomes related to ischemic stroke in their pathophysiology. These were coronary artery disease , heart failure and atrial fibrillation. Genetic associations with risk for coronary artery disease were measured on 60,801 cases and 123,504 controls and taken from the Coronary ARtery DIsease Genome wide Replication and Meta-analysis (CARDIOGRAM) plus The Coronary Artery Disease (C4D) consortium (CARDIoGRAMplusC4D),25 Genetic association estimates to proxy these traits were taken from a genome-wide association study (GWAS) on body mass index (BMI) measured on 694,649 subjects,26 lifetime smoking index measured on 462,690 subjects,27 and C-reactive protein (CRP) measured on 361,194 individuals in UK Biobank28.To investigate whether any genetic correlation between critical Covid-19 and the CVD outcomes was related to confounding factors, we further considered common risk factors to both, including obesity, smoking and chronic inflammation.g) of critical Covid-19 with the primary outcome ischemic stroke, and secondary outcomes coronary artery disease, heart failure and atrial fibrillation, using GWAS summary statistics data.30 We also estimated correlation with possible genetic confounders, including BMI, lifetime smoking index and CRP. We restricted our analyses to HapMap 3 single-nucleotide polymorphisms (SNPs), which are known to be well-imputed across most studies and utilized the pre-computed European LD-scores estimated using the 1000G reference panel, provided by the LDSC creators. For each set of summary statistics, the SNP-specific sample size information was used. If not available, we assumed that all SNPs had the same sample size for that trait, defined as the total sample size for continuous phenotypes or as the sum of cases and controls for case/control phenotypes. By default, LDSC also removed variants that were duplicate, strand-ambiguous, not SNPs (e.g. indels), with p-values not between 0 and 1, with alleles that did not match with the 1000G reference panel, and with low effective sample size or not included in all studies of a GWAS meta-analysis (if such information was available) for traits with no effective sample size information. After estimation of the genetic correlation across all phenotypes, we corrected for multiple hypothesis testing using the Benjamini and Hochberg false discovery rate (FDR).31 FDR-corrected p-values <0.05 were considered statistically significant.We performed LDSC to estimate the genetic correlation (rr2<0.01) at p-value<5×10−6. In sensitivity analyses, we applied a more stringent threshold and considered only genome-wide significant genetic variants .Genetic variants were selected based on associations with critical Covid-19. In our main analysis, we selected uncorrelated genetic variants  via a pathway other than through the exposure . Pleiotropy can cause heterogeneity in the MR estimates obtained by different variants employed as instruments, which was assessed using the Q-statistic and the respective heterogeneity p-value.33 A Mendelian randomization estimate with p-value<0.05 for the main IVW analysis was deemed to represent supportive evidence, given that MR was only performed to follow up positive LDSC findings.For CVD outcomes that showed evidence of genetic correlation with critical Covid-19 in LDSC, MR analysis was performed to estimate the association of genetically predicted liability to critical Covid-19 with that outcome using the random effects two-sample inverse-variance weighted (IVW) method.34 and MR-Egger35 to compare the MR estimates between different MR models. Each of these methods provides a statistically consistent estimator of the true causal estimate under different assumptions. The intercept of the MR-Egger model represents a test for directional pleiotropy and we included this in sensitivity analyses.35We performed sensitivity analyses with pleiotropy-robust two-sample summary-level MR approaches, including the weighted median MR12 including obesity (BMI),26 lifetime smoking index,27 and chronic inflammation (estimated using CRP). Multivariable MR includes all the respective genetic associations in a joint model to account for genetic confounding.36 While univariable MR measures the total estimate of an exposure, multivariable MR measures the direct estimate of the exposure independent of other risk factors (i.e. pleiotropy or genetic confounders) in the model.37 In the multivariable MR model, we selected instruments based on the primary exposure of critical Covid-19. We compared the multivariable MR model with the univariable MR model using likelihood ratio test to evaluate if accounting for the pleiotropic pathway provides a better model fit than the univariable MR model.We further performed multivariable MR to adjust for potential pleiotropic pathways via cardiometabolic risk factors that are known to affect risk of both Covid-19 and CVD,r2<0.01) associated with the CVD outcome at a p-value <5 ×106− were selected as instruments.For CVD outcomes that showed evidence of genetic correlation with critical Covid-19 in LDSC, bidirectional MR was also performed to investigate for any association of genetic liability to that CVD outcome with risk of critical Covid-19. Uncorrelated genetic variants (38MR estimates are expressed as odds ratios (OR) per unit increase in the logOR of the exposure for binary traits. All analyses were performed using the ieugwasr (version 0.1.5) and MendelianRandomization (version 0.5.0) R packages.g=0.29, FDR-p-value=4.65×10−3)  and CRP ). We did not observe evidence for genetic correlation between critical Covid-19 and the CVD outcomes  . Criticaoutcomes , and thep-value=0.03). Restricting to ischemic stroke subtypes, there were similar MR estimates for cardioembolic stroke , large artery stroke  and small-vessel stroke . For the MR estimates generated by different variants, we observed heterogeneity greater than would be expected by chance only for cardioembolic stroke , but none of the other considered ischemic stroke categories.We selected 31 uncorrelated genetic variants as instrumental variables for liability to critical Covid-19. These are detailed in Diagnostic scatterplots for ischemic stroke outcomes are presented in p-value threshold based on genome-wide significance to select genetic variants as instrumental variables. We identified 9 uncorrelated genetic variants that associated with critical Covid-19 at genome-wide significance . This MR analysis based on fewer variants generated consistent estimates to the main analysis, but with wider confidence intervals that crossed the null, reflective of lower statistical power. Results are displayed in As an additional sensitivity analysis, we used a more stringent p-value=0.01) and small-vessel stroke . Considering reported Covid-19  versus controls from the general population, this was associated with increased risk of any ischemic stroke  and large artery stroke .We further considered other Covid-19 definitions . GeneticThere was no strong evidence to support that genetic liability to any of the considered ischemic stroke outcomes was associated with increased risk of critical Covid-19, as illustrated in In this study, we used cross-trait LDSC to explore the genetic correlation of critical Covid-19 with ischemic stroke, other CVD outcomes, and risk factors common to both. We identified a genetic correlation between critical Covid-19 and ischemic stroke, and performed MR analyses that found genetic liability to critical Covid-19 to be associated with increased risk of ischemic stroke. Notably, there was no evidence to support that these associations were attributable to shared risk factors, such as obesity, smoking and chronic inflammation. Furthermore, there was no MR evidence that genetic liability to ischemic stroke increases risk of critical Covid-19.3 that Covid-19 is associated with 21-fold increased odds of in-hospital stroke compared to patients without Covid-19,6 and that stroke is the most common neurological/neuropsychiatric complication of Covid-19.4 On the contrary, other studies have demonstrated a reduced rate of hospital admissions with stroke during the first wave of the pandemic compared to one year before.9 Two main hypotheses have been proposed as explanations for these contrasting findings. The first is that the incidence of stroke declined during the first wave of the pandemic and that Covid-19 is not mechanistically associated with stroke, and the second is that the observed reduction in stroke presentations was due to a higher proportion of people with mild strokes not reaching stroke services.39 We have leveraged large-scale genetic data to identify that liability to critical Covid-19 is associated with increased risk of ischemic stroke. This is consistent with the host response in Covid-19 contributing to increased ischemic stroke risk. Mechanisms that increase risk