Chimeric antigen receptor–engineered T cells for liver cancers,progress and obstacles

Abstract

Chimeric antigen receptor–engineered T cells therapy has become the hottest topic of immunotherapy, as its great successes achieved in treating refractory hematological malignancies. These successes also paved the road to novel strategies of treating various solid tumors including liver cancer. Many specific proteins can be expressed aberrantly in liver cancers; therefore, a series of experimental and clinical researches exploring chimeric antigen receptor–engineered T cells and liver cancer are in progress, acquiring obvious antitumor effect and revealing its feasibility in treating liver cancer. However, lots of challenges and obstacles are emerging simultaneously, such as low infiltration, side effects, safety of chimeric antigen receptor–engineered T cells, and limited data of studies or clinical trials. Researchers have been working out many innovative ways to directly stroke these obstacles, theoretically or practically. This review focuses more on the progress and obstacles from chimeric antigen receptor–engineered T cells therapy to treat liver cancer, summarizing new breakthroughs in shooting those obstacles, meanwhile, hoping to provide enlightenment to this promising immunotherapeutic method.

Keywords

Chimeric antigen receptor immunotherapy liver cancer tumor-associated antigen off-target effect cytokine release syndrome

Introduction

Liver cancer is one of the most common malignancies worldwide, and it accounts for the third leading causes of cancer-related mortality. Currently, the hepatocellular carcinoma (HCC) mortality has been decreased with the advanced progression of surgical resection and adjuvant chemotherapy. However, the overall prognosis of liver cancer is poor with 5-year overall survival rate less than 12%. Moreover, for the majority of patients are diagnosed at an advanced stage, potentially curative therapies including chemotherapy, chemoembolization, ablation, external beam radiotherapy, and proton beam therapy are frequently ineffective. Even sorafenib as the first clinically approved target drug therapy could only extend overall survival by 2–3 months.¹ Hence, new treatment strategies to prolong survival and to minimize the risk of adverse response are desperately needed for patients with liver cancer.²

Tumor immunotherapy is a promising and novel treatment strategy, as it changes the treatment object from tumor itself up to the level of the whole immune system. Several immunotherapies, such as tumor antigen therapy, immune checkpoint inhibitor, and adoptive cell transfer immunotherapy (ACT), have been used in treating liver cancer and provided critical advantages in the improvement of prognosis.³ ACT, which utilizes the efficient antitumor activity of “domesticated” cell to treat malignant tumors, has emerged as an effective treatment for some malignancies. Currently, the use of genetic engineering approaches to insert antigen-targeted receptor of definite specificity into T cells has greatly extended the potential capability of ACT. As a result, chimeric antigen receptor–engineered T cells (CAR-T) therapy, a breakthrough of ACT, has made great progress in early-phase clinical studies treating CD19-positive hematological malignancies.^4,5 Meanwhile, this promising therapy is sparking great interest by clinical doctors and basic scientist searching for novel therapeutic strategies for liver cancer, as its potential characteristic of specifically recognizing tumor-associated antigens (TAAs) and eliminating tumor cells in a non-Human Leukocyte Antigen (HLA)-restricted manner. In this review, we focus on the progress and obstacles from CAR-T to treat liver cancer, evaluating more recent improvements, describing current clinical impacts, and discussing the future prospects of this novel approach.

Adoptive CAR-T cell therapy

Adoptive T cell therapy has the property of killing tumor cells that express specific antigens. The original application of this therapeutic strategy can be dated back to 1988, when Rosenberg utilized adoptive therapy with tumor-infiltrating lymphocytes (TILs) to treat patients with metastatic melanoma.⁶ Inspired by Rosenberg’s success, subsequent studies demonstrated its clinical potential in various solid tumors, and its efficiency has been proven in some tissue including ovarian cancer (OC), renal cell carcinoma (RCC), colorectal cancer, pancreatic cancer, HCC, cholangiocarcinoma, and gastric cancer (ClinicalTrials.gov identifier NCT01174121). Although these achievements are encouraging, the majority of patients did not meet the condition of TIL therapy, since tumor reactive lymphocytes did not exist in all patients.⁷ To overcome this limitation, genetic introduction of T cell receptor (TCR) and chimeric antigen receptor (CAR) into autologous T cells, termed gene-engineering of T cell, can provide an alternative and made T cell therapy more available for more patients and multiple types of cancer. Rapoport et al.⁸ achieved sustained antigen-specific antitumor effects in myeloma with NY-ESO-1-specific TCR-engineered T cell, and Morgan et al.⁹ demonstrated objective regression of patients with metastatic melanoma with TCR redirected ones. TCR-restricted T cell possess the capability of recognizing an antigen efficiently, but there are still disadvantages limiting its applications, for instance, the restriction to HLA manner and low affinities of TCRs.¹⁰ Fortunately, CAR-T therapy as an alternative can overcome these limitations.

