Capreolus pygargus bedfordi, whose genome was 16,357 bp long. There were 13 protein-coding genes (PCG), two ribosomal RNA genes (12S rRNA and 16S rRNA), 22 transfer RNA genes, and one control region. Nine PCGs started with ATG, while NAD2, NAD3, and NAD5 genes commenced with ATA, and ND4L began with GTG. ND6 and eight tRNA genes were encoded on the L-strand. These results provide newer molecular information, which contribute to its molecular and phylogenetic studies, and genetic diversity conservation.Due to the multiple causes, the population of roe deer has declined significantly. In this study, we analyzed the complete mitogenome of  Capreolus) belongs to Cervidae, including two species: the European roe deer (Capreolus capreolus) and the Siberian roe deer (Capreolus pygargus). The Chinese roe deer belongs to Siberian roe deer, which has two subspecies: Capreolus pygargus pygargus and Capreolus pygargus bedfordi  was downloaded from NCBI as the reference sequence to design primers (13 pairs). All primers were used to amplify the complete mitogenome of roe deer. Detecting products by gel electrophoresis and then bidirectionally sequenced those products. Sequences were edited and compared by SeqMan Software  and stored in Sichuan Agricultural University Museum (number 000758). The total genomic DNA was extracted by the standard phenol–chloroform method , 22 transfer RNA genes and 1control region (D-loop). The base composition of the sequence included 33.5% A, 30.02% T, 23.14% C, 13.34% G with an apparently AT biased (63.52%). For all protein-coding genes (PCGs), 5  had incomplete stop codons, 8 had complete stop codons, of which ATP8 ended with TAG, CYTB with AGA, and the remaining 6 with TAA. Nine PCGs started with ATG, while NAD2, NAD3, and NAD5 genes commenced with ATA, and ND4L began with GTG. ND6 and eight tRNA genes were encoded on the L-strand while others on the H-strand. The lengths of 12S rRNA and 16S rRNA were 955 and 1569 bp, respectively, and separated by the tRNA-Val gene. In the end, the D-loop was 1143 bp in length.The total length of the complete mitogenome sequence was 16,357 bp (GenBank accession number MN813763). There were 13 protein-coding genes (PPG), two ribosomal RNA genes  were used to establish a maximum-likelihood (ML) phylogenetic tree in MEGA6  sister branch. This result provides more molecular and evolutionary information about this species.The complete mitogenome of


RET proto-oncogene. In this review, we propose “5P” strategies for management of MEN2: prevention, prediction, personalization, psychological support, and participation, which could effectively improve clinical outcomes of patients. Based on RET mutations, MEN2 could be prevented through prenatal diagnosis or preimplantation genetic testing. Identification of pathogenic mutations in RET can enable early diagnosis of MEN2. Combining RET mutation testing with measurement of serum calcitonin, plasma or urinary metanephrine/normetanephrine, and serum parathyroid hormone levels could allow risk stratification and accurately prediction of MEN2 progression, thus facilitating implementation of personalized precision treatments to increase disease-free survival and overall survival. Furthermore, increased awareness of MEN2 is needed, which requires participation of physicians, patients, family members, and related organizations. Psychological support is also important for patients with MEN2 to promote comprehensive management of MEN2 symptoms. The “5P” strategies for management of MEN2 represent a typical clinical example of precision medicine. These strategies could effectively improve the health of MEN2 patient, and avoid adverse outcomes, including death and major morbidity, from MEN2.Multiple endocrine neoplasia type 2 (MEN2) is a neuroendocrine cancer syndrome characterized by medullary thyroid carcinoma, in combination or not with pheochromocytoma, hyperparathyroidism, and extra-endocrine features. MEN2 syndrome includes two clinically distinct forms subtyped as MEN2A and MEN2B. Nearly all MEN2 cases are caused by germline mutations of the  RET) proto-oncogene, with the exception of two families having germline mutations in ESR2 or MET gene that are predisposed to MTC  is a neuroendocrine cancer syndrome characterized by medullary thyroid carcinoma (MTC), in combination or not with pheochromocytoma (PHEO), hyperparathyroidism (HPTH), and extra-endocrine features . MEN2 had to MTC , 7. OverRET gene, located on chromosome 10q11.2, contains 20 exons and encodes a tyrosine kinase (TK) receptor (https://www.ncbi.nlm.nih.gov/gene/5979). To date, ~90 pathogenic variants are associated with MEN2 (www.arup.utah.edu/database/MEN2/MEN2_welcome.php)  . Most REso occur , 9. The RET extracellular domain  category for developing hereditary aggressive MTC, whereas patients with RET mutations in codons 609, 611, 618, or 620 are in the “moderate risk” (ATA-MOD) category  and HPTH (20–30%), compared to mutations in codons 609 , 611 , 618 , or 620  (RET C634 mutation (96.4%), although one patient with a V804M mutation (1.8%) and another with a C611Y mutation (1.8%) have been reported , C618 (27–30%), C611 (5–40%), and C609 (15–17%)  and 11 (codon 634), which encode the cysteine-rich domain (CRD) of the category  (2). The13%; 2%) . In addireported . The prereported . Individ(15–17%) . It shouRET mutations in tandem involving V804M in exon 14 and either Y806C, S904C, E805K, or Q781R are present in a rare group of patients with MEN2B  and are of paternal origin (RET M918T mutation are in the “highest risk” (ATA-HST) category for developing aggressive MTC, whereas patients with A883F belong to ATA-H category; Of these, ~50% of MEN2B patients develop PHEO . Doul origin , 31. Patlop PHEO . A recen studies , 34. Mor studies .prevent, predict, personalize, offer psychological support, and participate, which together can be used to improve clinical outcomes for patients with MEN2. In the subsequent sections we describe these individual strategies  who wish to have offspring. Non-invasive prenatal screening (NIPS) of MEN2 using cell-free fetal DNA from maternal plasma was successfully used for couples in which only the father was RET mutation carrier by Macher et al. in the year of 2012  and preimplantation genetic testing for monogenic disorders (PGT-M), can be provided to these patients to prevent MEN2 transmission   should be performed for MEN2B cases. The entire RET coding region should be sequenced when the initial test is negative or if the patient's phenotype is inconsistent with the RET mutation that was initially identified  in four families  , 16. In CLA, HD) . For MENof MEN2B , 5. Germividuals : (1) all regions –59. Recefamilies . These rN2 cases , 59, 60.RET mutations testing with measurement of bCtn/MN/NMN levels can be used to accurately predict disease progression and prognosis. The levels of bCtn were shown to be closely correlated with MTC burden, with initially bCtn levels >40 pg/mL be considered local nodal metastases, >500 pg/mL be considered distant metastases, and >3,000 pg/mL be considered wide-spread metastases and an inability to cure the patient despite aggressive surgery  and free plasma or 24-h urinary metanephrine/normetanephrine (MN/NMN) , 61. Thu surgery . In addi surgery . Recentl surgery , 65. Ide surgery . Meanwhi surgery .RET allele before MTC develops or while it is clinically unapparent and confined to the gland, is currently the most effective method for preventing or curing MEN2-related MTC. The timing of thyroidectomy is mainly based on the perceived clinical behavior of the specific RET mutation causing MEN2-related MTC , PTT should be performed in the first year of life, which was in accordance with findings by recent study conducted by Castinetti et al. (including our group) demonstrating that PTT performed when patients were 1 year old or younger was associated with a high probability of cure (20 patients) . Giv. GivRET n levels , 68 (Tabn levels . For MENatients) . Early catients) , 5. For atients) , 56, 57.RET-defined MEN2 families. Over the past 20 years, the prognosis of these patients has greatly improved  reported in over 25% and “permanent” hypoparathyroidism (>6 months) in up to 12% of all adult patients . In lineoutcomes , 74, 79.RET-driven cancers partially due to the off-target adverse effects, the next-generation highly potent selective RET inhibitors show improved efficacy and less toxicity. Two such drugs, pralsetinib and selpercatinib, have demonstrated remarkable clinical efficacy and safety in phase I/II trails , acquired non-RET alterations (MDM2 amplification and NRAS Q61K), and activation of bypass signaling  were known mechanisms involved (P = 0.03) (not published). Thus, screening and exclusion of PHEO in female MEN2 patients who plan to become pregnant is critical.In addition, after PTT or TT, long term patient follow-up is indicated. The doubling time of bCtn and CEA are powerful prognostic indicators of MTC progression, especially the bCtn doubling time . Levels I trails , 84. Alttant MTC . In additant MTC . The mecinvolved . The optinvolved   . There a = 0.03) , 90. Fir = 0.03) . Of note = 0.03) , 94. PHEin situ on a vascular pedicle or total parathyroidectomy with a heterotopic autograft .–99.96–99https://www.amend.org.uk/), was associated with a substantial reduction in psychosomatic complaints  are required to promote effective management of MEN2. The physicians should be trained to increase the knowledge of MEN2, such as optimal timing and extent of surgery, to improve the long-term disease-free survival rate of patients with MEN2. Patients and their families should be aware of the risk of MEN2 and the importance of prevention, early diagnosis and early normalized treatment from recognized patient support groups to avoid misleading information obtained from digital media. They should also be encouraged to follow intervention strategies as early as possible and the children in their families should receive age-appropriate information in a friendly and caring way. More efforts, such as strengthening MEN2 treatment infrastructure; promoting of RET screening and MEN2-related research programs ; introducing relevant policies to provide subsidies or reimbursements for expensive costs of therapy; developing a more detailed MEN2 database  including the information of diagosis, treatment, and follow-up of the patients worldwide based on full respect for the privacy of patients and their families, are also indispensable to effectively avoid and reduce the likelihood of unfavorable clinical outcomes due to MEN2.During the whole process, full participation of physicians, patients and family members, government, scientific researchers, pharmaceutical companies, and non-governmental organizations which provide resources for the medical team that will help inform management  to promote the development more potent and specific therapeutic strategies . In summary, MEN2 can be managed using an approach that involves 5Ps: prevention, prediction, personalization, psychological support, and participation. The occurrence of MEN2 could be  of MEN2 , 105.X-PQ conceived and carried out the 5P strategies. The manuscript was written and approved by X-PQ, S-YL, Y-QD, Y-LS, C-MX, and M-JY. 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.


