The aim of this study was to improve the intratumoral accumulation of an antibody–drug conjugate (ADC) and minimize its off-target toxicity, SKB264, a novel anti-trophoblast antigen 2 (TROP2) ADC that was developed using 2-methylsulfonyl pyrimidine as the linker to conjugate its payload (KL610023), a belotecan-derivative topoisomerase I inhibitor. The preclinical pharmacologic profiles of SKB264 were assessed in this study.in vitro and in vivo pharmacologic profiles of SKB264, including efficacy, pharmacokinetics–pharmacodynamics (PK-PD), safety, and tissue distribution, were investigated using TROP2-positive cell lines, cell-derived xenograft (CDX), patient-derived xenograft (PDX) models, and cynomolgus monkeys. Moreover, some profiles were compared with IMMU-132.The In vitro, SKB264 and SKB264 monoclonal antibody (mAb) had similar internalization abilities and binding affinities to TROP2. After cellular internalization, KL610023 was released and inhibited tumor cell survival. In vivo, SKB264 significantly inhibited tumor growth in a dose-dependent manner in both CDX and PDX models. After SKB264 administration, the serum or plasma concentration/exposure of SKB264 , total antibody , and KL610023 in cynomolgus monkeys increased proportionally with increasing dosage from 1 to 10 mg/kg. The linker stability of SKB264 was significantly enhanced as shown by prolonged payload half-life in vivo . At the same dose, SKB264’s exposure in tumor tissue was 4.6-fold higher than that of IMMU-132.Compared with IMMU-132, the longer half-life of SKB264 had a stronger targeting effect and better antitumor activity, suggesting the better therapeutic potential of SKB264 for treating TROP2-positive tumors. TROP2, a trophoblast cell-surface antigen, also known as tumor-associated calcium signal transductor (TACSTD2) is overexpressed in a variety of human epithelial cancers, including breast, lung, gastric, colorectal, pancreatic, prostatic, cervical, head-and-neck, and ovarian carcinomas , 2. Elevvia the coupling sites on the reduced inter-chain disulfide bonds. TL033, a novel drug-linker intermediate, contains the toxic payload KL610023 , which is a topoisomerase I inhibitor belotecan derivative with a bystander effect, and can arrest cell cycle at the G2/S stage after its internalization, leading to cell death  as IMMU-132. The mAb is conjugated with seven to eight molecules of novel payload ll death . Moreove, KL610023, and the linker of SKB264 are developed by Kelun-Biotech . IMMU-132, a reference ADC used in this study, was purchased from Immunomedics, Inc. .SKB264 mAb is a humanized anti-TROP2 mAb with reference to the same amino acid sequence as IMMU-132. SKB264 is a novel anti-TROP2 ADC, in which SKB264 mAb is conjugated with seven to eight molecules of novel payload named KL610023 2. All the cell lines were examined by short tandem repeat authentication via Genegle  and tested for mycoplasma using a mycoplasma detection kit .NCI-N87 (human gastric carcinoma), BxPC-3 (human pancreas adenocarcinoma), and Calu-3  were purchased from ATCC (American Type Culture Collection). Both HCC1806 (human breast adenocarcinoma) and NCI-H23  were purchased from CoBioer . The TROP2-overexpressing NCI-H23 (TROP2+) line, which is a subtype of NCI-H23 and constructed by Kelun-Biotech, and other cell lines, including NCI-N87, BxPC-3, HCC1806, and CI-H23 , were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Calu-3 cells were cultured in Minimum Essential Medium (MEM) supplemented with 10% FBS and non-essential amino acids. All cell lines were maintained at 37°C in 5% COFlow cytometry (FCM) was used to detect TROP2 expression in tumor cell lines. First, cells were incubated with the following antibodies on ice for 30 min: 1 μg/ml phycoerythrin (PE)-conjugated mouse IgG2a, k isotype control, or PE-conjugated anti-human TROP2 antibody . After rinsing, the labeled cells were analyzed by FCM (BD Accuri C6). The following gating strategy was used for FCM. In the FSC/SSC plot, cells were selected using a gate for high FSC cells except for cell debris. Then, single cells were selected using a gate for a single slope in the FSS-A/FSC-H plot. In the PE-A plot, nonspecific binding was excluded by isotype using a gate with little PE-A cells.An enzyme-linked immunosorbent assay (ELISA) was used to evaluate the binding and dynamics affinity. Human TROP2 protein (1 μg/ml)  was coated on a 96-well plate overnight at 4°C. The plate was then blocked by Super block reagent (ScyTek Inc. Logan UT) at room temperature for 1 h. Diluted SKB264, SKB264 mAb, or isotype control (human IgG1 antibody) was added for incubation at 37°C for 1 h. After washing, the diluted  goat anti-human IgG Fc-horseradish peroxidase (HRP)  was added for further incubation for 45 min at 37°C. Finally, TMB solution was added for absorbance measurement at 450 nm using a SpectraMax Plus384 Microplate reader .Ka) and dissociation (Kd) rate constant values were calculated by fitting analysis assuming a Langmuir binding model (KD (equilibrium dissociation constant) values.Surface plasmon resonance (SPR) was an alternative method to evaluate dynamic affinity, in which human TROP2 protein was captured with goat anti-human IgG Ab immobilized onto a CM5 sensor chip . Serial titrations of SKB264 or IMMU-132 ranging from 0.0823 to 2.22 nM and running buffer were individually injected over the surface, which was captured human TROP2 for an association phase of 120 s, followed by a 600-s dissociation time. Kinetic parameters were calculated using the BIA evaluation software with the average values of two datasets collected on separate days. Association , NCI-N87 , BxPC-3 , Calu-3 , NCI-H23 , and NCI-H23 . After overnight incubation, the diluted testing items were added respectively. After 72 h, cell viability was evaluated using a CellTiter-Glo Luminescent Cell Viability Assay , following the manufacturer’s instruction.All animal experiments in this study were conducted in the laboratory fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. The contents and procedures related to animal experiments in the study complied with the relevant laws and regulations in the experimental animal use and management and the relevant requirements of the Institutional Animal Care and Use Committee (IACUC). The study protocol was approved by IACUC .3, mice were randomized into five groups (n = 8) and subsequently administered by intravenous (i.v.) injection with the testing item twice a week for six times. SKB264 treatments were performed at doses of 1, 3, and 10 mg/kg in the HCC1806 xenograft model and 0.3, 1, and 3 mg/kg in the NCI-N87 xenograft model. Irinotecan (30 mg/kg) treatment was an arm of the standard of care in the CDX models. The percentage of tumor growth inhibition (TGI) was defined as %TGI = [1 − (Ti − T0)/(Vi − V0)] × 100%, to evaluate the antitumor efficacy in vivo, where T0 and Ti were the mean tumor volume of the treatment group on the days of randomization and measurement, respectively, and V0 and Vi were the mean tumor volume of the control group on the days of randomization and measurement, respectively. Body weight changes and mortality were recorded to evaluate the tolerability of the testing items.The CDX models of HCC1806 (breast cancer) and NCI-N87 (gastric carcinoma) cells were developed by subcutaneously engrafting tumor cells into female BALB/c nude mice . When the average tumor volume reached about 120 mm® breast cancer xenograft model, which utilized primary BR1282 breast cancer derived from a female patient, was provided by Crown Bioscience Inc. , and four gastric cancer PDX models, namely, 0406022, A11068, 0501116, and A19058, were provided by Personal Oncology . The BR1282 PDX model was confirmed to be TROP2-positive (high expression) by pathological examination. The four gastric cancer PDX models  were also confirmed to have high, moderate, low, and negative TROP2 expression, respectively. Immunohistochemistry (IHC) analysis of the paraffin-embedded PDX tissue section was performed with anti-TROP2 antibody , following the set procedure of the Bond RX Fully Automated Research Stainer . NDP2.0-HT, a next-generation viewer software (Hamamatsu), was used for the image analysis. After primary tumor tissue harvested in “stock mice”, the tumor fragments (2 to 3 mm in length) were implanted subcutaneously into the right flank of BALB/c nude (BR1282) or NCG (four gastric cancer PDX models) mice purchased from GemPharma . Mice were randomized in five groups when the average tumor volume reached about 80–120 mm3 (n = 8 or 6). The tumor-bearing mice were treated with SKB264 via i.v. injection at doses of 0.5, 1.5, and 5 mg/kg for BR1282 and 1, 3, and 10 mg/kg for gastric cancer PDX models twice a week for six times. Irinotecan, paclitaxel, or paclitaxel  were the standard of care in the PDX models. Tumor volume and body weight were measured twice a week. Tumor weight was measured at study termination. Body weight changes and mortality were recorded to evaluate the tolerability of the testing items.The HuPrimen = 3/sex/group). Groups #1, #2, and #3 were treated with 1, 3, and 10 mg/kg of SKB264, respectively, via 15 min i.v. infusion once. Group #4 received 0.24 mg/kg of unconjugated KL610023 via i.v. infusion once. The serum concentrations of SKB264, Tab, and KL610023 were continually measured up to 672 h post-dose.The PK properties of SKB264 and Tab were determined by ELISA, for which the lower limit of quantitation was 7.813 ng/ml. The serum concentration of KL610023 was measured by a validated liquid chromatography tandem mass spectrometry (LC/MS-MS), for which the lower limit of quantitation was 0.100 ng/ml. A total of 24 cynomolgus monkeys were randomized into four groups (n = 6 mice/group) according to body weights and tumor volumes. After receiving a single i.v. injection of [3H]KL610023, at 0.083, 4, 24, and 72 h, as well as a single i.v. injection of [3H-KL610023]SKB264, [3H]SKB264-mAb, or [3H-mAb]SKB264 at 1, 24, 168, and 336 h post-dosing, blood, tumor tissue, and 16 types of normal tissues were collected from the euthanized mice.A total of 96 female nude mice bearing human HCC1806 tumor cells were divided into 16 groups (3H]KL610023 or TRA (ng Eq./g).Plasma samples were directly quantified by liquid scintillation counting (LSC) without further manipulation; however, the blood and tissue samples were digested with 1N of KOH at 90°C prior to quantification by LSC. The tissue residue approach (TRA) for blood, plasma, and tissues was converted to nanogram equivalent of [n = 5 animals/sex/group). The experimental