of ischemic stroke in patients with Covid-19 are complex,15 and include systemic inflammation and endotheliopathy.42 Covid-19 can trigger a cytokine storm with upregulation of pro-inflammatory cytokines and chemokines such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and IL-6.15 Endothelial inflammation can induce a microvascular and macrovascular endotheliopathy that contributes to a pro-thrombotic state.40To date, studies assessing the incidence of ischemic stroke during the Covid-19 pandemic have produced contrasting findings. On one hand, some studies demonstrate that the likelihood of stroke is seven-fold higher in people with Covid-19 than with influenza,43 Moreover, the REMAP-CAP, ACTIV-4 and ATTACC trials have recently reported that therapeutic doses of anticoagulation do not improve clinical outcome and may increase bleeding for people with Covid-19 in the critical care setting. Previous work using an MR approach anticipated a beneficial effect of IL-6 receptor inhibition on both risk of ischemic stroke and severe Covid-19.45 More recently, clinical trials have demonstrated that IL-6 receptor inhibition can improve outcomes in patients hospitalized with Covid-19.46 Targeting the deleterious host immune response through similar approaches may also help to reduce the risk of ischemic stroke and should be further evaluated.While prophylactic low molecular weight heparin is used to prevent thromboembolism in patients with Covid-19, more targeted approaches to prevent strokes are yet undefined.11 This is reinforced by data that demonstrate the reduction in stroke admissions observed in some centers during the first wave of the pandemic was driven mainly by a reduction in presentations with minor stroke syndromes.11 People with minor stroke are at high risk of early recurrence47 and public health messaging should encourage people to attend stroke services if they have any symptom of stroke during the Covid-19 pandemic.Our findings also support the hypothesis that few patients with minor strokes reached stroke services during the first wave of the Covid-19 pandemic.6Our current study has strengths. We have made efficient use of existing large-scale data resources to address an important clinical issue in the context of the rapidly evolving global pandemic. A key strength of MR analysis is the use of randomly allocated genetic variants to help overcome environmental confounding, which is analogous to randomization of treatment allocation in clinical trials. This has helped to overcome some of the limitations of previous observational studies  assessing the relationship between Covid-19 and ischemic stroke.Our work also has limitations. A series of modelling assumptions are made when using MR, in particular, that the genetic variants do not affect the considered outcomes through pathways independent of the exposure. While this can never be completely excluded, we employed methods that are robust to genetic confounding (pleiotropy) in a series of sensitivity analyses (including pleiotropy-robust MR methods and accounting for measured pleiotropy using multivariable MR) and the estimates were consistent with our main analyses. We cannot be certain that genetic associations with liability to critical Covid-19 accurately reflect the pathophysiological process that actually occurs during critical Covid-19. For example, while genetic predisposition may place an individual at increased liability to critical Covid-19, it is not possible to determine from our analyses whether that factor is involved in the pathophysiological response to Covid-19.In conclusion, we have found genetic evidence that liability to critical Covid-19 is associated with increased risk of ischemic stroke. Our results are consistent with the host response in critical Covid-19 underlying this relationship, and support the evaluation of strategies to mitigate this.Supplement 1


Reactivation of hepatitis B virus (HBV) after treatment with bortezomib-based regimens in HBV-positive patients with multiple myeloma (MM) has been reported in the past few years. Nevertheless, there is evidence of inhibition of HBV replication by bortezomib in transgenic mice. However, there is still no clinical evidence that bortezomib inhibits HBV.A 55-year-old MM patient with a family history of MM, who was also a chronic HBV carrier, achieved HBV clearance after treatment with a bortezomib-based regimen in combination with anti-HBV drugs.The diagnosis was MM with chronic carrier of HBV.He received bortezomib-based regimen for MM as well as entecavir as a prophylaxis to prevent HBV reactivation.This patient achieved HBsAg and HBV-DNA clearance after 2 months and the remission was maintained during the next 2 years. He also achieved complete remission of MM and underwent consolidation therapy with autologous hematopoietic stem cell transplantation.This is the first case of MM with HBV clearance after receiving a bortezomib-based regimen combined with anti-HBV drug. Research on related mechanisms might provide new suggestions and hope for better management of HBV positive patients with MM and for the treatment of HBV patients. This patient was a known carrier of HBV for more than 20 years. His elder brother had also been diagnosed with multiple myeloma (subtype IgG/kappa) in 2008 at the age of 56 years. Before initiation of treatment for multiple myeloma, the patient was seropositive for HBsAg (39 IU/mL), anti-HBe, and anti-HBc. Alanine aminotransferase (ALT) was 64 U/L  and aspartate aminotransferase (AST) was 69 U/L . Serum HBV-DNA was 161 IU/mL.The patient received a 3-week cycle of PCD . Entecavir was administered as a prophylaxis against HBV reactivation, and HBV-DNA was monitored since the start of PCD therapy. The patient tolerated the chemotherapy regimen well, and there were no obvious adverse effects. After 1 cycle of therapy, the serum HBV-DNA was undetectable. HBsAg turned negative after 2 months and was also negative during the 2 years of follow-up. However, anti-HBe and anti-HBc were still positive. After 4 courses of PCD therapy, the patient's immunofixation electrophoresis turned negative, and the free light chain returned to normal, indicating a stringent complete response (sCR). Subsequently, his peripheral blood hematopoietic stem cells (HSCs) were collected and he was consistently treated with high-dose chemotherapy followed by autologous HSC transplantation as consolidation treatment, 10 months after the initial diagnosis of multiple myeloma. The patient continues to remain in sCR at 26 months of follow-up. During subsequent treatment and follow-up, HBsAg continued to be negative and HBV-DNA continued to fall below the detection limit.3Bortezomib is a proteasome inhibitor that is currently used in the treatment of multiple myeloma, mantle cell lymphoma, Waldenström macroglobulinemia/lymphoplasmacytic lymphoma, and other plasma cell-related diseases. The rate of reactivation of the herpes zoster virus was significantly higher in patients receiving bortezomib,This case is unique in 2 ways: first, the patient and his elder brother were diagnosed with multiple myeloma at a similar age; second, this patient achieved HBsAg clearance after receiving bortezomib-based regimen combined with anti-HBV drug. To our knowledge, this is the first case report about HBsAg clearance following bortezomib-based regimen combined with entecavir as antiviral therapy.Clearance of HBsAg is one of the important signs of HBV clearance. Possible causes of achieving HBsAg negativity include elimination of HBV and mutation of HBsAg antigen gene, due to which HBsAg is not expressed.HBsAg clearance is related to many factors, including HBV subtypes, treatment strategies, and certain genetic polymorphisms in patients.[Multiple myeloma is thought to have an inherited genetic component that might have existed about a century ago. Eight families with 2 or more first-degree relatives with multiple myeloma have been reported in 1974.Antiviral prophylaxis for HBV reactivation is recommended during the treatment of HBV patients with hematological malignancies, including multiple myeloma. Lu et al4This case suggests that anti-HBV virus treatment while receiving bortezomib therapy might achieve clearance of HBV infection in some HBV-positive patients with multiple myeloma. However, this hypothesis needs further validation. Further research is necessary to understand the detailed mechanism. These mechanisms can bring a new hope for the treatment of HBV infection.We thank Dr. Jinfan Li, from the Department of Pathology the Second Affiliated Hospital of Zhejiang University School of Medicine, for the kind help in pathology diagnosis and drafting the manuscript.ZX and LY designed the study, ZX, LX, WW, TJ, and XY collected the data, ZX and XY wrote the manuscript. All authors have read and approved the final manuscript.