CAR is an artificial protein complex consisting of an ectodomain, a hinge, a transmembrane domain, and an endodomain. The ectodomain, which commonly derives from a single-chain variable fragment (ScFv), is responsible for recognition of TAA in a non-HLA-restricted manner. Therefore, the immune escape induced by downregulated expression of HLA molecular can be suppressed by CAR-T cells. The hinge and transmembrane domain make the CAR fixable and stable to guarantee CAR’s function, as Moritz and Groner¹¹ demonstrated that introduction of a hinge between ectodomain and transmembrane can enhance the engagement of CARs and antigens on cell membrane. The endodomain deriving from CD3 provides stimulating signals for activation of T cell. Currently, the modular structure has been extended from first-generation that was attached with a CD3 chain singly to second- and third-generation CARs which was attached with two or three costimulatory domain, such as CD28, CD134, ICOS, and CD137.¹² These extra costimulatory domains can provide costimulatory signals to allow T cell to pass through multiple checkpoints that regulate activation, proliferation, differentiation, and survival after receptor engagement under physiological conditions.¹³ Moreover, costimulatory elements can enhance the activity and persistence of CAR-T cells, as preclinical data of homological malignancies have suggested that costimulatory elements provide a more potent signal enhancement not only on T cells proliferation but also on survival, and same conclusion was confirmed in ovarian tumor models.¹⁴ More recently, the fourth generation CAR-T, termed as TRUCKs, was developed; this novel CAR-T was derived from normal ones which are additionally redirected with a CAR inducible expression cassette. Therefore, TRUCKs can constitutively produce and secrete transgenic therapeutic proteins like cytokine in locally restricted pattern upon the engagement of CARs and targets. Then, these therapeutic proteins deposit in the targeted lesion to exert its antitumor activity.^15,16

The CAR-T therapy is attracting more attention for its great success in treating homological malignancies. Davila et al. at Memorial Sloan Kettering Cancer Center reported on 16 patients with B cell acute lymphoblastic leukemia that they treat with anti-CD19 CAR-T cells, and 14 patients achieved complete response, accounting for 88% complete response rate. What’s more they found that this therapy was effective even in high-risk patients with Philadelphia chromosome-positive (Ph+) disease.¹⁷ Encouraged by these successes, researchers have devoted their effort to explore the potential clinical value of CAR-T for treating solid tumor. As early as 2006, Kershaw et al.¹⁸ treated 14 patients with metastatic OC with first-generation anti-FRα CAR-T; unfortunately, no promising result was achieved. There was another trial using CAR-T cell targeting carbonic anhydrase IX (CAIX), which is overexpressed on renal cell.¹⁹ Then, in recent trials, researchers demonstrated that anti-mesothelin CAR-T therapy can lead to complete remission in relapsed or refractory malignancies and malignant pleural mesotheliomas or pancreatic cancer.²⁰ The application of CAR-T therapy for solid tumor is still in the initial stage, but more and more achievements were reached, and we believed that CAR-T can be perfect for various tumors including liver cancer.

TAA of liver cancer

The first step we need to face is defining the specific TAAs. As we all know, CD19 which expressed throughout B cell development and presented on almost all B cell malignancies has been detected as an excellent TAA for generating specific CAR-T cells. In this case, we wonder what the standard for defining an ideal TAA is. Marcela et al. summarized the requirements for discriminating a suitable TAA for engineering CAR-T: (1) definite targets must be expressed on the cellular surface of definite tumors; (2) ectopic expression of the target must not be present in the essential organs or cell type, even at a low level; and (3) the target must be expressed on all the tumor cells, or alternatively, the target must be requisite for the maintenances of tumorigenic phenotype.²¹

