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Combined therapy of molecular targeted drugs and immunosuppressive agents for hepatocellular carcinoma and other combined therapies
Hepatocellular carcinoma: Targeted drugs and immunosuppressive agents. Translational therapy means that patients who are inoperable but have potential surgery can be transformed into an operable treatment mode through comprehensive measures to control tumor progression and even reduce clinical stage, improve liver function.
The incidence of hepatocellular carcinoma (HCC) ranks the fourth among malignant tumors in some countries, and the third in mortality. 70%~80% of patients have lost the opportunity of surgery when they are diagnosed, and the recurrence rate is as high as 70% in 5 years after surgery. .
Molecular targeted drugs have opened a new chapter in the systemic treatment of advanced HCC, such as sorafenib, levatinib, regorafenib, bevacizumab, ramucirumab, etc. . Recently, immunotherapy has been applied to patients with advanced HCC.
Clinical trials such as Checkmate-040 and KEYNOTE-224 [4-5] have proved the safety and effectiveness of immunotherapy in patients with advanced HCC. This article reviews the latest research progress in the combination of molecularly targeted drugs and immunosuppressive agents in the treatment of HCC, the application of other combination therapies in HCC, and the future development direction and challenges of HCC combination therapy.
1. Immunotherapy combined with targeted therapy
Vascular abnormalities are an important feature of solid tumors including HCC. This abnormality stems from the increased levels of factors such as vascular endothelial growth factor (VEGF). Tumor vascular abnormalities cause the tumor microenvironment (TME) to be in a state of hypoxia and acidosis.
By increasing the accumulation, activation and expansion of immunosuppressive regulatory T lymphocytes (Treg), and inhibiting the maturation of dendritic cells, it leads to tumor specificity.
The antigen presentation and activation of sexual cytotoxic T lymphocytes (CTL) are impaired, and abnormal endothelial cells (EC) with immunosuppressive phenotypes expand.
At the same time, in the state of TME, programmed cell death protein 1 (PD-1) )/Programmed cell death protein 1 ligand 1 (PD-L1) pathway is activated, and PD-L1 is up-regulated on CTL, EC and tumor cells.
In summary, the use of anti-VEGF molecular targeted drugs to improve tumor vascular status and anti-PD-1/PD-L1 immunosuppressive agents to inhibit tumor immune escape response theoretically have a synergistic anti-cancer effect .
1.1 “A+T” combination [Bevacizumab + Atezolizumab]
Based on the initial effective results of the phase Ib clinical trial GO30140 , the phase III, open-label, randomized study IMbrave150 trial  studied 501 HCC patients who had not received systemic treatment. The results of the study showed that the median survival time (mOS) of the two groups of patients (“A+T” vs Sorafenib) was less than 13.2 months, the 12-month OS rate was 67.2% vs 57.6%, and the median was none.
Progressive survival (mPFS) “A+T” group was extended by 2.5 months (HR=0.59, 95%CI: 0.47~0.76, P＜0.001), and the objective response rate (ORR) was 27.3% vs 11.9% (RECIST 1.1 standard) , P＜0.001), disease control rate (DCR) was 73.6% vs 55.3%, and the proportion of patients with duration of remission (DOR) ≥ 6 months was 87.6% vs 59.1%.
During the course of treatment, the most common grade 3 or 4 adverse events in the “A+T” group and sorafenib group were hypertension and were safe and controllable. Compared with the sorafenib group, the “A+T” group had Lower incidence of grade 5 adverse events (4.6% vs 5.8%). The study also released the data of the Chinese subgroup.In the state of the overall patients with multiple adverse prognostic factors, the OS of the Chinese subgroup “A+T” group patients did not reach, and the mOS of the sorafenib group was 11.4 months.
In terms of PFS, the “A+T” group also had a significant prolongation compared with sorafenib (5.7 months vs 3.2 months), and the Chinese subgroup reduced the risk of death by 56% (OS stratification HR=0.44, 95%CI: 0.25~ 0.76), while the global study is only reduced by 42%, this combination therapy seems to bring greater benefits to Chinese patients. The study introduced the quality of life of patients. Most Chinese patients with hepatitis B and liver cirrhosis have poor quality of life. The median time for quality of life of patients treated with sorafenib monotherapy is 3.6 months. The “A+T” combined treatment was 11.2 months. The quality of life in the “A+T” group deteriorated significantly longer than that of mPFS, and patients had a better quality of life during the treatment.
1.2 “Cola” combination [Lenvatinib + Pembrolizumab]
At the ASCO meeting in 2020, the Phase Ib open, single-arm, multi-center study KEYNOTE-524  updated the latest study data on 100 patients with stage B or C HCC in Barcelona. The mOS of the 100 patients was 22.0 months, the mPFS was 8.6 months (95%CI: 7.1 to 9.7, RECIST v 1.1 per IIR assessment), and the DCR was 88%. According to the RECIST 1.1 standard, the ORR was 36.0%, of which 11 cases were complete remission (CR), 35 cases were partial remission (PR), and mDOR was 8.6 months. Studies have shown that 95% of patients have treatment-related adverse events.