Epithelial splicing regulatory protein 1 (ESRP1) has been described as an RNA-binding protein involved in cancer development. However, the expression and regulatory network of ESRP1 in cutaneous malignant melanoma (CMM) remain unclear.  From the sequencing data of 103 CMM samples in The Cancer Genome Atlas database, the expression level of ESRP1 and its correlation with the clinicopathological characteristics were analyzed using the Oncomine 4.5, Gene Expression Profiling Interactive Analysis (GEPIA), and UALCAN tools, while LinkedOmics was used to identify differential gene expression with ESRP1 and to analyze Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Gene enrichment analysis examined target networks of kinases, miRNAs, and transcription factors. Finally, TIMER was used to analyze the relationship between ESRP1 and tumor immune cell infiltration.  We found that ESRP1 was lowly expressed in CMM tissues, and a low level of ESRP1 expression correlated with better overall survival. Expression of this gene was linked to functional networks involving the condensed chromosomes, epidermal development, and translation initiation. Functional network analysis suggested that ESRP1 regulated ribosome metabolism, drug metabolism, and chemical carcinogenesis via pathways involving several cancer-related kinases, miRNAs, and transcription factors. Furthermore, our results suggested that ESRP1 played an important role in regulating tumor-associated macrophage polarization, dendritic cell infiltration, Treg cells, and T cell exhaustion.  Our study demonstrates ESRP1 expression, prognostic value, and potential regulatory networks in CMM, thereby shedding light on the clinical significance of ESRP1, and provides a novel biomarker for determining prognosis and immune infiltration in CMM. Melanoma, a common malignant tumor originating from skin melanocytes, is characterized by high invasiveness –3. Accor3, encoding 682 amino acids. As a member of the hnRNP family, ESRP1 plays a vital role in organ formation, including craniofacial and epidermal development, branching morphogenesis of the lungs, and salivary gland development. Recent studies have found that ESRP1 regulates the alternative splicing of multiple genes, including CD44, CTNND1, ENAH, and FGFR2, thereby affecting intercellular adhesion, cytoskeleton, and cell migration [Epithelial splicing regulatory protein 1 (ESRP1) was previously called RBM35A. The gene is located on chromosome 8q22.1, with a sequence length of 2046 bp and a relative molecular weight of 78 × 10igration . Hence, igration . HoweverIn this study, we aimed to systematically explore the gene expression, prognostic values, immune correlations, and potential functions of ESRP1 in CMM. The correlation between ESRP1 levels, clinical parameters, and tumor immune infiltration was comprehensively analyzed. Moreover, we also explored the prognostic value and functions of ESRP1 in CMM. These findings suggest that ESRP1 plays an important role in the clinical prognosis and immune regulation of CMM.http://www.oncomine.org) is a large oncogene chip database and integrated data mining platform, containing 715 datasets and 86733 samples that is established for collecting, standardizing, analyzing, and delivering cancer transcriptome data [P value of 0.05, a fold change of 2, and a gene rank in the top 10%.Oncomine 4.5 (ome data . In the http://gepia.cancer-pku.cn), a freely available comprehensive web-based tool, analyzes expression data at the transcriptional level with 9,736 tumors and 8,587 normal samples from TCGA and GTEx projects. GEPIA was used to analyze the expression and prognostic value of ESRP1 in melanoma.GEPIA  is a newly developed interactive web server for facilitating tumor subgroup gene expression analyses based on data from TCGA and MET500 [UALCAN (d MET500 . The corhttp://www.linkedomics.org) is a flexible, user-friendly portal providing analysis and comparison of cancer multiomics data across 32 TCGA tumor types [LinkedOmics (or types . We firshttp://www.genemania.org) is a flexible portal that can analyze the functions of gene lists and find neighboring genes by constructing a protein-protein interaction (PPI) network [GeneMANIA ( network . GeneMANhttp://www.genemania.org) is an immune infiltrates analysis tool that can provide various analyses with a dataset of 10,897 samples [TIMER ( samples . ESRP1 e samples –19. The P < 0.05). Data from Riker et al. [P value of 9.12E-5 and a fold change of -4.472 (P < 0.05). We then analyzed the correlation between the level of ESRP1 and clinicopathologic features in melanoma. We found no significant difference in the subgroup analyses by age, gender, weight, and race . However, there was no significant difference between the high ESRP1 level group and the low ESRP1 level group with regard to disease-free survival (P = 0.18).We also explored the significance of ESRP1 in the prognosis of patients with CMM. Consequently, we found that the CMM patients in the higher ESRP1 level group had poor overall survival, while patients in the low ESRP1 level group had good overall survival , DMKN , and GPR1 . However, a strong negative correlation was obtained between ESRP1 and the expression of RGS8 , SLC22A6 , and OGG1 . GSEA was performed to analyze the GO functional enrichment. The results demonstrated that the expression of ESRP1 is linked to functional networks involving the condensed chromosome, epidermis development, and translational initiation , G protein-coupled receptor kinase 3 (GRK3), protein kinase cAMP-activated catalytic subunit beta (PRKACB), protein kinase cAMP-activated catalytic subunit gamma (PRKACG), and protein kinase, X-linked (PRKX) . The topMoreover, GeneMANIA was used to construct a protein-protein interaction (PPI) network to reveal correlations among genes for the kinases CDK1, miRNA-138, and ETF_Q6. As a result, the gene set enriched for kinase CDK1 was involved in the regulation of mitosis, nuclear division, organelle fission, microtubule cytoskeleton organization, and chromosome segregation . The genP = 1.76e − 08), CD8+ T cells , CD4+ T cells , macrophages , neutrophils , and dendritic cells .As shown in In order to analyze the potential of ESRP1 as an immune biomarker in CMM, we further analyzed the association between ESRP1 and immune cells. As expected, after adjusting for purity, the data demonstrated a strong association between ESRP1 levels and most immune biomarkers of a variety of immune cells and different T cells in CMM .Specifically, the expression level of ESRP1 was significantly associated with most marker sets of monocytes, TAMs, and M2 macrophages, revealing that ESRP1 may mediate macrophage polarization in CMM. Moreover, low ESRP1 expression is related to high infiltration levels of DCs in CMM. A significant correlation was obtained between ESRP1 expression and expression of DC biomarkers such as HLA-DPB1, BDCA-1, BDCA-4, and CD11c, thus demonstrating a strong relationship between ESRP1 and DC infiltration. In addition, ESRP1 expression negatively correlated with FOXP3, CCR8, STAT5B, and TGFB1 in CMM for Treg cells. Furthermore, ESRP1 expression negatively correlated to PDCD1 (PD-1), CTLA4, LAG3, TIM-3, and GZMB in CMM for T cell exhaustion. Previous studies have demonstrated the significant role of PD-1, CTLA4, and TIM-3 in the immunotherapy of various types of cancers –23. ThusCMM is a highly malignant cancer, and metastatic melanoma often leads to a poor prognosis. Nevertheless, its immunogenicity allows intervention via immunotherapeutic strategies such as cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed death-1 (PD-1) inhibitors, which are considered important for the treatment of malignant melanoma , 24. RecESRP1, as a member of an RNA-binding protein family, is an exquisitely epithelial cell-type-specific splicing factor that regulates splicing genes involved in tumor progression , 27. EarTo gain more information about the potential functions of ESRP1 and its regulatory network, we performed target gene analyses of tumor data from public databases. We found that ESRP1 mRNA levels were significantly downregulated in CMM tissues compared to those in normal tissues. Patients with low ESRP1 expression had relatively good overall survival. Related functional networks are involved in epidermal development, translation initiation, ribosome metabolism, drug metabolism, and chemical carcinogenesis. This is consistent with the physiological function of ESRP1 . ESRP1 sSox2 in CDK1-overexpressed cells can significantly inhibit CDK1; hence, the CDK1-Sox2 interaction is a potential therapeutic target in cancer [Enrichment analysis found that ESRP1 in CMM is associated with a network of kinases, including CDK1, GRK3, and PRKACB. These kinases regulate mitosis, cell cycle, and cell proliferation , 33. In n cancer .miRNAs are small noncoding ribonucleic acid molecules that affect biological processes, including cell proliferation, differentiation, and migration , 36. OurWe found that the top five important transcription factor target networks are ETF, E2F, EN, USF, and CEBPB. ETF, E2F, and SP-1 participate in the cytokine-independent proliferation of mouse hepatocytes . FurtherAnother important aspect of our study is that ESRP1 is negatively related to infiltration of DCs and Treg cells such as FOXP3. It is well known that DCs can promote tumor metastasis by upregulating Treg cells and downregulating CD8+ T cell cytotoxicity . FOXP3 pOur study provides a multilevel evidence for the role and potential of ESPR1 as a molecular marker in CMM. However, further studies are required to validate our findings and thus promote the clinical utility of ESRP1 serving as a prognostic indicator or immunotherapy target in CMM.In summary, our study highlights the potential utility of ESRP1 status in predicting response to checkpoint blockade immunotherapy and could be a prognosis biomarker in patients with CMM.