The emergence of human intratumoural immunotherapy (HIT-IT) is a major step forward in the management of unresectable melanoma. The direct injection of treatments into melanoma lesions can cause cell lysis and induce a local immune response, and might be associated with a systemic immune response. Directly injecting immunotherapies into tumours achieves a high local concentration of immunostimulatory agent while minimising systemic exposure and, as such, HIT-IT agents are associated with lower toxicity than systemic immune checkpoint inhibitors (CPIs), enabling their potential use in combination with other therapies. Consequently, multiple HIT-IT agents, including oncolytic viruses, pattern-recognition receptor agonists, injected CPIs, cytokines and immune glycolipids, are under investigation. This review considers the current clinical development status of HIT-IT agents as monotherapy and in combination with systemic CPIs, and the practical aspects of administering and assessing the response to these agents. The future of HIT-IT probably lies in its use in combination with systemic CPIs; data from Phase 2 trials indicate a synergy between HIT-IT and CPIs. Data also suggest that the addition of HIT-IT to a CPI might generate responses in CPI-refractory tumours, thereby overcoming resistance and addressing a current unmet need in unresectable and metastatic melanoma for treatment options following progression after CPI treatment. In approximately two-thirds of all cases of primary cutaneous melanoma, disease spread begins with locoregional metastasis, with about 50% of patients developing the first metastasis in regional lymph nodes.5 These intratumoural immunotherapies can cause cell lysis, either directly or indirectly, and promote the induction of a local immune response, and might also be associated with the generation of a systemic immune response.19 Importantly, direct injection of a therapeutic agent into the tumour maximises its concentration at the disease site, promoting a tumour-specific immune response while reducing systemic exposure.2 There are anticipated benefits of combining intratumoural therapy with systemic immunotherapies such as CPIs. Not only do they offer different modes of action, but they also have non-overlapping toxicity profiles.20The need for additional treatment options for unresectable locoregional disease coupled with the accessibility of this type of metastasis has led to increased interest in immunostimulatory agents that can be injected directly into the tumour.21Here, we review the latest data on the development of human intratumoural immunotherapy (HIT-IT), as a single-agent strategy and in combination with CPIs, for unresectable melanoma, alongside practical aspects involved in administering and assessing the response to this therapeutic approach. Although some chemical and local physical and radiation strategies might be defined as intratumoural therapies  these therapeutic options are not immunotherapies and are thus considered to be outside the scope of this review.21 The ability to generate a local complete response and a durable response  is important to enable its use as a monotherapy, since it demonstrates the agent’s effectiveness when used on its own.23 Likewise, local disease control should translate into clinical benefit .22 Ideally, HIT-IT should stimulate a systemic immune response leading to regression of uninjected tumours at locoregional and distant metastatic sites.21 The initiation of a systemic immune response can occur through a variety of mechanisms, including the enhanced release and presentation of tumour antigens, immune cell trafficking and activation and inhibition of immunosuppressive pathways.27 It is anticipated that HIT-ITs will reach high concentrations in injected lesions, increasing the local bioavailability.28 Furthermore, by minimising systemic exposure, HIT-IT should be associated with low toxicity compared with systemic immunotherapies.30 Moreover, HIT-ITs might be able to reverse resistance to systemic immunotherapies.33 These attributes indicate that HIT-ITs could be used both as monotherapies and as part of a combination strategy,28 and data from the past 5 years suggest synergy between intratumoural and systemic immunotherapies (as described in more detail below).39An ideal HIT-IT should elicit a measurable biological effect, reflective of the mechanism of action, that should lead to an objective response in the injected tumour.44 Interleukin-2 (IL-2) is included in guidelines as a systemic treatment for unresectable metastatic melanoma,44 and intratumoural administration has also been investigated.47Talimogene laherparepvec (T-VEC) is the only treatment with regulatory approval for intratumoural administration in unresectable metastatic melanoma, and is recommended in current clinical practice guidelines.49 It selectively infects and replicates in tumour cells, which not only leads to cell lysis but also to the release of GM-CSF. This recruits dendritic cells, which, in turn, process and present tumour antigens to cytotoxic T lymphocytes (CTLs), thereby inducing a systemic tumour-specific immune response.49 T-VEC is approved in Europe for the treatment of adults with stage IIIB, IIIC and IVM1a unresectable melanoma with no bone, brain, lung or other visceral disease, and in the USA for the local treatment of unresectable cutaneous, subcutaneous and nodal lesions in patients with melanoma recurrent after initial surgery.T-VEC is a genetically modified oncolytic virus that expresses granulocyte-macrophage colony-stimulating factor (GM-CSF).n = 436), which demonstrated that the durable response rate (objective response lasting ≥6 months) and overall response rate were significantly higher following treatment with intralesional T-VEC than with subcutaneous GM-CSF in patients with unresectable and/or metastatic stage IIIB–IV melanoma .22 This implication is also supported by a prospective Phase 2 trial in which T-VEC led to a significant increase in the number of CD8+ T cells, effector and memory cytotoxic lymphocytes (CTLs), natural killer cells, and CTLs expressing PD-1 and CTLA-4 (indicative of immune activation) in uninjected lesions.50The approval of T-VEC was based on data from the OPTiM Phase 3 trial , they are largely limited to injected lesions, which suggests that intratumoural IL-2 does not elicit a strong systemic effect—at least, not at the doses and regimens that have been studied.30 Intratumoural IL-2 is generally well tolerated.IL-2 is a proinflammatory cytokine that can activate CD821 Figure Many agents are being developed for intratumoural use, including other oncolytic viruses and peptides, pattern-recognition receptor (PRR) agonists, immune CPIs and cytokines (see Table 54 Coxsackievirus A21 (CVA21) is an enterovirus that preferentially infects tumour cells, leading to cell lysis, which appears to provoke a systemic antitumour immune response even in the absence of the virus encoding an immune component such as GM-CSF or IL-2.55 In a single-arm Phase 2 trial, CVA21 led to durable responses lasting 6 months or more.56 No Phase 3 trials are currently planned for CVA21 monotherapy in melanoma since the future of these agents is most likely in combination, particularly with immunotherapies. In this regard, trials are ongoing with CVA21 in combination with other therapies (see below). Canerpaturev (formerly HF-10) is a spontaneously occurring, replication-competent mutant strain of herpes simplex virus type 1 that causes lysis of infected cells.57 Similar to CVA21, canerpaturev is being assessed in combination therapy; no monotherapy trials are ongoing. Other oncolytic viruses are in early clinical development as monotherapy and/or in combination therapy: RP1 and RP2, engineered strains of the herpes simplex virus; ONCOS102, an adenovirus engineered to express GM-CSF; JX-594 (Pexa-Vec), a GM-CSF-expressing poxvirus; and CF33-hNIS, a chimeric poxvirus encoding a human sodium iodide symporter (hNIS).61Given that the only currently approved HIT-IT is an oncolytic virus, it is not surprising that other oncolytic viruses are undergoing development for the treatment of unresectable and metastatic melanoma.62 PRRs are costimulatory molecules that recognise pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide and other bacterial and viral components, as well as damage-associated molecular patterns (DAMPs) resulting from cellular stress, apoptosis and necrosis.62 Recognition of PAMPs/DAMPs leads to upregulation of the transcription of genes involved in inflammatory responses, which encode proinflammatory cytokines, type-I interferons, chemokines and, antimicrobial proteins.63 Activation of TLR-9 signalling in plasmacytoid dendritic cells induces production of interferon-α and tumour necrosis factor (TNF)-α, which promotes leucocyte migration and induces synthesis of antimicrobial peptides and cytokines, and promotes phagocytosis in macrophages.64 Three TLR-9 agonists  are in clinical development in combination therapy (see below).66 RIG-I-like receptors are cytosolic PRRs that detect viral and endogenous RNA, triggering binding to the mitochondrial antiviral signalling protein (MAVS) and resulting in type-I interferon production.62 MK4621 (formerly RGT-100), a synthetic RNA agonist of the RIG-I pathway, has been shown to have antitumour activity in mouse models.67 STING is an endoplasmic reticulum transmembrane protein involved in recognition of cytosolic DNA. In tumours, STING pathway activation leads to interferon-β production and T-cell response.62 Cyclic dinucleotides have been found to act as immune adjuvants by activating STING, in turn stimulating a proinflammatory immune response;68 Phase 1 trials of two intratumoural STING agonists, ADU-S100 and MK-1454, are ongoing.71Another class of HIT-IT in development comprises the PRR agonists, which include Toll-like receptor (TLR) agonists, stimulator of interferon genes (STING) agonists and retinoic acid-inducible gene I (RIG-I)-like receptor agonists.21 The activation of CD40 on antigen-presenting cells initiates their maturation and ability to activate CD8+ T cells. Modulation of this pathway in melanoma is being evaluated in a Phase 1/2 trial of an intratumoural CD40 agonist, APX005M.72 The feasibility of utilising approved anti-PD-1 systemic therapies as intratumoural agents was demonstrated in a 2018 pilot study; further studies are needed to determine the efficacy of this approach.73 Similarly, intratumoural administration of the anti-CTLA-4 antibody ipilimumab plus IL-2 demonstrated the induction of both local and systemic immune responses in a Phase 1 trial; no dose-limiting toxicities were reported.27 A Phase 1/2 trial assessing intratumoural ipilimumab plus systemic