The most common grade 3 treatment-related adverse event is hypertension (17%). Leukopenia is the only treatment-related grade 4 adverse event, and 3 cases are treatment-related. The five grades of adverse reactions were acute respiratory failure, abnormal liver function and intestinal perforation. The phase III clinical trial of lenvatinib combined with pembrolizumab-LEAP-002 (NCT03713593) is underway. It is expected to enroll 750 patients. The primary endpoints are PFS and OS, and the secondary endpoints include ORR, DOR, and DCR. At present, the project has been enrolled in 172 research centers around the world and is under observation and follow-up.
Comparing the data published by the “Coke” combination and the “A+T” combination, although the “Coke” combination is currently only in phase I clinical trials, mPFS, ORR, DCR, etc. all show certain advantages, and the mOS of the “Coke” combination is as high as In 22 months, if LEAP-002 achieves gratifying results, the “Coke” combination may become a first-level expert recommendation in another first-line treatment strategy for advanced HCC after the “A+T” combination.
1.3 Apatinib (Apatinib) + Carrelizumab (Camrelizumab)
An open-label, dose escalation (phase Ia) and expansion (phase Ib), multi-center application of apatinib combined with carrelizumab in the treatment of advanced HCC, gastric or esophagogastric border cancer safety and effectiveness clinical The study  included a total of 18 patients with HCC, of which 17 patients were in Barcelona stage C. The results of the study showed that among the 16 patients with evaluable efficacy, 8 patients achieved PR, ORR reached 50%, DCR was 93.8%, mOS was 3.4 months, and the average follow-up period of 7.8 months, mPFS was 5.8 months.
Among 43 patients, 33 patients who received 250 mg of apatinib showed good tolerability. Adverse reactions of grade 3 and above with an incidence of ≥10% included hypertension and elevated AST. The treatment of apatinib combined with carrelizumab showed controllable adverse reactions and showed significant efficacy in patients with advanced HCC. The phase II clinical trial of apatinib combined with carrelizumab for the treatment of advanced HCC has been completed (NCT03463876), and the data has not yet been announced.
The Phase III clinical trial SHR-1210-III-310 (NCT03764293), which is the application of apatinib combined with carrelizumab and sorafenib as the first-line treatment in patients with advanced HCC, is also under recruitment. In addition, a number of clinical trials related to immune combined targeted therapy are under recruitment or experimentation.
2. Dual immune combination therapy
Under physiological conditions, an immune tolerance system composed of antigen presenting cells (APC), Treg and inhibitory cytokines in the liver maintains immune balance. However, in TME, immune checkpoint molecules such as PD-1/PD-L1, cytotoxic T lymphocyte-associated protein 4 (CTLA-4)-CD80/86 on APC, Treg and other cells have varying degrees of overexpression [11 ].
A study  found that T lymphocytes in tumor tissues with high levels of immune checkpoint molecules are depleted, forming an immunosuppressive microenvironment in tumor tissues. CTLA-4 is mainly expressed on activated T lymphocytes and Tregs. Under the stimulation of T lymphocyte antigen receptors, CTLA-4 competes with CD28 on the plasma membrane for B7 ligand to inhibit T lymphocyte activation.
PD-1 is mainly expressed on CD8+ T lymphocytes, similar to the CLTA-4 receptor, PD-1/PD-L1 is also a member of the CD28 superfamily, and can be used as a co-inhibitory signal for T lymphocyte antigen receptor (TCR). CTLA-4 and PD-1/PD-L1 have similar mechanisms in the formation of tumor immune tolerance. Simultaneous inhibition of CTLA-4 and PD-1/PD-L1 can play a role in the different life cycles of effector T lymphocytes.
Regulate the role of immunosuppression . A series of preclinical research data of solid tumors [14-15] also show that compared with single treatment, dual immune combination therapy has a synergistic effect. The dual-immune combination therapy has gradually become a hot spot for the treatment of the HCC system, and multiple combination therapies are underway (Table 1).
2.1 Ipilimumab + Nivolumab
The results of the cohort 4 study  of the CheckMate-040 study showed that the ORR of the combination therapy was 31%, which was twice (14%) that of nivolumab monotherapy. There were 7 cases of CR, and the mDOR was 17.5 months. , DCR is 49%, and the 24-month OS rate is 40%. Subgroup analysis found that regardless of the expression level of PD-L1, patients in all treatment groups observed clinical benefit.
Group A (nivolumab 1 mg/kg, ipilimumab 3 mg/kg, once every 3 weeks), group B (nivolumab 3 mg/kg, ipilimumab 1 mg/ kg, once every 3 weeks) and group C (nivolumab 3 mg/kg, once every 2 weeks; ipilimumab 1 mg/kg, once every 6 weeks) mOS were 22.8, 12.5 and 12.7, respectively Months. 37% of patients have grade 3 to 4 treatment-related adverse reactions, which are reversible and controllable.
