The last 3 years have seen the emergence of promising targeted therapies for the treatment of hepatocellular carcinoma (HCC). Sorafenib has been the mainstay of treatment for a decade and newer modalities were ineffective and did not confer any increased therapeutic benefit until the introduction of lenvatinib which was approved based on its non-inferiority to sorafenib. The subsequent success of regorafenib in HCC patients who progress on sorafenib treatment heralded a new era of second-line treatment and was quickly followed by ramucirumab, cabozantinib, and the most influential, immune checkpoint inhibitors (ICIs). Over the same period combination therapies, including anti-angiogenesis agents with ICIs, dual ICIs and targeted agents in conjunction with surgery or other loco-regional therapies, have been extensively investigated and have shown promise and provided the basis for exciting clinical trials. Work continues to develop additional novel therapeutic agents which could potentially augment the presently available options and understand the underlying mechanisms responsible for drug resistance, with the goal of improving the survival of patients with HCC. Unfortunately, a significant proportion of HCC patients present with intermediate and advanced stage disease often, despite diligent surveillance, and curative treatments are frequently not possible.4 In these patients, systemic therapy remains essential and its pivotal role and potential have stimulated considerable research over the past decade. In this review, we examine recent advances in targeted therapy and discuss the impact this has had on the management of HCC. We also provide an overview of the most important areas of HCC research including novel clinical trials and technical platforms which promise to facilitate substantial progress within the next decade.Primary liver cancer remains a major problem for all health care systems worldwide and is associated with a significant clinical, economic, and psychological burden. Hepatocellular carcinoma (HCC) accounts for ~90% of cases of non-metastatic tumors of the liver.9 ushering in the era of systemic treatment. Subsequently, virtually all trials were centered around sorafenib and it was used as a control with which novel first-line agents were compared and evaluated in an attempt to improve the prognosis of patients with HCC. Unfortunately, despite a number of trials which compared these novel agents including sunitinib,10 brivanib,11 cediranib,12 linifanib,13 dovotinib,14 and immune-checkpoint inhibitors (ICIs) to sorafenib, none achieved the predefined primary end points .23 Noticeably, the application of sorafenib in clinical practice displays significant regional variations and incompliance with guidelines besides its usage as first-line therapy. It is common that initially unresectable HCCs got downstaged after sorafenib treatment and underwent curative-intent surgery28 and locoregional therapies before sorafenib were commonly encountered in real-world settings.30The success of SHARP and Asia-Pacific trial promoted the approval of sorafenib as first-line targeted therapy for advanced HCC,31 Other agents such as interferon,32 selumetinib,33 capecitabine,34 tegafur-uracil,35 gemcitabine and oxaliplatin (GEMOX),37 and gemcitabine alone38 have also been evaluated but to date no treatments involving combinations containing sorafenib have succeeded in phase III trials.The clinical benefit of sorafenib however remains modest and the complex molecular pathogenesis of HCC stimulated the investigation of combinations of sorafenib with other molecular targeting drugs. Sorafenib has been combined with anti-angiogenic agents, MEK/ERK pathway inhibitors, mTOR pathway inhibitors, histone deacetylase inhibitors, EGF/EGFR pathway inhibitors, and HGF/c-Met pathway inhibitors.39 did not improve progression free survival (PFS).40 It was also shown that the addition of sorafenib did not confer any survival benefit in patients with unresectable HCCs, who had already responded to TACE.41 Contrasting with these findings, retrospective studies from China have shown that combination therapy with sorafenib and TACE increased OS by more than 50% compared with TACE alone,53 which was supported by the findings from a number of other groups.55 Recently, the TACTICS trial, a randomized, multi-center prospective trial from Japan reported an improved PFS for TACE plus sorafenib compared with TACE alone ,56 although this trial used a redefined PFS . The TACTICS trial also used time to any cause of death plus OS as primary endpoints (results not reported) and compared with sorafenib monotherapy, TACE plus sorafenib was only superior in controlling tumor progression and did not prolong OS.58Since sorafenib and TACE are both recommended therapies for advanced HCC, it is reasonable to expect that their combined use would confer benefits when compared with monotherapy. Results however highlighted regional differences and the heterogeneity of the trial protocols. In TACE 2, the multi-center, randomized, placebo-controlled, phase 3 European trial, when compared with TACE alone the addition of concurrent sorafenib, unlike the SPACE trial,59 and TACE plus intensity-modulated radiotherapy combined with sorafenib.60 In the SARAH study, selective internal radiotherapy with yttrium-90 resin microspheres did not produce any survival benefit compared with sorafenib in locally advanced and inoperable HCC , and did not meet the primary endpoint of OS.61 Similarly, the addition of selective internal radiation therapy to sorafenib did not result in a significant improvement in OS compared with sorafenib alone.62 Bettinger et al.63 however did demonstrate that stereotactic body  radiotherapy employed as monotherapy (SBRT) was able to improve OS compared with sorafenib and SBRT with TACE also provided improved OS and PFS when compared with sorafenib and TACE in combination.64 In a recent trial of hepatic arterial infusion chemotherapy , He et al.65 reported that sorafenib plus HAIC with FOLFOX improved OS compared with sorafenib alone in advanced HCC when portal vein invasion was present, which was supported by other studies.67 Although the SCOOP-2 trial found sequential HAIC with cisplatin and sorafenib did not improve the survival benefit compared with sorafenib alone this is likely to have resulted from the study being underpowered for the primary and secondary endpoints.68The acceptance of sorafenib as the standard to which other newer agents and non-surgical interventions are compared has resulted in studies comparing its use as monotherapy with TACE plus external beam radiotherapy69 and other western studies.70 Explanations for the negative outcome in the STORM trial include high-dose modification rates, short treatment durations, and the enrollment of patients who were not at high risk of tumor recurrence .71 Consistent with this viewpoint, Wang et al.72 reported no case of recurrence during the sorafenib dosing period whereas 4/14 patients suffered recurrence of their tumor within 7 months of discontinuation of sorafenib72 and persistent sorafenib intake following postoperative recurrence improved OS.73 Considering that patients who respond to sorafenib may belong to limited clinical or biological subsets, the effectiveness of sorafenib in an unselected population cohort supports its use in the adjuvant setting. A number of studies from the Far East including China, Japan, and Korea include patients with HCCs who are treated with hepatectomy despite their tumors being outside Barcelona Clinic Liver Cancer Classification (BCLC) guidelines, and although the results are difficult to compare due to heterogeneity of the protocols the results are positive. Sorafenib significantly reduces tumor recurrence in BCLC stage C patients75 and increases disease-free survival (DFS),76 and Zhuang et al.77 demonstrated that adjuvant therapy increased disease free survival (DFS) and OS. Sorafenib treatment following hepatectomy significantly prolonged the OS of advanced HCC rather than intermediate HCC.78 In addition to BCLC stage, patients who underwent hepatectomy and were pathologically diagnosed with microvascular invasion (MVI) also benefited from adjuvant sorafenib treatment.79 In line with these results, a large recent study with propensity score matching analysis also demonstrated that sorafenib significantly improved overall and recurrence-free survival following resection.80 The results from these studies which include all eligible patients suggest that more precise stratification would enable the identification of those patients who will benefit most from the use of adjuvant sorafenib and those in where additional treatment is not appropriate. Ongoing trials are attempting to evaluate the role of sorafenib in patients with MVI following radical resection (NCT02867280 and NCT02537158).Due to the high recurrence rates following hepatectomy for HCC, approaches to adjuvant therapy has been extensively investigated although previous attempts, including the use of anti-viral agents, have been largely unsuccessful. Based on the palliative use and success of sorafenib its potential in the adjuvant setting was investigated and improved survivals following surgery anticipated. Unfortunately, this has not been demonstrated and it failed to reduce postoperative tumor recurrence in the STORM trial81 Although not approved for long, further multi-center data from “real-world conditions” confirmed the efficacy of lenvatinib, regardless of previous tyrosine kinase inhibitor (TKI) therapies83 and lenvatinib monotherapy demonstrated anti-tumor activity for more than 4 years in unresectable HCC when portal vein invasion was present.84 In intermediate-stage HCC patients with tumors exceeding the up-to-seven criteria, for whom TACE is not helpful, lenvatinib could provide significant longer OS (37.9 vs. 21.3 months) and PFS (16.0 vs. 3.0 months).85 Lenvatinib pharmacokinetics in HCC is affected by body weight87 and a sufficient dose  is required to achieve a good therapeutic effect and consequently improved outcomes and prognosis are associated with the preservation of liver function which reduces the number of patients who need to discontinue their treatment.91 With lenvatinib, unlike other TKIs, there are issues with thyroid toxicity and surveillance for thyroid abnormalities during treatment is important.92 Hypothyroidism is not unusual and there are also fewer common reports of thyrotoxicosis and destructive thyroiditis.93 From a health economics standpoint however, lenvatinib is more cost effective than sorafenib.95Following the approval of sorafenib for use in the treatment of HCC it takes more than a decade before the second first-line targeted agent for HCC emerged. Lenvatinib was approved for the first-line therapy in advanced HCC following the results of the REFLECT trial, a randomized phase III non-inferiority trial published by Kudo et al.Still, sorafenib displays limited anti-tumor activity and some initially sorafenib-sensitive would eventually succumb to the disease, indicating the acquired resistance to sorafenib reduces its beneficial effects and an urgent need for second-line therapy.96 The RESORCE trial was a randomized, double blind, placebo-controlled, and phase III trial demonstrating the effectiveness of regorafenib in patients who had progressed on sorafenib treatment. This