animals were administered with 0.9% NaCl injection (negative control) or SKB264  via i.v. infusion once every 2 weeks for four doses . The mice were treated with SKB264 or IMMU-132 at doses of 1, 3, and 10 mg/kg twice a week for six times via i.v. injection. To evaluate the PK-PD of ADC, when the average tumor size reached approximately 270 mm3, mice were randomized into three groups (n = 5/time point). The mice received a single i.v. injection of control hIgG1, SKB264, or IMMU-132 (3 mg/kg), respectively. Blood samples (from five mice) were collected at 1 h, 4 h, 8 h, 24 h, 48 h, 96 h, 192 h, or 336 h post-dosing to detect the concentration of payload at each time point. The rate of TGI  was determined to evaluate the antitumor efficacy of ADC. Body weight changes and mortality were also recorded to evaluate the tolerability of the testing items.Female BALB/c nude mice  were inoculated with 2×10in vitro stability of SKB264 and IMMU-132 in monkey and human plasma, the release rate of payload from SKB264 and IMMU-132 at the concentration of 50 μg/ml at 37°C up to 144 h was examined by measurement of payload concentration in plasma using LC/MS-MS, with a lower limit of quantitation was 2.00 ng/ml.To evaluate the t1/2, Tmax, Cmax, AUC0-t, and AUC0-∞. The in vitro data were analyzed and displayed using software PRISM 5.0 (GraphPad). The half maximal effective concentration (EC50) and half maximal inhibitory concentration (IC50) were calculated using a nonlinear regression model with sigmoidal fitting. The in vitro antitumor activities of drugs were evaluated by a two-tailed paired t-test.Phoenix WinNonlin 8.1 was used to calculate the PK parameters in the non-compartment model, including via a sulfonyl pyrimidine-CL2A-carbonate linker. The payload is conjugated with the antibody through irreversible nucleophilic aromatic substitution by tris (2-carboxyethyl) phosphine hydrochloride (TCEP HCl), a reducing agent that can reduce the interchain disulfide bounds. The drug-to-antibody ratio (DAR) of SKB264 was approximately 7.4, which was determined by the reversed-phase chromatography (RPC), and might be the theoretical maximum drug loading number for conventional interchain cysteine conjugation , highly expressed TROP2, while NCI-H23  was TROP2-negative  and 1.360–5.996 nM (KL610023). NCI-H23 (TROP2+) cells were more sensitive to SKB264 than parental cells (TROP2−). These results indicate that SKB264 exhibits tumor suppression ability mainly through its payload terminal. Taken together, after SKB264 endocytosed through its interaction with TROP2 on cell surface, the releasing free KL610023 can inhibit tumor cell proliferation.To evaluate the effect of SKB264 on tumor cell proliferation, negative Figure 3In vivo antitumor activity of SKB264 was evaluated using both the CDX model and the PDX model. In the xenograft tumor model of NCI-N87 cells . Similar to the NCI-N87 xenograft model, in the HCC1806 xenograft model , respectively, on the 24th day after first dose. Furthermore, high TROP2 expression in BR1282 tumor xenograft (derived from the tumor tissue of a breast cancer patient) was found . Overall, our study demonstrated high efficacy (in dose-dependent manner) of the in vivo antitumor activity of SKB264 in both CDX and PDX models.To explore a minimum level of TROP2 to exert an antitumoral activity, four gastric cancer PDX models with different TROP2 expression levels were transplanted into mice. As shown in 50 of the affinity of SKB264 and SKB264 mAb with monkey TROP2 was 4.363 ng/ml and 3.399 ng/ml, respectively. These results correlated with the affinity of SKB264 and SKB264 mAb with human TROP2 protein. However, SKB264 and SKB264 mAb bound to rat and mouse TROP2 protein with low affinity , indicating that cynomolgus monkey was an appropriate species for preclinical pharmacokinetic and toxicological analysis of SKB264 because of the similar binding affinity to human TROP2. Therefore, we performed the pharmacokinetic experiments in cynomolgus monkeys.Firstly, the affinities of SKB264 and SKB264 mAb to TROP2 of different species were evaluated by ELISA. The ECt1/2: 24–57 h) (t1/2: 5.7 h) after a single intravenous administration at a dose of 0.24 mg/kg . The clearance of SKB264 was slow after a single injection  and would be quickly cleared  was conducted. In detail, 40 cynomolgus monkeys were randomized into four groups to receive saline or different doses of SKB264  via i.v. infusion. The first dosing day was defined as Day 1. Five monkeys  in the group of SKB264 (75 mg/kg) treatment were euthanized due to their status of moribundity on Days 10–24. The cause of the moribundity appeared to be the deteriorating condition of the animal, the symptoms of which included loss of body weight, reduced food consumption, bone marrow toxicity, and intestinal toxicity. Histopathological examination found several abnormalities in in the group of SKB264 (75 mg/kg), including intestinal villi atrophy, chronic inflammation, intestinal gland dilatation, thymic cortex lymphopenia and cysts, bone marrow hematopoietic cell reduction, skin hair follicle atrophy, pigmentation and other morbid conditions, and changes in the corresponding clinic pathology indicators. In the SKB264 (50 mg/kg) group, there were also similar changes, but its incidence or severity was less or low. However, in the SKB264 (25 mg/kg) group, only the lesions in the skin and thymus were observed, most of which could be recovered or unobserved at the end of the recovery phase, except for the lesions in thymus. Although gastrointestinal toxicity and bone marrow toxicity are typical dose-limiting factors related to the clinical application of topoisomerase I inhibitors, in our study, the observed SKB264 effect on the intestines was very slight, even in the SKB264 (50 mg/kg) group. The dramatic decrease of side effects was presumable due to less off-target toxicity. TROP2-negative cells and the PDX model were less sensitive to SKB264, probably because it mainly targets and releases payload in TROP2-positive cells or tissues (Cmax of SKB264 after the first dosing in the SKB264 (75 mg/kg) group, but they were higher in females than the males after the last dosing. Under the experimental conditions, the highest non-seriously toxic dose (HNSTD) of SKB264 was 50 mg/kg in monkeys following the repeated administration. The SKB264 HNSTD in monkeys provides evidence of preclinical safety of this ADC, which meets the requirement of further clinical trial. Therefore, those data are acceptable for entry into human clinical trials.The  tissues 5. The bKd values of 0.3083 nM and 0.2935 nM, respectively, demonstrating that SKB264 and IMMU-132 have similar affinity for TROP2  was 48.12%, 70.64%, and 153.27%, respectively  or IMMU-132 (3 mg/kg) after a 13-day single administration was 123.47% and 21.05%, respectively, indicating that the inhibitory effect of SKB264 on tumor growth was stronger than that of IMMU-132 after a single administration at the same dose Table 4.The release rates of the payload from IMMU-132 in cynomolgus monkeys and human plasma were almost 100% after 48-h incubation, while only 70% payload was released from SKB264 after 144-h incubation Figure 8Cancer is characterized by the uncontrollable growth of abnormal cells, causing the disease that seriously affects human health and quality of life . It is rvia an irreversible covalent binding with disulfide reduced cysteine, by which the problem that the payload falls off easily from ADC can be fixed. Moreover, the use of toxic payload KL610023 further increases the stability of SKB264 as well. We hope to enhance the targeting ability of SKB264, reduce its off-target toxicity, and obtain a broader therapeutic window through the innovative design of SKB264.Since TROP2 is also expressed in multiple normal tissues, concerns have been raised on the specificity of anti-TROP2 ADC . Anti-TRin vivo and in vitro experiments were conducted in the present study (in vitro) to TROP2 protein and their ability to bind to TROP2 expressed on HCC1806 and NCI-N87 cells, followed by being internalized, suggesting that KL610023 conjugation cannot affect the affinity of SKB264 mAb. On the other hand, the ability of tumor targeting and internalization of SKB264 was mediated by SKB264 mAb. Furthermore, the results of cytotoxic assay showed that the antitumor effect of either SKB264 or KL610023 was almost the same, but the SKB264 mAb did not affect the survival of tumor cells, suggesting that KL610023 is the main cytotoxic factor to kill tumor cells. In in vivo experiments, SKB264 could significantly improve the TGI rate in both CDX and PDX models in a dose-dependent manner and also manifest better antitumor effect in vivo, as compared to that of IMMU-132 at the same dose. As to the mechanism of its action, SKB264 targets TROP2-positive tumor cells and mediates endocytosis via SKB264 mAb. After internalization, SKB264 releases its payload (KL610023) to kill tumor cells. However, there is variation in the process of SKB264 internalization and KL610023 release, which might be due to the difference in the factors related to intracellular trafficking pathways, for example, clathrin- or caveolin-mediated endocytosis, or lysosomal enzyme activities  polymerase (PARP) inhibitor is an attractive therapeutic approach, because both of them target the DNA damage response (DDR) pathway with promising results from preclinical studies , 37. HowIn summary, we developed SKB264, a novel TROP2-targeting ADC using several patented technologies. Our study showed the remarkable efficacy of SKB264 in the nonclinical TROP2-expressing xenograft models, with an acceptable safety profile and an excellent therapeutic window in animal studies, suggesting that SKB264 treatment may be a promising therapeutic therapy for treating gastric carcinoma and breast cancer in the clinical setting. With its highly promising results in preclinical studies, SKB264 has been applied in clinical trials, hopefully to provide clinical benefit for patients with gastric carcinoma, lung cancer, and breast cancer.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC).YZC: Conceptualization, formal analysis, investigation, methodology, and writing—original draft. XY, QT, XH and YC: Conceptualization, methodology, and writing—original draft. YP and HL: Formal analysis, methodology, and writing—review and editing. MX, YJ, JX and YT: Investigation and writing—review and editing. XZ and HS: Project management and writing—review and editing. All authors contributed to the article and approved the submitted version.