Overall, embryos cultured under physiological O2 tension (5%) have improved development compared to embryos cultured under atmospheric O2 conditions (20%). The mechanisms responsible for this remain unclear. This study aimed to evaluate the effect of physiologic (5%) or atmospheric O2 (20%) tension on the microscopic ultrastructure of pre-implantation mouse embryos using Transmission Electron Microscopy (TEM). Embryos flushed out of the uterus after natural mating were used as the control. For use as the control, 2-cells, 4-cells, morulae, and blastocysts were flushed out of the uterus after natural fertilization. In vitro fertilization (IVF) was performed using potassium simplex optimized medium (KSOM) under different O2 tensions (5% and 20%) until the blastocyst stage. After collection, embryos were subjected to the standard preparative for light microscopy (LM) and TEM. We found that culture in vitro under 5% and 20% O2 results in an increase of vacuolated shaped mitochondria, cytoplasmic vacuolization and presence of multi-vesicular bodies at every embryonic stage. In addition, blastocysts generated by IVF under 5% and 20% O2 showed a lower content of heterochromatin, an interruption of the trophectodermal and inner cell mass cell membranes, an increased density of residual bodies, and high levels of glycogen granules in the cytoplasm. In conclusion, this study suggests that in vitro culture, particularly under atmospheric O2 tension, causes stage-specific changes in preimplantation embryo ultrastructure. In addition, atmospheric (20%) O2 is associated with increased alterations in embryonic ultrastructure; these changes may explain the reduced embryonic development of embryos cultured with 20% O2.Assisted Reproductive Technologies routinely utilize different culture media and oxygen (O Preimplantation embryos show an incredible ability to adapt to different culture conditions and result in viable offspring. However, it is becoming apparent that preimplantation embryos may respond differently to different culture conditions. For example, mouse embryos cultured in different media may result in offspring with altered intrauterine growth  or abnor2) is known to profoundly affect the development of preimplantation embryos. Overall, in vitro fertilization and culture has been performed in either 5% O2 (physiologic) or 20% O2 (atmospheric) [2 concentration in the uterus [2 concentration in the oviducts of mammals has been found to range from 5% to 8.7%, while in the uterus ranges from 1.5% to 8% [2 tension in the oviduct ranges from 2.7% to 8%, and 1.5% to 2% in the uterus [2 [2 [2 in vitro corresponds to the O2 tension existent in the female reproductive tract [2 tension (5%) showed higher cleavage, implantation, pregnancy and live birth rates [2 showed a decreased number of normal-appearing mitochondria, an increase in vacuoles and an increase in mitochondria containing vacuoles, and were therefore considered abnormal compared to in vivo embryos. Vacuole formation is a sign of oocytes and pre-implantation embryo stress, frequently identified by electron microscopy (EM) [In addition, the effect of different oxygen concentration (Ospheric) ,5. Theree uterus . The O2 5% to 8% ,7. In mie uterus . Recentlterus [2 . Many stus [2 [2 ,11,12, mve tract ,14. Mammth rates ,16. Howeopy (EM) . In embropy (EM) . In addiopy (EM) .2, using in vivo embryos flushed out of the uterus as controls. Furthermore, we focused our analysis on the blastocyst stage, since embryo transfer at this stage is common, with increased occurrence of healthy singleton live birth [2 concentrations can affect, to what extent and at which stage, the ultrastructure of murine embryos cultured in vitro.In this study, we used light (LM) and transmission electron microscopy (TEM) to describe the morphological changes occurring in pre-implantation mouse embryos , following in vitro culture under physiologic (5%) and atmospheric 20%) concentrations of O% concentve birth . These dAll materials were purchased from Sigma Chemical Co.  unless stated otherwise.All experiments were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco (IACUC number AN153175-03F). All mice were kept in the University of California, San Francisco (UCSF) animal facility, with regulated temperature conditions of 21 to 23°C, a 12L:12D cycle, and ad libitum access to water and food. On average, to generate mice at each developmental stage, 5 females were mated with males (FB group) or eggs from 5 females were used to perform IVF with sperm from one male.2 in humidified air with 20% oxygen (IVF 20% group), or 5% CO2 and 5% oxygen at 37 °C (IVF 5% group). Control embryos (C) at different phases of development were flushed out of the uterus. Embryos in 2-cells, 4-cells, morula and blastocyst (n = 5/stage) stages were analyzed in triplicate.In vitro fertilization was performed as previously described. For each experiment, 5 Carworth Farms (CF1) female mice (6 weeks old) were super-ovulated by injecting 5 IU PMSG and, 42–46 h later, 5 IU hCG as described . In brieFor each experiment, 5 CF1 female mice (8–9 weeks) were super-ovulated by injecting 5 IU PMSG and, 42–46 h later, 5 IU hCG. The CF1 females were then mated to B6D2F1/J males. The next morning, after the vaginal plug was identified (the presence of the plug was considered at day 0.5 of pregnancy -E0.5), the uteri were removed and placed in a petri dish, and each horn was flushed using a 1ml insulin syringe loaded with warm culture medium. The resulting flushed embryos were collected. The collections of various developmental stages from the IVF and control groups were based on the morphology of the embryos, and were about as follows: 2-cell, C-24–28 h, IVF 38 h; 4-cell, C-30–40 h, IVF 48-50 h; morula, C-60–70 h, IVF 75–85 h; blastocyst, C-82–84h, IVF 108–110 h. Five embryos for each developmental stage were collected in triplicate, washed in PBS and immediately fixed in 2.5% glutaraldehyde  in phosphate-buffered saline (PBS). Fixed specimens were stored at 4 °C for 2–5 days and then processed as previously described ,24,25,26By LM, 2-cell embryos cultured in vitro under 5% and 20% oxygen tension presented morphological features similar to the control 2-cell embryos. Blastomere were well-preserved, and the zona pellucida had a medium thickness of 2 µm. The cytoplasm showed several vacuoles in all groups A, inset.By TEM, blastomeres of all the groups appeared well preserved. Blastomeres were spherical or ovoid and of equal size, with large, rounded nuclei B. The bl2) showed dilation of the SER and Golgi membranes, resulting in: 1) evident vacuolization of the cytoplasm, and 2) progressive vacuolization of the mitochondria, caused by the swelling and blebbing of their membranes  (not shown).TEM micrographs showed blastomeres of all the groups had a round-to-ovoid shape with large roundish nuclei, delimited by an intact electron-dense nuclear membrane. Chromatin was uniformly distributed. Nucleoli were frequently found in all groups D. MicrovSER was tubular in appearance in all groups. Mitochondria were spherical or oval, showed dense granular matrices and presented a few arch-like cristae. Compared to the 2-cell embryos, mitochondrial matrices were less dense, and had increased cristae. Several mitochondria were closely associated with SER elements. Mitochondria often showed evident vacuoles or dense bodies in their matrices E,F, partWhen analyzed by LM, blastomeres of the morulae of the control and IVF 5% groups appeared well stained and preserved, with wide inter-blastomeric spaces A, inset.By TEM, in the control and IVF-5% groups, rounded blastomeres contained round or slightly oval nuclei, delimited by intact nuclear membranes. Inside the nucleus, chromatin was dispersed with patches of heterochromatin and euchromatin, with clearly noticeable nucleoli A,B,E.Differently to the other groups, the IVF-20% group showed compacted blastomeres of polyhedral and irregular shape, containing round/oval nuclei. Both external and internal cells showed a peculiar polygonal shape D,E.In all groups, but especially in IVF embryos, several cells showed different grades of electron density. Intercellular contacts were more common compared to the previous developmental stages, with the appearance of specialized junction, especially at the apical limits of the outermost cells. Microvilli appeared larger and more abundant than in the previous stage, and projected into the perivitelline space and into the cleavage of the intercellular clefts A,E. RemnIrregularly shaped (tubular or round) mitochondria were generally distributed around the nuclei and, frequently, close to vacuoles. Mitochondrial electron-density was higher than in other embryo stages C. NumeroBlastocysts, as examined by LM, appeared roughly spherical, with well-preserved nuclei, and showed an amorphous zona pellucida (thickness 1–1.5 µM) A, inset.By TEM, the cells of the trophectoderm (TE) formed a single continuous layer of flattened cuboidal polyhedral cells A. Cells 2 embryos  increase of vacuolated shaped mitochondria, (2) increase in vacuoles in the cytoplasm, (3) presence of multi-vesicular bodies, and (4) presence of advanced compaction in morulae in the IVF 20%.2, may be responsible for altering mitochondrial morphology  [2 concentration, and mitochondrial dysfunctions could represent the crucial factor predisposing to embryo developmental failure [These morphological data are in agreement with our previous results, thus confirming that embryo culture, in particular at 20% Ohondria) . Table 3hondria) ,53. Impohondria) . The prehondria)  probablyhondria) . It is w failure ,58.In addition, IVF blastocysts showed some other minor findings; specifically, deep invaginations in the nuclear surface and lower content of heterochromatin, and interruption to the TE or ICM cell membrane.2 [2 showed 264 mis-expressed genes, while blastocysts cultured in KAA and 20% O2 showed a stunning 2133 altered genes. Pathways more commonly altered in the atmospheric oxygen conditions included RNA post translational modification, energy production, molecular transport, cell growth and organismal development [More importantly, heterochromatin was frequently observed in both ICM and TE nuclei of control and IVF 5% blastocysts, whereas the euchromatin was prominent in the IVF 20% blastocysts. Heterochromatin contains silent genes , while e2 ,37. For elopment .The presence of more or less abundant nuclear invaginations is associated with nuclear size reduction. They reflect a mechanism for the adaptation of the nuclear envelope and its lamina to a shrinking nuclear size during preimplantation development .2 concentrations, alter the normal metabolism of the developing embryos [Another important finding is that IVF embryos showed higher levels of glycogen than the control. Glycogen is physiologically present in the cytoplasm of the pre-implantation embryo. Biochemical and histochemical studies indicate that glycogen synthesis occurs mainly during the cleavage stage . Cytopla embryos ,64.Among the limitations of this paper, several findings  were qualitative and not quantitative in nature. Future work should exactly quantify the relative abundance of each component.Finally, IVF groups contained numerous residual bodies. The presence of these structures indicates increased cytoplasmic degradation and increased autophagy: in many cell types, autophagic vacuoles and related structures are the structures used for digestion of damaged organelles and mitochondria by the cell ,66.2, results in important morphological alterations. The morphological alterations found in IVF embryos reflect the reduced development of preimplantation mouse embryos during in vitro culture. We can further speculate that these morphological changes might be associated with significant changes in pre-implantation embryo metabolism.In conclusion, our results indicate that the stress of culture, especially in the presence of atmospheric O2 concentrations, and therefore optimal O2 exposure or the addition of an antioxidant in extended culture may be a key factor for improved embryo development.Overall, our data support the interpretation that preimplantation embryos are vulnerable to the environmental disturbances induced by high O