study finally confirmed the potential of second-line agents and ushered in the era of second-line and sequential therapy.97 Regorafenib provided survival benefit regardless of the rate of disease progression during prior sorafenib treatment or since the last sorafenib dose.98 This was confirmed by Yoo et al.99 in a retrospective study of safety and efficacy in Korean patients where data were consistent with those from the RESORCE trial. Regorafenib was even shown to be effective in patients with HCC recurrence following liver transplantation with a median OS of 12.9 months following regorafenib initiation and 38.4 months following sorafenib initiation  for the sorafenib followed by regorafenib sequential therapy.100Initial attempts to discover effective second-line agents were unsuccessful and mirrored attempts to develop first-line agents which were superior to sorafenib.101 In clinical practice only ~30% of patients are eligible for second-line regorafenib treatment.102 Good liver functional reserve and ECOG performance status during sorafenib treatment contributed to the efficacy and better outcomes of subsequent treatment,104 including lenvatinib.105 This may in part be due to the RDI required to achieve a clinically significant improvement in prognosis.106 This is supported by the demonstration that the new liver reserve function biomarker, albumin-bilirubin grade (ALBI),107 successfully identified regorafenib candidates and that in the selected cohort a median OS of 15.6 months was achieved compared with 6.8 months for non-candidates.108 Even in patients not eligible for regorafenib, the ones with an ECOG-PS score of 0, the absence of MVI, and TTP (time to progression) ≥4 months could still have acceptable postprogression survival.109 Long-term treatment with regorafenib has also been shown to reduce angiogenesis and improve portal hypertension (PHT) and acute administration ameliorates portal haemodynamics, suggesting that it may be especially suitable for patients with PHT and preserved liver function.110However, not all patients who progress on sorafenib are candidates for second-line therapy.111 and was consequently approved in the EU and USA. There remains a paucity of data however from real-world clinical practice examining the sequential treatment utilizing cabozantinib as the second-line agent, it is a costly option associated with frequent high-grade adverse events. Consequently, several studies have addressed the cost-effectiveness of cabozantinib using the cost and utility data extracted from the CELESTIAL trial. The conclusion from these studies is consistent and confirms that at its current cost point, the gain of quality-adjusted life-years for cabozantinib  and the incremental cost-effectiveness ratio  mean that it is not a cost-effective treatment option for patients with sorafenib-refractory HCC,114 compared with regorafenib .116Cabozantinib is another small molecule inhibitor of the tyrosine kinases which are implicated in the progression of HCC and the acquired resistance to sorafenib. Cabozantinib blocks the receptors involved in oncogenesis and angiogenesis including VEGFR 1, 2, 3, hepatocyte growth factor receptor (MET), AXL and the angiopoietin receptors TIE-2, RET, c-Kit and FLT-3 in vitro and in vivo. In CELESTIAL trial, cabozantinib achieved the primary endpoint with median OS of 10.2 months compared with 8.0 months for the placebo group117 subgroup analysis found survival benefit in patients with AFP of 400 ng/ml or higher.121 This was later confirmed in the REACH-2 trial,122 which led to the approval of ramucirumab as second-line treatment for advanced HCC. REACH-2 is the first positive phase 3 trial in patients with HCC performed in a biomarker-selected patient cohort and more recent findings demonstrated that AFP-enriched HCCs displayed significant activation of VEGF which suggests the underlying mechanism of action and confirms the potential value of biomarker-driven clinical trials.123Ramucirumab is a fully human recombinant IgG1 monoclonal antibody targeting the VEGF2 receptor. Although ramucirumab failed to meet its primary endpoint as second-line treatment in the REACH trial,124 and KEYNOTE-224125 studies evaluated the safety and efficacy of nivolumab and pembrolizumab in patients with advanced HCC refractory to previous sorafenib treatment, which established the basis for accelerated approval by the FDA as second-line treatment. Subanalysis of CheckMate-040 data validated the safety and efficacy of nivolumab in Asian cohort.126 In an international real-world cohort study, ICIs have showed promising efficacy and safety in advanced HCCs as systemic first-/second-/third-/fourth-line treatment, with median OS and PFS of 11.0 and 4.6 months respectively127 and an excellent response to anti-PD-1 therapy has also been described in case report.128 Although the subsequent phase III KEYNOTE-240 trial did not meet its pre-specified statistical significance in respect of improved PFS and OS, the results were consistent with previous KEYNOTE-224.129 The KEYNOTE-394 presently underway in Asian patients may clarify the role of pembrolizumab in cases of advanced HCC with a viral background (NCT03062358). Recently, CheckMate-459, the multi-center phase III randomized sorafenib controlled trial evaluating nivolumab as first-line treatment for advanced HCC, failed to achieve its endpoints (ESMO 2019) but nivolumab did prolong OS (16.4 vs. 14.7 months) and achieve long-time disease control, less adverse events (AEs) and survival benefit regardless of the level of PD-L1 expression. Furthermore, nivolumab improved the survival of HCC patients whose etiology was HBV/HCV and did not reactivate hepatitis. Camrelizumab , is an anti-PD-1 inhibitor from China investigated for the treatment of Hodgkin lymphoma and HCC. It has been shown to have antitumor activity in previously treated Chinese patients with advanced HCC in a multi-center, open-label, parallel-group, randomized, phase II trial (NCT02989922),130 providing evidence for the effectiveness of PD-1 therapy for HBV related HCC in Chinese patients. The results from other trials investigating novel ICIs including durvalumab, avelumab, tislelizumab, sintilimab, tremelimumab, ipilimumab, spartalizumab, and toripalimab will hopefully yield positive results and provide further options for the treatment of patients with HCC, particularly those who have relapsed on first-line treatments.ICIs stand as the mainstream of immunotherapy. The CheckMate-040131 demonstrated that lenvatinib and regorafenib could affect the expression of PD-L1 and real-time PCR results suggested that the mRNA expression of PD-L1 in the lenvatinib group was significantly higher than that in the control group, while its expression in the regorafenib group was significantly lower. When combined with anti-PD-1, lenvatinib can modulate cancer immunity in the tumor microenvironment and enhance antitumor activity.133 In July 2019, the FDA announced its approval of the first combination therapy employing the TKI lenvatinib with the ICI pembrolizumab based on the results from the KEYNOTE-524/Study 116 (NCT03006926) for the treatment of HCC. Recently, results from Study 117  showed marginally better results for lenvatinib with nivolumab than lenvatinib with pembrolizumab. MET-mediated phosphorylation leads to a decreased expression of PD-L1 using the combination of MET inhibitors tivantinib and capmatinib, anti-PD1 and anti-PDL1 produced an additive effect which slows the growth of HCCs in mice.134 Clinically, based on the results from the experimental arm A of the GO 30140 study (NCT02715531), the FDA approved atezolizumab plus bevacizumab as breakthrough therapy for untreated advanced or metastatic HCC.135 Individual case studies also reported promising results for the use of combined TKI and anti-PD1/PD-L1 agents for advanced HCC.138 Such results were confirmed in the phase III trial IMbrave 150 study (NCT03434379) which reported that atezolizumab combined with bevacizumab resulted in better OS and PFS than sorafenib in patients with unresectable HCC.139 Other combination therapies include Galunisertib with nivolumab (NCT02423343), spartalizumab with and without capmatinib (NCT02795429), FGF401 with spartalizumab (NCT02325739), regorafenib with pembrolizumab (NCT03347292), cabozantinib with nivolumab (NCT03299946), avelumab with axitinib (NCT03289533), ramucirumab with durvalumab (NCT02572687), and XL888 with pembrolizumab  occur frequently during treatment with ICIs and the clinical consequences can be significant.144 apatinib still displayed promising anti-tumor effects in sorafenib-resistant HCC,147 where portal vein invasion was present,148 when metastases have occured,150 and for unresectable and relapsed HCCs.152 Combination therapy in studies utilising apatinib with TACE have achieved better clinical effectiveness than TACE alone, with tolerable AEs.161 Recently, the combination of apatinib with the anti-PD-1 monoclonal antibody camrelizumab achieved partial response rates of 50%.153 The results of other ongoing trials including the phase III trial comparing TACE and apatinib with sorafenib as first-line treatment for locally advanced or metastatic and unresectable HCC (NCT 03764293) and the adjuvant apatinib after hepatectomy for the prevention of tumor recurrence (NCT03722875 and NCT03261791) will hopefully prove effective and add to the presently available therapeutic options.Despite abovementioned targeted drugs, novel agents have been continuously under development Table . Of note154 The combination of bevacizumab and erlotinib has been extensively evaluated as first-155 or second-line in advanced HCCs,162 but unfortunately the heterogeneous nature of the results precludes firm conclusions and recommendations. Recently, a single-arm meta-analysis of prospective studies found that combination therapy with bevacizumab and erlotinib used as second-line treatment was associated with a favorable PFS  and OS , suggesting that future well-designed and sufficiently powered large-scale RCTs should be able to identify the potential contribution of these agents.163These promising results have stimulated the investigation of other new agents, the combinations of agents and regimens, which have been thoroughly discussed in a recent review from Zhu and Sun.166 especially with the potential ability to abrogate the emergence of sorafenib resistance167 and sensitize HCC to regorafenib treatment.168 A number of CKD inhibitors are presently undergoing evaluation including palbociclib (NCT01356628), milciclib (NCT03109886), and ribociclib (NCT02524119). The anti-MET monoclonal antibody emibetuzumab exhibited the greatest antitumor activity in HCC when combined with ramucirumab and had an excellent safety profile169 and for HCC with high MET expression there was an almost 3-fold increase in PFS (8.1 vs. 2.8 months) relative to those with low MET expression, suggesting the potential for further biomarker-driven clinical trials. Rigosertib is a synthetic benzyl styryl sulfone small molecule inhibitor which has been used in the treatment of monomyelocytic leukemia and due to its activity as a RAS- and PLK1-signaling inhibitor, it was investigated in HCC patients who demonstrate upregulation of PLK1 during tumor development and HRAS expression in advanced HCC. High expression levels of PLK1 are also significantly