An increasing number of autoimmune encephalitis (AE)-associated autoantibodies have been successfully characterized. However, many cases of AE remain unexplained on account of unknown antibodies. The aim of the present study was to identify a novel antibody against collapsin response mediator protein 2 (CRMP2) in suspected AE patients.A patient’s serum and cerebrospinal fluid samples tested negative for known AE antibodies; however, strong immunolabel signals were observed in the neuronal cytoplasm of the cortex, hippocampus, and Purkinje cells on rat brain sections. Immunoprecipitation from the rat brain protein lysate, followed by mass spectrometry analysis, was used to identify the targeting antigen. Western blotting and cell-based assay with antigen-overexpressing HEK293T cells were used for antibody specificity, epitope, IgG subtype determination, and retrospective study.An antibody against CRMP2, a synaptic protein involved in axon guidance, was identified. The immunostains of the patient’s samples on rat brain sections were eliminated by pre-absorption with HEK293T cells overexpressing CRMP2. The samples specifically immunoreacted with CRMP2, but not with CRMP1, CRMP3, CRMP4, and CRMP5. The C-terminus of CRMP2 with 536 amino acids contained the epitope for antibody binding. The subtype analysis showed that the anti-CRMP2 antibody was IgG4. Furthermore, a screening of 46 patients with neurological disoders and neuro-cytoplasm immunostainings on rat brain sections resulted in the identification of anti-CRMP2 antibodies in a case of encephalomyelitis. The two patients responded well to immunotherapies.This study discovered that a novel anti-CRMP2 antibody was associated with suspected AE and thus should be included in the testing list for AE. Vα2δ), and glutamate kainate receptor subunit 2 (GluK2), respond well to immunotherapy  encompasses a large category of inflammatory disorders mediated by immune responses against neuronal intracellular antigens, cell surface, or synaptic antigens, which, in some cases may be accompanied by neoplasia. Intracellular antigens include Hu , Ri (ANNA2), and Ma2 in the nucleus and Yo , amphiphysin, glutamate decarboxylase 65-kDa isoform (GAD65), and Kelch-like protein 11 (KLHL11) in the cytosol, which cause a T cell-mediated immune response. As it has been previously reported, some of these cases respond to immunotherapy . Most AEotherapy . Thus, iotherapy –12. Consi.e., CRMP1–5, which are cytosolic microtubule-associated phosphoproteins and have important roles in dendrite and axonal guidance, regulating migration and synaptic dynamics (The family of collapsin response mediator proteins (CRMPs) comprises five homologous members, dynamics . Structudynamics . CRMPs adynamics . Auto-Abdynamics . Previoudynamics . Abs to dynamics .e.g., anti-CRMP1/2 Abs have been reported in autoimmune retinopathy patients  patient was admitted due to persistent dizziness, visual rotation, recurrent nausea and vomiting, and fever that lasted for 8 days. The patient also reported feeling unstable while walking and was suffering from slurred speech for 1 day. The fever was mild, and the highest body temperature was 38.5°C. On the first day, some medications were given by a local clinic to relieve the vomiting and fever. Three days later, the dizziness worsened and was accompanied by recurrent nausea and vomiting that started after the patient underwent a root canal treatment for chronic pulpitis. The patient was admitted to a local hospital on the next day. The cranial computed tomography (CT), magnetic resonance image (MRI), and magnetic resonance angiography (MRA) results were normal. Anti-platelet treatment and antibiotics were given to treat the dizziness and fever. However, the symptoms deteriorated with slurred speech and difficulty walking. The patient’s past medical history was not remarkable, except for glucose-6-phosphate dehydrogenase deficiency without symptoms. The patient was allergic to many products, including shrimp and albumin. On admission to our center, the physical examinations revealed signs of cerebellum damage, including scanning speech, nystagmus, and bilateral finger–nose test instability. Slight neck stiffness was noticed.2O with only a slightly elevated white blood cell count of 38 cells/µl (90% monocytes). The CSF glucose  and protein  were within the normal range. The results of screenings of paraneoplastic antigens and serological autoantibodies, including anti-nuclear, anti-thyroid peroxidase, and anti-thyroglobulin Abs, were all negative. The serum anti-Mycoplasma pneumoniae (M.P.) IgM tests resulted positive two times within the first week after admission and weakly positive at 2 weeks later. The serum anti-Widal’s O test was positive with a titer of 1:80 on admission and conversed to negative at 2 weeks later. The next-generation sequencing  of CSF sample detected 64 unique reads against M.P. The CSF test was negative for AE Abs, but positive by tissue-based assay (TBA).The CSF examination revealed a normal opening pressure of 148 mmHM.P. infection and possible secondary immune-mediated encephalitis. Azithromycin, doxycycline, intravenous methylprednisone , and intravenous immunoglobin  were prescribed. Levodopa and clonazepam were also given to control the myoclonus. The treatment successfully relieved the mild dizziness and opsoclonus. The brain MRI was repeated, and the result was not remarkable with mild white matter degeneration . It was conducted as immunofluorescence with antigen overexpressing HEK293T cells by transfection with corresponding plasmids, which contain the full-length human cDNAs of each antigen fused with GFP in vector pcDNA3.1-C-eGFP. Anti-Hu, Yo, Ri, Ma2, PCA2, amphiphysin, and CRMP5 Abs were tested with blot assay by Nanfang Precision Ltd. . All the known auto-Abs tested were negative. The samples were further analyzed by TBA of immunostaining on rat brain sections and showed strong positive immunoreactive signals in the cytoplasm of neurons. It was considered with unknown Abs and applied for further Ab identification.P1’s CSF and/or serum samples were screened for known AE auto-Abs, including anti-NMDAR, LGI1, Caspr2, GABAFor the retrospective study to find more anti-CRMP2 antibody-positive patients, we reviewed 1,473 patients admitted to our Neurology Department between January 1, 2018 and December 31, 2020 Figure 2Frozen tissue sections, 5 μm in thickness, from the brain of adult SD rat or C57BL/6 mouse were prepared and performed by IHC (or TBA) with CSF, sera, and secondary Abs as previously reported . The dilFresh whole-brain protein lysates of adult SD rats and Western blotting were processed as reported . A lysisPrimary cortical neurons were taken from 18- to 19-day-old C57BL/6 mouse fetuses and cultured for 14 days as reported . Ice aceThe HEK293T cells were transfected with plasmids pcDNA3.1-CRMP1/2/3/4-Flag, pcDNA3.1-CRMP5-eGFP, pcDNA3.1-CRMP2 T1 (1-141 aa of isoform 1)/T2 (1-36 aa of isoform 2)-eGFP, and pcDNA3.1-CRMP2 T3 (142-677 aa of isoform 1)-Flag separately for 48 h. The protein coding sequences in the plasmids referred to mRNAs of CRMP1: NM_001014809.3, CRMP2 (isoform 1): NM_001197293.3, CRMP2 (isoform 2): NM_001386.6, CRMP2 (isoform 3): NM_001244604.2, CRMP3: NM_006426.3, CRMP4: NM_001197294.2, and CRMP5: NM_020134.4. The full-length isoform 1 of CRMP2 was used unless specified otherwise. The transfected cells were fixed with ice acetone for 10 min, washed with PBST, blocked with 10% goat serum, and then incubated with the patient’s serum or rabbit anti-CRMP2 Ab (1:200) overnight at 4°C. After washing, the appropriate secondary Abs were incubated at room temperature for 1 h, including DyLight 488-labeled goat anti-human IgG, DyLight 550-labeled goat anti-human IgG , FITC-labeled anti-human IgG1/2/3/4 , and/or Alexa Fluor 594-labeled goat anti-rabbit IgG secondary Abs (1:200). The fluorescence images were taken by using LSM980 confocal microscope (Zeiss) or IX73 inverted microscope (Olympus).BR, AMPAR, DPPX, DNER, D2R, mGluR5, GAD65, and IgLON5 Abs by CBA and anti-Hu, Yo, Ri, Ma2, PCA2, amphiphysin, and CRMP5 by blotting assay. However, the immunofluorescence assay with P1’s CSF (The serum and CSF samples were collected from a patient suspected of AE (P1). The patient was negative with the known auto-Abs, including anti-NMDAR, LGI1, Caspr2, GABAIP complex pulled down from rat brain protein lysate by P1’s CSF was blotted with P1’s CSF, which revealed a band above 70 kDa  carrying truncated CRMP2 were constructed as schematically indicated and applied to the CBA assay , decreased glucose of 2.64 mmol/l, elevated protein level of 0.80 g/l, and normal chloride level of 123 mmol/l. The CSF pathogen screenings for tuberculosis (TB) culture, TB-SPOT, X-pert, bacteria, and fungi cultures were all negative. Abs including AE panels, anti-aquaporin 4 (AQP4), anti-myelin oligodendrocyte glycoprotein (MOG) all resulted negative. Brain MRI with contrast revealed multiple patchy abnormal signals in the white matter area of the bilateral cerebral hemispheres and brain stem  were not remarkable, while the spine MRI showed long-segment spinal cord lesions from the medulla to the C6 segment  and intravenous MP pulse therapy . Following the treatment, the patient’s chest pain and numbness gradually improved. A lumbar puncture was repeated 20 days later, which revealed that the WBC dramatically decreased to 20 cells/µl, while the glucose and protein returned to normal. The patient was followed up regularly in our clinic, and the steroids were tapered. The anti-TB therapy was discontinued after 6 months of treatment. A repeated brain MRI revealed the dramatic decrease of the abnormal signals in the white matter area of the bilateral cerebral hemispheres and brain stem . Currently, these patients are free of neoplasms, and they will be continuously followed up in the future. Therefore, the etiology of encephalitis remains unclear.In this study, we identified IgG4 auto-Ab specifically against CRMP2 in the sera and CSF samples of two patients with encephalitis or myelitis. The P1 patient dominantly presented signs of cerebellar encephalitis, including dizziness, slurred speech, and instability while walking. The P2 patient had encephalomyelitis, with the major complaint indicative of myelitis being chest pain and limb numbness. There was not enough evidence to support CRMPs are highly expressed during embryonic development and significantly decrease after birth, while each CRMP has a different subcellular localization . In the Crmp2 gene knockout, it was found that lack of CRMP2 leads to defects in axon guidance, axon pruning in the hippocampus and visual cortex, and altered dendritic spine remodeling  to regulate Ca2+ and Na+ neuronal homeostasis . The patients/participants provided their written informed consent to participate in this study.YHu, SP, YW, and KX designed the study and drafted the manuscript. DW and SW took care of the index patient and were responsible for the clinical data preparation. YHe and GL performed most of the experiments. YP provided cultured cells and neurons, HJ and YP were responsible for the other anti-CRMP2 Ab-positive patients. FX prepared the mouse and rat brain sections. YHuang took confocal images. QW performed Western blotting. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China , Guangdong Provincial Scientific and Technologic Progression Fund (2016A020215182 to YHu), Natural Science Foundation of Guangdong Province (2019A1515011760 to YW), President Foundation of Nanfang Hospital (2019B007 to DW and 2020B008 to KX), and the Medical Science and Technology Foundation of Guangdong Province (A2021151 to KX).A patent (ZL202110461611.6) is authorized and a PCT patent (PCT/CN2021/138864) is pending for the assay to detect the anti-CRMP2 Ab in autoimmune encephalitis or encephalomyelitis .The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We evaluated he effects of molecular guided-targeted therapy for intractable cancer. Also, the epidemiology of druggable gene alterations in Chinese population was investigated.The Long March Pathway  is a non-randomized, open-label, phase II trial consisting of several basket studies examining the molecular profiles of intractable cancers in the Chinese population. The trial aimed to 1) evaluate the efficacy of targeted therapy for intractable cancer and 2) identify the molecular epidemiology of the tier II gene alterations among Chinese pan-cancer patients.In the first stage, molecular profiles of 520 intractable pan-cancer patients were identified, and 115 patients were identified to have tier II gene alterations. Then, 27 of these 115 patients received targeted therapy based on molecular profiles. The overall response rate (ORR) was 29.6% (8/27), and the disease control rate (DCR) was 44.4% (12/27). The median duration of response (DOR) was 4.80 months , and median progression-free survival (PFS) was 4.67 months . In the second stage, molecular epidemiology of 17,841 Chinese pan-cancer patients demonstrated that the frequency of tier II gene alterations across cancer types is 17.7%. Bladder cancer had the most tier-II alterations (26.1%), followed by breast cancer (22.4%), and non-small cell lung cancer .The Long March Pathway trial demonstrated a significant clinical benefit for intractable cancer from molecular-guided targeted therapy in the Chinese population. The frequency of tier II gene alterations across cancer types supports the feasibility of molecular-guided targeted therapy under basket trials. HER-2 overexpression in the breast or gastric cancer, BRAF V600E mutation in melanoma, EGFR or ALK in lung cancer)  . The cho cancer) . The unaTMB]; >10 mut/Mb])  –11.BRAF mutations, all of whom received vemurafenib. In non-small cell lung cancer (NSCLC) patients, the objective response rate (ORR) was 42% after multiline therapy, which is a more favorable response when compared with an ORR of 7% reported for standard, second-line treatment with docetaxel. In a cholangiocarcinoma cohort, the disease control rate (DCR) was 62%, which was also better than second-line chemotherapy (49.5%)  with multiple lines of therapy, as well as those without an effective standard of care (including rare cancers) . There i (49.5%) . In addi (49.5%) .Previous basket trials have mainly focused on non–East Asian populations. It is known that gene alterations driving cancer progression differ significantly across ethnicities . Both thThe druggable gene alterations were defined to be either tier I gene alterations or tier II gene alterations. Tier I gene alterations have the strongest clinical significance, and are supported by level A evidence  and level B evidence (well-powered studies with consensus from experts in the field). Tier II gene alterations are of potential clinical significance, and are supported by level C evidence  and level D evidence  .The Long March Pathway  is a non-randomized, open-label, phase II trial that combines multiple basket studies of intractable pan-cancer patients from China. The trial was conducted in 2 main stages. The first part of the Long March Pathway trial included 2 steps. In the first step, 520 intractable cancer patients were recruited prospectively to identify druggable gene alterations. Intractable cancer patients, considered to be those patients who had no standard of care according to the National Comprehensive Cancer Network (NCCN) guidelines  older than 18 years with any type of metastatic solid tumor were eligible for this trial. Additional eligibility requirements included measurable, or evaluable, lesions .Genetic profiling was determined using a next-generation sequencing (NGS) platform  in a clinical laboratory improvement amendment (CLIA)–approved laboratory. The protocols were described previously , 21. BriEGFR mutation/amplification, ALK fusion, BRAF V600E mutation, BRCA1/2 mutation, CDK4/6 amplification, CDKN2A/2B deletion mutation, ERBB2 amplification/mutations, FGFR1/2/3 fusion, MET amplification, PIK3CA mutation, RET fusion, ROS1 fusion).Patients enrolled in the trial were eligible for the second step of the first stage if 1, or more, druggable gene alterations were identified after genetic profiling screening . Patients with ERBB2 alterations were treated with pyrotinib . Patients with CDK4/6 amplifications or CDKN2A/2B deletion mutations were treated with palbociclib . Patients with BRCA1/2 mutations were treated with olaparib . Patients with BRAF mutations were treated with vemurafenib . Patients with EGFR mutations were treated with a first-generation EGFR tyrosine kinase inhibitor . Patients with PIK3CA mutations were treated with everolimus . Patients with EGFR amplifications were treated with nimotuzumab (100 mg intravenous injection once per week). Patients with FGFR1/2/3 fusions or mutations were treated with erdafitinib . Patients received treatment for 2 cycles and were then evaluated for treatment response. Patients with objective response or stable disease (SD) continued the targeted therapy, with repeat evaluations every 2 cycles for the first 24 weeks, followed by evaluations every 12 weeks until tumor progression or unacceptable toxicity.Based on their options, patients with the above druggable gene alterations were able to receive a molecular-guided targeted therapy. Patients with In the second stage of the trial, we conducted a multi-center study to determine the possibility of the above described molecular-guided targeted therapy in pan-cancer. The molecular epidemiology of the druggable gene alterations were evaluated in a larger cohort of 17,841 pan-cancer patients across 16 principal tumors types. In this stage, cancer types included NSCLC, SCLC, breast cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, biliary system cancer, ovarian cancer, uterus cancer, prostate cancer, bladder cancer, kidney cancer, malignant melanoma, head and neck tumors, and sarcoma. All samples were obtained from fresh tissue or formalin-fixed paraffin-embedded tumor tissue, and they were profiled in a CLIA-certified NGS laboratory using the OncoScreen 295 or OncoScreen Plus 520 cancer-related gene panels . Molecular epidemiology of the tier II gene alterations in the pan-cancer patients were also determined based on genetic profiling. The trial was approved by the Ethics Committee of Changzheng Hospital (2017SL016). All participants provided written informed consent to take part in the study.The primary study endpoints for the Long March Pathway trial were ORR. Treatment response was evaluated by the investigator according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 . SecondaAmong the cancer patients who underwent multiple-line therapy, PFS2/PFS1 ratios were also analyzed. PFS2 was defined as the PFS after genomics-guided targeted therapy. PFS on prior therapy (PFS1) was defined as the time interval from the most recent prior treatment to progression (as defined by RECIST), or clinical progression. A PFS2/PFS1 ratio of greater than 1.3 was considered indicative of a treatment benefit, in keeping with the definition of clinical benefit in basket trials . AdditioP value <0.05 was considered statistically significant.We used a Simon’s minimax 2-stage design with a type I error of 0.10 and 90% power to test the null hypothesis of an ORR of 10% or lower versus the alternative hypothesis of one 30% or higher . The regEGFR mutation/amplification, ALK fusion, BRAF V600E mutation, BRCA1/2 mutation, CDK4/6 amplification, CDKN2A/2B deletion mutation, ERBB2 amplification/mutations, FGFR1/2/3 fusion, MET amplification/mutation, PIK3CA mutation, RET fusion, and ROS1 fusion) were identified in 115 patients (115/520). Overall, 196 out of 520 patients had tier 1 or tier 2 gene alterations, and 115 of 196 had tier II gene alterations , EGFR, ERBB2, FGFR1/2/3, MET, PIK3CA, RET, and ROS1.In the second phase (Molecular epidemiology investigation), a total of 17,841 pan-cancer patients across 16 common tumor types were analyzed to evaluate the frequency of tier I and tier II gene alterations based on the genetic profiling of each patient. The druggable gene alterations included ALK fusions, ERBB2 mutations, and amplification, PIK3CA mutations, cell cycling pathway mutations , MET amplification, BRCA1 mutations, and EGFR amplification. The most frequently used targeted agents were pyrotinib for HER2 alterations, crizotinib for ALK fusions and MET amplification, everolimus for PIK3CA mutations, nimotuzumab for EGFR amplification, olaparib for BRCA1 mutations and Osimertinib for EGFR mutation.A total of 27 patients with tier II gene alterations received targeted therapy on the basis of molecular profiles. Clinical characteristics of patients receiving targeted therapy are reported in ) . Among the 27 patients receiving targeted therapy on the basis of molecular profiles, 13 patients discontinued treatment due to cancer-related death or poor performance status before the first response evaluation; the patients were identified as non-responders. No patient was dead due to the toxicity of targeted therapy. The median duration of follow-up was 8.3 months (range 2.4–55.0 months). Among the additional 14 patients who received response evaluations, 8 patients showed a partial response, and 4 patients showed stable disease. Overall, ORR was 29.6% (8/27) and DCR was 44.4% (12/27) . FigureEGFR, KRAS, and PIK3CA gene alterations were the most common, while CDK pathway , FGFR1/2/3, BRAF, RET was uncommon across pan-cancer. The frequency of the 16 druggable gene alterations across 16 common tumor types were shown in KRAS mutations were observed in colorectal or pancreatic cancer patients, and PIK3CA mutations mainly occurred in breast cancer and uterine cancer.  were variably represented. This variation in the distribution of gene alterations thus warrants further investigations in other ethnic populations.To further explore the possibility of molecular-guided targeted therapy across cancer types, the mutational landscape of metastatic cancer was analyzed using an NGS panel (256 or 520 gene panel) in 17,841 Chinese pan-cancer patients to detect the frequencies of druggable targets. The frequency of tier II gene alterations across pan-cancer outside of current FDA indications was 17.7%, suggesting that clinicians or physicians can consider various molecular targeted therapies across different tumor types with shared genetic features for the treatment of patients, especially for intractable cancer. In 2017, the mutational landscape of metastatic advanced cancer in 10,366 patients from the Memorial Sloan Kettering Cancer Center (MSKCC) was published. The study demonstrated that 27% of patients had at least 1 actionable alteration apart from targets with levels 1 and 2A evidence according to whether mutations were FDA-recognized biomarkers. On comparing the distribution of actionable alterations between the MSCKCC and Long March Pathway cohorts, it was evident that while gynecological cancer and biliary cancer in the Chinese population harbored fewer alterations, lung cancer patients had more alterations than the MSKCC cohort. Some types of cancer, including breast cancer and bladder cancer, showed higher frequency of druggable targets in both cohorts Similarly, some gene alterations, including BRAF V600E were found to respond well to vemurafenib, while those with colorectal cancer did not  could not be evaluated, while the association of targeted agents type and the concomitant alterations with a response to targeted therapy were also not further assessed . Thus, the further exploration of the application of basket trials in a larger patient cohort is recommended.Our results have validated the use of targeted therapy on the basis of molecular profiles in clinical practice and have shown that the discussed methods can help guide treatment choices for intractable cancer. However, several limitations require clarification for accurate interpretation of the results. First, the ratio of patients who received off-label targeted therapy was low, and 23.5% (27 of 115) of patients received targeted therapy based on an individual molecular profile. The low ratio may be attributed to the high cost of targeted therapy and off-label treatment, which medical insurance does not cover. Second, a few of patients  failed to undergo response evaluation. Most patients discontinued the targeted therapy due to poor performance or cancer-related death during treatment. These patients were defined as non-responders to off-label targeted therapy. This might have affected the accuracy of efficacy evaluation. Third, the small sample size limited the efficacy evaluation of different druggable targets. For example, only 8 targeted therapies were assessed under multiple basket trials. The efficacy of molecular-targeted therapy against another 5 druggable targets (assessed . In addiIn conclusion, the Long March Pathway trial demonstrated a significant clinical benefit for intractable cancers using targeted therapy based on molecular profiles in the Chinese population. The frequency of tier II gene alterations across a variety of cancers supports the feasibility of molecular-guided targeted therapy under basket trials and may provide novel insights into the optimal clinical treatment of intractable cancers.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Ethics Committee of Changzheng Hospital. The patients/participants provided their written informed consent to participate in this study.X-DJ and Y-SZ designed the study. X-DJ, B-DQ, ZW, KL, YW, YL, W-XQ, M-MW, L-YY, HL, Y-YX, X-MQ, MW, MJ, L-CX, YZh, HY, X-QC, YZe, JS, W-YZ, Q-QD, WJ, S-FL, J-JZ, HS, S-HY, Z-JG, TL, P-JZ, X-WD, and W-FL recruited patients for this study and collected data. X-DJ, B-DQ, ZW, KL, YW, YL, W-XQ, M-MW, L-YY, and Y-SZ did the analyses. X-DJ, B-DQ, SB, AK, KM, RC, MA, CA, FP, and Y-SZ interpreted the data and wrote the manuscript. All authors have agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors contributed to the article and approved the submitted version.