Prior analyses suggest that the effect of a genetic risk score (GRS) on fasting plasma triglyceride levels increases with the percentile of the triglyceride distribution. Postprandial lipemia is well suited for testing quantile-dependent expressivity because it exposes each individual’s genotype to substantial increases in their plasma triglyceride concentrations. Ninety-seven published papers were identified that plotted mean triglyceride response vs. time and genotype, which were converted into quantitative data. Separately, for each published graph, standard least-squares regression analysis was used to compare the genotype differences at time ABCA1, ANGPTL4, APOA1, APOA2, APOA4, APOA5, APOB, APOC3, APOE, CETP, FABP2, FATP6, GALNT2, GCKR, HL, IL1b, LEPR, LOX-1, LPL, MC4R, MTTP, NPY, SORT1, SULF2, TNFA, TCF7L2, and TM6SF2. The effect size for these polymorphisms showed a progressively increasing dose-response, with intermediate effect sizes at intermediate triglyceride concentrations. Quantile-dependent expressivity provided an alternative interpretation to their interactions with sex, drugs, disease, diet, and age, which have been traditionally ascribed to gene-environment interactions and genetic predictors of drug efficacy .Consistent with the phenomenon, genetic effect sizes increased (P≤0.05) with increasing triglyceride concentrations for polymorphisms associated with Quantile-dependent expressivity applies to the majority of genetic variants affecting postprandial triglycerides, which may arise because the impaired functionalities of these variants increase at higher triglyceride concentrations. Purported gene-drug interactions may be the manifestations of quantile-dependent expressivity, rather than genetic predictors of drug efficacy. The majority of a person’s day is spent in the postprandial state, which is characterized by the elevation of triglyceride-rich lipoproteins (TRL) . ZilversPostprandial lipemia is the consequence of the relative rates of intestinal fat absorption, TRL synthesis and lipolysis, intervascular lipid transfers, and plasma clearance of TRL remnants . FollowiQuantile-dependent expressivity describes an effect of the genotype on the phenotype that depends upon the level of the phenotype . Using qTo test this hypothesis, quantitative data were extracted from the postprandial response graphs from 97 published papers out of 128 identified as potentially relevant through Pubmed search of genetics and postprandial triglycerides or oral fat tolerance test and literature cited within each paper  4–131].–131.4–13APOA2 -265T/C genotypes (rs5082). For each genotype, average triglyceride concentrations are presented for the fasting state at time 0, and the postprandial states at 1, 2, …, 6, 8.5 and 11 hours thereafter. The average triglyceride concentration across genotypes, and average triglyceride difference between genotypes, were determined for each time point  and used to create the quantile-dependent expressivity graph of -5). Therefore, consistent with the hypothesis of its quantile-dependent expressivity, the APOA2 -265T/C effect size increased with increasing plasma triglyceride concentrations.The primary analyses are graphical as illustrated in Apo E isoforms are the most-reported genetic modifier of postprandial triglyceride concentrations, with heightened responses reported for both E2-carriers , 51,104 447X polymorphism is in complete linkage disequilibrium with the HindIII polymorphism (rs320) . El. El18]. 1 and an average triglyceride of X1(t) and genotype “2” a frequency of (1- P1) and an average triglyceride of X2(t) then the average triglyceride for the total sample at time t was P1*X1(t) + (1-P1)*X2(t). For three genotypes with frequencies of P1, P2, and 1-P1-P2 and average triglycerides of X1(t), X2(t) and X3(t), respectively, the average triglycerides at time t was calculated as P1*X1(t) + P2*X2(t) + (1-P1-P2)*X3(t). In the case of a rare genotype vs. unaffected controls, the average triglyceride concentration was taken as the mean triglyceride concentration of the unaffected controls.The analyses presented in this paper are based exclusively on the published graphs of postprandial triglyceride responses over time. Of the 128 published papers, we identified 97 papers providing plots of postprandial triglyceride lipoprotein differences between genotypes at four or more time points . The fig2adj) are presented to assess the level of correspondence between the average triglyceride differences between genotypes vs. average triglyceride concentrations at each time point, including the fasting (baseline) value. Although all of the regression models include only one explanatory variable, the adjusted R2 was used to penalize the R2 for the small number of observations (time points) used in the model. The slopes are presented with their standard error, and their significances based on the degrees of freedom .Linear regression analyses of the genetic effect (dependent variable) versus mean triglyceride concentrations (independent variable) were performed using JMP . The regression models are based on the mean triglyceride values presented in the published figures and not individual subject responses. Adjusted coefficients of determination Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Gallbladder cancer (GBC) is rare, but is the most malignant type of biliary tract tumor. Unfortunately, only a small population of cancer patients is acceptable for the surgical resection, the current effective regimen; thus, the high mortality rate has been static for decades. To substantially circumvent the stagnant scenario, a number of therapeutic approaches owing to the creation of advanced technologic measures  have been intensively innovated, which include targeted therapy, immunotherapy, and nanoparticle-based delivery systems. In the current review, we primarily focus on the targeted therapy capable of specifically inhibiting individual key molecules that govern aberrant signaling cascades in GBC. Global clinical trials of targeted therapy in GBC are updated and may offer great value for novel pathologic and therapeutic insights of this deadly disease, ultimately improving the efficacy of treatment. The other factors, which associated with chronic inflammation and disease pathogenesis, such as hepatobiliary stones, liver flukes, and Clostridium frequently observed in these areas, also constitute the other high-risk factors of bile tract cancer (BTC) including GBC.7 In addition to these regionally limited factors, plenty of ubiquitous risk factors have been documented globally and taken into account seriously, which include gallstone, gender, age, obesity, reproductive factors, race, primary sclerosis cholangitis, gallbladder polyps, congenital biliary cysts, typhoid, Helicobacter pylori infection, alcohol intake, smoking, fatty liver disease, unhealthy diet, and environmental exposure to specific chemicals.10 As a result, the early protection from carcinogenesis has been enforced in clinical practice and the occurrence of this disease only accounts for 1.2% of all cancers diagnosed in the world.Gallbladder cancer (GBC) is the most common cancer of the biliary tract system and ranked as the top six in general gastrointestinal tract neoplasms worldwide.12 The non-surgical therapies engaged in patients were primarily composed of chemotherapy and radiotherapy. However, over the past decades, additional therapeutic strategies have been continuously renovated, given rapid discoveries of the advanced technology, including next-generation sequencing (NGS), whole-exome sequencing (WES), RNA-sequencing (RNAseq), and single-cell isolation, as well as characterization that have fundamentally opened a novel view enabled to globally identify genetic and epigenetic features and key molecules as potential therapeutic targets. In particular, specific target treatment, immune therapy, vaccine therapy, biotherapy, and nanoparticles have been intensively developed in the preclinical and clinical trials. In the present review, we will focus on the targeted regimen and immune therapy as these treatments have recently received considerable attention with the hope of improving quality of life and overall survival (OS) of GBC patients in the clinic.Currently, radical resection is the most effective strategy to potentially cure GBC. Unfortunately, the population falling into this operational course is largely limited, as a large number of patients (>70–90%) can only accept non-surgical treatment. Such unfavorable outcome is because of the atypical clinical symptoms at earlier stages, contrary to the noticeable symptoms that emerge in most cases with advanced disease.15 Although there are limited data to define a standard regimen or definitive benefit, the combined therapy regimens of FOLFOX ,16 CAPOX ,18 GC (gemcitabine and cisplatin),19 and Gemox 20 still remain the mainstream chemotherapy programs in clinical trials. It is noticeable that some clinical trials have shown that combination chemotherapy with fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) yield promising results in patients with BTC.22 However, none of the single program has been widely offered, since unexpected responses were deliberately taken into account, such as systemic toxicity, insufficient drug responses, and drug resistance.23 Therefore, there are currently a large number of preclinical and clinical researches that are adding significant efforts in order to define the overall benefit of drug treatment even in the presence of adversary responses, which can be otherwise governed at a minimal level. For example, transplantation of freshly resected patient tissue of GBC in mice as a mini patient-derived xenograft (mini-PDX) model to examine individual drug sensitivity and select more effective drugs from combined agent trials for the guidance of clinic drug-selective treatment in the patient. They tested gemcitabine, oxaliplatin, 5‐fluorouracil, nanoparticle albumin‐bound nab‐paclitaxel, and irinotecan after surgery, and found that patients in the PDX-guided chemotherapy group had significantly longer median OS  15.9–21.3 months) and disease-free survival  than patients in the conventional random drug-selective chemotherapy group with mOS   3.18; 95% CI 1.47–6.91) and DFS  . Thus, mini-PDX may devise the regimes of chemotherapy to improve the outcomes; in particular, providing an optimal opportunity for personalized medicine.25Chemotherapy involves agents used for non-specific inhibition of tumor cell proliferation usually via blockade of DNA synthesis, which has been extensively engaged in the treatment of a variety of cancers. National Comprehensive Cancer Network has provided two options for GBC treatment: single-agent therapy, which is fluoropyrimidine or gemcitabine-based treatment, and multiagents regimen, which includes oxaliplatin, cisplatin, and capecitabine.26 miR-122,27 miR-223,28 miR-31,30 miR-30a-5p,31 lncRNA-HGBC,32 lncRNA-PAGBC,33 lncRNA PVT1,34 and lncRNA GBCDRlnc1.35 Concomitantly, advanced nanotechnology employing various modified materials that offer optimal cargos to efficiently deliver RNA molecules has also given rise to promising benefit in the chemotherapy of GBC. Cai and co-workers37 found that nanomaterial-induced photothermal therapy in combination with chemotherapy and chloroquine inhibited GBC cell proliferation. It is worthwhile monitoring the efficacy of this novel delivery system with RNA molecules as an alternative therapy for GBC in future.In addition to intervention of most chemotherapeutic agents on DNA synthesis, over the past decade, there has been growing research work focusing on RNA molecules that controls oncogene or tumor suppressor gene expression. It is emerging that microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) act as indispensable factors to coordinately manipulate multiple gene expression underlying carcinogenesis, in sharp contrast to the traditional paradigm that non-coding RNAs are noisy and non-functional in the regulation of gene expression. Multiple laboratories including ours have reported that miRNAs and lncRNAs mediate the proliferation, invasion, and chemotherapy resistance of GBC, and serve as new therapeutic targets for the treatment of advanced GBC, including miR-125b-5p,38 For instance, a phase II study led by the Southwest Oncology Group (SWOG) investigated the clinical outcomes of extrahepatic cholangiocarcinoma and GBC treated with adjuvant chemotherapy GC followed by chemoradiation (combined capecitabine and radiotherapy) in a single-arm study. The 2-year OS was 65% for all patients (67% for R0 and 60% for R1), the mOS time was 35 months, and only 14 patients had local recurrence, demonstrating the feasibility and benefit of adjuvant chemotherapy followed by chemoradiation therapy for GBC.39 Thus, the combination of radiotherapy is well tolerated, and has a considerable effect; however, it still needs to be confirmed by large-scale, multicenter randomized case–control phase III clinical trials.Albeit