correlated with poor patient survival and the multiple effects of rigosertib could be beneficially employed to produce a therapeutic “dual-hit” approach in selected patients.170 Donafenib is a novel multi-kinase inhibitor which is similar to sorafenib, displaying comparable or better safety and efficacy when treating advanced HCC in phase 1b trial and phase 3 studies using sorafenib as the control (NCT02645981).171 There are ongoing trials evaluating novel agents such as anlotinib, another multi-kinase inhibitor which is orally administered and targets VEGFR, fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptors (PDGFR), and c-kit (NCT02809534). Tivozanib is another oral inhibitor of VEGFR-1/2/3 with promising activity against HCC in vivo (NCT01835223) and TRC105, which despite demonstrating clinical activity and being well tolerated in HCC patients following sorafenib, has not to date met prespecified criteria and its development in HCC continues as combination therapy with sorafenib (NCT02560779).Preclinical evidence for cyclin-dependent kinase (CDK) targeting therapies in HCC has showed promise and supports their investigation,182 Although the molecular classification of HCC has been widely reported , celecoxib (COX-2/Wnt inhibitor), sirolimus (mTOR inhibitor), and cabozantinib, respectively, with a reduction of des-gamma-carboxy prothrombin and AFP following treatment.196The continued belief that the future lies with personalized treatment which will be made possible through the rapid developments in next generation sequencing and the precision medicine that it underpins, have encouraged the development of novel trial designs.TSC1 and TSC2 have been shown to confer sensitivity to the mTOR protein. Aurora kinases are known to be oncogenic and overexpressed in a variety of tumors including colon, breast and prostate cancer and HCC tumor and other genetic mutations are known to affect the response to tyrosine kinase inhibitors and amplification of the MET gene is associated with hypersensitivity to cabozantinib which inhibits the tyrosine kinases c-Met and VEGFR2.198 The Wnt/β-catenin and Akt/mTOR pathways have been investigated and are reported to be co-activated in 14.4% of HCCs with the result that inhibition of the Jak/Stat pathway has therapeutic potential.199 Considering the wide range and type of genetic alterations which may act as potential targets,185 it is reasonable to believe that with an appropriate and well-designed trial protocol it should be possible to identify and validate specific biomarkers which will predict the response to specific targeted agents. Thus, we can prevent the use of treatments which can have no therapeutic effect, and may be associated with significant, avoidable toxicity.Approaches to developing biomarker-driven targeted therapy strategies have also been examined in vitro and inactivating mutations in 200 and tumor heterogeneity and clonal evolution are the underlying mechanisms  within tumors. This heterogeneity explains the attraction of targeted therapies and the search for biomarkers which can reliably predict the response to different agents. Nevertheless, it is also clear that the potential for this degree of heterogeneity renders the task of identifying single useful biomarkers or combinations of biomarkers extremely complex. On the other hand, the heterogeneity that has been identified does explain the, often quite dramatic, differences in the response to different therapeutic agents or combinations of agents.Drug resistance remains the principle cause of treatment failure during the use of targeted therapies203 Within the tumor, intratumoral heterogeneity (ITH) has been found at genomic,204 epigenetic,205 transcriptional206, and protein level.208 Considering the level and extent of the intratumoral heterogeneity it is not surprising that the response to single treatment is equally heterogenous and irrespective of the efficacy of a single agent the most favorable outcome that can be reasonably expected is the eradication of a small portion of the total tumor cell burden leaving resistant clones surviving and responsible for progression.209 Although trunk mutational events in HCC are less heterogeneous210 and it is reported that single region sample could effectively recapitulate the genomic or proteomic features of HCC,213 which seems to shed light on overcoming drug resistance by ITH, tumor evolution due to selective pressure from targeted therapy brings new challenges.214 Thus, we have to find new ways to circumvent tumor heterogeneity and tumor evolution.Unlike other primary cancers, multifocal lesions in liver is not uncommon whether derived from synchronous carcinogenesis or intrahepatic metastases. When multiple tumors occur, despite the fact that they originate from genetically similar cells and the potential etiology is the same, the lesions differ significantly (often very significantly) from each other demonstrating genomic alterations, varied biological behavior and loco-regional tumor microenvironment diversity which produces the well described differences in response to therapeutic agents.215 Single-cell whole-genome sequencing has been used to examine the diverse modes of clonal evolution in HCC and relate this to tumor morphology. Variations are seen to occur early in tumor development but subsequently demonstrate stability during tumor progression. These findings suggest that treatment strategies could be developed based on the knowledge of tumor morphology.216Fortunately, several novel techniques including single-cell sequencing, liquid biopsy, circulating tumor DNA (ctDNA), patient-derived cell-lines (PDC), patient-derived organoid (PDO), and patient-derived xenografts (PDX) should enable us to track cancer evolution in HCC. Compared with bulk tumor tissue sequencing, single-cell sequencing provides higher sensitivity and specificity, delineating tumor biology and characterization of cancer stem cell heterogeneity to understand the cellular diversity of HCC.217 In gastrointestinal cancers, ctDNA has been shown to outperform single-lesion tumor biopsies in discovering alterations which produce clinically relevant resistance and the mechanisms responsible for resistance to multiple agents.218 Novel alterations and parallel evolution which result in treatment-associated resistance are common in ctDNA, although the occurrence of multiple treatment related alterations in the circulation confound attempts to integrate the results into clinical protocols.219 When tumor biopsies are not available genetic profiling is possible using ctDNA which can provide a similar level of accuracy in respect of identifying somatic mutations and has the added advantage of being able to also detect de novo mutations.220 To date, few studies had examined the value of ctDNA to guide targeted therapy in HCC patients although preliminary results have demonstrated the feasibility of ctDNA in circumventing ITH,212 delineating tumor evolution,222 efficiently capturing mutations indicative of targeted therapy,223 and dynamically revealing genomic change during pharmacological treatment.224ctDNA represents a non-invasive, dynamic method to profile the tumor genome, predict treatment response, monitor disease progression, and help elucidate the mechanisms of drug resistance.227 An example of this approach is our recent study using a patient-derived xenograft where we established 103 stable and transplantable xenograft lines that could be serially passaged, cryopreserved and revived. These lines maintained the diversity of HCC and the essential features of the original specimens at the histological, transcriptome, proteomic, and genomic levels. Using this model, we explored the predictive markers for sorafenib response and found that mitogen-activated protein kinase kinase kinase 1 (MAP3K1) might play an important role in sorafenib resistance and sorafenib response is impaired in patients with MAP3K1 down-expression.228 The combination of ctDNA sampling, next generation sequencing and PDX modeling has been applied to the clinical management of melanoma and facilitated personalized treatment.229 This approach using combinations of investigative techniques may also be applicable in the management of HCC and allow us to improve our understanding of the problems and opportunities for targeted therapy, tumor clonal evolution and interactions and facilitate the implementation of precision, personalized treatment  and PDX. PDO and PDX closely mimic hepatocarcinogenesis and preserve the tumor microenvironment making them excellent preclinical models for drug screening, biomarker development and research into the alterations and mechanism responsible for drug resistance.vide supra) further complicates these decisions. We propose that the discovery and validation of novel biomarkers to reliably predict the response to treatment and define suitable candidates for a specific targeted agent, improving response rates and limiting avoidable toxicity in those who are unlikely to benefit, should be the focus of future HCC research.Several issues remain to be determined in the near future Fig. . The besIn the clinical setting, we should ensure that we adhere to established treatment protocols particularly in respect of sequential targeted therapies or when a change of therapeutic agent is indicated. It is not uncommon for patients to be switched from sorafenib to lenvatinib, from regorafenib to lenvatinib, or from sorafenib to lenvatinib and to regorafenib when drug resistance develops, and these changes may be instigated by an oncologist or the patient themselves. While many patients do clearly benefit from these treatment changes, there is no evidence base underpinning many of these changes and this practice is undesirable as it confounds data collection at best and potentially exposes the responsible clinician to criticism especially where serious adverse events occur.An iterative approach to targeted therapy has provided a wealth of data and encouraging results particularly in those patients where resistance to sorafenib develops. Although the understanding and management of HCC has changed dramatically due to the extensive basic and clinical research which has occurred over the last decade, HCC sadly remains a devastating disease which has a ubiquitous, enormous impact on health care systems across the world. The advances over this period however mean that patients can be comforted by the knowledge that there is a huge international effort underway from oncologists, hepatologists, and basic scientists to fully understand the mechanisms which are providing more rapid progress and ensure that the prognosis continues to improve.