Recently, researchers have been devoting themselves in the field of exploring the “perfect antigen,” and they really made some progression. Transferrin receptor (TfR/CD71) as a selective target for malignancy therapy has attracted spotlight due to its abnormal expression in malignant tissues compared with normal ones. Ye et al. constructed human chimeric antibody against TfR termed as D2C and confirmed that D2C has the characteristics of tumor-specific affinity to human liver cancer SMMC-7721 in vitro and in vivo. This evidence suggests that TfR may likely to be served as a TAA for certain liver cancer cells.²²

GPC3 has been demonstrated as a promising liver cancer–specific target in multiple studies, due to its overexpression in HCC and limited expression in normal tissues. Therefore, antibodies and peptide vaccine targeting GPC3 has been detected for treating patients with HCC.²³ GC33, a novel recombinant humanized antibody against GPC3, significantly inhibit the growth of GPC3-positive human HCC xenografts, but no effect was detected in GPC3-negtive HCC xenografts in mice model.²⁴ Sawada et al.²⁵ conducted a phase I trial of GPC3-derived peptide vaccine for advanced HCC, in which they demonstrated that this vaccine was well tolerated, and measurable immune response was detected in 30 out of 33 patients. These data, taken together, revealed that GPC3 could be a therapeutic target for HCC and even an antigen for engineering CAR-T.

Carcinoembryonic antigen (CEA), a cell surface glycoprotein associated with carcinomas, has been shown to be predictive of metastatic potential of patients with hepatic metastases from colorectal cancer. Frampas et al.²⁶ constructed colonic cancer model in mice and evaluated the impact of tumor location on tumor antigen direct targeting CEA with radiolabeled anti-CEA antibodies, and they revealed a high biodistribution of radiolabeled anti-CEA antibody. Moreover, Burga et al. have constructed CAR-T targeting CEA to detect its efficiency in mice model with liver metastasis (LM). And they demonstrated that regional infusion of anti-CEA CAR-T in mice with LM can delay tumor progression.²⁷ In conclusion, CEA is also a candidate TAA for liver cancer immunotherapy.

MUC1 is a transmembrane glycoprotein, whose overexpression has been widely reported in several malignant cancers, including liver cancer.²⁸ Therefore, MUC1 becomes an ideal target of immunotherapy for liver cancer, due to its overexpression. For example, Ma et al. have engineered first- and third-generation CAR-T against MUC1 to treat MUC1 overexpressed liver cancer. Their results reveal its potential of serving as TAA for liver cancer.²⁹

Other TAAs have also been explored as potential target for immunotherapy. Alpha-fetoprotein (AFP) is positive in 60%–80% of HCC, MAGE-A1 in approximately 70%, NY-ESO-1 in approximately 45%, heat shock protein 70 (HSP70) in approximately 71.9%.³⁰ Detection of all these TAAs are in progress, and we believe that more appropriate will be defined in the future research.

Preclinical and clinical development of CAR-T for liver cancer

As demonstrated earlier, there are several TAAs expressed on the surface of liver tumor cells, and these tissue-restricted antigens constitute potential targets for engineering CAR-T cells.

In 2005, researchers constructed human–mouse chimeric antibody-targeted transferrin receptor against liver cancer SMMC-7721. Subsequent studies demonstrated that this artificial antibody displayed a tumor-specificity distribution and revealed a strong antitumor effect in treating human liver cancer SMMC-7721 in vivo and in vitro.²² Initial studies using first- and third-generation CAR targeting MUC1 demonstrated that these two types of CAR-T cells can recognize MUC1 molecules and eliminate MUC1 overexpressed tumor cells specifically without damaging other normal hepatic cells. Moreover, the third-generation CAR-T presented superiority in cell proliferation, interleukin (IL)-2 secretion, and the killing effect of hepatic carcinoma cells compared to the first generation.²⁹ Gao et al. constructed first- and third-generation CAR-T cells targeting GPC3 first. In their studies, they demonstrated that the redirected GPC3-targeted CAR-T cells could efficiently lyse the four GPC3-positive HCC cell lines (HepG2, Hep3B, PLC/PRF/5, HUH7), but not the GPC3-negtive SK-HEP-1 cell line in vitro. Furthermore, they confirmed that third-generation GPC3-targeted CAR-T cells could eradicate HCC xenografts with high level of GPC3 expression and efficiently suppress the growth of HCC xenografts with low-GPC3 expression.³¹ Based on this discovery of great significance, Renji Hospital in China set a phase I clinical trial named “Autologous T Cells Redirected to GPC3 for Treating Patients With Advanced Hepatocellular Carcinoma” (Clinical Trials, NCT02395250) to appraise the safety and efficacy of GPC3-targeted CARs, which is also the first clinical trial exploring CAR-T therapy to treat liver cancers globally.