nivolumab, an anti-PD-1 antibody, is currently recruiting patients.74Immune CPIs, such as TNF receptor superfamily agonists (e.g. CD40) and immunoglobulin superfamily antagonists (e.g. PD-1 and CTLA-4), are also in development as intratumoural agents.5 Subsequently, cytokine fusion proteins and plasmids expressing cytokines have been developed with the aim of increasing efficacy.76 For example, the immunocytokine fusion protein L19–IL-2 (Darleukin) is a targeted form of IL-2 that recognises the extra domain B of fibronectin, which is expressed in cancer-associated blood vessels and extracellular matrix but absent from almost all healthy tissue.75 In a single-arm Phase 2 trial, L19–IL-2 resulted in local responses, including local complete responses.75 Daromun, another investigational treatment, combines L19–IL-2 and L19–TNF. In a single-arm Phase 2 trial, Daromun led to objective responses in both injected and uninjected lesions. A pivotal trial of Daromun is being conducted in the neoadjuvant setting.77Cytokines (e.g. IL-2 and GM-CSF) were among the first HIT-ITs to be assessed in melanoma.78 Tavokinogene telseplasmid was given orphan drug status by the US Food and Drug Administration (FDA) in 2017 for the treatment of unresectable metastatic melanoma.76Tavokinogene telseplasmid  is a synthetic plasmid encoding the cytokine IL-12, which, when delivered to melanomas in a highly localised manner, leads to a proinflammatory response, resulting in T-cell recruitment and activation. In a Phase 2 trial, tavokinogene telseplasmid demonstrated induction of an antitumour immune response and a high disease control rate in melanoma.81 IFx-Hu2.0 is a plasmid DNA encoding the streptococcal membrane protein, Emm55, and the first in human Phase 1 study of intratumoural use in melanoma is in progress.82 Preliminary laboratory data from the first three patients in the trial suggest the treatment may be associated with decreases in tumour cells and formation of an immune response.83 mRNA-2416 is a novel lipid nanoparticle therapeutic agent encoding the TNF receptor ligand OX40L. Results so far delivered from an ongoing Phase 1/2 study indicate that intratumoural mRNA-2416 monotherapy is well tolerated and elevates PD-L1 levels and proinflammatory activity.84Several other novel HIT-ITs have shown promising preclinical antitumour activity and are entering clinical trials. AGI-134 is a glycolipid that recruits pre-existing endogenous anti-Gal antibodies to the injected lesion, leading to complement activation and enhanced tumour antigen processing, whereas IMM60 is a non-glycolipid that activates invariant natural killer cells, leading to an antitumour immune response. A Phase 1/2 trial of AGI-134 is currently recruiting patients, and trials of IMM60 are anticipated.87 for example, despite promising efficacy in Phase 2 trials, the plasmid-DNA-based intratumoural immunotherapy velimogene aliplasmid (a plasmid–lipid complex comprising the DNA sequences encoding HLA-B7 and β2 microglobulin) either provided no benefit or reduced survival compared with standard of care in Phase 3 trials.86 This result highlights the need for caution when interpreting data from single-arm, non-randomised trials in small and highly selected patient populations, as well as emphasising the importance of mechanistic tumour biology research to enable rational drug and clinical trial design.Although many HIT-ITs have been investigated, most data at present originate from Phase 2 trials only. Such data are difficult to interpret, because early-stage efficacy might not reflect the results of Phase 3 trials:As noted, many intratumoural agents in development are being assessed in combination with systemic CPIs, reflecting the likely future clinical application of intratumoural therapy.7 only a subset of patients initially respond, and a substantial proportion of responders subsequently develop resistance and relapse.88 Data suggest that therapeutic responses to immunotherapy can, to some extent, be predicted by the presence of tumour immune cell infiltration. Three tumour immune profiles correlate with response to CPIs: inflamed or ‘hot’ tumours exhibiting immune cell infiltration are likely to respond, whereas ‘cold’ immune-excluded tumours with immune cells surrounding, but not infiltrating, the tumour, and immune desert tumours, characterised by a complete lack of immune cells, are less likely to respond. HIT-ITs that elicit a local immune response have been shown to promote immune cell infiltration into the tumour in both injected and uninjected lesions.50 Consequently, by altering the tumour microenvironment and converting a non-responsive ‘cold’ tumour into a responsive ‘hot’ tumour, HIT-IT might enhance response to systemic immunotherapies.38Although CPIs provide an effective therapeutic approach as monotherapy in melanoma,90 Indeed, a 2018 Phase 2 trial of T-VEC showed that this therapy leads to an increase in the number of CTLs expressing PD-1 and CTLA-4.50 Blocking PD-1 or CTLA-4 might restore suppressed antitumour immune responses and enhance the ability of T cells  to recognise and kill tumour cells.91 CPIs might also enhance the systemic effects of some HIT-ITs.50 Therefore, combination therapy might result in improved clinical activity beyond what would be expected with either agent alone. Additionally, owing to the low toxicity of HIT-IT, combination therapy has the potential to be tolerated at effective doses.On the other hand, CPIs might also enhance the response to HIT-IT. Cancer cells can activate immune checkpoint pathways to downregulate the response to local immunostimulation, thus limiting the response to intratumoural monotherapy.+ T-cell infiltration into the tumour, in both injected and uninjected lesions, and increased the numbers of systemic circulating CD4+ and CD8+ T cells, combination therapy was associated with greater clinical benefit than that seen previously with either agent alone, with no additional toxicity.92 Longer-term follow-up (median 36.8 months) of the Phase 1b part of the trial suggests that this combination induces a high rate of complete responses (43%), with a 3-year survival rate of ~70%.93 The same combination is currently being evaluated in the large randomised Phase 3 MASTERKEY-265/KEYNOTE-034 trial.94Given the therapeutic promise of combining intratumoural and systemic immunotherapies, several combination trials have been conducted or are ongoing Table . Data in66 Pembrolizumab is also being investigated in a Phase 2 trial with intratumoural tavokinogene telseplasmid95 and in a Phase 1b trial with the oncolytic virus CVA21. A Phase 2 trial is also planned to investigate pembrolizumab with CVA21.96 Apart from T-VEC plus pembrolizumab, none of these combinations is currently being assessed in Phase 3 trials.Systemic pembrolizumab is being assessed in combination with various other HIT-ITs, with promising early results. Interim data from a Phase 1b/2 trial combining the intratumoural TLR-9 agonist SD-101 with pembrolizumab demonstrated that the combination is well tolerated and leads to increased tumour immune cell infiltration, as well as inducing tumour shrinkage, in injected and uninjected lesions—including those in distant metastases.97 the results suggest that this combination is tolerable and might have greater efficacy than either agent alone.99 Other ipilimumab and intratumoural combinations have shown efficacy in Phase 1b or 2 trials, including with the oncolytic viruses CVA21 and canerpaturev.100 However, a Phase 2 trial of ipilimumab combined with intratumoural IL-2 reported no objective responses.2 Currently there are no ongoing or planned Phase 3 trials of any of these combinations.T-VEC has also been assessed in a randomised Phase 1b/2 trial in combination with ipilimumab versus ipilimumab alone;88 Data from the past 3 years—particularly from studies using TLR-9 agonists—indicate that treatment regimens incorporating a HIT-IT can lead to responses in patients who have previously received, or who have progressed following, anti-PD-1 therapy  to ipilimumab revived the immune response in injected and uninjected anti-PD-1-resistant tumours.32 Likewise, in a Phase 1b/2 trial, addition of the TLR-9 agonist SD-101 to pembrolizumab restored tumour sensitivity to PD-1 inhibition in refractory tumours; responses were seen in both injected and uninjected lesions.33 A Phase 2 trial of T-VEC plus pembrolizumab is ongoing in patients with advanced melanoma whose disease progressed following anti-PD-1 therapy.103Early phase data indicate that the addition of a HIT-IT to an anti-PD-1 agent might restore response and fundamentally overcome resistance to anti-PD-1 therapy. In a Phase 1b trial, the TLR-9 agonist CMP-001 was able to overcome resistance to PD-1 inhibition when combined with pembrolizumab.104For future analyses on the sequence of HIT-ITs, it will be important to ascertain if the response to these agents differs among patients who progress following initial response to CPI treatment compared with those who never respond to CPI treatment and/or patients who have previously received a CPI and discontinued for other reasons (e.g. poor tolerability). This will help to identify the most appropriate HIT-IT to use in different clinical situations. Another interesting area for future research is the potential to give HIT-IT to patients who experience progression despite adjuvant CPI therapy, as many of these patients progress with accessible locoregional disease only.105 The combination of MEK inhibition and T-VEC has shown increased melanoma tumour cell death in vitro106 and a Phase 1 trial of dabrafenib, trametinib and T-VEC is ongoing.107There is also interest in combining HIT-ITs with BRAF and MEK inhibitors such as dabrafenib and trametinib. This approach aims to specifically target the BRAF driver mutation, which is present in around 50% of malignant melanomas.From a practical perspective, HIT-IT can only be administered to patients with lesions that are visible, palpable or detectable by ultrasound or other imaging techniques. Key considerations for the selection of patients for HIT-IT monotherapy are described in Box Box 1. Patient selection for HIT-IT monotherapySeveral key considerations exist for HIT-IT monotherapy patient selection:124tumour location, leading to unreasonable morbidity (e.g. cutaneous head and neck melanoma)risk of surgical complicationspresence of numerous in-transit lesionsdisease recurrence despite multiple surgical interventionsWhen surgery is no longer an option owing to a number of reasonsfor whom systemic therapies are contraindicated or poorly toleratedwith slowly progressing disease or locoregional progression but stable visceral metastases who wish to avoid systemic therapywho wish to