HCMV antigens from the immediate early, early, and late phase of viral replication, and cellular MHC class I molecules were detected simultaneously or in serial sections by immuno-peroxidase and immuno-alkaline phosphatase techniques. Investigated organs included lung, gastrointestinal tract, and placenta. Colocalization of MHC molecules with sites of viral replication as well as MHC expression in individual infected cells were analyzed. To detect immune effector cells at sites of viral replication, leukocytes, CD8+ lymphocytes, and HCMV antigens were stained in serial sections. While strong MHC class I expression was detected in the cells surrounding infected cells, it appeared downregulated in the majority of infected cells themselves, particularly in the late replication phase. Despite significantly reduced MHC class I signals on infected cells, sites of infection were infiltrated by inflammatory cells that consisted predominantly of CD8+ lymphocytes. The extent of inflammatory infiltrates was negatively correlated with the extent of HCMV infected cells. Taken together, our findings indicate that HCMV can downmodulate MHC class I expression in vivo, whereas cytokines originating from infiltrating immune effector cells probably up regulates MHC class I expression in noninfected bystander cells. The presence of cytotoxic lymphocytes in close contact to infected cells may reflect control of viral spread by these cells despite MHC class I downmodulation.Based on cell culture data, MHC class I downregulation by HCMV on infected cells has been suggested as a means of immune evasion by this virus. In order to address this issue  Human cytomegalovirus (HCMV), also known as human betaherpesvirus 5, is distributed worldwide with seroprevalence rates between 45 and 100%  against various viral proteins were used. MAb El3 (Biosoft) is directed against the non-structural immediate early antigens (pUL123 and pUL122) which are detectable throughout the replication cycle  that was raised against free class I, HLA-B locus heavy chains  was used, which is directed against leukocyte common antigen (CD45). For identification of cytotoxic lymphocytes, antibody clone C8/144B (Dako) was used, which is directed against CD8.Tissue sections were deparaffinized and endogenous peroxidase was blocked by incubation with 0.6% hydrogen peroxide in methanol for 20 min at room temperature (RT). Tissue sections were then rehydrated with graded ethanol and digested with 0.1% protease  in phosphate-buffered saline (PBS) for 5 min at RT.For detection of HCMV early antigen, sections were pretreated with 0.1% protease type 24 (Sigma) in PBS for 5 min at RT. For detection of HCMV immediate early or late antigen, MHC class I molecules or leukocyte common antigen, sections were pretreated with antigen retrieval solution (Dako). For detection of CD8, sections were boiled with 1 mmol/l EDTA solution (pH = 8.0) in a pressure cooker. To prevent nonspecific binding of antibodies, rabbit non-immune serum was added for 20 min. Non-immune serum was then removed and tissue sections were subsequently incubated with primary antibodies, secondary antibodies, and staining reagents to visualize the antigen of interest.For brown staining, tissue sections were first incubated with the primary antibody of choice for 90 min at RT, followed by incubation with biotinylated rabbit anti-mouse Ig-antibody (Dako) for 60 min at RT and lastly incubated with streptavidin-biotin-peroxidase complexes (Dako) for 20 min at RT. Sections were rinsed in PBS for 5 min after each incubation step. Staining was performed using 0.6% DAB in PBS.For red staining, tissue sections were first incubated with the primary antibody of choice for 90 min at RT, followed by incubation with biotinylated rabbit anti-mouse Ig-antibody (Dako) for 60 min at RT and lastly incubated with streptavidin-biotin-alkaline phosphatase complexes (Dako) for 20 min at RT. Sections were rinsed in 0.05 mol/l Tris (pH = 7.6) for 5 min after each incubation step. Staining was performed using Fast Red TR/Naphthol AS-MX Tablets (Sigma).Finally, tissue sections were counterstained with hematoxylin, mounted in glycerol gelatin, and analyzed with a Polyvar light microscope (Cambridge Instruments).t-test function  of Excel.Quantitative analyses of signal intensities were done using the “measure” function of ImageJ software  this indicates a strong downmodulating effect of HCMV infection. While in most infected cells no red signal at all could be discriminated, a minority of infected cells showed weak to normal signals as compared to adjacent cells, indicating that MHC class I downmodulation occurred later than immediate early viral gene expression and/or was not always complete . These cTo differentiate the stages of HCMV replication in infected cells, we applied antibodies directed against immediate early and late viral proteins to serial sections of the tissues. The first section was immunoperoxidase-stained for an early viral nuclear protein, the second was stained by double labeling or immediate early antigen and MHC class I as described before, and the third section was immunoperoxidase-stained for a late viral nuclear protein. All infected cells will display immediate early antigen, those that have proceeded beyond the immediate early phase will display early antigen in addition and only those that entered the late productive phase of viral replication will display the late antigen. This correlative analysis of adjacent sections showed that MHC class I signals were detectable in most cells in the immediate early phase of replication but a few of these cells lacked MHC class I signals already at this replication phase . Cells iTaken together, this first set of experiments showed that clear signs of MHC class I downmodulation can be detected in various HCMV-infected tissues. This occurs principally in the late phase. A subset of infected cells in each stage of infection continued to show HLA expression, albeit at lower levels as demonstrated by a reduction in the intensity of staining.During the analysis of tissue sections that were double stained for infected cells and MHC class I molecules, it became obvious that the expression of MHC class I molecules was not homogenous throughout the tissue sections as there were areas with higher and lower signal intensity . A closeTo test whether this impression was true, we analyzed the placental specimen more in detail. Placenta was chosen, because it consists of numerous separated villi that could be easily distinguished in the tissue sections and counted as individual “events.” To exclude that the initial immune peroxidase staining of viral immediate early antigen interfered with the subsequent immuno-alkaline phosphatase staining of MHC class I molecules in a way that higher signal intensity was falsely amplified, we reversed the order of the staining steps.p = 0.004), corroborating the initial impression of elevated MHC class I expression at sites of infection , a pan-leukocyte marker expressed in cells of the myeloid and the lymphoid lineage , the secp = 0.045) with focal spread of HCMV being more probable in the absence of dense leukocyte infiltrations. An illustration of the extreme situations  with individual staining of CD45 and HCMV immediate early antigen in serial sections is provided in All villi that contained infected cells  were categorized for the degree of infection and the degree of inflammatory infiltration. Concerning infection, we distinguished single infected cells, several infected cells dispersed in the villus or clusters of three or more infected cells indicating focal spread. Leukocyte infiltrates were regarded weak if there were single leukocytes in the villus, medium if there were several leukocytes and strong if there were dense infiltrates present . The chiThis immunohistochemical analysis of selected cases of HCMV infection in different organs and conditions provides novel information on the balance between the virus and the host's immune response on the tissue level, which may also provide an idea of how HCMV manages to establish a lifelong persistence despite the remarkable frequency of CD8+ T cells that are directed against this virus. While reports on the viral immunoevasins US2, US3, US6, and US11 highlight the capability of HCMV to escape from cytotoxic T-cell responses, analyses of the host's T cell response emphasize the fact that HCMV can be well controlled by antigen-specific CTLs. Our findings can reconcile both notions as they show a remarkably strong MHC class I downmodulation in HCMV-infected cells but also infiltrates by CD8+ lymphocytes which obviously can limit viral spread in the tissue.The simple fact that HCMV-infected cells in the late replication stage can be detected in tissues despite the presence of cytotoxic T-cells strongly suggests that they escaped antiviral responses at least for a few days, as this is the time frame HCMV needs to proceed to the late stage. Together with the findings that such late stage infected cells were typically mostly negative for MHC-class I signals and that cytomegalic cells, suggestive of being in the late stage, were often in direct contact with CTLs, it is tempting to imagine a scenario, in which MHC class I downmodulation actually allows late stage infected cells to withstand detection and lysis by CTLs and to continue producing viral progeny. On the other hand, given the finding that uninfected cells in the adjacent tissue strongly express MHC class I molecules, it is conceivable that transfer of virus to these cells might result in at least some degree of antigen presentation and control by the CTLs that have already been attracted to such sites. While we cannot formally prove that the CTLs in the surrounding of infected cells are HCMV-specific this is highly probable considering similar findings in the murine cytomegalovirus model under well-controlled conditions, where the epitope-specificity of the infiltrating lymphocytes has been demonstrated , but conclusions on a causal relationship between these factors will remain speculative. Yet, as controlled treatment with virus and virus-specific lymphocyte in varying concentrations and kinetics is not possible as HCMV is restricted to the human host, retrospective analysis of tissues from infected individuals is the best option that is available. Second, our analysis only visualizes the presence of MHC class I molecules but cannot evaluate their functionality. Hence, we can only address the effects of US2 and US11, which target MHC class I molecules for degradation, but not US3 and US 6, which block the translocation of peptide-loaded MHC complexes or translocation of peptides into the ER, respectively. And third, antibody HC10 detects a subset of MHC class I molecules including all HLA-B and HLA-C variants and a number of HLA-A variants and these were found clearly downmodulated. We cannot formally exclude that certain alleles of HLA-A that are not detected by this antibody behave different from the molecules that were visualized. However, given the overlapping effect of US2 and US11 on the various HLA-A alleles, it is unlikely that an additional visualization of those alleles would change the overall pattern found with HC10. Lastly, the low number of tissues analyzed and the lack of information regarding the HLA types of the respective tissues excludes any conclusions on whether HCMV downmodulation is a regular finding and whether the degree of downmodulation depends on the HLA alleles. Certainly, many important questions are still open, .While the widely used MHC class I-specific antibody W6/32 is limited to use in frozen sections, antibody HC10 has the advantage that it can also be used in sections from formalin-fixed paraffin-embedded tissues (Stam et al., Our finding that depletion of MHC class I molecules in infected cells was most prominent beyond the immediate early stage of infection fits with initial reports on early expression kinetics of US2 and US11 and their mode of action, which results in degradation of MHC class I molecules in the