In addition to MUC1 and GPC3 presented above, researchers had also explored anti-epithelial growth factor receptor 2 (EERB2) and anti-carcinoembryonic (CEA) CARs to treat liver cancers. Morgan et al. treated an advanced colon cancer patient with LM through giving anti-ERBB2 CARs containing both humanized Herceptin ScFv fragment and optimized costimulatory signaling domains intravenously, the patient rapidly suffered an acute respiratory distress syndrome and died in 5 days. The analysis about the fatality revealed that the transfer of highly active anti-ERBB2 T cells recognized ERBB2-expressed normal lung cells and released a series of inflammatory cytokines, which caused pulmonary toxicity and edema followed by a cascading cytokine storm, resulting in multi-organ failure.³² Furthermore, a phase I clinical trial related to hepatic immunotherapy tested the safety of transhepatic arterial anti-CEA CAR-T cells infusion for unresectable CEA positive LMs; the results revealed an increase in neutrophil and lymphocyte ratios (NLR) after treatment correlated with increased CEA levels, suggesting that systemic variations of NLR and inflammatory cytokines can reflect the response to CAR-T activity within the intrahepatic space. The test also demonstrated the safety of anti-CEA CAR-T with encouraging signals of clinical activity in a heavily pre-treated population with large tumor burdens.³³ However, another research exploring anti-CEA CARs proposed that the antitumor efficacy could be inhibited through the expansion of liver myeloid-derived suppressor cells (L-MDSC) in mice, and infusing anti-CEA CAR-T cells with agents that targeted L-MDSC is a rational strategy for future clinical trials.²⁷ Objectively, the data in the research above are limited by the small number of samples not only in the phase I trial but also in the murine exploration, which decreased their clinical values.

The problems and potential solutions of CAR-T to treat liver cancers

The lack of specific antigens of liver cancers make the normal tissues which express the same antigen get attacked under CAR-T therapy, resulting in severe toxic effects, that is, the so-called Off Target Effects. Besides, the shorter survival duration of CAR-T cells in vivo, the fewer number of CAR-T cells that transfer to the solid cancers location and the inhibitive tumor microenvironment are limiting the antitumor effects of CAR-T in liver cancers.^18,34 Therefore, all these negative factors should be deeply researched and overcome in the development of therapeutic strategies for CAR-T to treat liver cancers.

Fortunately, all these limiting factors can be controlled or even removed to a certain degree as the further exploration progress. Grada et al.³⁵ had demonstrated that tandem CAR (TanCAR), a novel bispecific CAR can be used to enhance the specificity of CAR-T cells. In regard to the lower infiltration of CAR-T cells in solid cancers, Craddock et al.³⁶ and Moon et al.³⁷ had proved that the transfer ability of CAR-T cells can be enhanced by expressing functional chemokine receptors like C-C chemokine receptor type 2 (CCR2), though the direct causes of low infiltration remain unrevealed. Some investigators proposed that it is possibly because of the expression of vascular endothelial growth factor (VEGF) that suppresses T cells infiltration in the tumor microenvironment.³⁸ Moreover, selecting specific T cell subsets like Central Memory T cell (TCM) for genetic modification may also enhance persistence and activity, but the clinical trials using this approach have just been started.^39,40 Researchers had also demonstrated that not only combining CAR-T cells with immune checkpoint inhibitors or perhaps by additional engineering cotransfecting cytokines like IL-2 and IL-12 or silencing inhibitory molecules like factor-associated suicide (Fas) might protect the T cell from the inhibitory tumor microenvironment, but the vascular normalizing doses of antiangiogenic intervention can meliorate the immunosuppressive tumor microenvironment and thus enhance the effect of immunotherapy.^41–45 Except for these limiting factors, the expensive costs of production, the increased potency of second- and third-generation CARs, coupled with the lack of truly tumor-specific antigenic targets, has also increased the risks of severe toxicities.⁴⁶ Therefore, what is more challenging for CAR-T to treat liver cancers is about its clinical safety and products commercialization.