preserve systemic therapies for later treatment lines in the event of disease progressionAs an alternative to systemic therapy in patientsTo ensure accurate drug delivery, response and evaluation, lesion mapping using clinical evaluation  should be conducted to identify and measure lesions for injection. Lesions that will not be injected should also be mapped to enable the assessment of systemic responses. Ultrasound provides an accessible option for the mapping of most lesions , as well as allowing the measurement of 3D tumour volume, and can be more accurate than palpation for the determination of lesion size. Computed tomography (CT), positron emission tomography (PET-CT) and, in rare cases, magnetic resonance imaging can also be considered for mapping deep lesions or those located in the extremities. Before initiating HIT-IT, it is important to record the tumour load throughout the patient, the tumour volume available for injection and the total volume of agent injected before response evaluation. Both injected and uninjected lesions  should be measured before treatment and compared with previous measurements: a lesion tracking sheet and high-resolution photographs could facilitate lesion tracking.21 Gutzmer et al.108 and Harrington et al.109. Briefly, most cutaneous, subcutaneous and superficial lymph node lesions can be injected under ultrasound guidance.21 Multiple injections can be administered to a single lesion, and multiple lesions can be injected at the same visit.21 The choice of whether to use the same needle for all lesions or individual needles for each lesion might depend on the type of HIT-IT used and the risk of drug exposure to the patient or healthcare professional.41 Intratumoural injections should be performed by a trained healthcare professional .21It is beyond the scope of this review to detail handling and administration for HIT-IT; these topics have been covered extensively by Marabelle et al.,110 The precision of the technique is an advantage. Only cells that are exposed to both the plasmid and the electrical field will undergo transfection, enabling local, targeted delivery.110Electroporation is sometimes employed for delivery of tavokinogene telseplasmid and other plasmids. After injection of the plasmid into the lesion, application of electroporation pulses locally permeabilise and transduce the cells.111 Although HIT-IT is generally associated with low toxicity, adverse events such as fatigue, chills and pyrexia have been reported in clinical trials and it is important to manage the patient’s expectations of tolerability to ensure that they remain on treatment.112As mentioned, most reported adverse events associated with HIT-IT have generally been mild and might not require active treatment. Injection-site reactions are frequent, but they tend to resolve within 24–48 h and can be helped with ice and pain relief. Local anaesthetic can be given ~30 min before the procedure. Careful wound care is important to help to avoid skin infections. In the event of cellulitis, oral or intravenous antibiotics should be administered as required and blood samples taken for culture.113 Indeed, pseudoprogression, in which a tumour appears to grow as a result of a treatment effect rather than true disease progression, has been reported to occur in patients receiving HIT-IT;18 in such cases, discontinuing treatment at the point of apparent initial disease progression might not be appropriate. The immune-related response criteria (irRC) and immune-related (ir)RECIST partially ameliorate these issues by requiring confirmation of progressive disease by consecutive imaging assessment at least 4 weeks from the date of the first documentation and by incorporating the measurement of new lesions into the sum of lesions.116 irRECIST further attempts to harmonise data collection and to clarify response measurement that allows for pseudoprogression. New lesions are assessed separately. Importantly, the progressive disease thresholds for irRECIST are aligned with RECIST v1.1, allowing comparisons to be made between trials and historical data.Current trials of HIT-ITs have used a range of response criteria. Thorough and consistent assessments of response will be required in future clinical trials to properly evaluate the plethora of HIT-ITs in development. The Response Evaluation Criteria in Solid Tumors (RECIST v1.1) were designed to measure responses to cytotoxic agents, and might not be suitable for evaluating HIT-ITs: according to RECIST v1.1, new lesions define progressive disease, but the appearance of new lesions can precede prolonged disease stabilisation in response to immune checkpoint blockade.21 We are in agreement with the ESMO recommendations that injected lesions are evaluated separately from uninjected lesions, to characterise systemic effects in clinical trials.21 As a result, the evaluation of HIT-IT in clinical trials is more complex than for conventional therapies, due to the need to differentiate lesions . Furthermore, visceral lesions must be considered in the response criteria; responses to HIT-IT have been reported in uninjected visceral lesions, and advances in imaging guidance techniques now allow visceral lesions to be directly injected.31 Such complexity in response assessment requires specially trained radiologists.European Society for Medical Oncology (ESMO) recommendations on the development of HIT-ITs propose that, for clinical trials, RECIST v1.1 is used for the overall assessment of tumour response, iRECIST is used for assessing responses of injected and uninjected tumours, and the duration of response for both injected and uninjected lesions should be reported.These assessment criteria are currently likely to be too complicated and not relevant for real-world practice, in which treatment decisions are based on patient-level responses, and harmonisation on how to evaluate tumour response to HIT-IT is still awaited. As HIT-IT becomes more widely used, the link between formal response evaluation, clinical benefit and decision-making in practice will evolve further.The efficacy and tolerability of HIT-IT for the treatment of melanoma provide proof-of-concept for this modality. Early data indicate the existence of a synergy between HIT-IT and CPIs, and it is likely that future clinical use will focus on the combined use of these agents; indeed strategies that combine treatments that have different modes of action without overlapping toxicities are likely to feature in future research. Furthermore, there is a high unmet need in unresectable and metastatic melanoma for treatment options following progression after PD-1 inhibition, and data indicate that adding HIT-IT to systemic agents can lead to responses in anti-PD-1-refractory tumours, thereby overcoming resistance.118 Additionally, although it has been widely assessed in melanoma, HIT-IT could be considered for use in any tumour that is injectable (including under image guidance).21 Consequently, a plethora of HIT-ITs are in early-stage clinical development for the treatment of a range of solid tumour types and lymphoma.The development of HIT-IT has focused on unresectable disease; however, there is now interest in the efficacy of this approach in the neoadjuvant setting, and early data suggest that neoadjuvant HIT-IT could prevent recurrence following surgery in patients with resectable melanoma.


The purpose of the present trial is to determine whether opening co-existing chronic total occlusions (CTOs) using percutaneous coronary interventions (PCIs) improves cardiac function in patients with multi-vessel disease (MVD). Patients with MVD are defined as having at least one additional major vessel exhibiting no less than 75% stenosis combined with the presence of a CTO artery.Patients will be prospectively recruited who meet the following criteria:(1)angiographically diagnosed with MVD concurrent with CTO;(2)previously diagnosed with myocardial infarction, or suffered myocardial necrosis of the CTO-territory determined using cardiovascular magnetic resonance (CMR).Patients presenting with no necrosis of myocardial tissue in the territory of the CTO will be excluded. Recruited patients will be randomized into 2 groups: those undergoing PCI of only the non-CTO artery (non-CTO PCI group), and those undergoing PCI of the non-CTO artery concurrently with the CTO artery (CTO-PCI group). The primary outcome will be the change in cardiac function evaluated via CMR at a 12-month of follow-up appointment, which will be compared to a baseline measurement. Secondary outcomes include occurrence of major cardiac events, CMR-assessed myocardial viability in the CTO-supplied territory, and quality of life assessed by Seattle angina questionnaire, Patient Health Questionnaire 9 and Generalized Anxiety Disorder Scale-7 after 12-month follow-up.The SOS-moral trial will provide data necessary to determine whether to open concurrent CTOs among MVD patients with CMR-detected necrotic myocardial tissue. The difference was most significant in young patients and those with acute ST-segment elevated myocardial infarction (STEMI). Another study found that ischemic cardiac dysfunction was associated with increased mortality among patients with CTO lesions within the first 1 to 2 years of identification of the disease. Percutaneous coronary intervention (PCI) for CTO is performed to relieve symptoms, improve cardiac function, decrease the need for subsequent coronary artery bypass graft (CABG), and improve quality of life (QOL).A chronic total occlusion (CTO) is defined as a coronary artery occlusion for a period ≥3 months, which can be detected by angiogram.