early and late replication phase (Jones et al., Regarding the finding of elevated MHC class I expression at sites of HCMV infection, various explanations are conceivable. In principle, this could reflect that MHC class I high expressing cells are more susceptible to infection and hence infection is more pronounced at sites of higher MHC class I expression. However, this is highly unlikely, as no data are available in support of this explanation. It is more plausible to assume an immune reaction in response to infection with infiltrating lymphocytes (Böhm et al., The negative statistical correlation that we found in placental tissue between the density of infected cells and the density of infiltrating leukocytes suggests that the immune response had an antiviral effect in the respective tissue, as it is hard to imagine how less infection could cause larger infiltrations. Alternatively, a common cause could be responsible for both restriction of infection and attraction of leukocytes but this also appears less likely than a contribution of the infiltrates to restriction of viral spread. This explanation for the reciprocal correlation is supported by a previous finding in the murine CMV model that the presence of virus-specific CTLs restricted MCMV spread in liver tissue whereas a mutant lacking the respective epitope did not attract lymphocytes and formed large foci of infected cells (Böhm et al., Regarding the question of how specific CTLs can be induced despite the expression of viral immunoevasins that can block MHC class I-mediated antigen presentation, several explanations are possible. Cross presentation by professional antigen presenters like dendritic cells upon uptake of material from lysed infected cells may be central for priming of a CTL response (Arrode et al., All datasets generated for this study are included in the article/supplementary material.Ethics approval was not required for this study since tissue samples were provided by the Cooperative Human Tissue Network, a National Cancer Institute supported resource. Other investigators may have received samples from these same tissue specimens.FG, GJ, DS, and CS designed the project. DS provided reagents. FG and CS performed experiments and evaluated the data and drafted the manuscript, which was critically revised by GJ and DS. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Circadian rhythms are internal manifestations of the solar day that permit adaptations to predictable environmental temporal changes. These ~24-h rhythms are controlled by molecular clockworks within the brain that are reset daily to precisely 24 h by exposure to the light–dark cycle. Information from the master clock in the mammalian hypothalamus conveys temporal information to the entire body via humoral and neural communication. A bidirectional relationship exists between mood disorders and circadian rhythms. Mood disorders are often associated with disrupted circadian clock-controlled responses, such as sleep and cortisol secretion, whereas disruption of circadian rhythms via jet lag, night-shift work, or exposure to artificial light at night, can precipitate or exacerbate affective symptoms in susceptible individuals. Evidence suggests strong associations between circadian rhythms and mental health, but only recently have studies begun to discover the direct interactions between the circadian system and mood regulation. This review provides an overview of disrupted circadian rhythms and the relationship to behavioral health and psychiatry. The focus of this review is delineating the role of disruption of circadian rhythms on mood disorders using human night shift studies, as well as jet lag studies to identify links. We also review animal models of disrupted circadian rhythms on affective responses. Lastly, we propose low-cost behavioral and lifestyle changes to improve circadian rhythms and presumably behavioral health. Thus, optimal timing of physiological events is coordinated by these internal timekeepers. Endogenous circadian rhythms have a period of ~24 h and are reset on a daily basis to precisely 24 h through exposure to light–dark cues. Over the past century, however, the boundaries between day and night have been obscured by the widespread adoption of electric light at night. As a result, behavioral health and psychiatric consequences of circadian disruption by light at night are becoming increasingly apparent2. The focus of this review is delineating the role of disruption of circadian rhythms on mood disorders using human night shift studies, as well as jet lag studies to identify links. We also review animal models of disrupted circadian rhythms on affective responses. Finally, we propose low-cost behavioral and lifestyle changes to improve circadian rhythms and presumably behavioral health.Life on this planet is adapted to the 24-hour (h) solar day. Over evolutionary time, the predictable daily cycles of light and dark have been internalized in the form of circadian rhythms. Circadian  is a circadian rhythm sleep disorder associated with working outside of the typical 800 to 1700 h shifts18. Because circadian rhythms do not instantaneously reset, for several days they may remain more closely entrained to the original time zone than the current time zone; the lag in synchronizing these internal rhythms to the current photic (light) and non-photic  cues results in disturbed sleep, daytime fatigue, hormone profiles, gastrointestinal issues, and changes in mood. These symptoms are all manifestations of a misaligned circadian system. People who regularly cross multiple time zones, such as international flight crews, often report persistent jet lag symptoms; negative symptoms typically increase with the number of time zones crossed and are worse for travelers flying in an eastern direction than a western direction due to the necessity to advance the body’s clock20. Several studies have suggested that mood changes, especially dysphoric mood, are an important aspect of jet lag21. For example, a study of five men experiencing 7-h phase shifts (one westward shift and one eastward shift) over the course of a month reported that the eastward shift significantly disrupted sleep and elevated anxiety and depression scores20.In addition to light at night, circadian rhythms can be disrupted by another modern convenience, jet travel across time zones. Jet lag, also called jet lag disorder, is a transient sleep problem that arises when an individual travels across multiple time zones22. Indeed, social influences on sleep time commence as children begin day care or school— parents often enforce a set bedtime and awakening during the school week, but then allow their children stay up later and sleep later to resolve sleep debts on weekends and vacations22. A large scale epidemiological study confirmed that both sleep timing and duration are substantially challenged by work and school schedules or other social events. To align sleep and wake times with social obligations, 80% of the population uses alarm clocks on school or work days23. Early school and work schedules are particularly difficult for individuals with late chronotypes (i.e. “evening larks” with late sleep periods).Although scientific studies have focused primarily on the health consequences of “jet lag” caused by air travel across time zones, a much greater proportion of the population regularly experiences ‘social jet lag’, which is a related phenomenon in which individuals remain in their time zone but significantly shift their sleep-wake patterns several days per week22, reflecting reduced sleep during school or work days. The profound mismatch between the “enforced” sleep duration on school/work days and the variable sleep duration on days off demonstrates the strong effect of social times on daily sleep behavior. Although only ~20% of individuals in Europe and North America work night shifts, and a much smaller proportion of individuals experience chronic jet lag, virtually everyone experiences circadian disruption provoked by social jet lag, and the associated change in the timing of light exposure.Social influences on the timing of human sleep becomes apparent when comparing sleep–wake behavior on work and free days. The vast majority of people display clear differences in both sleep duration and timing; sleep duration shortens significantly (by ~2 h/night) from the age of 10–1726. Notably, neural systems associated with affect, such as the limbic brain regions, monoamine neurotransmitters, and the hypothalamic–pituitary–adrenal axis (HPA), are all under circadian regulation31. Thus, it is reasonable to expect that widespread disruption of circadian rhythms would contribute to the prevalence of mood disorders33.In sum, lifestyle changes that have occurred in response to technological advances over the past century have resulted in unprecedented changes in the timing and duration of light exposure, which in turn has the potential to desynchronize circadian rhythms; increased prevalence of disrupted circadian rhythms strongly correlates with the increased incidence of mood disorders34. The ipRGCs constitute a small fraction of the larger class of retinal ganglion cells, but their expression of melanopsin, a photopigment, makes them exquisitely photosensitive35. Blue light (~480 nm) most strongly stimulates the melanopsin photopigment ipRGCs, whereas red light (>600 nm) has minimal effect35. The characteristics of sunlight change across the day and in response to the atmosphere; typically, sunlight contains more short (blue) wavelengths during the day and then shifts to longer (red) wavelengths toward sunset. The sensitivity spectrum of melanopsin may be an adaptation to the natural solar cycle, so that ipRGCs are tuned to discriminate daylight from evening, allowing better entrainment of circadian rhythms9. When exposed to light in the activating spectrum, ipRGCs propagate neural impulses via the retinohypothalmic tract directly to the circadian master clock, the mammalian suprachiasmatic nucleus (SCN) of the hypothalamus. This monosynaptic pathway conveys photic information via the release of excitatory amino acids, namely, glutamate. Activation of the SCN via light results in Ca2+ influx and activation of intracellular signaling cascade; Ultimately, leading to increased expression of Period genes to set the molecular clock36. In addition, ipRGCs have other direct and indirect targets throughout the brain, including mood-related structures 37.In addition to the image forming photoreceptors (rods and cones) in the retina, light is also detected by specialized photoreceptor cells in mammals. This third class of photoreceptors, called intrinsically photosensitive retinal ganglion cells (ipRGCs), are engaged in several non-image-forming functions, including circadian phototransduction39. In the primary feedback loop, circadian locomotor output cycle kaput (CLOCK) and brain and muscle ARNT-like protein 1 (BMAL1) proteins form heterodimers in the cytosol of cells of the SCN. The CLOCK-BMAL1 complex translocates to the nucleus and binds to regulatory elements of the DNA containing E-boxes, which in turn activate expression of period  and cryptochrome (CRY1 and CRY 2) genes. PER and CRY proteins then heterodimerize and translocate into the nucleus, where they repress their own transcription by acting on the CLOCK-BMAL1 complexes. In