Hepatocellular carcinoma (HCC) is an aggressive human cancer and is caused as consequences of chronic liver diseases. Although HCC is more common in patients with cirrhosis, there is increasing evidence that this cancer may develop in the setting of noncirrhotic nonalcoholic steatohepatitis (NASH), even simple hepatic steatosis may progress to carcinogenesis development. NASH is associated with obesity, hyperlipidemia, insulin resistance, and type 2 diabetes (T2D), which are becoming emerging risk factors for the development of HCC, as well as for cardiovascular disease. In this review, we discuss the current molecular data supporting the link between HCC and NASH, with a focus on metabolic alterations, genetic and epigenetic drivers, including current therapeutic strategies for NASH and HCC prevention and treatment.Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is the sixth most frequent cancer in the world, being the third cause of cancer-related deaths. Nonalcoholic steatohepatitis (NASH) is characterized by fatty infiltration, oxidative stress and necroinflammation of the liver, with or without fibrosis, which can progress to advanced liver fibrosis, cirrhosis and HCC. Obesity, metabolic syndrome, insulin resistance, and diabetes exacerbates the course of NASH, which elevate the risk of HCC. The growing prevalence of obesity are related with increasing incidence of NASH, which may play a growing role in HCC epidemiology worldwide. In addition, HCC initiation and progression is driven by reprogramming of metabolism, which indicates growing appreciation of metabolism in the pathogenesis of this disease. Although no specific preventive pharmacological treatments have recommended for NASH, dietary restriction and exercise are recommended. This review focuses on the molecular connections between HCC and NASH, including genetic and risk factors, highlighting the metabolic reprogramming and aberrant epigenetic alterations in the development of HCC in NASH. Current therapeutic aspects of NASH/HCC are also reviewed. Furthermore, Obese males had a higher risk of primary liver cancer than obese females (p = 0.027)  allele in the PNPLA3 gene was significantly associated with elevated hepatic fat levels (p = 5.9 × 10−10) and liver inflammation (p = 3.7 × 10−4). In addition, the allele was more commonly seen among Hispanics (0.49) compared to non-Hispanic whites (0.23) and African Americans (0.17) [p < 1.0 × 10−9) [Obesity and insulin resistance are considered the major contributing factors to the development of hepatic steatosis. However, the heterogeneous prevalence of hepatic steatosis between races/ethnicities suggests other influencing factors, such as genetic variance . A study < 0.05) . Genetic < 0.05) ,62. The s (0.17) . Similars (0.17) . Furthers (0.17) . A meta- × 10−9) . PNPLA3  × 10−9) ,67. In t × 10−9) . In line × 10−9) . TherefoPNPLA3 I148M and TM6SF2 E167K gene variants are major determinants for interindividual differences in liver steatosis and susceptibility to NASH.Other SNPs are peroxisome proliferator-activated receptor gamma coactivator-1-alpha (PPARGC1A) rs8192678 GA/AA genotypes and transmembrane 6 superfamily member 2 (TM6SF2) rs58542926 T allele, which have been confirmed as heredity risk factors associated with NASH in Asia population ,70. PPARPNPLA3, TM6SF2, GCKR, and MBOAT; meanwhile, HSD17B13 variants decreased the risk of HCC [Ethnicity has been associated with the risk of HCC. Genetic polymorphisms in several genes have been associated with an increased risk of HCC, including variants in k of HCC ,79,80. Hk of HCC ,81,82. Ak of HCC . Moreovek of HCC . A systek of HCC . Donati k of HCC . These fTM6SF2 mutations are frequently found in patients with NASH [YWHAZ gene plays an important role in driving HCC tumor progression according to the analysis results [Genetic predisposition and gene expression represent a significant risk regarding disease progression to NASH and HCC. Next-generation sequencing provides a better understanding of genetic factors related to the initiation and progression of hepatic cancer in recent years. ith NASH . In addiith NASH . Desterkith NASH . The YWH results ,95. TERT promoter, TP53, and CTNNB1 [AXIN1, ARID1A, ARID2 and RB1, BAP1, NFE2L2, KEAP1, TSC1/TSC2, MLL2, ALB (Albumin), and APOB mutations), help define some of the core deregulated pathways in HCC [TERT promoter (56%), CTNNB1 (28%), TP53 (18%), and ACVR2A (10%) were the most frequently mutated genes [TP53, RB1, and higher VEGF-binding pathway compared with European Americans, while IL-17 was mutated only in European American population. Additionally, three putative oncogenic genes, including TRPM3, SAGE1, and ADAMTS7, were mutated exclusively in the Asian population [DGAT1, FASN, LPL, IRS2, and YWHAZ), which are implicated in lipid synthesis, insulin resistance, and cancer progression [The most frequently mutated genes in HCC cases were reported by the Cancer Genome Atlas (TCGA) Research Network, including the d CTNNB1 ; and alos in HCC . In lineed genes . Interespulation . It was gression ; thus, tMtor, Sdk1, Braf, Pik3cb, Ctnnd1, Ctnna3, Akt3, and Nos [Ryr1 and Sdk1), 8 genes mutated in 3/5 NASH-HCCs , and 71 genes mutated in 2/5 NASH-HCCs  [Mtor and Sdk1 may also function as cancer drivers in NASH-HCC [Several mutated genes were identified in a murine NASH-associated HCC model, which has also been reported as drivers in other human cancers, including  and Nos . Liang end Pclo) . These fNASH-HCC . Ryr1, Ryr2, Cacna1d, Cacna1h, P2rx1, and Itpr1) and all encoding calcium channel proteins [Insr), and some other well-known cancer-related genes , with Mtor and Hras1 commonly mutated. Other 75 mutated genes were expected to dysregulate 15 specific metabolic signaling pathways, including 6 associated with lipid metabolism [To investigate the signaling networks with mutations in NASH-HCCs, Liang et al. conducted KEGG pathway analysis; and they found seven pathways significantly enriched by gene mutations in three or more NASH-HCCs, which include metabolic, insulin and calcium signaling, cell adhesion and tight junction molecules, ABC transporters, and axon guidance . In addiproteins . Furthertabolism . Anothertabolism . MTOR , SDK1 (cell adhesion molecule), and three calcium signaling genes  [RYR1, RYR2, CACNA1B, CACNA1E, CACNA1H, CACNA1I, GRIN2C, ATP2A2, ATP2B4, SLC8A1, and ITPR3). The nine RYR1 mutations identified in both animal models and human NASH-HCCs were located across the whole gene, with 3 stop gains or truncating mutations inferring likely loss-of-function mutations [In 37 human NASH-HCCs, 21 genes were recurrently mutated in at least two human NASH-HCCs. These included CACNA1H) . Moreoveutations . TherefoNOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis, and mice treated with GKT13783 increased insulin sensitivity [Brg1), a chromatin remodeling protein, is upregulated in NASH and primary hepatocytes exposed to free fatty acids. Furthermore, Brg1 contributes to the transcription of pro-inflammatory mediators possibly by regulating the interaction between NF-κB and its co-factor MRTF-A; and liver injury and hepatic inflammation are attenuated in hepatocyte-specific Brg1 knockout mice fed with a methionine- and choline-deficient diet (MCD) [Xbp1-deleted macrophages reduced steatohepatitis by decreasing the expression of nucleotide-binding domain leucine-rich repeat-containing receptor (NLR) family pyrin domain containing 3 (NLRP3) and secretion of pro-inflammatory cytokines [PPARA, PPARGC1A, and CNPB) [LPL, CD36, and SREBF2), long-chain fatty acid import (transporters SLC27A4 and CD36), and triglyceride biosynthesis  [Ctnnb1 and Myc [Wnt gene expression in NASH could be related to the genetic background , comorbidities, and other factors implicated in NASH pathogenesis.Development and progression of NASH and HCC is a multistep event, which involves dysregulated gene expression implicated in metabolism signaling, inflammation, oxidative stress, and several signaling pathways involved in hepatocarcinogenesis, including dysregulation of proliferation, differentiation, and neoangiogenesis. Several upregulated genes have been found in the progression of NASH, for example, levels of nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase 4 (NOX4) were found to increase in patients with NASH compared with controls . NOXs arsitivity . Brahma-et (MCD) . In liveet (MCD) , which ret (MCD) . Hepatocytokines . Desterknd CNPB) . The genand LPL) . The expand LPL) , or the  and Myc . The conBCL2A1, an anti-apoptotic player, usually implicated in cancer progression, as well as YWHAZ, YWHAH, and IRS2, which participates in the development of insulin resistance as well as hepatocellular carcinoma. In addition, six genes are involved in cancer and inflammatory processes  and lipid droplet formation and metabolism  and were significantly increased in NASH patients [LPL, VLDLR, LIPA, ANXA2, and PLEK; lipid accumulation ; and metabolism (PPARA and BCL2A1). Additionally, five genes were associated with poor prognoses such as FASN, DGAT1, LPL, IRS2, and YWHAZ. Four of these genes are implicated in lipid metabolism regulation and one in liver cancer [Apoa4, Anxa2, Serpine1, Igfbp1, and Tubb2a, which are involved in the development of NASH and HCC [Apoa4 is involved in the regulation of multiple metabolic pathways such as lipid absorption, lipid metabolism, and glucose homeostasis, and the overexpression of APOA4 promotes lipid accumulation in the liver [Anxa2 gene encodes calcium-dependent phospholipid-binding protein ANXA2, and is a key contributor to several hallmarks of cancer, including cancer cell proliferation, inhibition of apoptosis, cell adhesion, invasion, metastasis, and angiogenesis. Several studies have shown an upregulation of ANXA2 in human hepatocarcinogenesis [Other genes upregulated were patients . In addir cancer . Another and HCC . Apoa4 ihe liver . The Anxogenesis ,112. TheNon-coding RNAs (ncRNAs) are groups of RNAs with little or no protein-coding potency which can affect diverse cellular functions and participate in pathological processes. Based on length, ncRNAs are classified into micro RNAs , long noncoding RNAs , small noncoding RNAs , and recently emerged circular RNA (circRNA), noncoding RNAs formed with a covalently closed loop. p < 0.05). Moreover, significant correlations between miR-122-5p and unfavorable lipid profiles, including hs-CRP and miR-34a-5p were also reported [HNF4α, PPARA, and SIRT1 expressions, which are associated with higher triglycerides