the benefit of adjuvant radiotherapy (ART) is evident in many other types of cancers, it remains unclear in GBC, since there is a lack of strong evidence of improved endpoints. Up to date, no standard regimens are available for the selection of ART; thus, combination therapy of ART with chemotherapy is usually engaged in the clinic. Some of the clinical settings have yielded encouraging results.40 Since then the efficacy of targeted therapy has been extensively investigated in multiple cancers to specifically block a number of molecular targets that are keenly associated with tumor cell proliferation, differentiation, migration, cancer stemness, vascular angiogenesis, and antitumor immune responses.42 A great volume of drugs for targeted therapy have been created, which mainly consist of small molecules and immunized antibodies. The small molecules with molecular weight <900 Da can be readily transported into cells to inactivate specific proteins or enzymes, thus inhibiting tumor cell growth,43 while therapeutic antibodies specifically bind to cell membrane receptors or their ligands to regulate cell proliferation or apoptosis.44 Some drugs were developed to target extracellular molecules that mediate angiogenesis or immune reaction in the tumor microenvironment, resulting in inhibition of tumor growth, angiogenesis, and metastasis.46 With regards to recently intense clinical research on varied specific agents that intervene intracellular signaling pathways dysfunctional in GBC .49 HER2 has been well appreciated to play a crucial role in cancer biology and is an essential functional partner for the other family member receptor binding orchestrating a heterodimer, while HER2 itself forms a homodimer as cognate ligand binding.50 HER2/HER3 interaction leads to PI3K/Akt phosphorylation and downstream signaling activation that mediates cell polarity, cell adhesion, and cell cycles.52 HER2 can also trigger mitogen-activated protein kinase (MAPK) signaling.53 Many elegant mechanistic studies on these signaling pathways reported previously were not elucidated here.HER2, also known as ErbB2, is a member of HER family, which includes HER1, HER2, HER3, and HER4, also called ErbB1 (EGFR), ErbB2, ErbB3, and ErbB4, respectively.55Abnormality of HER2 with gene overexpression and/or activated mutations has been reported in multiple cancers, such as breast cancer, colon cancer, lung cancer, stomach cancer, and BTC.56 first reported that overexpression of HER2 in the basal layer of biliary tract epithelium in transgenic mice led to the development of gallbladder adenocarcinoma at the age of 3 months. In human GBC, HER overexpression was found between 9.8 and 12.8%.57 Since 2014, we have added notable effort to gain GBC WES and identify potential mutations of ERBB family. In Fig. 58 Supporting the evidence, these activated mutations of HER2/3 in GBC cell lines resulted in a significant increase in cell proliferation and tumor development in animals,59 underscoring the essential role of HER2/3 mutations in the development of GBC. In concert with our findings, Sebastian and co-workers60 also recently demonstrated the high alteration frequency (12.6%) of HER2 in GBC and the S310 of HER2 as a hot-spot mutation.Kiguchi et al.61 found that HER2+ SNU-2670 and SNU-2773 GBC cell lines were more sensitive to trastuzumab, dacomitinib, and afatinib than HER2− BTC cell lines. In addition, in the mouse xenograft model of SNU-2670, trastuzumab as a monotherapy and in combination with gemcitabine demonstrated a stronger antitumor effect and greater cell apoptosis than gemcitabine treatment. Iyer et al.62 discovered that treatment of GBC cells isolated from patients with ErbB2-specific short hairpin RNA (shRNA), EGFR-specific shRNA, or afatinib can inhibit the invasiveness of GBC cells. Interestingly, Wang et al.63 showed that gemcitabine/5-fluorouracil increased the expressions of total and phosphorylated forms of HER2 in GBC cells, thus enhancing the cytotoxicity of trastuzumab, suggesting that sequential therapy with gemcitabine/5-fluorouracil followed by trastuzumab perhaps devises a novel and promising therapeutic strategy aiming at HER2− GBCs that are currently short of targeted drugs.There was growing research evidence reported from preclinical and clinical therapeutic trials in GBC. Ah-Rong and co-workers64 reported that a GBC case harboring HER2 mutation on the primary and metastatic site underwent HER2-targeted treatment with lapatinib and capecitabin. After two cycles of treatment, contrast computed tomography imaging showed a decrease in the size of tumor emboli and hepatic lesions. Likewise, Prieto et al.65 demonstrated favorable benefit of 5-year survival without recurrence after treatment with chemotherapy and trastuzumab on a stage IV GBC patient who presented progression during first-line chemotherapy treatment following radical savage surgery. It is emerging that the insufficiency of GBC cohorts enrolled in the studies is the primary obstacle for the clinical trial practice. In 2009, Ramanathan et al.66 reported a phase II study with lapatinib on patients with advanced biliary tree and hepatocellular cancer previously treated with no more than one line of chemotherapy. Of 17 BTC patients, 5 patients with GBC did not achieve any improvement with the treatment. In 2019, Harding et al.67 operated another phase II SUMMIT “basket” trial, a multi-histology, open-label, phase II “basket” study for patients who harbored somatic HER2 mutations and received neratinib . A total of 19 BTC patients containing 8 GBCs were recruited for a single-arm trial with neratinib monotherapy at 240 mg oral daily. Calculating all BTC patients as a whole, the confirmed objective response rate (ORR) was 10.5% (95% CI 1.3–33.1) and clinical benefit rate was 31.6% (95% CI 12.6–56.6), including two confirmed partial response (PR) and four patients achieving stable disease. mPFS was 1.8 months (95% CI 1.0–3.7). These data indicate that HER2-directed targeted therapy represents a novel and tolerable treatment approach for advanced BTCs that express somatic HER2 mutations.Inagaki et al.We have recently begun a clinical trial named “A Multicenter, Open-label, Randomized, Controlled Study of Target Therapy Based on Tumor Molecular Profiling With GEMOX in Resectable GBC Patients Monitored by ctDNA.” . A total of 54 GBC participants will be enrolled from multiple hospitals and the feasibility, efficacy, and safety of target therapy with afatinib will be evaluated and the outcomes of this study will be reported in 2 years.71 There are five distinct VEGF family members in a mammal: VEGF-A , VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PLGF). VEGFR family consists of RTK member, including VEGFR1, VEGFR2, and VEGFR3, as well as the non-tyrosine kinase co-receptors neuropilin-1 (NP-1) and NP-2. The tyrosine kinase receptors are composed of an extracellular ligand-binding region with distinct binding affinities for individual VEGF ligands, seven immunoglobulin-like loop domains, and a cytoplasmic catalytic domain.73 VEGF-A, VEGF-B, and PLGF are mainly involved in angiogenesis, while VEGF-C and VEGF-D regulate lymphangiogenesis. VEGF-A and VEGF-B display the rigorous ability to interact with VEGFR1 and VEGFR2 expressed on vascular endothelial cells and vascular smooth muscle cells.74 VEGF-C and VEGF-D have high affinity to bind to VEGFR3 expressed on lymphoendothelium, stimulating lymphangiogenesis.75VEGF/VEGFR axis plays a key role in both physiological and pathological vascularization in diseases such as tumor angiogenesis.77 instead, VEGFR1 regulates epithelial cell differentiation and migration.78 In endothelial cells, VEGFR1 is recognized as a decoy to regulate free VEGF-A as it binds to and activates VEGFR2.79 The binding of VEGF to VEGFR2 leads to phosphorylation of VEGFR2 at Tyr951 and Tyr1175, in which phosphorylated Tyr951 regulates vascular permeability mediated by SRC tyrosine kinase activation,80 whereas phosphorylated Tyr1175 of VEGFR2 recruits phospholipase Cγ (PLC-γ) and activates downstream of both MAPK cascade and PI3K/AKT pathway, stimulating endothelial cell proliferation and survival.81 Activated VEGFR3 induces the RAS/MAPK/ERK pathway and the PI3K-AKT/PKB pathway, leading to increased differentiation, migration, proliferation, and survival of lymphatic endothelial cells.83 Intriguingly, VEGFR3 was also documented to fenestrate VEGF-A/VEGFR2 signaling, participating in angiogenesis.84VEGFR1 was expressed on many types of cells, including endothelial cells, epithelial cells, inflammatory cells, and cancer cells. Interestingly, VEGFR1 does not seem to regulate endothelial cell migration or proliferation;85 Tumor necrosis factor-α (TNF-α)-induced ERK1/2-AP-1 pathway-dependent transcriptional activation of VEGF-D, leading to lymphangiogenesis and lymphatic metastasis of GBC.86 Likewise, receptor-interacting protein 1, a multifunctional protein in the TNF-α signaling pathway, was highly expressed in GBC and promoted lymphangiogenesis and lymph node metastasis via nuclear factor-κB-mediated transcriptional activation of VEGF-C.87 Dual-specificity MAP kinase phosphatase1 (DUSP1/MKP1) suppressed VEGF expression, abrogating angiogenesis in GBC mouse model.88 In addition, overexpression of miR-1 in GBC cells inhibited VEGF-A mRNA expression.89In addition to vast research evidence revealing the regulatory role of the VEGF–VEGFR axis, the cooperative or independent impacts of the axis-associated molecules in GBC have also been concomitantly explored. For example, HIF‑1α was found to promote tumor cell migration via upregulation of VEGF-A in GBC; this effect was inhibited by metformin.90 and VEGF-A was overexpressed to serve as an independent prognostic factor of survival in GBC.91 Recently, Xu et al.92 found that VEGF was notably elevated in the serum of patients with GBC and VEGF promoted angiogenesis, cell proliferation, and invasion, but inhibited apoptosis in GBC cells. Inconsistent with the data, Zhang et al.93 reported that the expression of estrogen receptor 1 (ER1) or VEGF-A alone was not correlated with OS of GBC patients. However, combined high expression of VEGF-A with ER1 predicted poor prognosis for GBC patients, suggesting that VEGF-A combined with hormone receptor ER may provide a biomarker for GBC prognosis. Resembling tumor angiogenesis, lymphoangiogenesis was known to play a central role in GBC metastasis. In a case–control study with 50 patients of GBC, 10 samples of normal gallbladder tissues and 19 samples of chronic cholecystitis, VEGF-C and -D were overexpressed in GBC tissues relative to normal or inflammation tissues.94 Strongly supporting this evidence, a number of independent studies have found that serum and tumor VEGF-C levels were increased in patients with GBC compared with healthy donors. In addition, the elevated serum VEGF-C was positively correlated with decreased OS and increased lymph node metastasis.96 In a large cohort study with 195 GBC patients and 300 healthy serum samples, polymorphisms of c.