–12 However, another study could not demonstrate benefits of CTO-PCI when assessing the occurrence of major adverse cardiac events (MACE); among patients with CTO of a single coronary artery, PCI did not reduce cardiac mortality and MACE when compared to optimal medical therapy (OMT). The prospective, open-labeled, randomized DECISION-CTO trial revealed that OMT as an initial strategy was non-inferior to PCI with regard to the composite endpoint of death, MI, stroke, or any revascularization within a 3-year follow-up period. A multicenter, open-labeled, randomized, controlled EuroCTO trial showed that PCI for CTO resulted in a significant improvement of health status compared with OMT alone, rather than resulting in the reduction of MACE.Previous studies revealed that for both CTO of a single coronary artery and MVD with concurrent CTO, even with well-developed collateral circulation, successful CTO-PCI is associated with reduced long-term cardiac mortality and reduced need for CABG, which is an advantage that can persist 2 to 5 years following PCI. Hence, the effect of CTO-PCI on cardiac function merits investigation. An early study that recruited patients with at least 1 CTO and a LVEF of ≤ 40% found that CTO-PCI decreased left ventricular end-systolic volume and increased LVEF. Another study, with a small sample size, showed that successful CTO-PCI improved regional wall motion. The EXPLORE trial showed that routine CTO-PCI did not result in an overall benefit with respect to LVEF and lower left ventricular end-diastolic volume (LVEDV) within 4 months, when compared with a non-CTO PCI strategy in an unselected cohort of patients with STEMI and concurrent CTO. An extended examination of the EXPLORE trial also did not reveal beneficial effects associated with routine early CTO-PCI on LVEF within a 1-year period.The presence of a CTO and not MVD alone has been reported to be independently associated with long-term mortality and impaired left ventricular eject fraction (LVEF).Myocardial viability affects CTO-PCI outcomes. With respect to myocardial viability, the myocardium in the CTO territory can be divided into groups as follows: complete survival, partial survival, and non-viable myocardium. We predict that conflicting conclusions drawn by previous studies can be partially attributed to the failure to consider the status of myocardial tissue or the burden of ischemia.At present, US and European guidelines provide Class IIa or IIb recommendations for PCI for the treatment of CTO. The recommendation comes despite the fact that the rate of successful CTO-PCI is more than 90% at experienced centers and can be increased by training of operators working within clinical practices, especially those in small centers. These facilities include a large portion of patients presenting with CTO combined with MVD who receive treatment involving exclusively non-CTO PCI or incomplete revascularization. The purpose of the present trial is to compare the effect of CTO-PCI combined with non-CTO PCI versus non-CTO PCI with respect to cardiac function, myocardial viability, and MACE among patients with MVD and necrotic myocardial tissue. Cardiovascular magnetic resonance (CMR) will be used to assess the viability of the myocardium, which facilitates identification of patients who will benefit from complete revascularization procedures.22.1This SOS-moral study is a randomized controlled trial with a multi-center design. The protocol was approved by the ethics committee of Beijing Anzhen Hospital  and has been registered at ClinicalTrials.gov (identifier: NCT 03372785).Patients meeting the inclusion criteria will be categorized into two groups at a ratio of 1:1 according to a randomized digital table used to randomly assign patients to the CTO-PCI group (PCI of both the non-CTO artery and a concurrent CTO artery), or the non-CTO PCI group (PCI of only the non-CTO artery). The randomized digital form and interactive voice response mode will be applied to facilitate the randomization procedure. The randomization sequence will be produced and managed by an independent staff member. Before intervention, myocardial viability and cardiac function of patients will be evaluated using CMR. Cardiac function will be assessed using transthoracic echocardiography and CMR. The Seattle angina questionnaire (SAQ), Patient Health Questionnaire 9 and Generalized Anxiety Disorder Scale-7 will be used to evaluate patient QOL. Cardiac function, QQL, and MACE will be observed within 12 months of follow-up. A flow chart outlining the study design is depicted in Figure 2.2Patients in the CTO-PCI group receive PCI treatment involving both the CTO artery and the non-CTO artery. Patients of the non-CTO PCI group will only have PCI performed on the non-CTO artery. To ensure safety of all patients, they will be monitored, and those with persisting clinical symptoms occurring within one month of OMT will undergo PCI of CTO artery.2.3We will enroll patients hospitalized for stable or unstable angina pectoris in Beijing Anzhen Hospital, The First Affiliated Hospital of Dalian Medical University and Beijing Friendship Hospital from April of 2018 to December of 2021 meeting the selection criteria. All subjects/guardians enrolled in the trial should clearly understand the research requirements and intervention procedures, and each will be required to sign a written informed consent agreement before participating in any type of examination or intervention. All participating research centers should obtain all informed consent forms from patients before the initiation of intervention. Personal information will be kept confidential before, during, and after the trial.2.3.1To meet inclusion criteria, patients should: be 18 to 80 years old; be diagnosed with single CTO concurrent with MVD detected using coronary angiography (at least one other major vessel should have exhibited no less than 75% stenosis); previously have been diagnosed with myocardial infarction, or suffer from myocardial necrosis in the territory of CTO artery determined by CMR; present with an LVEF above 35% determined using transthoracic echocardiography; present with CTO located in an epicardial coronary artery with a reference diameter of ≥ 2.5 mm; and comply with all the evaluations and follow-up protocols.2.3.29/L or > 700 × 109/L and white blood cell counts < 3 × 109/L; with active bleeding or bleeding tendencies ; severe coexisting conditions, including severe renal function dysfunction , severe heart failure (NYHA classification III–IV), acute infectious diseases and immune disorders; tumors; surgery within 3 months; a life expectancy less than 12 months; pregnancy or planning to become pregnant; history of allergy or adverse reactions to aspirin, clopidogrel, ticagrelor, stains, contrast material, anticoagulant, or stents. Patients will also be excluded if they cannot tolerate dual antiplatelet treatment; are unable to communicate due to cognitive impairment, auditory, or visual impairment; are participating in another trial for medication or an apparatus and in which the main endpoint has not been reached, or plan to participate in a clinical trial within 12 months of the intervention.Patients will be excluded if they have suffered from acute myocardial infarction within the previous 3 months; a lesion located in the left main artery (stenosis ≥50%); rheumatic valvular disease; severe arrhythmia; a history of revascularization within the non-CTO artery; multiple vessels with CTO (more than one CTO artery); lesions unsuitable for PCI; severely abnormal hematopoietic systems, such as platelet counts <100 × 102.42.4.1Experienced operators have been designated to ensure that the success rate of CTO-PCI is above 80%. Procedural success is defined as resulting in residual stenosis of <10% with thrombolysis in myocardial infarction grade 3 flow, in the absence of dissection and thrombosis. The use of a DES and specialized devices will be left to the discretion of the operator.2.4.2Patients included in both CTO-PCI group and non-CTO PCI group will be administered aspirin (300 mg) and one P2Y12 receptor antagonist (300–600 mg clopidogrel or 180 mg ticagrelor) before PCI unless they have been treated with these antiplatelet agents previously. Calcium antagonists, nitrates, sodium nitroprusside, and GP IIb/IIIa receptor antagonists will be prepared and administered in any case in which no-reflow occurs in the PCI procedure. After PCI, all patients will be routinely administered one P2Y12 receptor antagonist (clopidogrel 75 mg/d or ticagrelor 90 mg twice daily), aspirin (100 mg/d) and statin once daily for 12 months. Other medications such as beta-blockers, calcium antagonists, and nitrates will be administered according to the clinical status of each patient, considering factors such as heart rate, blood pressure, and cardiac function.2.52.5.1CMR will be performed using a Siemens 3.0T whole-body MR scanner . A 32-element matrix coil will be utilized for data collection, and electrocardiographic gating will be utilized to acquire all sequences. First, the morphologic data from the heart will be acquired with the short-axis plane (from the base to the apex) and long-axis plane of the left ventricle (LV). Next, 0.05 mmol/kg Magnevist contrast agent will be injected intravenously with a high-pressure syringe at 5 mL/s. The first-pass perfusion image (8-mm sections with no intersection gap) will be acquired when magnevist is injected. LGE imaging (8-mm sections with no intersection gap) will be acquired 10 minutes after the intravenous administration of gadolinium  using a 2-dimensional, phase-sensitive, inversion-recovery, breath-hold sequence.CMR will be utilized to assess myocardial infarct size, left ventricular mass, LVEF, LVEDV, left ventricular end-systolic volume (LVESV), cardiac output, stroke volume, segmental wall thickening, and myocardial viability in the territory supplied by the artery with CTO.2.5.2Transthoracic echocardiography (Philips ie33 GE vivid 7) will be utilized to determine left ventricular end diastolic diameter, left ventricular end systolic diameter, LVEF, and segmental wall thickening.2.6Patients will be followed-up via outpatient consultation 1, 6, and 12 months following PCI. A follow-up appointment 12-months post-PCI will be conducted at the hospital. Basic characteristics including gender, BMI, waist circumference, comorbidities, medication history, blood pressure, and heart rhythm both at baseline and 1-year post-PCI will be documented by an independent physician. Blood tests assessing routine blood counts, liver and kidney function, blood lipid profiles, fasting blood glucose, glycosylated hemoglobin, hsCRP, and BNP will be recorded at baseline (before angiography) and at 1-year post-PCI. CK, CK-MB, and cTNI levels will be collected at baseline, one day after intervention, one week after intervention, and at 1-year post-PCI.2.6.1The primary endpoint of this SOS-moral study will be difference in cardiac function observed between baseline and 1-year post-intervention, which will be determined by CMR and transthoracic echocardiography.2.6.2Secondary endpoints involve the occurrence of MACEs, defined as all-cause mortality, cardiac death, a first or recurrent, non-fatal, acute myocardial infarction, target lesion revascularization (PCI or CABG), stroke, heart failure and cardiac rehospitalization within 1, 6, and 12 months post-PCI. Other secondary endpoint will include changes in myocardial viability in the territory supplied by the CTO artery between the baseline and 12 months post-PCI, to be determined by CMR. In addition, QOL changes at 1, 6, and 12-month follow-up appointments will be assessed by comparing with baseline values using the SAQ, patient health questionnaire 9 and Generalized Anxiety Disorder Scale -7. Finally, the volume of contrast used during the procedure and the total