mice, activation of CLOCK-BMAL1 occurs in early morning leading to the transcription of PER and CRY protein in the early afternoon and subsequent repression of CLOCK-BMAL1 transcription in the evening/night39. In an interacting feedback loop, CLOCK-BMAL1 complexes activate expression of nuclear receptors, REV-ERB α and RORα. Their protein products feedback to regulate BMAL1 by competitively binding retinoic acid-related orphan receptor response elements in the BMAL1 promoter40. REV-ERBs repress the transcription of BMAL1, whereas RORs activate it. These two loops form the basis of the molecular clock, but a complex network of interacting genes and post-translational modifications ensure that the process takes ~24 h to complete39. This transcriptional–translational feedback loop is the basis of the intrinsic daily circadian rhythm.The SCN molecular clock comprises a set of transcriptional–translational feedback loops that drive rhythmic 24-h expression of the core canonical clock components41. Therefore, light serves as the major entraining (synchronizing) cue to precisely maintain synchrony with the environment. Light exposure during the night phase shifts the clock transcription-translations cycle by rapidly inducing expression of Per1 or Per2, depending on whether the light occurs during the early or late night44. Phase shifting can be useful for adapting to changing seasonal day lengths, or more recently travel to a new time zone, but abrupt changes or inappropriate timing of light exposure can be problematic for daily life. For example, the use of electronics during the night can unintentionally phase shift the circadian rhythm, leaving it decoupled from the natural environmental light–dark cycle45, with potentially dysregulated physiology and behavior.In the absence of environmental signals, the molecular clock will continue to produce ~24-h or circadian rhythms. People living in caves or bunkers without light or temperature cues will maintain circadian sleep–wake and body temperature rhythms, although they begin to free-run with a rhythm slightly longer than 24 h48. Aberrant light exposure that disrupts these rhythms may drive downstream dysregulation of circadian rhythms in peripheral systems.The SCN functions as the central circadian oscillator, although similar intracellular clock mechanisms are expressed in other brain regions, as well as in peripheral tissues. Clocks throughout the body remain synchronized with one another by responding to signals from the SCN, either through direct neural inputs or indirect signals such as humoral, behavioral, or other physiological rhythms. For example, timing of feeding and body temperature can help synchronize peripheral oscillators49. Cells in the periphery exposed to melatonin respond in ‘night-mode’, whereas cells not exposed to melatonin respond in ‘day-mode’. The secretion of melatonin is tightly controlled by light; <3 lux of light exposure at night is effective in suppressing the onset of melatonin secretion and shortens melatonin secretion duration in humans51. Thus, via suppression of melatonin rhythm, light at night has the potential to substantially alter physiology and behavior.The two most prominent endocrine manifestations of circadian rhythms are the daily cycles of melatonin and glucocorticoids . Time of day information is transmitted to the pineal gland from the SCN to regulate production and secretion of the indole amine, melatonin. Melatonin is derived from serotonin via two enzymatic steps, and then secreted from the pineal gland at night in both diurnal and nocturnal animals. Circulating melatonin is also a cue to help entrain clocks in peripheral organs via several interactions with the molecular clock mechanism, including phase-resetting clock genes53. The reverse pattern occurs in nocturnal mammals. The functional significance of increased glucocorticoid concentrations during the active period and decreased concentrations during sleep is related to the hormone’s important role in glucose homeostasis. Glucocorticoids also subserve several physiological and behavioral responses to stress. Given the wide range of glucocorticoid effects on biological processes that are crucial for survival, it should not be surprising that glucocorticoid concentrations are tightly regulated by negative feedback at each level of the HPA axis. Indeed, dysregulated glucocorticoid secretion is associated with several debilitating health conditions, including major depressive disorder54. Because light exposure is an important zeitgeber for the circadian rhythm of cortisol in humans, exposure to light at night could dysregulate the HPA axis, in turn increasing the prevalence of cortisol-associated mood disorders55.Additionally, the circadian system regulates glucocorticoid secretion from the adrenal glands, such that concentrations tend to peak in the morning just before awakening and decrease throughout the day in diurnal animals, including humans56. Studies of light at night exposure in diurnal rodent species generally yield similar affective responses to nocturnal rodents58. Another important difference between humans and individuals of some rodent species is the production of pineal melatonin. Although both nocturnal and diurnal species produce melatonin during the dark phase, several common laboratory strains of mice have no detectable pineal melatonin rhythms. Nonetheless, studies using Swiss Webster mice  vs. Siberian hamsters  have reported similar effects of light at night on affective responses61. This observation suggests that suppressed melatonin by nighttime light is not the only or even primary mechanism, but still may be an important contributor in humans and a potential point of intervention.Most of the evidence supporting the effects of atypical light exposure on affective responses has been gleaned from animal models, particularly rodents, because of the ease with which light exposure can be controlled. One advantage of using rodents is that the most common lab species are nocturnal, and so exposure to light at night occurs during their active and awake phase. Thus, light at night does not directly alter sleep in nocturnal species. This is important because most effects of disrupted circadian rhythms on human mood are attributed to sleep disruption, but studies using nocturnal species demonstrate that this is not the only cause as sleep remains intact when animals are exposed to dim light at night63. Mice exposed to alternating cycles of 3.5 h light and 3.5 h dark (termed T7), which do not significantly affect cycling clock gene expression or sleep, develop depressive-like symptoms63. In contrast, mice lacking the melanopsin gene are protected against such depressive responses, suggesting the behavioral changes occur because ipRGCs project either directly or via the SCN to brain regions involved in mood regulation. In addition, there are several indirect pathways affected by light at night that may contribute to mood disturbances37.Accumulating evidence demonstrates both direct and indirect connections between artificial light at night and mood regulation. First, exposure to light at night can directly affect mood through ipRGC projections to brain regions involved in affect64. Diagnosis of MDD requires these symptoms to persist for at least two weeks and interfere with normal daily functions64. MDD affects vast numbers of people worldwide; the Global Burden of Disease Consortium examined the global incidence and prevalence for 328 diseases in 195 countries from 1990–2016, MDD ranked within the top ten leading causes of disease burden in all but four countries examined65. The incidence of MDD worldwide is on the rise; the number of people diagnosed with depression increased by ~18% from 2005 to 201566. Notably, rates of MDD correlate with modernization of society67; which may reflect the increased circadian disruption  or the interaction between circadian disruption and other environmental factors experienced in modernized countries.Major depressive disorder (MDD) is characterized by alterations in mood, typically increased sadness and/or irritability that is accompanied by at least one of the following psychophysiological symptoms: alterations in sleep, sexual desire, or appetite, inability to experience pleasure, slowing of speech or actions, crying, and suicidal thoughts70. For example, in a population of ~4000 South Koreans, the prevalence of MDD among shift/night workers was significantly increased relative to daytime workers68. Among a Brazilian cohort of ~36,000 workers, night shift work was significantly associated with MDD only in females70. No association was reported between shift work and MDD in a French study69. If all types of depression are combined, then a clear association emerges between shift work and depression73. A meta-analysis of 11 studies concluded that night shift workers are 40% more likely to develop depression relative to daytime workers13.There are few human studies that have examined the link between circadian disruption and MDD specifically, as most human data combine all types of depression. Nonetheless, studies examining the association between shift work and MDD have produced varied results74. One such study, examined the amount of social jet lag  in patients with MDD and healthy individuals. The authors concluded that there was no association between the amount of social jet lag and severity of depressive symptoms. Additionally, patients with MDD demonstrated no differences in social jet lag relative to healthy people74. However, in common with shift work, generalizing depression  results in a link between jet lag/social jet lag and depression79.Human studies examining the effects of jet lag or social jet lag on MDD are sparse80. Patients with MDD typically express more severe symptoms in the morning, which is considered to be associated with a more severe form of depression80. Disruptions of biological rhythms underlie hallmarks of MDD; specifically, alterations in sleep/wake states , social rhythms, hormone rhythms (reduced amplitude in melatonin and cortisol rhythms), and body temperature rhythms  are seen in patients with MDD81. Clinical studies demonstrate that the severity of MDD is correlated with the degree of misalignment of circadian rhythms82. Further, examination of circadian patterns of gene expression within postmortem brains of patients with MDD demonstrate reduced amplitude, shifted peaks, and altered phase relationship between genes, particularly in canonical clock genes27.Human clinical data support a strong interaction between MDD and circadian processes. Symptoms of depression demonstrate diurnal variations; patients exhibit symptoms in a morning-worse or evening-worse pattern83. Morning bright light therapy phase advances the rhythm in melatonin and the degree of phase advancement is correlated with the amelioration of depressive symptoms84. Additionally, administration of antidepressants  result in a phase advancement of circadian rhythms 86. Wake therapy directly alters sleep-wake rhythms by increasing the amount of SWS, reducing the latency to REM sleep, and reducing the duration of REM sleep87. Thus, successful treatment of MDD with chronotherapies, as well as diurnal variation in symptoms provide evidence that circadian disruption may underlie this disease.Most successful treatments of depression, bright light