*237C > T and g.43737830A > G of VEGF gene were associated with the disease development, indicating that VEGF polymorphisms may offer a valuable marker to predict the susceptibility of carcinogenesis.97 In line with clinic trials, blocking VEGF-C by short interfering RNA or a neutralizing antibody in cultured GBC cells inhibited tumor cell proliferation and invasion. Indeed, using orthotopic xenograft models, Lin et al.98 found that inhibition of VEGF-D led to the suppression of lymphangiogenesis and lymphatic metastasis.It is well appreciated that microvessel density correlated with cancer progression, metastasis, and prognosis in GBC99 A similar single-arm phase II trial (NCT00356889) of bevacizumab in combination with erlotinib but no traditional cytotoxic drugs in patients with unresectable BTC demonstrated a response rate of 18.4%, mOS of 9.9 months, and time to progression (TTP) of 4.4 months.100 In addition, atrial of multicenter phase II study (NCT01007552) of bevacizumab in combination with GC in advanced BTCs reported a 24% PR, 8.1 months of median PFS (mPFS), and 10.2 months of mOS.101A phase II study (NCT02053376) suggested promising efficacy of regorafenib (inhibitor of VEGFR1-3) in chemotherapy-refractory advanced/metastatic BTC, which demonstrated that mPFS was 15.6 weeks, mOS was 31.8 weeks, PR was 11%, and stable disease was 44% with a disease control rate of 56%. During the courses of these clinical trials, adverse reactions included hypertension (23%), hyperbilirubinemia (26%), hypophosphatemia (40%), and hand–foot skin reaction (7%).102 Some of trial failure events were also documented. Sorafenib, a multi-kinase inhibitor of VEGFR2/3, B-Raf, PDGFR-β, and C-Raf, showed a minimal level of drug efficacy in advanced BTC in a non-randomized phase II clinical trial with an ORR of 2%, the rate of stable disease at 12 weeks of 32.6%, PFS of 2.3 months (range: 0–12 months), and a mOS of 4.4 months (range: 0–22 months).103 Agreeing with this study, a multicenter, multinational phase II study (NCT01082809) revealed that sunitinib (inhibitor of multiple RTKs including VEGFR) monotherapy showed marginal efficacy in metastatic BTC patients. The median TTP was 1.7 months, the ORR was 8.9%, and the disease control rate was 50.0%.104 VEGFR2 antagonist vandetanib monotherapy or chemo combinations did not yield noticeable benefits in PFS in advanced BTC in a phase II trial (NCT00753675).105 Furthermore, a phase I trial (NCT02443324) of ramucirumab, a fully humanized monoclonal VEGFR2-targeted IgG antibody, reported that OR rate was 4%, and mPFS and mOS were 1.6 months and 6.4 months, respectively, in advanced BTC.106 A similar new phase II study of ramucirumab in patients with advanced BTC is presently ongoing (NCT02520141). Although the discrepancy from these individual clinical studies on angiogenic blockade remains to be mechanistically deciphered, a number of potential factors may be taken into account. First, monoclonal antibodies  usually have higher specificity to bind to single proteins compared to small-molecule inhibitors that display a broader binding ability to block multiple proteins/kinases, likely eliciting off-target reactions. Second, some of the individual drug resistance cannot be neglected, as acquired drug resistance may rapidly develop in some patients who were not timely evaluated for the dynamic alterations of targeted receptors/molecules expression during the therapy. In addition, the divergent expression levels of VEGF/VEGFR and/or polymorphisms in GBC should be evaluated with respect to susceptibility to specific blockers. Finally, substantially longitudinal, case–control studies with large cohorts may be essential to establish the benefits of anti-angiogenic blockers. Nevertheless, at present, the combination regimen with angiogenic-targeted antibodies and other chemotherapeutic agents offers the optimal means to treat GBC.As a variety of anti-angiogenic inhibitors, including antibodies and small molecules, were frequently engaged in multiple cancer patients, these inhibitors have also been increasingly employed in the clinical practice for GBC patients. A multicentric phase II study of VEGF antibody bevacizumab (NCT00361231) in combination with gemcitabine and oxaliplatin in advanced BTC with a single-arm trial demonstrated that response rate was 40% and mPFS was 7 months, and OS was 12.7 months.107 Like HER2, EGFR activation triggers multiple intracellular downstream signaling cascades, including ERK/MAPK, PI3K-AKT, SRC, PLC-γ1-PKC, JNK, and JAK-STAT pathways,109 thus mediating cancer proliferation, angiogenesis, cell motility, adhesion, and metastasis.111 Elevated expression levels of EGFR were identified in non-small cell lung cancer (NSCLC) and malignant gliomas.113 In addition, constitutively activated mutations of EGFR existed in multiple cancers,115 thereby EGFR serves as a diagnostic and prognostic cancer biomarker, and also a potential target for cancer treatment.118EGFR, also commonly known as ErbB1 or HER1, was discovered to be associated with cancer development when v-ErbB oncogene of the avian erythroblastosis virus was observed in transformed chicken cells.120 TKIs primarily include gefitinib, erlotinib, and afatinib, which have the ability to bind to the ATP-binding pockets on the intracellular catalytic kinase domain of RTKs, committing to the disruption of downstream signaling.121 To date, three generations of EGFR-TKI drugs have been evolutionarily devised to fight against mutation activity of EGFR.124 Erlotinib and gefitinib, representing the first generation of these TKIs, have the ability to compete reversibly with ATP binding at the tyrosine kinase domain of EGFR. As point mutation of EGFR T790M developed during the treatment of NSCLC patients with the first-generation EGFR TKIs,127 drug resistance emerged. To improve the efficacy, the second-generation EGFR TKIs such as afatinib and dacomitinib were created, which irreversibly inhibit ATP binding at the tyrosine kinase domain.128 In some of the clinical trials with afatinib, the drug failed to reach a level by which it effectively abolishes activity of T790M mutant EGFR.130 Subsequently, the third-generation TKIs osimertinib and olmutinib were formulated.124 Both drugs exhibited robust inhibition on the mutation activities of EGFR, as a favorable responses to osimertinib and olmutinib were achieved in 50–60% of patients with the T790M mutations.132 Thus, these two drugs have been approved as the second-line treatment drugs of patients who resist to the first-generation EGFR TKIs.Identical to the earlier description regarding VEGF-targeted drugs, drugs that were developed to target EGFR mainly involve humanized monoclonal antibodies against the EGFR extracellular domain and small molecules of tyrosine kinase inhibitors (TKIs). Typical antibodies are cetuximab and panitumumab that are able to prevent EGFR from activated dimerization, thus inhibiting the downstream signaling.135 Elevated expression of EGFR in GBC tissues was positively correlated with poor prognosis of the patients.136 We also found that somatic mutations of EGFR were quite low ranging from 2.5 to 3.9% in GBC patients.59The overexpression population of EGFR in GBC was observed between 44 and 77% of patients in different independent studies.137 reported a metastatic GBC case who received a combination treatment with gemcitabine (1000 mg/m2) on days 1 and 8 every 21 days and daily erlotinib (100 mg). The disease remained for 18 months with no progression after 12 cycles of combination therapy followed by maintenance with only erlotinib for 6 months. This result indicated the potential effective responses of EGFR-TKI on GBC therapy. Philip et al.138 reported a phase II study of erlotinib in 42 patients with advanced biliary cancer, in which 16 cases were GBCs. The overall confirmed response rate was 8%  and the median TTP was 2.6 months (95% CI 2–4 months), while EGFR level was not associated significantly with clinical outcome. Lubner et al.100 published results of a multicenter phase II trial testing a combination of bevacizumab with erlotinib in 53 patients with unresectable biliary cancer . Twelve percent of patients (95% CI 6–27) had a confirmed PR. mOS was 9.9 months and TTP was 4.4 months. El-Khoueiry et al.139 reported ineffective outcomes in the phase II SWOG study on sorafenib and erlotinib in patients with 14 advanced GBCs and 20 cholangiocarcinomas. The combination of the two drugs resulted in two unconfirmed PRs  with a mPFS of 2 months (95% CI 2–3), and a mOS of 6 months (95% CI 3–8 months) in the single-arm study. Cai et al.140 employed a meta-analysis to evaluate a combination therapy of EGFR-targeted drugs  with GEMOX  in 612 BTCs. The combination of GEMOX and EGFR-targeted therapy demonstrated improved PFS  compared with GEMOX alone, although OS was not significantly different. They also found that cholangiocarcinoma exhibited significantly greater benefits with 44% reduction from targeted therapy than non-cholangiocarcinoma including GBC and ampulla of Vater carcinomas that showed only 6% reduction in cholangiocarcinoma risk. Nonetheless, more clinical settings with combined regimens targeting EGFR are essential to offer a powerful tool for clinical practice.A variety of therapeutic trials targeting EGFR in GBC patients have been completed, but endpoints were varied. Mody et al.141 RAS/RAF/MEK/ERK is the most common pathway in MAPK signaling by which a variety of cancerous cells promote cell proliferation, death, differential, cell cycle progression, apoptosis, survival, metastasis, metabolism, and angiogenesis.144 A number of membrane receptors and intracellular proteins are directly or indirectly able to activate RAS, which includes K‐Ras, H‐Ras, and N‐Ras family members.145 Once the RAS protein is activated, it recruits RAF kinase family members such as Araf, Braf, or Craf to the plasma membrane and elicits downstream MEK.147 MEK has dual-specific serine/threonine and tyrosine kinase activity and share 80% sequence homology in MEK1 and MEK2.148 Activated MEK1/2 by RAFs induce phosphorylation of ERK1 or ERK2; then, pERK1 or pERK2 dimerizes and translocates to the nucleus, where it activates transcription factors to regulate gene expression.150 Approximately 40% of all human cancers involve altered MAPK pathway, including mutations of BRAF (~10%) and RAS (~30%).151 In GBC, KRAS point mutations were detected between 0 and 41% and BRAF gene amplifications existed in 5% patients.157MAPK signaling pathway is a crucial intracellular signal transduction that regulates varied cellular activities and is frequently dysfunctional in cancer.160 Gln25His polymorphism of KRAS gene was identified to connect with GBC pathogenesis.161 Consistent with this result, KRAS rs61764370 polymorphism was intimately associated with risk and prognosis of cancers in 307 healthy controls and 541 GBCs in Indians.162 With regard to BRAF mutation frequency in GBC, it is still inconsistent, as no BRAF mutations were reported in the United States and Chile patients, but 33% of patients with BRAF mutations were present in Europe.158 However, the role of MEK/ERK in GBC has been relatively confirmed. MEK1/2 inhibitor trametinib inhibited GBC cells’ proliferation, migration, and invasion in a dose- and time-dependent manner, and induced GBC cell apoptosis in vivo and in vitro.163 Likewise, Horiuchi et al.164 demonstrated that MEK inhibitor U0126 abolished tumor liver invasion and increased survival of nude mice bearing human GBC cells.16 In addition, some traditional Chinese medicines, such as bufalin, pachymic acid, and artemisinin, had the ability to block GBC cell proliferation and invasion via interrupting MEK/ERK signaling.166 MiR-663a impaired MAPK/ERK pathway via altered regulation of EMP3 to suppress GBC progression.167 In contrast, lncRNA MALAT1, SLC25A22, and miR-101 augmented GBC cell proliferation through activating the MAPK/ERK pathway, leading to metastasis.170There was ample convincing evidence demonstrating that KRAS mutations mediate carcinogenesis of BTC by multiple research groups.171 reported that two cases yielded better therapeutic efficacy with PFS6months after receiving BRAF/MEK inhibitors treated for secondary GBC. Consistent with the report, Yu et al.172 also published an advanced secondary GBC case with a successful combination therapy with BRAF and MEK inhibitors after surgical excision, as there was no evidence of metastasis with PFS for 14 months and OS for 26 months after 8 months of treatment. Interestingly, a phase II study of GEMOX in combination with EGFR inhibitor cetuximab declared that KRAS mutations did not affect the difference in ORR and PFS between GEMOX and combination with EGFR inhibitor  in GBC,174 suggesting that the addition of cetuximab to gemcitabine and oxaliplatin did not seem to enhance the activity of chemotherapy in patients with GBC. In a phase II study of trametinib , the first generation of MEK1/2 inhibitor approved by Food and Drug Administration,175 trametinib showed safer and more effective drug responses than single gemcitabine treatment. Trametinib led to 10.6 months of PFS (95% CI 4.6–12.1), 20.0% of 1-year OS, 65% of stable disease, and 35% of PD in 20 Japanese patients with advanced BTC refractory to gemcitabine‐based therapy (NCT01943864).176 Similar results were obtained in SWOG S1310 study that recruited 44 patients (32% GBC patients) for trametinib treatment. The ORR of trametinib therapy was 10% (95% CI 0–23) vs. 8% (95% CI 0–19) seen in fluoropyrimidine therapy and the mPFS in trametinib therapy was 3.3 months in contrast to 1.4 months in fluoropyrimidine therapy.177 Some drug candidates with notable on-target adverse and toxicities events were limited to extensive clinical trials, including CI-1040 and PD0325901.179 Selumetinib , a second generation of MEK1/2 drug, was developed for the selective and uncompetitive small-molecule inhibitor of MEK1/2.180 A recent multi-institutional phase II study (NCT00553332) of selumetinib demonstrated that the drug response is of acceptable tolerability in patients with metastatic BTC, and that mPFS and mOS in selumetinib-treated cases were 3.7 months (95% CI 3.5–4.9 months) and 9.8 months (95% CI 5.97–not