cost of medical care covering OMT and the equipment for coronary artery disease therapy from the day of enrollment  to the last follow-up  will be evaluated. Perioperative myocardial infarction will be evaluated by assessing CK, CK-MB, and cTNI levels;2.6.3The safety of the perioperative period, including acute coronary artery occlusion, acute vascular perforation, acute stent thrombosis, acute myocardial infarction, and cardiac death of the two groups assessed, will be compared.2.7 Two-hundred sixteen cases recruited to the study will allow for the use of a 1-tailed test with a significance level (α) set to 0.025, and power (1-β) of 0.90. Ultimately, it is estimated that 240 patients will be randomized, and a 10% loss is expected to occur throughout the 1-year follow-up period.PASS 15.0 will be utilized to calculate sample size. In the study, 226 individuals are predicted to be recruited in the trial. This sample size was calculated using the optimal efficiency test. The main purpose of this trial is to investigate the effect successfully opening concurrent CTO lesions on cardiac function in patients with MVD. LVEF has been taken into consideration for the calculation of sample size. Previous studies revealed a 6.5% mean difference between the two experimental groups and a 9% standard deviation with respect to LVEF. The optimal boundary value for LVEF was set to 4%.2.8In the full analysis set, all randomized patients will be kept in the treatment group to which they were originally randomized. Moreover, patients without protocol deviation will be included in the per protocol set. Patients of the per protocol set will be included in baseline information analysis rather than in primary analysis if the CTO-PCI fails. Patients will not be included in the primary analysis if the MACEs occur.t-test will be used to make comparisons between two independent samples. Median and 4-quintile intervals will be used to express variables that are not normally distributed, and the Mann–Whitney U-test will be used to compare differences. Categorical variables will be presented as frequencies and percentages, and the differences between groups will be assessed using a chi-square test. Survival analysis will be performed using a Kaplan–Meier curve and Cox regression analysis. A 2-sided P-value < .05 will be considered statistically significant.Continuous variables normally distributed will be presented as means ± standard deviation, and student 2.9Inclusion and exclusion criteria will be enforced strictly to minimize the potential for the inclusion of confounders. An experienced cardiovascular interventionist (CTO-PCI >50 cases/yr) has been designated to ensure that the success rate of CTO-PCI is above 80%. The number PCI performed within each of the centers participating in this study is least 3,000 cases. An expert committee has been established to ensure interventional safety and prevent study-associated complications. At least two senior experts in PCI (PCI >3000 cases per year) will assist to discern MACEs throughout the follow-up period. Two experienced specialists will acquire CMR images and collect data using specific equipment and software. Two independent investigators will be responsible for the creation of protocols and supervising follow up, and they will communicate with the enrolled participants throughout follow-up. The case report form will be completed by independent investigators. All data obtained will be documented in the database by two independent investigators according to guidelines governing data management. A special inspector will be responsible for tracking, monitoring and reviewing the research process, and it will not be possible to alter data at will. The database will be inactive after cross validation, followed by statistical analysis. The staff that perform analysis will be blinded to the intervention.3,10 however, there remains a lack of evidence from a randomized control trial, which can be sued to conclusively determine whether the success of opening concurrent CTO lesions improves cardiac function among patients presenting with MVD but not STEMI.This trial aims to investigate the effect of CTO-PCI combined with non-CTO PCI on cardiac function in patients with MVD. Patients with MVD in a EuroCTO trial received non-CTO PCI before randomization; participants in the DECISION-CTO trial were not limited to those with MVD; the EXPLORE study recruited patients with STEMI. Consequently, a number of studies have investigated the clinical effects of successful CTO-PCI;–25 An early meta-analysis found that successful recanalization of a CTO resulted in an overall improvement of 4.44% absolute LVEF points, reduced remodeling, and improved survival (OR: 0.52). However, the EXPLORE trial showed that in acute STEMI patients with concurrent CTO, CTO-PCI did not increase global LVEF or decrease LVEDV assessed by CMR 4-month post-PCI compared to patients receiving treatment in the non-CTO artery. Interestingly, a subgroup analysis of the EXPLORE trial showed that CTO-PCI in patients with a concurrent CTO in the LAD was associated with significantly higher LVEF levels after 4-month post-PCI compared with non-CTO PCI, suggesting that CTO-PCI may potentially benefit high-risk patients. According to Chung, the benefit of CTO-PCI is controversial with respect to cardiac function and survival among the MVD patients with concurrent CTO. This may partly be because myocardial viability affects the alteration of cardiac function produced by CTO-PCI. An in-depth analysis in the EXPLORE CMR study revealed that CTO-PCI was associated with the greater recovery of regional systolic function relative to baseline values 4-month post PCI in dysfunctional myocardial tissue supplied via CTO. The benefit was most evident in dysfunctional but viable segments . The investigators opined that the benefit was probably attributable to the recovery of stunned myocardial tissue affected by the acute interruption of blood supply, as well as by the prevention of arrhythmia. Taken together, the data suggest that varying levels of myocardial viability in patients included in previous studies may be associated with reported variations in cardiac functioning observed after successful CTO-PCI treatments. Therefore, myocardial viability should be considered.Early studies reported that successful recanalization CTO can both improve LVEF and ventricular wall movement assessed by CMR, and can improve QOL. Differences in myocardial viability may contribute to the variable prognosis of CTO-PCI. A previous study including 32 CTO patients presenting with significant inducible perfusion deficits found that recanalization was associated with decreased ischemic burden, improved QOL, and reversed remodeling. The effect of CTO-PCI on cardiac function among patients with necrotic myocardial tissue in the territory supplied by the CTO combined with MVD requires further investigation. The present SOS-moral study excludes those patients with complete myocardial survival in the CTO artery territory. This distinction will facilitate the investigation of the effects of actively opening concurrent CTO lesion in MVD patients with necrotic myocardial tissue identified via CMR in the territory supplied by the CTO, aiming to provide reliable evidence for use in clinical practice.Previously, 128 patients were observed using CMR, and among the myocardial regions that corresponded to 149 CTO lesions, only 11.5% presented as transmural myocardial infarction. In fact, 58.6% of the myocardial segments showed no delayed enhancement. CMR has been used to assess myocardial viability throughout the previous decade. According to Cardona et al., CMR accurately detects myocardial viability in patients with reduced LVEF. We previously demonstrated that, using PET as gold standard, CMR has high consistency, sensitivity, and specificity for the detection of myocardial viability compared with PET in CTO patients. Therefore, in the SOS-moral trial, CMR will be used to evaluate both the amount of viable myocardial tissue and level of cardiac functioning.CMR is an appropriate approach for the evaluation myocardial viability and cardiac function. The method is accurate, safe, and provides great spatial resolution as well as sensitivity.4SOS-moral will sort myocardial viability before randomization to avoid the effect of viability of myocardial on cardiac function. Hence, the real benefits of CTO-PCI on cardiac function among these patients without 100% survival viable myocardial will be assessed. Furthermore, the present trial strictly recruits patients with MVD and concurrent CTO. SOS-moral will provide evidence for the value of CTO-PCI among these selected patients. Nevertheless, there remain several potential limitations in this study. First, SAQ may be affected by factors other than recanalization of CTO artery during the study period. Second, patients with high risk such as those with left main artery disease stenosis ≥50% and severe arrhythmia are not included. Patients with left main artery disease are not eligible because the PCI procedure needs to be individualized. Because cardiac function might be affected by the severe arrhythmia, those with severe arrhythmia will be excluded. Therefore, the outcomes are unable to include all patients at high risk. Third, in a limited study period, the study may be not powered to detected MACE differences between the two groups. Therefore, further studies including high-risk patients and with longer follow-up periods are needed in the future.The authors wish to thank Ya Yang and Yueli Wang for their contribution in performing transthoracic echocardiography. The authors highly thank for Wenyi Zhang and Haoran Xing, who help recruit patients. We gratefully acknowledge Jiahui Li for performing follow-up evaluations of patients, as well as Xin Zhao and Nan Nan for collecting data. The authors also wish to thank the patients who participated in the study and staff from the 3 centers who supported the study.Xiantao Song and Rongchong Huang contributed to the study design. Jinfan Tian and Dongfeng Zhang contributed to draft the manuscript. Xueyao Yang contributes to the patients’ follow-up. Minduo Zhang and Yuan Zhou perform the intervention implementation and patients’ recruitment. Lijun Zhang contributes to CMR perform. Yi He and Hongzhi Mi help to CMR technology supervision. Huijuan Zuo contribute to the data monitor. Xiantao Song and Min Zhang contribute to the endopoint adjudication.Conceptualization: Xiantao Song and Rongchong Huang.Data monitor: Huijuan Zuo.Date management: Jinfan Tian and Dongfeng Zhang.Endpoint adjudication: Xiantao Song and Min Zhang.Investigation: Xueyao Yang, Minduo Zhang and Yuan Zhou.Technology supervision: Yi He and Hongzhi Mi.