accumulation and liver steatosis [Micro RNAs are involved in the regulation of gene expression by suppressing or degrading their translation; and regulate a wide spectrum of biological processes, including lipid synthesis, fatty acids and glucose catabolism, inflammation, cell differentiation, proliferation, apoptosis, and necrosis, which have been known to be epigenetically deregulated in NAFLD, NASH, and HCC . MiR-122teatosis . In additeatosis . Anotherteatosis . Overexpteatosis . In maleteatosis . Anotherteatosis . In viscteatosis .p < 0.05) either in simple steatosis or NASH. Indeed, miR-19a/b and miR-125b were correlated with biomarkers of atherosclerosis [p < 0.05), and the expression levels of miR- 195-5p, miR-130b-3p, miR-16-5p, miR-19a-3p, miR-19b-3p, miR-27a-3p, miR-93-5p were suppressed (p < 0.05) in the group of patients with NASH [PPARA gene, which may induce a decrease in fatty acid oxidation and an increase in lipogenesis in the liver [IL1B gene in NASH patients, and it has been shown that an increase in IL-1β production leads to the activation of fibrogenic responses in HSCs, including inflammation and secretion of tissue inhibitor of metalloproteinase-1 [VCAM1 gene, and pharmacological inhibition or genetic deletion of VCAM1 in liver endothelial cells reduced liver inflammation, injury, and fibrosis in mice with NASH [Pirola et al. also reported that miR-122, miR-192, miR-19a, miR-19b, miR-125b, and miR-375 were upregulated > 2-fold  belong to non-coding RNA without or with limited protein-coding capacity. lncRNAs are generated through a pathway similar to that for protein-coding genes ,142. Thed NFKBIA . Metastad NFKBIA . Hepaticd NFKBIA . Anotherd NFKBIA . In addid NFKBIA . In termd NFKBIA . Maternad NFKBIA . lncRNAsResearch circular RNAs (circRNAs) have garnered much interest recently due to their potential to serve as biomarkers or therapeutic targets. The majority of the current studies on circRNAs in liver diseases are focused on HCC and hepatitis, with limited information available for NASH . Jin et −/−) showed enhanced hepatic lipid accumulation, increased hepatocellular apoptosis, and accelerated fibrosis, which aggravated NASH. However, the lack of OPN lowered systemic inflammation, prevented HCC progression to less differentiated tumors, and improved overall survival [Obesity is a strong independent risk factor for the development of cardiovascular disease and also confers an elevated risk of both hepatic and extra-hepatic complications ,151. NASsurvival . NASH mainly appears when the rate of liver non-esterified fatty acids (NEFA) uptake surpasses its capacity for esterification into triglycerides . When NAOxidative stress and lipotoxicity play an important role in the progression of NASH and fibrosis . The prop53 tumor suppressor gene, which is observed in HCC [TP53 gene [Sirt3 knockout mice were documented to exhibit NASH. Both SIRT1 and SIRT3 are critical for redox state, epigenetic modification, and hepatic lipid metabolism to maintain the homeostatic equilibrium [Oxidative stress is also elevated due to defects in redox defense mechanisms involving glutathione (GSH), catalase, or superoxide dismutase (SOD), which induces several events related to NASH and carcinogenesis, including DNA damage, tissue remodeling, and alterations in gene expression such as mutations in the d in HCC ,170. ForP53 gene . Oxidatiilibrium ,173. + T cells are selectively lost, these cells are important for antitumor surveillance and are susceptible to oxidative stress due to increased mitochondrial mass [Excessive accumulation of fructose obtained by the Western diet induces uric acid, which generates ROS both in hepatocytes and adipocytes. Xanthine oxidase (XO) catalyzes the reaction converting xanthine into uric acid, and XO acts as an electron donor for oxygen, thus generating ROS and inducing a pro-inflammatory signaling cascade through the release of cytokines and oxidative stress in the liver . ROS proial mass . TherefoHepatic insulin resistance and obesity are both well-established conditions, which induce systemic changes with chronic low-grade inflammation. Excessive fructose intake promotes the cascade of inflammatory pathways in hepatocytes and adipocytes, which may contribute to the activation of stress hormones involved in the hypothalamus-pituitary-adrenal, thus synthesizing immunosuppressors such as glucocorticoids . NASH onNLRP3 inflammasome is an intracellular multiprotein complex implicated in the production of mature IL-1β, promotion of metabolic inflammation, and progression of NASH in both mice and patients . NLRP3 i+ concentration increases ROS levels and promotes the mutagenesis of both nuclear DNA and mitochondrial DNA (mtDNA), thus promoting the activation of oncogenes or the inhibition of onco-suppressors, and altering mtDNA replication [Mitochondria are vital for cell homeostasis because they provide energy requirements through oxidative phosphorylation (OXPHOS) and ATP synthesis, also regulate redox status, β-oxidation, tricarboxylic acid cycle (TCA), ketogenesis, and glucidic/lipidic metabolism . The exclication . The mutlication .Mitochondrial biogenesis (mitobiogenesis) is the process by which cells renew from pre-existing ones, involving fusion and fission events, since they cannot be generated de novo . In the Autophagy is a lysosomal degradative pathway necessary for cell survival due to providing energy in stressful conditions. Autophagy plays a critical role in the progression of NASH and HCC. In hepatic tissue, autophagy suppresses protein aggregate, lipid accumulation, oxidative stress, chronic cell death, and inflammation. As a selective autophagy, lipophagy is a mechanism of lipid metabolism and, in hepatocytes, suggests that impaired lipophagy may contribute to the development of NASH . CD36 prThe induction of autophagy through the increased expression of liver-specific Atg7 in ob/ob mice improved metabolic stress and reduced hepatic steatosis. The loss of beclin-1, an autophagy gene, leads to HCC development. The autophagy inhibited in NASH leads to the accumulation of p62/SQSTM1 (p62), an autophagic substrate hypothesized to be implicated in the formation of the Mallory-Denk bodies (MDB). MDB are present in ballooned hepatocytes and are markers for NASH diagnosis . The imp+ T cells) in tumor tissues [The innate and adaptive immune mechanisms are important components of steatosis, insulin resistance, inflammation, and fibrosis during NASH progression. Innate immune cells, such as macrophages are essential players in the progression of NASH and have been intensively investigated . In the  tissues . Therefo tissues .+ T cells to hepatocytes [+ T cells have been also reported accumulated in the liver of patients with NASH, correlating with elevated frequency of blood CD8+ T cells expressing perforin, IFN-γ, and TNF-α, thus leading to NASH-related HCC. Experimental depletion of CD8+ T cells in animal models improves NASH and NASH-associated HCC, restoring hepatic insulin sensitivity, decreased liver damage, and reduced fibrosis [+ T cells have a potential role in promoting NASH through the release of pro-inflammatory cytokines such as IFN-γ [+ T cells in NASH, whole-body IFN-γ deficiency attenuates infiltration of intrahepatic macrophages as well as disease progression in a mouse model [+ T cells in hepatocarcinogenesis was demonstrated in several animal models [Adaptive immunity involves T and B cells which play a role in the triggering and progression of NASH. Mechanistically, livers with NASH may accumulate B cells with increased pro-inflammatory cytokine secretion and antigen presentation ability following activation through the innate adaptor myeloid differentiation primary response protein 88 . B cell atocytes . In NASHatocytes . CD8+ T fibrosis . CD4+ T as IFN-γ . Supportse model . In addil models . Th17 cel models . TNF receptor superfamily member 4  deficiency inhibits Th1 and Th17 differentiation, as well as suppresses monocyte migration, antigen presentation and M1 polarization. TNFRSF4 levels in serum are positively associated with NASH, and it is a key regulator of both intrahepatic innate and adaptive immunity and is involved in the development of NASH . Additio+ T cells accentuate liver damage through the production of IFN-γ, generating a permissive microenvironment favoring the emergence of transformed NASH hepatocytes. By contrast, CD4+ T cells exhibit protective effects in limiting both tumor initiation and tumor progression [Several studies indicate that natural killer T (NKT) cells display detrimental effects during NASH development, and this cells can be activated in response to elevated levels of IL-12, type I interferons and IL-1 during NASH . NKT celgression . TherefoThe liver plays a central metabolic role and metabolic disturbances are early events implicated in the development of chronic hepatic diseases and HCC . In NASHCarbohydrate metabolism is a complex biochemical process, which ensures a constant supply of energy to living cells. Glycolysis metabolizes glucose to pyruvate, following to its transformation to either lactic acid (anaerobic glycolysis) or to acetyl-CoA (aerobic glycolysis), which is utilized in the tricarboxylic acid cycle (TCA) and subsequently occurs oxidative phosphorylation. Activated HSCs were reported to shunt their glycolysis pathway towards the production and accumulation of lactate, despite increased expression of the lactate transporter MCT4 and increased lactate efflux ,227. IntHK2 is expressed in skeletal and cardiac muscle and adipose tissues [Metabolic reprogramming is widely recognized as one of the hallmarks of cancer ,195. The tissues . The enz tissues ,231, due tissues . With re tissues . The tum tissues . In resp tissues . Additio tissues . The enz tissues . Mechani tissues .Gluconeogenesis, a reverse process of glycolysis, synthetize glucose from pyruvate, lactate, glycerol, and gluconeogenic amino acids . In HCC Lactate is the main carbon source for the TCA cycle in cancer cells . PyruvatLiver is main organ in lipid metabolism and generates apolipoproteins, endogenous lipids, and lipoproteins . AccumulWith respect to lipolysis in HCC, three major enzymes are involved in this process to release of fatty acids, such as adipose triacylglycerol lipase (ATGL), hormone-sensitive lipase and monoacyglycerol lipase (MAGL) . MAGL prFASN blocked the growth of HCC in vitro [SREBP1 in HCC cells induced growth arrest and apoptosis, as well as inhibited the migration and invasion of tumor