available), respectively.181A number of divergent therapeutic approaches in treatment with MAPK pathway blockers have been employed to unveil the potential targets for GBC patients. Giannini et al.184 A wealth of oncogenic research evidence has established the notion that PI3K/AKT/mTOR pathway is one of the most key signaling pathways remarkably upregulated in a broad spectrum of cancers that involve breast cancer,185 NSCLC,186 gastric cancer,187 hepatocellular carcinoma,188 colorectal cancer,189 pancreatic cancer,190 cholangiocarcinoma,191 and GBC.192 Transmembrane growth factor receptors VEGFR, EGFR, insulin growth factor receptor 1, G protein-coupled receptors, and RAS proteins are capable of activating PI3K that phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 subsequently binds to phosphoinositide-dependent kinase 1 (PDK1), thus phosphorylating and activating the serine/threonine kinase AKT.194 PTEN (phosphatase and tensin homolog), a tumor suppressor, is a phosphatase that dephosphorylates PIP3 into inactive PIP2, thereby dampening AKT and PDK1.196 Activated AKT can phosphorylate and activate mTOR or indirectly promote mTOR activity by phosphorylation and inactivation of tuberous sclerosis complex 1/2, a mTOR inhibitor. mTOR exists in two distinct complexes: mTOR complex 1 (mTORC1), which is composed of mTOR, Raptor, mLST8, and PRAS40; mTOR complex 2 (mTORC2) that consist of mTOR, Rictor, Sin1, and mLST8. mTORC1 activates S6 kinase 1  and promotes dissociation of eukaryotic initiation factor 4E binding protein 1 from eIF4E, stimulating cell growth and protein synthesis.198 As relatively limited understanding is available of mTORC1, the mTORC2 complex, insensitive to rapamycin, regulates actin cytoskeleton activity and controls AKT phosphorylation at Ser 473.200PI3K/AKT/mTOR signaling pathway is well known to participate in various biological and physiological cellular processes, including cell growth, mobility, differentiation, metabolic activity, and apoptosis.201 found that 90% of Pten+/− mice with a high level of phosphorylated AKT developed GBC, highlighting an active role of PI3K/AKT signaling in the transformation of gallbladder epithelial cells. Leal et al.202 demonstrated that phospho-mTOR was positive in 64.1% of GBC patients and in 24% of chronic cholecystitis cases and that a high phospho-mTOR level in immunohistochemical analyses predicted poorer prognosis in patients with advanced GBC. PI3KCA mutations in GBC were differently reported in individual studies in the world. While no PIK3CA mutations were found in Brazil patients, 12.5%, 16.9%, and 21.4% of GBC with PIK3CA mutations were identified in USA, Japan, and Chile, respectively.205 We previously found that PIK3CA mutations E545K occurred in ~5.9% of GBC, and that these patients exhibited a worse prognosis.206 Epigenetic alteration of PTEN also contributes to the development of GBC as 30% GBCs exhibited PTEN promoter hypermethylated.207Like other types of cancers that revealed important pathological impacts of the PI3K/AKT/mTOR pathway in cancer growth and metastases, mechanistic insights of GBC pathogenesis have advanced our knowledge that this pathway predominantly contributes to the initiation and progression of GBC. Lunardi et al.208 EIF3D stabilizes GRK2 kinase that activates PI3K/AKT signaling pathway, rendering GBC cells invasive.209 Exploiting the same pathway, Nectin-4 and STYK1 promote GBC cell proliferation, metastasis, and tumor growth.211 In addition, ubiquitin protein ligase E3 component N-recognin 5 decreases the degradation of PTEN/PI3K/AKT signal pathway, facilitating tumor growth.212 Given the growing evidence that lncRNAs emerge to regulate gene expression, a number of laboratories have paid considerable attention in the identification of novel lncRNAs that prompt GBC progression. We recently discovered that lncRNA-HGBC stabilized by HuR promotes GBC cell proliferation, migration, and invasion by regulating miR-502-3p/SET/AKT axis.32 Analogous to our findings, PABPC1-stabilized lncRNA-PAGBC acted as a miRNA sponge to activate the PI3K/AKT/mTOR pathway, committing to tumor growth and metastasis.33 Specificity protein 1-induced lncRNA LINC00152 up-regulates PI3K/AKT pathway and contributes to GBC cell growth and tumor metastasis.213 Jin et al.214 demonstrated that miR-143-3p targets ITGA6 to inhibit PI3K/AKT pathway, thus suppressing GBC growth and angiogenesis. Overall, the identification of a great number of key factors has pointed to novel targets potential for therapy in GBC. Several inhibitors targeting the PI3K/AKT/mTOR pathway, including A66, Wortmannin, and LY294002, have been demonstrated to inhibit GBC cell proliferation, migration, and invasion both in vitro and in vivo.217 In addition, rapamycin, RAD001, and AZD8055 reported by Leal et al.218 were able to block mTOR and inhibit the growth and migration of GBC cells in vitro. In a transgenic mouse model, rapamycin can also inhibit the incidence of GBC.219 OSI-027 blocked mTOR, enhancing the sensitivity of GBC cells to 5-fluorouracil.220 Some of the traditional Chinese medicines, such as bufalin, liensinine, and dioscin, also received striking attention capable of inhibiting GBC cell proliferation and inducing cell apoptosis via targeting the PI3K/AKT pathway.222Accumulating research evidence, not limited to PI3K, AKT, and mTOR, has also unveiled tumor-promoting function of important molecules that manipulate activation of the PI3K/AKT/mTOR pathway. For instance, M2 macrophages secrete CCL18 to promote GBC cell migration and invasion via activating PI3K/AKT pathway.2, Cohort I) or gemcitabine plus cisplatin  in cancers. In this clinical setting, 10 patients with cholangiocarcinoma or GBCs were abstracted for the Cohort III trial treated with three drugs. The results showed that six patients remained stable, whereas four developed a progressive stage, suggesting that the three-drug combination course may offer favorable benefit to GBC.223 A multi-institutional phase II study of MK-2206 (NCT01425879) demonstrated that MK-2206, a single-agent targeting AKT, exhibited acceptable tolerability in eight patients with advanced, refractory BC.224 Regarding the multiple drug candidates available from the preclinical trials described early, it is worthwhile interrogating if some of these agents function to block the PI3K/AKT/mTOR pathway in GBC patient, thereof offering additional opportunities for patient treatment.225In the clinic, a phase I trial (NCT00949949) was performed to determine the maximum tolerated dose (MTD) of mTOR inhibitor everolimus (5 mg) combined with either gemcitabine  was comparable to breast cancer (23%), urothelial cancer (20%), and pulmonary squamous cell carcinoma (27%).239 Mody et al.240 found that 12% patients with tumor cell-expressing PD-L1 and 55% patients with tumor-infiltrating lymphocyte (TIL)-expressing PD-1 in a total of 203 GBC patients were associated with corresponding genetic aberrations and tumor mutational burden (TMB) status. Likewise, Lin et al.241 showed that 18% of 66 GBC patients were positive for PD-L1 expressed by tumor cells. Ha et al.242 measured the serum level of the soluble form of PD-L1 (sPD-L1) in 158 advanced BTC patients, and found that patients with high sPD-L1 (≥0.94 ng/mL) showed decreased OS than patients with low sPD-L1 . Kim et al.243 observed that the mOS of low and high PD-L1 expression of 101 primary GBC cases was 50.13 ± 3.14 and 27.88 ± 6.69 months , respectively, and the mPFS was 49.48 ± 3.29 and 23.33 ± 7.47 months , respectively. These individual studies underscore that PD-L1 serves as an independent marker for the prognosis of GBC. Not surprisingly, there was a limited observation that did not fully support these earlier findings. Neyaz et al.239 recruited 174 cases of GBC and found that mOS in PD‐L1‐negative (PD-L1−) and PD‐L1-positive (PD-L1+) cases were 12.0 and 14.0 months, respectively (P = 0.546). Another study showed that PD-1+ in TILs and PD-L1+ in tumor cells did not correlate with OS or PFS, but a high density of CD8+ TILs in PD-L1− tumors was positively correlated with OS (P = 0.002) and PFS (P = 0.014), indicating that CD8+ TILs is an additional marker for disease prognosis, except PD-L1 and PD-1. Although we do not have sufficient knowledge to explain the inconsistency, the research focusing on tumor immune checkpoint proteins has opened a new era to explore their potential roles in mediating tumor ability to escape immune surveillance.There were accumulating literature reports analyzing the relationship between expression levels of PD-L1 and/or PD-1 and clinical outcomes. Elevated PD-L1/PD-1 levels were correlated with poor survival of cancer patients with NSCLC,245 TMB-H and MSI-H are potentially useful for assessing neoantigen presentation and viability of immune checkpoint inhibition.247 Weinberg et al.248 reported that PD-L1 overexpression was seen on GBC tumor cells from 19 of 237 (8.0%) tumors using IHC. Increased MSI-H and TMB-H were, respectively, seen in 1 out of 104 tumors (1.0%) and 6 out of 104 tumors (5.8%) using NGS. All of these results suggested that PD-L1 in GBCs is a therapeutic marker for immune checkpoint blockade (12%). In 2019, Kong et al.249 reported a successful immune therapy case that expressed strong PD-L1 expression (≥50%) and gave rise to substantial responses to nivolumab immunotherapy after the failure of multiple lines of therapies. Indeed, multiple clinical studies have demonstrated the efficacy of PD-L1/PD-1 inhibitors in the treatment of GBC. In a multicenter, case-controlled phase I trial named MakotoUeno (ID: JapicCTI-153098), 30 unresectable and recurrent BTC patients were enrolled into two cohort trials: patients received nivolumab only (240 mg every 2 weeks) as the monotherapy; patients received nivolumab (240 mg every 2 weeks) and cisplatin (25 mg/m2) plus gemcitabine (1000 mg/m2) as combined regimens.250 The results showed that the mOS was 5.2 months (90% CI 4.5–8.7) vs. 15.4 months (90% CI 11.8–not estimable), and mPFS was 1.4 months (90% CI 1.4–1.4) vs. 4.2 months (90% CI 2.8–5.6), respectively, for the first and second cohort trials. In a single-arm phase II study of nivolumab in BTC patients (NCT02829918), the mPFS was 3.68 months (95% CI: 2.33–5.98) and the mOS was 14.24 months (95% CI 6.64–NA).251 In addition, 6- and 12-month OS was obtained in 71.4% and 52.3% populations and 6- and 12-month PFS was seen in 35.2% and 24.1%, respectively. These results indicated that nivolumab is a manageable and effective agent for BTC patients. In the KEYNOTE-028  and KEYNOTE-158  study, BTC patients received pembrolizumab treatment.253 In KEYNOTE-028 study, objective response rate (ORR) was 13.0%  and median duration of response (DOR) was not reached . mOS and mPFS were 6.2 months (95% CI 3.8‒10.3) and 1.8 months (95% CI 1.4‒3.7), respectively. Furthermore, ORR of PD‐L1+ patients (n = 61) and PD‐L1− patients (n = 34) was 6.6% (4/61) and 2.9% (1/34), respectively. In KEYNOTE-158, ORR was 5.8%  and median DOR was NR (range 6.2–23.2+ months). mOS and mPFS were 7.4 months (95% CI 5.5–9.6) and 2.0 months (95% CI 1.9–2.1), respectively. The results showed that pembrolizumab provides durable antitumor activity, regardless of PD-L1 expression. Unlike KEYNOTE‐028 study, the trail (NCT02443324) recruited both PD-L1+ and PD‐L1− patients, in which 46.2% of PD-L1 patients underwent tumor recurrence or metastasis. ORR was 4%. mOS and mPFS were 6.4 and 1.6 months, respectively. PD‐L1+ patients had improved OS compared with PD‐L1− cases (11.3 vs. 6.1 months), but there is no difference in mPFS (1.5 vs. 1.6 months).106 All the results suggest that PD-1/PD-L1 inhibitors are safe and effective in treating GBC.Treatment of GBC patients with PD-1/PD-L1 inhibitors has emerged as a promising strategy for targeted therapies Table .244,245 260 In a phase I study led by Xie’s group (NCT01853618) showed that PFS and OS were 3.4 months (95% CI 2.5–5.2) and 6.0 months (95% CI 3.8–8.8),261 which declared that tremelimumab ) is a potential treatment strategy for patients with advanced BTC. A phase I study (NCT01938612) evaluated durvalumab (anti-PD-L1 mAb) with/without tremelimumab in Asian GBC patients. Median DOR was 9.7 and 8.5 months for single durvalumab and durvalumab + tremelimumab treatment, respectively. mOS was 8.1 (95% CI 5.6–10.1) months and 10.1 (95% CI 6.2–11.4) months for the single and dual treatment, respectively, suggesting that conjunction of anti-PD-L1/PD-1 with anti-CTLA-4 therapies may hold promising efficacy for patients with GBC. Thus, comprehensive trials utilizing anti-CTLA-4 antibody combined with anti-PD-L1/PD-1 antibodies in