Off-target effects

As previously stated, Morgan reported a death case of colon cancer with LM induced by anti-ERBB2 CAR-T cells for off-target attack on normal lung cells. Also, in 2013, Lamers et al.⁴⁷ reported that in his treating of metastatic RCC through CAR-T cells against carboxy-anhydrase-IX (CAIX), the patients appeared different level of hepatotoxicity, and the reason was the ectopic attack of biliary epithelial cells induced by anti-CAIX CAR-T cells.

Now two methods have been worked out. The first method is the utilization of inhibitory CAR (iCAR) which relied on two different antigens.⁴⁸ Nonspecific antigen A can be expressed on either tumor cells or normal cells while antigen B can only expressed on normal ones. The gene-modified T cells can both express a CAR as antigen A recognition element and an iCAR as antigen B recognition element. Normal cells expressing both antigen A and B, when they are recognized by CAR-T cells, iCAR will produce an inhibitive signals through Programmed Death-1 or Cytotoxic T Lymphocyte-associated Antigen-4 to attenuate the T cell active signals come from CAR, which prevent T cells from activating and attacking normal cells and thereby avoid off target attacks. Nevertheless, when the modified T cells meet tumor cells that only express antigen A, the signals produced by CAR will activate T cells and therefore release granzyme and perforin to kill the tumor cells.

Another promising method is based on combinatorial antigen recognition with balanced signaling in which nonspecific antigens A and B can both express on tumor cells while normal ones only express one of them.⁴⁹ The gene-modified T cells can express both a first-generation CAR with CD3ζ as antigen A recognition element and a third-generation CAR with chimeric costimulatory receptors (CCRs) like CD28/CD137 as antigen B recognition element. When the T cells meet the normal cells that express only antigens A or B, the former cells can only provide a low-affinitive activating signals from CD3ζ without costimulatory signals from CCR while the latter cells just provide costimulatory signals without T cells activating signals from CD3ζ, which in any case provide insufficient T cells activating signals and thus protect normal cells. However, if recognize tumor cells that express both antigen A and B, the activating signals from A and the costimulatory signals from B can work together to active T cells and therefore release granzyme and perforin to kill the tumor cells. The key point of this method is the diversity of affinity in which CD3ζ holds a lower affinity with its target antigen while CCR holds a high affinity, only when the CD3ζ and CCR are binding together with their target antigens can the T cells be activated.

In addition, preclinical animal models that permit assessment of antigen-specific toxicities may also provide further insight into viable antigen targets or opportunities to evaluate strategies that minimize antigen off-target toxicities, such as the utilization of defined T cell subsets.⁵⁰

Cytokine release syndrome

After infusing abundant highly reactive CAR-T cells, especially the second and third generations, a bunch of inflammatory cytokines can be released to the blood circulation that may be related to the leakage of costimulatory signals or the lower threshold of T cells activation. This phenomenon can result in fever, hypotension, hypoxia, neurologic changes, and even acute respiratory distress syndrome or multiple organ dysfunction syndrome; this range of clinical and laboratory findings has been termed a cytokine release syndrome (CRS).⁵¹ Predicting CRS is difficult due to the limited utilization of CAR-T cells to treat solid cancers in clinical practice, which may also be different from those observed in leukemia and lymphoma, but we can also take it as a reference.

Some investigators had concluded a severe CRS requiring further intervention and a mild CRS, the former one including the presence of fever for three consecutive days, 75-fold increase of at least two of seven serum cytokines, that is, interferon γ (IFN-γ), IL-6, IL-5, IL-10, human FMS-like tyrosine kinase 3 ligand (Flt3L), granulocyte–macrophage colony-stimulating factor (GM-CSF), and Fractalkine, and one clinical sign of toxicity such as hypotension, hypoxia, or neurologic disorder. While on the contrary, mild CRS including low-grade fevers and limited cytokine increases or absent CRS requires only routine observation. Furthermore, the investigators proposed increases in serum C-reactive protein (CRP) levels (>20 mg/dL) as a surrogate marker for severe CRS, reflecting a strong correlation between CRP and IL-6 levels. Anyhow, CRS-associated toxicities require intensive medical management including support with vasoactive pressors, mechanical ventilation, and antipyretics.¹⁷ To date, the utilization of glucocorticoid or targeting block signal transduction pathways of increasing cytokines can help control the CRS.