cells, whereas the overexpression of SREBP1 promoted cellular proliferation, suggesting that SREBP1 may be a therapeutic target for HCC [The enzymes of lipogenesis are increased in various cancers, including HCC, such as ATP citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), acyl-CoA synthease (ACS) and stearoyl-CoA-desaturase 1 (SCD1) . ACC is in vitro ,7. SCD cin vitro ,7. The ein vitro . SCD1 plin vitro . The tra for HCC ,7. Liver for HCC . Liver X for HCC ,253. Lip for HCC . Therefo for HCC . In well for HCC . Consist for HCC . TherefoCholesterol is important for cell structure and cell proliferation. The cholesterol content in the Western diet is 10-fold higher than in the Mediterranean diet . SeveralMethylation is a heritable enzyme-mediated chemical transformation and is catalyzed by DNA methyltransferases (DNMTs), which transfers a methyl group from S-adenosyl methionine (SAM) to carbon 5 of the Cytosine ring. DNA methylation mainly occurs in the Cytosine base (C) when is followed by a Guanine (G), thereby named CpG sites. In general, when methylation occurs in the gene promoter region results transcriptional repression, while methylation in the gen region promotes gene expression, as described in most cancers including HCC . The onlIGFPB2) are common reported in mice and patients with NAFLD and NASH [PFKL), DNL , and insulin signaling (PRKCE) in obese and T2D patients [PPARGC1A gene, and its promoter methylation is increased in adult patients with NASH compared to those isolated steatosis, correlating with insulin resistance, reduced mitochondrial DNA, and decreased gene transcriptional activity [PPARG promoter analyzed in circulating cell-free DNA obtained from patient plasma [PPARG recruit MeCP2, which promotes repressive H3K9me3-modifying enzymes to inhibit initiation of transcription. Second, transcriptional elongation is blocked by enhancer of zeste homolog 2 -mediated H3K27me3 modifications in the downstream coding region of PPARG gene, with expression of EZH2 being dependent on MeCP2. EZH2 is a histone H3K27 methylation leads to recruitment of the PRC1 Polycomb complex which causes chromatin condensation, and promotes hepatic fibrosis by repressing the transcription of PPARG. Conversely, MeCP2 has also been shown to stimulate the transcription of several pro-fibrotic genes through the control of ASH1, an H3K4/H3K36 histone methyltransferase that directly binds to the regulatory regions of αSMA, collagen 1, TIMP1 and TGF-β1 in activated HSCs, thus promoting their transcription [PNPLA3 promoter was hypermethylated in the livers of more severe (F3-4) fibrotic NASH, and was inversely correlated with mRNA levels, significantly with the GG genotype [CSL4, CRLS1, CTP1A, SIGIRR, SSBP1, and ZNF622 genes, which are differentially methylated in peripheral blood leukocytes of patients with NASH compared with those exhibiting simple steatosis. However, only differences in DNA methylation of ACSL4 were confirmed by pyrosequencing [Nutrition is closely related to DNA methylation since this epigenetic modification depends on the availability of SAM, which requires methyl donors from food  for its synthesis ,264. In and NASH . Additiopatients . As alreactivity . SIRT1-dactivity . In addit plasma . PPARγ icription ,273,274.cription . It was genotype . Smallerquencing .Gnmt) genes were inhibited and concurrently methylated in HCC, and also hypermethylated in NASH with advanced fibrosis. This result supports the hypothesis of simultaneous accumulation of CpG island methylator phenotype alterations in the disease process causing to HCC [CDKN2A, RASSF1, APC and SMAD6, while other studies described hypomethylated genes, such as IGF2, CCL20, and NQO1; as reviewed by [Alterations in DNA methylation and its machinery are commonly linked with cancer, including HCC. In cancer occurs three types of aberrations in DNA methylation: hypermethylation of the CpGIs in the promoter regions of tumour suppressor genes, altered expression of DNMTs, and global hypomethylation of genes and repetitive sequences, thereby leading to genomic instability and oncogene activation . A studyg to HCC . The majiewed by . SUV39H2 activity was investigated in knockdown mice fed a HFD, and they exhibited a less severe form of NASH compared with the control mice for this enzyme [Methylation of lysines on histones is regulated by several methyltransferases and demethylases, which is an important mechanism of epigenetic histone modifications. Generally, histone 3 lysine 4 methylation (H3K4me) is related with gene activation, while histone 3 lysine 9 methylation (H3K9me) and histone 3 lysine 27 methylation (H3K27me) lead in gene repression . With res enzyme . It was s enzyme . TherefoMOF knockdown promotes HCC growth, while its overexpression reduces HCC growth, partially through promoting of the SIRT6 expression [Histone proteins undergo various modifications such as acetylation, phosphorylation, methylation, ubiquitination, SUMOylation, and ribosylation, among which acetylations have been largely reported. Histone acetylation generated by the histone acetyltransferase (HAT) activates the gene transcription, whereas histone deacetylation catalyzed by histone deacetylase (HDAC) promotes the gene repression . p300 , in charge of the deubiquitination of both histones H2A and H2B. Histone deubiquitination is commonly associated to transcriptional activation, epigenetic regulation and cancer development . In HCC SUMOylation consist in the reversible addition of a small ubiquitin-like modifier (SUMO) group to proteins. This modification have profound effects on carcinogenesis and multidrug resistance in HCC. In the human genome the SUMO isoforms  are encoded. SUMO-1 has been found highly expressed in HCC cell lines and HCC specimens, and SUMO-2 expression is robustly correlated with patient survival rate . MoreoveH2A.Z.1 knockdown suppressed HCC cell growth, and reduced the metastatic potential of HCC cells by selectively regulating cell cycle components [macroH2A1 knockdown cell lines acquire cancer stem cells features, such as enhancement of the tumorigenic potential, slow proliferation, resistance to chemotherapy treatments, elevating in mRNA expression of reprogramming genes, and augmented glycolysis, reflecting the same phenotype of human undifferentiated and aggressive HCCs, which express a low level of macroH2A1 [Histone variants are non-canonical  variants of histones, showing one or a few amino acid differences and affect chromatin remodeling and histone post-translational modifications. For instance, in vitro studies demonstrated a potential oncogenic role for H2A.Z isoforms in hepatocarcinogenesis . In HCC mponents . Histonemponents . The promponents . It has acroH2A1 . FurtheracroH2A1 . PhosphoacroH2A1 . Taken tThere are currently no therapies indicated for use in patients with NASH, despite its prevalence and clinical significance. Although several drugs are in development with potential therapeutic and effective at reversing the histopathologic features of NASH, difficulties in identification of accurate biomarkers have retard the approval of effective therapeutics. A continuation are described summary current therapeutic strategies and pharmacological therapies for NASH.Lifestyle modification have been recommended as the main method for the nonclinical management of NASH, with several publications reporting on this. Since 1970, studies indicated that weight loss improves liver histology as evidenced by biopsies and liver enzyme levels . In geneLong-term exercise can prevent NASH development by promoting the phagocytic capacity of liver resident KCs and decreasing liver inflammation and fibrogénesis, including reducing risk of diabetes, hypertension and metabolic syndrome ,303,304.Oxidative stress is implicated in the progression of NASH and the hypothesis that an antioxidant agent as vitamin E (α-tocopherol) can attenuate liver histology has been tested in NASH experimental protocols . Severalp < 0.001), and there was also a reduction in hepatic steatosis (p < 0.001) and lobular inflammation (p = 0.004), but not with improvement in fibrosis scores (p = 0.12) [Thiazolidinediones (TZDs) including pioglitazone, troglitazone, and rosiglitazone are a family of anti-diabetic medications, and they are high-affinity ligands for PPARγ . Pioglit = 0.12) ; suggest = 0.12) . Conflic = 0.12) .Liraglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist. Liraglutide is effective at improving indices of glycemic control, and has been approved for use in the United States, Europe, and Japan for the treatment of T2D . Also, iOne of the first antidiabetic drugs evaluated in NASH was metformin, which is a first-line anti-diabetic drug of the biguanide family. Metformin reduces hepatic glucose production and promotes peripheral glucose utilization. It also attenuates insulin resistance via activation of the AMPK pathway and inhibits the mitochondrial glycerophosphate dehydrogenase shuttle with change in redox state . A compaObeticholic acid (OCA), a synthetic variant of natural bile acid chenodeoxycholic acid, is a ligand of FXR. In animal models, FXR activation reduces hepatic glucogenesis, lipogenesis, and steatosis . Clinicap = 0.02 for interaction), thus the I148M PNPLA3 risk variant limited this beneficial effect [Statins have anti-oxidant and anti-inflammatory effects due to their lipid lowering action . A prospl effect . Based ol effect . Additiol effect . A retrol effect .Omega-3 polyunsaturated fatty acids (PUFAs) include α-linolenic acid , stearidonic acid , eicosapentaenoic acid , docosapentaenoic acid , and docosahexaenoic acid . Several experimental and epidemiological studies have shown that the ω-3 PUFAs decrease the risk of cancer, which is also supported by several clinical studies . CapanniPentoxifylline (PTX), a methylxanthine derivative, is an inhibitor of TNF-α and decreases oxidative stress by increasing hepatic glutathione synthesis . Zein etHepatic fibrosis grade is the most important determinant of mortality in NASH patients . TherefoCenicriviroc (CVC), a C-C motif chemokine receptor-2/5 (CCR2/5) antagonist, has anti-inflammatory and antifibrotic effects, and also improves insulin sensitivity . CCR5 anPirfenidone (PFD) is an orally bioavailable pyridone derivative, which has antifibrotic, anti-inflammatory, and anti-oxidative effects, and clinically used for the treatment of idiopathic pulmonary fibrosis ,342. PFDThe development of HCC involves dysregulation of several biological processes such as cell cycle, apoptosis, and