GBC are currently underway, as this approach yielded noticeable benefits to other types of cancer patients. Fujiwara et al.262 employed a new therapeutic tool engineering a bifunctional fusion protein that targets PD-L1 and transforming growth factor-β (M7824) to treat Asian patients with advanced solid tumors (including 40% GBC patients) (NCT02699515). In this trial, three patients discontinued M7824 treatment due to treatment-related adverse events . This endogenous interruption targeting both molecules needs to be substantially evaluated in future. While multiple clinic settings with immune checkpoint therapies combined with other therapies wait for outcomes at present, we are fully confident with this novel approach able to yield greater benefits to GBC patients, ultimately improving life quality.Combined regimens of anti-PD-L1/PD-1 checkpoint agents with other therapies have also been increasingly practiced in clinic, including additional immunotherapies, chemotherapy, and targeted therapies.263 There are four major DDR pathways identified in the cells, for example, base excision repair, nucleotide excision repair (NER), double-strand break repair and mismatch repair (MMR).264 As the intracellular events participate in the pathogenesis of GBC, DDR pathways act an active role to contribute to the development of this disease. Fang et al.265 reported that a dual-specific phosphatase DUSP1 enhances the chemoresistance of GBC via the modulation of the p38 pathway and DNA damage/repair system. Suppression of cholesterol biosynthesis by lovastatin could inhibit GBC cell proliferation, possibly through attenuating the DDR process.266DDR can execute full repairing or elimination of damaged cells to protect host organisms against possible carcinogenesis.267 have clustered 20 “direct” DDR genes  and “caretaker” genes that regulate DDR process  in BTC  by hybrid capture-based NGS. BRCA-associated BTC is uncommon as BRCA1/2 mutations were detected in 4.0% of 353 GBC samples by Spizzo. However, they found a correlation of BRCA-mutant BTC with MSI-H/dMMR, which represented an additional predictive marker for response to checkpoint inhibition.268 Consistent with these reports, Javle et al.269 identified 7.8% BRCA2 or ATM mutations in 623 advanced GBC patients.Abdel-Wahab et al.270 Hwang et al.271 evaluated the effect of ERCC1 on treatment outcomes in advanced BTC patients treated with platinum-based chemotherapy, which is the 5′ endonuclease of the NER complex to prevent damage to DNA by NER. They found that mPFS and mOS were significantly longer in ERCC1− group than in ERCC1+ group of cisplatin-treated group . Baek-Yeol Ryoo et al.272 revealed that DDR gene mutations were found in 62.5% of BTC patients (including 20.2% GBC patients), and that DDR gene mutations associated with longer mPFS  and mOS  in patients with BTC treated with first-line platinum-based chemotherapy for unresectable or metastatic disease. These results indicate that mutations in DDR genes may serve as predictive biomarkers for the response to platinum-based chemotherapy in patients with BTC.The alterations of DDR genes increase the sensitivity of anti-cancer chemotherapy and radiation treatments. The recent researches suggest that specific DDR gene mutation or expression may have an impact on response to platinum-based chemotherapy in patients diagnosed with BTC.To date, clinical trials clarifying the efficacy of DDR inhibitors in GBC patients have not been reported, but poly (ADP-ribose) polymerase (PARP) inhibitors have been developed in monotherapy that is engaged for patients with HR deficiency  and also in BRCA-like tumors. At present, a clinical trial (NCT03878095) targeting PARP is waiting for final results.275 which regulate cell proliferation, metastasis, and drug resistance. Elevated MET was associated with poor prognosis in liver cancer, breast cancer, pancreatic cancer, gastric cancer, NSCLC, cervical cancer, and colorectal cancer.285 In GBC, MET overexpression ranged from 5 to 74% of patients, and was also associated with clinical poor outcome.290 The conclusion was further supported by different groups that found a similar correlation.288 In addition, NK4, an HGF inhibitor, inhibited tumor growth and invasion of GBC in animal models.294 Inconsistent with these findings, Kim et al.295 reported dissimilar results that no such correlation between MET expression and poor prognosis was significantly obtained. Up to date, there are three categories of MET inhibitors available in the clinic: small molecules targeting MET receptors , MET receptor mAbs , and antibodies against its ligand HGF .299 A clinical trial (NCT03027284) is currently settled to evaluate the feasibility and efficacy of MET inhibitors in the treatment of GBC.MET is an oncogene encoding tyrosine kinase receptor of the hepatocyte growth factor (HGF). Once HGF binds to MET, the receptor undergoes dimerization and induces downstream signaling pathways, such as PI3K/AKT, RAS/RAF/MEK/ERK, and Wnt/β-catenin signaling,301 Magnolol, an organic compound derived from Chinese traditional medicine, was found to upregulate P53, resulting in interrupting cell cycle progression at theG0/G1 phase and inducing mitochondrial-mediated apoptosis. However, this cell death effect was prevented by pretreatment with a p53 inhibitor pifithrin-a.302 Tian et al.303 also demonstrated that apoptosis-stimulating of p53 protein 2 activated p53 and recruited macrophages via PKC-ɩ/GLI1 pathway, committing to inhibition of metastasis.TP53 is an important tumor suppressor gene and its mutations are commonly detected in 50% of almost all human cancers, such as colon cancer, pancreatic cancer, breast cancer, and hepatobiliary cancer.TP53 is known to participate in the cellular DNA damage response, and induction of cell cycle arrest and apoptosis.304 Moreover, different types of TP53 mutations in GBC were defined in Japan, Chile, and Hungary.306 It is quite interesting to mechanistically understand the genetic variations associated with geographic difference.It is of note that the expression of p53 in GBC is ethnically related to different race populations. The TP53 mutations in Greek GBC patients were lower than those in Japan and Chile GBC patients. One-third of the north Indian patients with GBC have mutations in exons 5–8 of p53 gene.309 thus serving as a cancer biomarker. We previously analyzed GBC mutagenesis and found that TP53 was ranked as the top one in a large spectrum of mutated genes in GBC.228 In addition, Singh et al.310 demonstrated that p53 is an independent prognostic factor for the poor prognosis of GBC . Supporting this notion, a variety of studies also pointed to the mutated p53 as a prognostic marker of GBC.311 P53 expression together with other factors was engaged to predict poorer prognosis of GBC, such as cyclin D1, Ki-67, p16, and MSH2.312 Interestingly, some divergent evidence was also documented in the literature. One hundred and three (44.8%) of 230 GBC cases expressed mutant p53 protein that was not correlated with clinical parameters such as tumor growth.313 Likewise, Hidalgo Grau et al.314 reported p53 protein nuclear overexpression in 41 GBCs was not associated with poor histological differentiation, gallbladder wall invasion, or patient survival. Although these distinct conclusions are still controversial, mechanistic insights should be in parallel taken into account, as a variety of p53 mutation forms together with multiple phosphorylation sites coexist, implicating dissimilar roles played by p53 in tumor development, such as tumor-promoting effects and loss of tumor suppressor activity.Overexpression and high mutation frequency of p53 protein, which has tumor-promoting signature rather than cell apoptotic activity, were correlated with a poor survival of a broad type of cancers, including GBC,315 led to a phase II clinical trial of oncolytic adenovirus ONYX-015, which intervenes the p53 pathway in 19 patients with hepatobiliary tumors, in which 15 cases expressed p53 mutations, and 5 patients had GBCs.316 In this study, 16 patients responded to the viral product in intralesional treatment, while serious toxicities (>grade 2) were rarely observed. The comprehensive analyses of this approach for therapeutic efficacy and safety remain to be clarified.Although TP53 has the highest mutation rate in GBC, very limited clinical trials targeting mutated p53 were reported to evaluate potential therapeutic benefit. Makower et al.319 In an analysis of the comprehensive genomic profiling with NGS, CDKN2A/B gene is one of the most frequently mutated genes in 108 Chinese and 107 US GBC patients. The altered rate with 26% in Chinese patients was identical to 25% seen in US populations.320 More interestingly, the coincidence of ERBB2 genetic mutations with CDKN2A/B variations in US patients was stronger  than those in Chinese cohort , which suggests that CDKN2A/B alterations were significantly associated with distant metastases.321 Our previous study showed that CDKN2A/B mutation rate was ~5.9% in GBC.228 There were other reports supporting the notion that CDKN2A/B mutations mediate the pathogenesis of GBC.322 Collectively, all the data indicate that CDKN2A/B is a potential target for GBC therapy. It was also noted that Leiting et al.323 found that CDKN2A was not associated with the survival of BTC. Nonetheless, to date, no potential drug candidates have been explored to target CDKN2A/B in cancer treatment.CDKN2A/B, a cyclin-dependent kinase inhibitor 2A/B, inhibits CDK4 and CDK6, and also prevents pRB phosphorylation, thus leading to cell cycle arrest at the G1/S phase.324 The downstream signaling of KIT mainly involves MAPK, PI3K/AKT, and JAK/STAT pathways, thus participating in the regulation of cell proliferation, differentiation, growth, survival, and migration.327 Interestingly, the expression of KIT of patients with gallstones was lower in gallbladder tissue than that in healthy subjects.328 However, in GBC, the expression of KIT was elevated.329 At present, at least 16 KIT inhibitors are available for the blockade of its activity elevated in various types of cancers, including leukemia, prostate cancer, gastrointestinal stromal tumor, and renal carcinoma.333 Typical therapeutic blocking agents employed to target KIT include cabozantinib, dovitinib, masitinib, and pazopanib.336 At present, two additional clinical trials  wait for final results.KIT is a type III transmembrane RTK expressed in a variety of human cells.337 Therefore, these clinical attempts are anticipated to be of great benefits for the treatment devises. It is concomitantly worthwhile to note that a number of unexpected obstacles coexist and even persist throughout the entire clinical trials, resulting in the elimination of a majority of drug candidates, terminating trials, or leading to testing failure. These typical unfavorable events involve apparent drug side effects or off-target toxicities particularly in the multiple chemotherapeutic drug applications, and decreased responses or no responses due to rapid development of resistance. In addition, other scenarios cannot be ignored, including individual genetic difference, tumor gene mutations, and tumor heterogeneity that otherwise coordinately bypass the targeted therapy. Hence, the therapeutic efficacy of promising drug candidates together with potentially adversary impacts should be warily taken into account. To achieve overall benefits, it is highly recommended to exploit combination targeted therapies aiming at different key pathways underpinning cancer metastasis, which expectedly yield synergistic efficacy with minimal toxicities. Of note, the newly created tumor models transplanted directly with patient-derived tumors such as PDX/PDTX and patient-derived organoids have offered a great opportunity for drug sensitivity and/or resistance screening, as it is of paramount importance for devising sensitive treatment in personalized medicine. In addition, it is also emerging that the tumor immune therapy has paved an exciting novel therapeutic avenue to improve the efficacy of GBC treatment. In context with recent findings of the key roles played by PD-L1 immune checkpoint and other signaling molecules in GBC, we have begun a multicenter clinical phase II trial (NCT03768375) employing an umbrella design, which aims to evaluate the efficacy of different courses of treatment based on genomic and proteomic profiling combined with FORFIRINOX in advanced or recurrent GBC patients , proteomic, epigenetic, and metabolomic profiling that assist evaluation of genetic signature, drug action mechanisms, and therapeutic responses.nts Fig. . We expe