therapy, wake therapy, social rhythm therapy, and antidepressants  directly affect circadian rhythms88. Rats that received bilateral SCN lesions demonstrated reduced immobility. Thus, the authors concluded that SCN lesions have a protective effect in the induction of behavioral despair88. Similar conclusions were reached almost a decade earlier when Arushanyan and colleagues demonstrated reduced immobility during the forced swim test, in rats that received bilateral SCN lesions89. It may be difficult to draw definitive conclusions from these studies as testing of depressive-like behavior occurred during the inactive phase of the rats. SCN lesions likely disrupted the diurnal rhythm in activity; subsequent studies examining the role of the SCN in regulating depressive-like behavior have reached the opposite conclusion utilizing animals with an intact SCN84. Genetic disruption of circadian rhythms in the SCN via SCN-specific Bmal1-knockdown, or forced desynchrony of the SCN via exposure to a T22 light–dark cycle increased depressive-like behavior in mice and rats, respectively91.There is ample basic science evidence to support an association between disrupted circadian rhythms and depressive-like behavior. To assess the role of the SCN in regulating depressive-like behavior, Tataroglu and colleagues bilaterally lesioned the SCN and examined the effect on depressive-like behavior via forced swim tests92. Administration of agomelatine to rats exposed to 3 and 6 weeks of constant light prevented the increase in depressive-like behavior and restored diurnal corticosterone and melatonin rhythms respectively94. As with constant light, exposure to dim light at night induces depressive-like behavior in multiple species of rodents95. In female Siberian hamsters, exposure to 4 weeks of dim light at night  increases depressive-like responses and neuroinflammation with concurrent decreases in dendritic spine density within the hippocampus59. Treatment with a dominant-negative TNF inhibitor prevented the increase in depressive-like behavior59. Additionally, diurnal rats exposed to dim light at night (5 lux) for 3 weeks demonstrated increased depressive-like behavior and reduced dendritic length within CA1 and dentate gyrus58. Acute (3 nights) exposure of mice to dLAN (5 lux) is sufficient to induce alterations in clock genes and increase depressive-like behavior60. However, studies have also reported no association between LAN and mood in C57Bl/6 mice suggesting a potential strain specific effect of LAN97. Taken together, there is substantial clinical and basic science evidence to support a link between circadian rhythm disruption and major depressive disorder.Similar effects of increased depressive-like responses have been demonstrated in other rodent models of circadian disruption. Rats exposed to 8 weeks of constant light exhibit increased depressive-like behavior concurrent with loss of diurnal rhythms in activity, melatonin, and corticosterone98. Similarly, nurses with Shift Work Disorder display elevated anxiety scores on the Hospital Anxiety and Depression Scale99. However, rapid transitions to night shift work did not affect anxiety levels in a questionnaire study of nurses100. Jet lag, accomplished by undergoing a 7-h westward time shift by jet in five men preceding the study and, 1 month later, a 7-h eastward shift preceding was associated with disrupted sleep and elevated anxiety and depression scores, especially in simulated eastward travel20.Although several studies have suggested that night shift work and persistent jet lag provoke anxiety, more recent analyses suggest that the mood changes may reflect disturbed sleep, rather than disturbed circadian rhythms per se. For example, a longitudinal study of day shift workers without prior sleep disturbances who transitioned to rotating shift work schedules reported elevated anxiety along with disordered sleepClock gene display reduced anxiety-like behavior and are less fearful of aversive stimuli than wild-type mice101. Notably, clock regulates cholecystokinin (CCK) expression in the ventral tegmental area (VTA) and Δ19 mutation in the Clock gene is sufficient to induce manic-like behaviors102. In contrast, mice lacking both Per1 and Per2 display elevated anxiety-like behavior, whereas mice lack either Per1 or Per2 do not have altered anxiety-like responses103. Inhibition of Per1/Per2 expression in the nucleus accumbens (NAc) of wild-type mice also produces anxiety-like behavior, suggesting a causal role for these core clock components in the NAc for regulating anxiety.Studies of rodents revealed relationships among the circadian system and anxiety-like disorders. For instance, targeted disruption of canonical molecular clock components alerts anxiety-like behavior. Mice with a Δ19 mutation in the 92. However, the effects of light as a circadian disruptor are inconsistent across species108, and may depend on the developmental window during which circadian disruption occurs, as well as the type of light  and its intensity109. For example, exposure to dim light at night during early development in mice increases adult anxiety-like responses109, whereas exposure of adult mice to light at night reduces anxiety-like responses106. Furthermore, glucocorticoid concentrations are often reduced in hamsters and unaltered in mice exposed to light at night compared to dark nights, suggesting that the affective behavioral responses to atypical lighting are not the result of elevated corticosterone111. Mice housed in 20-h light–dark cycles, a paradigm that disrupts circadian rhythms, display reduced dendritic length and complexity in neurons of the prelimbic prefrontal cortex, associated with anxiety112. Obviously, the extent to which light exposure alters sleep differs among species of diurnal and nocturnal rodents113. Together these data provide modest evidence in support of an association between circadian rhythm disruption and anxiety.Other studies have investigated how environmental disruption of circadian rhythms  contributes to the development of anxiety-like behavior. For example, housing adult rats chronically in constant light induces anxiety-like behavioral responses114, however, no causal “smoking gun” gene has yet been identified. Genetic linkage studies have been equivocal115, yet modest associations have been reported between BD and multiple genes of the molecular circadian clock117. Additionally, treatment regimens for BD that normalize circadian rhythms have proven effective as treatments (see below), further implicating dysregulation in the circadian system in the pathology of this disease. Dysregulation of, or certain polymorphisms in, the afore associated genes involved in the circadian molecular clock may increase susceptibility to develop BD and also influence circadian phenotypes which could lead to relapse into episodes117. However, in a recent meta-analysis of 42 clinical studies on circadian rhythms and BD, the authors determined that, although circadian rhythm disruption is prevalent in BD, the cross-sectional research design of most studies precluded establishing a cause/effect relationship between circadian disruption and BD118. Thus, it remains undetermined whether circadian disruption is a primary pathophysiology of BD, or if it is secondary to other environmental and genetic factors. Nonetheless, a recent study has proposed the possibility of using circadian rhythms in buccal cell circadian clock gene expression and cortisol rhythms as biomarkers in BD patients for depression (phase delayed rhythms) and mania (phase-advanced rhythms)119.Bipolar disorder (BD) is identified by cyclic extreme mood swings between mania and depression separated by periods of normal affect. This brain disorder is divided into four categories (in decreasing order of severity of the symptoms); Bipolar I, Bipolar II, Cyclothymic, and Other. These extreme mood episodes differ greatly from the typical behavior of the person, and are concurrent with significant changes in sleep, activity, and energy levels. BD is a genetic disorder, with 85–89% heritability121. Disruption of circadian social rhythms, such as social jet lag, may also induce bipolar episodes. A major social disruptive event is associated with inducing mania, but not depression in BD patients122.Circadian disruption in the form of jet lag has been reported to induce bipolar episodes in susceptible people who fly across multiple time zones; east to west travelers with BD who then experience a phase delay in circadian rhythms at their destination are more likely to develop depression, whereas those traveling west to east who then experience a phase advance in their circadian rhythms are more likely to develop mania123. The approach of normalizing and stabilizing circadian rhythms, through lithium or other methods, has proven an effective therapy for ameliorating episodes of depression and mania, and for preventing relapse into these states125. Randomized placebo-controlled clinical trials have demonstrated that normalizing disrupted circadian rhythms with midday bright light therapy can resolve episodes of bipolar depression, whereas morning bright light therapy to treat depression in BP patients can induce mixed states127. Other clinical trials have reported that episodes of bipolar mania can also be treated effectively by normalizing disrupted circadian rhythms with enforced darkness; either through blue light blocking glasses128 or controlled environmental darkness129.Historically, treatment for BD symptoms has been discovered by serendipity (lithium) or informed by treatments developed for other disorders with shared state, such as depression (described above). Early studies reported that BD patients have a ‘fast running’ circadian clock, likely leading to chronic circadian disruption; treatment with lithium, which slows the molecular circadian clock, ameliorates the symptoms and stabilizes circadian rhythmicityIn sum, disrupted circadian rhythms appear to be both a state marker and a trait of BD. These disruptions of circadian rhythms can arise either via internal desynchrony or environmental desynchrony and can both predispose one to BD, as well as induce bipolar episodes, dependent on the phase relationship between the internal and external circadian rhythms. Resynchronization and normalization of circadian rhythms (chronotherapy) has proven effective in prevention and treatment of bipolar episodes.133. Most animal models of BD are endophenotype-based and probe the various states of BD from depression (see above) to mania-like behaviors  expression differentially express increased depressive-like or mania-like behaviors dependent upon day length134. However, the validation of DAT deficient mice as a model of state switching in BD remains controversial135. Regardless, altered dopaminergic signaling has been implicated in bipolar mania. One of the most well-established models of bipolar mania, the ClockΔ19 mutant mouse, has a fundamentally disrupted molecular circadian clock and restoration of a functional molecular circadian clock to the VTA, likely restoring dopaminergic regulation, ameliorates the mania-like behaviors101. Given that ClockΔ19 mutant mice have a genetically disrupted circadian clock and already recapitulate many mania-like behaviors based on their inherent internal circadian desynchrony, this model does not lend itself well