many other cellular pathways. Tumor cell progression is promoted by mutations in various proteins implicated in the regulation of cell cycle. Therefore, the research for the treatment of HCC is focused on proteins target such as CDKs or growth factors to suppress the tumor development . The proIn 2020, the combination of bevacizumab and atezolizumab was approved by the FDA, thereby is the new standard of care for the systemic first-line treatment in unre-sectable HCC . Atezolip = 0.173, respectively) [Sorafenib is a multikinase inhibitor targeting PDGF-A/B, VEGFR1-3, c-kit, RET, and various proteins of the kinase cascade, Ras, C-Raf, B-Raf, ERK, FLT-3, JAK/STAT as well as MAPK, contributing to its antitumor effect by decreasing tumor cell proliferation, inducing apoptosis, and suppressing angiogenesis ,348. In ctively) . In micectively) . In a huctively) , supportLenvatinib was approved by FDA in 2018, and is a multiple receptor kinase inhibitor targeting VEGFA, VEGFC, VEGFR1-3, fibroblast growth factor receptor (FGFR) 1-4, stem cell factor (SCF), PDGFRα, ret proto-oncogene (RET), and kitproto-oncogene (KIT) to reduce angiogenesis and lymphoangiogenesis ,348,350.Several drugs are licensed for second-line treatment of advanced HCC by FDA including regorafenib, cabozantinib, and ramucirumab, which are multikinase inhibitors .Regorafenib was approved in 2017 as a second-line oral drug for unresectable HCC . It has In 2019, the FDA approved cabozantinib for patients with HCC previously treated with sorafenib. It is a multikinase inhibitor targeting VEGFR1-3, MET, the TAM family of kinases , RET, ROS1, KIT, TRKB, FLT-3, and TIE-2, which are mostly involved in tumor growth, angiogenesis, and immune regulation ,348,370.Ramucirumab is an IgG-1 recombinant monoclonal antibody that inhibits VEGF-2 receptor (anti-angiogenic) and was approved in 2019 as a second-line option upon progression on sorafenib in those with elevated alpha-fetoprotein (AFP) ≥ 400 ng/mL  ,373. TheIn conclusion, NASH and HCC have a high prevalence in the world because they are closely associated with pandemic diseases such as obesity and diabetes, including metabolic syndrome and NAFLD. In the next decades these diseases will be challenge for the health care system and governments due to its high growing worldwide, especially for HCC. Although new therapies have emerged in the last decade, there not effective treatments for NASH and NASH-derived HCC, which could be related to incomplete understanding of the complexity of the mechanisms involved in the onset and progression of both NASH and HCC. To note, HCC tumors have a high heterogeneity and different genetic mutations as well as genetic disorders in the form of polymorphisms that could be determining effectiveness of current therapeutic treatments. Therefore, ongoing clinical trials should considered the most representative polymorphisms, genetic mutations, and molecular profiles to improve screening and surveillance strategies to identify biomarkers to guide personalized treatment for HCC in NASH early and delay its progression. Future directions of targeted therapy for NASH and NASH-associated HCC should be focused on several targets of pathological events driving the progression of disease and identification of biomarkers to predict treatment response of therapy. Currently, several trials are testing immune checkpoint inhibitors in combination with anti-angiogenic agents, and primary results are promising.


The advent of graphene opens up the research into two-dimensional (2D) materials, which are considered revolutionary materials. Due to its unique geometric structure, graphene exhibits a series of exotic physical and chemical properties. In addition, single-element-based 2D materials (Xenes) have garnered tremendous interest. At present, 16 kinds of Xenes  have been explored, mainly distributed in the third, fourth, fifth, and sixth main groups. The current methods to prepare monolayers or few-layer 2D materials include epitaxy growth, mechanical exfoliation, and liquid phase exfoliation. Although two Xenes  have not been synthesized due to the limitations of synthetic methods and the stability of Xenes, other Xenes have been successfully created via elaborate artificial design and synthesis. Focusing on elemental 2D materials, this review mainly summarizes the recently reported work about tuning the electronic, optical, mechanical, and chemical properties of Xenes via surface modifications, achieved using controllable approaches  to broaden their applications in various fields, including spintronics, electronics, optoelectronics, superconducting, photovoltaics, sensors, catalysis, and biomedicines. These advances in the surface modification of Xenes have laid a theoretical and experimental foundation for the development of 2D materials and their practical applications in diverse fields. Graphene, initially isolated from graphite via mechanical exfoliation, possesses a layered honeycomb structure and exhibits fascinating electrical and thermal properties . SubsequOver the past few years, many studies have focused on searching for other 2D Xenes with distinctive and exciting properties beyond graphene a 18,19],1918,19]Even though 2D Xenes have demonstrated great potential in numerous applications, their intrinsic properties usually constrain the expansion of these applications. For instance, despite the high mobility, the gapless band structure in graphene restricts its applications in electronic devices. Therefore, finetuning the properties of 2D Xenes plays an essential role in overcoming their intrinsic constraints ,35,36,37The material properties  of 2D Xenes are not only determined by their chemical compositions but are also strongly associated with the atom arrangement in the lattice. Due to the preferred orbital hybridization of various elements in the main group, 2D Xenes have been theoretically predicted or experimentally verified to possess allotropes with diverse crystal lattices . The rep1−ν☐ν; ν represents the vacancy concentration) with various vacancy concentrations and 2D aluminene with various forms Galleneneubstrate .Graphene. The sp2 hybridization of carbon atoms leads to the formation of flat honeycomb graphene with a σ bond between neighboring carbon atoms 118] but of 1.7 K f,g 52]..c of 1.7Silicene. Silicene, the silicon analog of graphene, was first predicted in a theoretical study . . 168]. BB-doping . With a B-doping . In addiB-doping . When axB-doping . MoreoveB-doping .Arsenene. Arsenene mainly has two kinds of allotropic structures, including buckled and puckered . T. T4 clusnochains b,c 24]..4 clustenochains d,e 177]4 clusternochains f, indicaf 30.8 K . Indeed,f 30.8 K . Meanwhif 30.8 K . In addif 30.8 K .Antimonene and bismuthene. The most stable structures of antimonene and bismuthene are α-form (puckered) and β-form (buckled), which are the monolayers of black and gray bulk allotropes, respectively. Monolayer β-Sb and β-Bi possess indirect bandgaps of 2.28 and 0.99 eV, respectively, whereas the direct bandgaps of monolayer α-Sb and α-Bi are 1.18 and 0.36 eV, respectively [ectively ,182,183.ectively ,185,186.Doping with 3d transition metal atoms for antimonene can lead to significant changes in the bandgap and the magnetic moment . For Cr-Selenene and tellurene. Selenene has been predicted to have three allotropic structures, including a 1T-MoS2-like structure (t-Se or α-Se), a tiled helical-chain structure (c-Se), and s square structure (s-Se) [2-like, rectangle, and 2H-MoS2-like structures, respectively [e (s-Se) . Both t-e (s-Se) . Howeverectively . It was ectively . Therefoectively ,196. MeaAs mentioned above, 2D Xenes possess various physical and chemical properties, such as flexibility, layer-dependent semiconducting, high carrier mobility, molecule and light sensitivity, topologically nontrivial band structures, etc.c of 30.8 K [2/graphene hybrids decorated by CdS nanocrystals can act as high-performance photocatalysts for H2 evolution under visible light irradiation [In order to utilize 2D Xenes more effectively, surface modifications become particularly important  to tune f 30.8 K . Iodine-f 30.8 K . Graphenf 30.8 K . MoS2/gradiation . Moreoveadiation . For exaadiation . Polyethadiation . OverallIn total, except graphene, 15 different elemental 2D materials of the main group elements have been experimentally created or theoretically predicted to date. In fact, 14 Xenes, including borophene, graphene, silicene, phosphorene, gallenene, germanene, arsenene, selenene, stanene, antimonene, tellurene, thallene, plumbene, and bismuthene, have been successfully grown on proper substrates using epitaxial methods. Although a lot of research, engineering, and development related to 2D Xenes—most of which were investigated theoretically—has been reported in recent years, experimental studies are still desperately required for the development of synthesis strategies and novel applications. Before realizing the surface modifications of 2D Xenes for the applications of interest, three significant challenges need to be overcome:(i) The synthesis strategies of 2D Xenes must not only ensure reliably large-scale production but also be tailored for the requirements of the application. For example, the applications of electronics and batteries demand low costs and scalable techniques, while plasmonics and spintronics require high fidelity and reproducible techniques. Hence, reliable synthesis approaches play a crucial role in functional design and practical applications.(ii) The strategies to enhance the environmental stability and mitigate the degradation of 2D Xenes must be taken into consideration for certain applications. For instance, an optoelectronic device based on black phosphorene must be concerned with ambient stability, requiring appropriate encapsulation or the passivation of the surface.(iii) Many of the predicted and fascinating properties of 2D Xenes require further efforts to find strategies for their implementation, which may offer opportunities to discover revolutionary technologies. In addition, the exciting and novel properties necessitate not just “one-off” research, but also statistical evaluation to ascertain their viability and accessibility on a commercial scale.Despite the challenges ahead, the exceptional properties of 2D Xenes will significantly impact future applications in various fields. It is hoped that this review will inspire more exhilarating discoveries and applications in the growing family of 2D Xenes.