Background: This study developed a novel inflammation score system to predict survival outcomes using preoperational inflammatory markers in hepatocellular carcinoma (HCC) after surgery.Materials and Methods: An inflammation score system was developed using five preoperative inflammatory markers based on the clinical data of 455 HCC patients (training cohort) receiving radical resection in the Eastern Hepatobiliary Surgery Hospital. The system was validated using a cohort from a different hospital . Kaplan-Meier curves and log-rank test were used to compare the survival of patients with different inflammation scores. A nomogram including inflammation scores for survival prediction was created to exhibit the risk factors of overall survival (OS).Results: The patients in the low-score group showed better OS and recurrence-free survival (RFS) in the training and external validation cohorts than those from the high-score group. Subgroup analysis showed that compared with patients in the training cohort from the high-score group, stage I (eighth TNM stage) patients in the low-score group exhibited better prognosis results, whereas the findings for Stage II and III patients were different. Multivariate Cox analysis revealed that high inflammation score is an independent risk factor of OS and RFS. The nomogram established using the inflammation score with the C-index value of 0.661  revealed a good three- and five-year calibration curves.Conclusions: The inflammation score system based on five preoperative inflammatory markers well predicted the survival of HCC patients after surgery, especially in those at the early stage (Stage I). Hepatocellular carcinoma (HCC) remains a significant health problem in the world; it is the sixth most common neoplasm and the third leading cause of cancer-related mortality worldwide Numerous studies have demonstrated that inflammation is a decisive component of tumor progression. Several inflammatory markers, such as modified Glasgow prognostic score, platelet-to-lymphocyte ratio (PLR), and neutrophil-to-lymphocyte ratio (NLR), have been proven to be significant prognostic factors in many cancers Nomogram is a novel tool or a new standard that predicts the OS of many cancer patients Data of 455 HCC patients from September 2010 to September 2012 as training cohort form the Eastern Hepatobiliary Surgery Hospital (EHBH) and 253 HCC patients from October 2010 to October 2013 as an external validation cohort form Longyan First Hospital were retrospectively reviewed. The inclusion criteria were as follows: (1) radical resection, (2) no anticancer treatment before surgery, (3) no history of other malignant diseases, (4) Child-Pugh grade A or B7 liver function, (5) no evidence of distant metastasis and major portal vein/hepatic vein invasion. Patients who received palliative tumor resection or combined other malignant diseases, had incomplete data, and failed to follow-up within 30 days from the date of surgery were excluded. Patients in the external validation cohort were assessed with the same inclusion/exclusion criteria. Informed consent was obtained from all the patients before surgery.All patients were routinely investigated with liver and renal function tests, blood routine tests, carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), serum alpha-fetoprotein (AFP), chest X-ray, abdominal ultrasound, contrast-enhanced computerized tomography (CT) scan, or/and magnetic resonance imaging (MRI) of the abdomen. We also specifically collected the data of pre- and post-operative platelets, neutrophils, and lymphocyte cell count. The preoperative diagnosis of HCC was based on the criteria of the American Association for the Study of Liver Diseases The inflammatory markers studied in this research contained aspartate aminotransferase (AST)-to-alanine aminotransferase (ALT) ratio (AAR), AST-to-lymphocyte ratio index (ALRI), PLR index (PLR), NLR ratio index (NLR), AST-to-neutrophil ratio index (ANRI), and AST-to-platelet-count ratio index (APRI).All patients underwent liver resection (LR) with the intention of complete removal of macroscopic tumors with adequate resection margins. The scheme of hepatectomy was carefully developed through a preoperative assessment. The patients were recommended for LR if they were in good general condition, with technically resectable tumors, and showed sufficient estimated volume of the future liver remnant. Histopathologic study of the surgical specimens was carried out independently by three pathologists, who came to a consensus by discussion if any controversy existed. Major hepatectomy was defined as the removal of ≥3 Couinaud liver segments Patient age at the time of operation, gender, and Child-Pugh classification were regularly measured. The tumor-related variables based on histopathological examination include tumor size, number, and presence of microvascular invasion (MVI). Tumor number was classified as solitary or multiple, and the largest diameter was measured for patients with multiple tumors.2) = weight (kg)0.425×height (cm)0.725×0.007184 Commonly used scoring systems contained the 8th edition of the American Joint Committee on Cancer (AJCC) All the patients were followed-up regularly after discharge using the following protocol. Briefly, the assessments were conducted once every two months in the first two years after surgery and then once every three to six months with blood routine tests, prothrombin time (PT), liver function tests, levels of CA19-9, CEA, AFP, and abdominal ultrasound. A contrast-enhanced CT scan or/and MRI was performed once every six months or earlier if tumor recurrence/metastasis was suspected. The diagnosis of HCC recurrence was similar to that of the initial disease diagnosis. Recurrent lesions were managed by a multidisciplinary approach as previously reported Time-dependent areas under the receiver operating characteristic (ROC) curve (AUC) of each point in different indicators were measured from 20 months to 100 months after surgery, thereby reflecting the performance in predicting OS at various time points. In this study, the five-year OS rate was used as an endpoint to stratify the values of AAR, ALRI, PLR, NLRI, ANRI, and APRI through the ROC curves. The cut-off point representing the highest Youden index (specificity + specificity-1) was selected as the optimal threshold value L, QU)] were used without normal distribution. The differences were compared by Student's t test and Mann-Whitney U test. Categorical variables were reported as the number of cases and percentages; the differences between the three groups were compared by the chi-square test. The Kaplan-Meier (KM) method was used to analyze OS and RFS to recurrence, and the differences were explored by the log-rank test. The Cox proportional hazard model was used to analyze the independent prognostic factors of prognosis. We used survival tree analysis to define the best cut-off value of inflammation score and then form two differentiated groups of patients (high-score group and low-score group) with different prognosis. The nomogram was built based on the results of multivariate Cox regression analyses of OS with enter method by using the rms package of R, version 3.2.0 (http://www.r-project.org/). We compared the nomogram's ability of survival prediction with other commonly used scoring systems, such as AJCC 8th TNM stage, Child-Pugh stage, BCLC stage, and so on by the time-dependent areas under the ROC curve of each point in different cohorts from 10 months to 60 months. Variables with P values < 0.05 in the univariate analysis were included in the multivariate analysis. Statistical analysis was performed using the SPSS® version 25.0 . R program version 3.2.0 (http://www.r-project.org/) was used, with P value less than 0.05 indicating statistical significance.Continuous variables with normal distribution were reported as the mean and standard deviation (mean ± SD), and the median and interquartile range [median , the median follow-up times were 56.2 and 31.4 months in the training and external validation cohorts, respectively. A significant difference in OS and RFS was observed between the two groups in the training cohort  rate and RFS (P=0.001) compared with those from the low-score group A and S1BThe variables listed in the Table Based on the results of multivariate analysis, we conducted a further analysis by establishing a nomogram with a C-index of 0.661  = 0.624-0.698) to predict one-, three- and five-year OS rates of an individual patient. The calibration curves of the nomogram for survival probability at three and five years after radical resection presented an optimal agreement between the prediction by nomogram and actual survival  are important immune cells found within tumors and cause antitumor immune responses A novel nomogram was developed in this study to predict the survival rates of HCC patients who suffered from the operation. The results suggest that this predictive nomogram includes four integrated predictors. Tumor number and major hepatectomy are regarded as important indicators reflecting the prognosis of HCC in studies Our study featured several limitations. First, the inflammation score system enrolled patients originated from one medical center only, thereby easily leading to bias. More external validation cohorts from other medical centers should be enrolled to further validate the findings. Second, neutrophil, platelet, and lymphocyte levels are easily influenced by infections, inflammation in other tissues, and medications before HCC treatment. These factors might interfere with the inflammatory marker measurements. Third, other meaningful inflammatory markers should be further investigated to clarify and support our conclusions.In conclusion, our results confirmed that the inflammation score system established in our hospital could be used as a novel significant predictor for prognostication in HCC patients, especially those at early TNM stages. This inflammation score system could be used to assist surgeons in identifying patients who might benefit from radical liver surgery.Supplementary figures and tables.Click here for additional data file.