to studying the effects of environmental circadian disruption on bipolar mania.There is a paucity of animal studies of BD mainly because there are no animal models that satisfy the validity criteria for modeling this complex multifactorial disorder136. Studies on circadian disruption in ICR mice, by inverting the LD cycle for 3 days, have reported that approximately half of the mice fail to recover normal sleep-wake cycles within 6 days after re-exposure to normal lighting conditions. The mice that took longer to re-entrain normal sleep-wake rhythms after circadian disruption also displayed greater quinpirole-induced hyperactivity, indicative of a more mania-like state, however depressive-like behavior (forced swim test) was not affected138. Based on these studies and human clinical data, the pathophysiology of environmental circadian disruption underlying bipolar mania may be via altered protein kinase c activity affecting neuronal signaling in frontal and limbic brain regions139.Circadian disruption by sleep deprivation can induce a manic-like phenotype in mice (increased aggression and hyperlocomotion), yet these effects are transient and behaviors return to baseline within 24 h141. The high heritability of SZ (~80%) suggests a substantial genetic risk, however, the lower concordance between monozygotic twins (~50%) demonstrates that other factors, such as environment, epigenetic modifications, or nutrition, impact illness risk142. SZ is characterized by positive symptoms , negative symptoms , and cognitive symptoms . Furthermore, the severity of SZ symptoms has been associated with the extent of sleep or circadian rhythm disruption145. Indeed, circadian disruption is a common prodrome of SZ145, although the nature of the relationship remains unclear, in part because circadian disruption has been studied less extensively in SZ than other mood disorders. There are case studies reporting the emergence and relapse of SZ/schizoaffective psychosis among individuals who have traveled across time zones, and therefore may be experiencing jet lag147, but no large scale, controlled studies have examined whether jet lag, social jet lag, simulated jet lag, or other forms of circadian disruption precipitate or exacerbate SZ symptoms.Schizophrenia (SZ) is a fairly rare , but severely disabling, mental disorder that typically emerges in the second or third decade of life148 demonstrated that subjects with SZ lost rhythmicity in most genes within the prefrontal cortex that were rhythmic in healthy controls. However, subjects with SZ gained rhythmicity in completely different set of genes not seen in healthy controls . Additionally, genes that have previously been implicated in SZ (i.e. BDNF and GABAergic-related transcripts) were differentially expressed in SZ subjects that died during the night148. Further, several studies have documented alterations among SZ patients in two key endocrine transducers of the circadian clock: melatonin and cortisol. As described above, melatonin typically begins to rise with the onset of darkness and peaks several hours later, whereas cortisol concentrations are typically low over night and begin to rise prior to waking. Studies of individuals with SZ who were not currently taking medication have revealed alterations in several aspects of the melatonin rhythm, including reduced nighttime melatonin concentrations151, blunted rhythmic amplitude150, and phase advance of the typical melatonin acrophase152. Among medicated SZ patients, a subset of individuals exhibit a phase delay153 or phase advance154 of an otherwise typical melatonin rhythm, whereas others experience a blunting of the rhythm with or without a phase shift143. Regardless of the direction, a “phase-shift” in the melatonin rhythm can result in uncoupling from the environmental day/night cycle, in turn desynchronizing physiological processes and the behaviors they subserve. Indeed, the breakdown of the typical relationship between the melatonin rhythm and the sleep-wake cycle, has led some researchers to propose that the sleep promoting properties of endogenous melatonin are compromised in SZ155. However, others have proposed that the social withdrawal that often occurs in SZ may contribute to circadian disruption by limiting exposure to zeitgebers that are important for keeping the clock entrained to the 24 h day143.In contrast, studies have demonstrated altered diurnal rhythms in subjects with SZ. Using postmortem brain tissues, Seney and colleagues152, although concentrations are often elevated relative to healthy controls157. Interestingly, hyper-reactivity of the HPA axis is observed in both individuals with SZ and individuals at high genetic risk for SZ, which may support the model of stress vulnerability in SZ158. Furthermore, altered regulation of the HPA axis in SZ is predictive of more severe psychotic and motor symptoms160. Fortunately SZ-associated HPA axis hyperactivity is responsive to treatment; 4 weeks of treatment with olanzapine produces cortisol concentrations that are comparable to healthy controls161.In contrast to melatonin, the cortisol circadian rhythm appears to remain more or less intact in SZRetinoic acid-related orphan receptor (RORβ), Per2, Per3, and Neuronal Pas domain protein 2), however, no associations remained significant after correcting for multiple comparisons, suggesting these individual polymorphisms in clock genes are unlikely to confer sizeable (>1.5 OR) risk for SZ162. An earlier and smaller study also identified modest associations for PERIOD3 and TIMELESS in patients with SZ163. Data collected from cultured skin and blood cells also support the conclusion of disrupted circadian rhythms in SZ. Specifically, fibroblasts enriched from skin biopsies of SZ patients exhibit loss of rhythmic expression of CRY1 and PER2 compared to cells from healthy controls, while rhythms in BMAL1, REV-ERBα persist in a manner comparable to the cells from healthy individuals164. Similarly, mononuclear blood cells from SZ patients experiencing their first episode of psychosis, have decreased expression of CLOCK, PER2, and CRY1 relative to non-SZ individuals164.As mentioned, there is a strong genetic component to SZ, and given the extensive disruption of circadian rhythms in this mood disorder, it is not surprising that several core clock genes have been implicated. In a study that examined 276 single nucleotide polymorphisms (SNPs) at 21 circadian genes, significant associations were detected for 8 SNPs from 4 circadian-related genes (early gene growth response 3 (Egr3)168. Additional support for this candidate gene is provided by Egr3-deficient mice (−/−), which display behaviors that are reminiscent of SZ and sleep less than WT controls, even though the circadian organization of the sleep-wake cycle remained intact169.Another gene that has been implicated in both SZ and circadian organization is immediate (Vipr2) is associated with increased risk for SZ171. Mice that are deficient in Vipr2 exhibit cognitive deficits associate with hippocampal dependent associative memory- which is reminiscent of SZ172. Under a 12:12 LD cycle, the VPAC2-KO mice adrenal clock gene rhythm and corticosterone rhythm was phase-advanced compared to WT mice and when the lighting was changed to constant darkness, the adrenal clock genes and corticosterone secretion became arrhythmic173. These data support the role of VIP in synchronization of internal rhythms. Importantly, the low concordance of SZ in monozygotic twins suggest that alterations in the core clock genes are not likely to be the cause of SZ, but they may affect disease progression, symptom severity, or efficacy of treatment.In humans, duplication of the vasoactive intestinal polypeptide receptor 174. With the prevalence of hand-held and wrist-worn mobile devices today, it may also be possible to leverage data from these devices to predict and monitor psychiatric state of users. Indeed, passively collected data from mobile phones can be used to monitor and predict real time behavioral indicators of depression and PTSD175. Furthermore, passively collected keystroke metadata may also be used to predict state changes found in BD176. In future studies, it might be informative to consider leveraging data from the devices’ sensors to monitor ambient light, at eye level, in association with the other data collected to investigate whether exposure to light at night is a predictive cofactor in affective disorder state.Although light at night from modern electronic devices may be responsible for the circadian disruption affecting the psychiatric diseases discussed above, these devices might also provide the data to help resolve some disease states and improve outcomes. A recent study analyzing digital logon data from nearly 15,000 students reported that circadian disruption  negatively affects learning outcomes, and furthermore that these data could be used to time educational activities to minimize impairments in performance caused by circadian disruption at the individual and population levelsAdditionally, future studies should focus on expanding both the clinical and basic science literature in relation to circadian disruption and behavioral health. There are numerous studies in humans examining the effects of shift work on behavioral and psychiatric health, however, the number of studies examining the effects of jet lag on mental health are modest. In 2017, commercial airlines carried more than 4 billion passengers (>50% of the world’s population). Hence, modern air travel has greatly increased the likelihood of experiencing disruption in circadian rhythms. Clinical and basic science research should reflect the increased prospect of experiencing circadian disruption via jet lag. Studies examining the effects of social jet lag on mental health are even more scarce. As noted, ~80% of the population suffers from social jet lag. Thus, it is essential to understand the consequences of social jet lag on behavioral health and appropriately manage them. Ultimately, the expansion of research in this field will broaden our knowledge and may result in novel treatments to improve patients’ quality of life and outcomes.177. However, rodent studies have demonstrated that even among healthy animals, experimentally induced disruptions of circadian rhythms can lead to affective changes. Targeted resynchronization of circadian rhythms improves symptoms of mood disorders. In sum, while circadian disruption may not be the sole cause of mood disorders, it may elicit or exacerbate symptoms in individuals with a predisposition for mental health disorders.Altered circadian rhythms are commonly reported among individuals with several psychiatric disorders, including major depressive disorder (MDD), bipolar disorder (BD), anxiety, and schizophrenia (SZ). However, the nature of the relationship between circadian rhythm disruption and psychopathology is poorly understood. The vast majority of clinical data are correlational. Thus, it remains unknown whether the relationship reflects: (1) causation in which circadian disruptions predisposes individuals to developing mood disorders, (2) causation in which the manifestation of mood disorder leads to circadian disruption, or (3) an absence of causation in which the association between circadian disruption and mood disorders reflects commonalities in underlying physiological processes