However, the application of glucocorticoid will suppress inflammation but terminate the antitumor activity of CAR-T cells at the same time. Targeting block is a potential way to treat CRS, researchers had reported that monoclonal antibody like tocilizumab could bind IL-6 receptors specifically to block signals transmitting without damaging CAR-T cells.^52,53 But unfortunately, due to the uncertificated cytokine detection methods and higher levels of inflammatory cytokines in tumor patients from their underlying diseases, it is difficult to diagnose and quantitatively determine the severity of CRS effectively and accurately, though more studies had suggested that CRP levels in serum can be recognized as a crucial indicator for CRS recently.⁵⁴

Aiming at maximizing the chance for therapeutic benefit from the immunotherapy while minimizing the risk for life-threatening complications of CRS, researchers had outlined a proactive management strategy that incorporates a five-grading system and treatment algorithm designed to administer early immunosuppression for patients of high risk while avoiding unnecessary immunosuppression due to the potential risk of diminishing antitumor efficacy, based on their treating experiences.⁵⁵ Besides, several investigators have developed CARs targeting fibroblast activation protein-α (FAP), which expressed on the surface of cancer-associated fibroblasts as a major component of the solid cancers stoma network, hoping to avoid the toxic effect.⁵⁶ Paradoxically, some articles had reported that CAR-T cells targeting murine FAP could induce severe bone marrow toxicity while some other ones had demonstrated its antitumor benefits in the absence of toxicities in mice, indicating that more researches are needed to explain why some FAP-specific CARs induce toxicities and others do not.^57,58

Improving the safety of CAR-T therapy

Suicide gene therapy, the genetic induction of a conditional suicide phenotype into donor T cells, is the mostly used manner to increase the safety of CAR-T. Gene products will generate cytotoxicity in some occasions and kill those CAR-T cells that express these modified genes, thus avoiding potential toxic effect. Presently, herpes simplex viral thymidine kinase (HSV-TK) gene, inducible caspase-9 (iC9) gene, and epithelial growth factor receptor (EGFR) blocking gene have been researched for CAR-T and have gotten some good achievements.^59,60 Nevertheless, suicide gene therapy is a therapeutic means rather than a preventive measure which makes it not easy to determine the optimum intervening juncture. In theory, suicide gene therapy should be used before the appearance of severe toxicity resulting from CAR-T cells infusion, but the reality is that not only it is a technical defect to identify the toxicity accurately but also there are less time for suicide gene to function due to the much suddenly happened toxicity reactions. What is worse, suicide gene therapy will eradicate the antitumor effect of CAR-T cells when avoiding toxicity reaction which make it struggling to improve the safety of CAR-T therapy.

Other methods like short-lived CAR-T cells infusion and incremental dose stepped infusion are still in research, but none of them can relieve the toxicity reaction without attenuating the antitumor effect of CAR-T cells. Alternatively, researchers had attempted to study if the CAR elements could be introduced in γδ T cells or natural killer (NK) cells, which are also highly cytolytic killer cells but lack an endogenous αβ TCR, exploring to maximize the chance for therapeutic benefits from the CAR-T therapy while meanwhile minimize the risk for life-threatening complications.^61–63

Conclusion

Immunotherapy has shown its advantages in treating liver cancer with various methods, including application of cytokine, tumor vaccines, immune checkpoint inhibitor therapy, and adoptive immunotherapy. These immunotherapies change the treatment objective from tumor itself up to the level of the whole immune system. As the newest and most promising immunotherapeutic strategy of adoptive cellular immunotherapy, CAR-T has already been applied to treat some solid tumor and homological malignancies. Just like the anti-CD19 CARs and anti-mesothelin CARs can lead to complete remission in relapsed or refractory B cell malignancies and malignant pleural mesotheliomas or pancreatic cancer, respectively, we believe that breakthrough progress will be made in the treatment and improvement of prognosis of liver cancer in the short run.

In summary, at this infant stage of clinical development, CAR-T cells offer much promises to seeing this transformative therapy being delivered to more patients, but amplifying clinical activities to test CAR-T cells against liver cancers and to identify optimal protocols and treatment programs is a major goal of clinical activities in the field currently.

Footnotes

Acknowledgements

We are so grateful to all the authors who helped in writing this review: K.L. as the first author wrote and edited the manuscript; Y.L. and J.W. contributed significantly to finish this paper as coauthors; L.L., the corresponding author, devoted himself so much reviewing and editing the manuscript; and K.L. and Y.L. contributed equally to this article.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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