2.2 “D+T” combination [Durvalumab + Tremelimumab]
A randomized, open, multicenter phase III clinical study of HIMALAYA The efficacy of devalumumab combined with trimelimumab in the first-line treatment of advanced HCC. As of September 2, 2019, each group of patients (T300+D vs T75+ The median follow-up time of D vs single agent D vs single agent T) was 11.7 vs 14.6 vs 8.9 vs 15.8 months, mOS was 18.7 vs 11.3 vs 11.7 vs 17.1 months, and mORR was 22.7% vs 9.5% vs 9.6%, respectively vs 7.2%, the proportions of grade 3~4 adverse reactions were 35.1% vs 24.4% vs 17.8% vs 42.0%, respectively.
Moreover, in the T300+D group, the combination was found to have a special proliferating T lymphocyte profile, and patients in objective remission showed high CTL counts. The trial is currently comparing the efficacy of T300+D and D and Sorafenib in the first-line treatment of advanced HCC (NCT03298451).
3. Chemotherapy-related combination therapy
Chemotherapy drugs are often considered immunosuppressive agents because of their toxic effects on immune cells. However, experiments  have found that chemotherapy can induce immunogenic cell death, release tumor-associated antigens and risk-related molecular patterns, both of which can be used in TME Recruiting immune cells while chemotherapeutic drugs also deplete bone marrow-derived suppressor cells and Treg , reducing the activity inhibition of tumor-infiltrating T lymphocytes, so when chemotherapy is used in combination with immunosuppressants, it can not only actively relieve The immunosuppression in TME can also recruit more immune cells for the action of immunosuppressants.
A prospective, single-arm, two-group, multi-center phase II clinical trial , carrelizumab (3 mg/kg, once every 2 weeks) combined with FOLFOX4 regimen or GEMOX regimen for the treatment of advanced HCC and In patients with cholangiocarcinoma, the primary endpoint is ORR, and the secondary endpoints include DCR, PFS, OS and so on.
The results showed that among the 34 evaluable patients in the HCC cohort, ORR was 26.5%, DCR was 79.4%, mPFS was 5.5 months, and mDOR was not achieved. The most common adverse reactions are neutropenia, leukopenia, thrombocytopenia and allergies, most of which are grade 1 to 2, well tolerated and safe and controllable. A phase III clinical study (NCT03605706) evaluating the role of carrelizumab in combination with FOLFOX4 and sorafenib or FOLFOX4 in the first-line treatment of advanced HCC is under recruitment.
At the ASCO conference in 2020, a retrospective study of lenvatinib combined with hepatic arterial infusion chemotherapy (HAIC) in the first-line treatment of advanced HCC  enrolled 24 patients with advanced HCC, with an ORR of 66.7% and a DCR of 79.2%. The mPFS was 8.1 months, and the OS rates at 6, 9, and 12 months were 91.7%, 83.3%, and 75%, respectively; another retrospective review of HAIC combined with apatinib and teriprizumab as the first-line treatment for advanced HCC In the study , a total of 6 newly-treated advanced HCC patients were enrolled, and the DCR of the final patient reached 100%. According to the RECIST assessment, all 6 patients reached PR.
4. Chimeric antigen receptor T lymphocyte immunotherapy (CAR-T) related combination therapy
CAR-T is an improved T lymphocyte immunotherapy. For CAR-T, TME’s hypoxia, high acid, low nutrition, and oxidative stress conditions are not conducive to survival and proliferation. In vitro experiments have shown that it can improve tumor oxidation. Under conditions such as stress, the proliferation ability of CAR-T can be improved, which can reduce the failure of CAR-T caused by immunosuppressive cells in TME . Currently, one of the most popular strategies to improve CAR-T failure in TME Even with the use of immune checkpoint inhibitors, a combination of drugs targeting PD-L1, PD-1 and CTLA-4 has also been found to be effective [23-24].
Wu et al.  analyzed the combined effects of GPC3-CAR-T and sorafenib in immunocompetent mouse HCC model and immunodeficient mouse HCC model and found that sorafenib can induce the secretion of macrophages in TME IL-12, thereby increasing the activity of CAR-T, and in the study of immunodeficient mice, it was found that sorafenib and CAR-T synergistically promote the apoptosis of liver cancer cells and enhance the anti-tumor effect of CAR-T. The synergistic treatment of sorafenib and CAR-T involves multiple mechanisms.
This experiment provides a basis for clinical trials of multi-kinase inhibitors combined with CAR-T in solid tumors. In a related study on combination therapy with immune checkpoint inhibitors, Guo et al.  confirmed that blocking PD-1 not only enhances the anti-tumor activity of CAR-T against HCC, but also improves the survival and infiltration of CAR-T in tumors. This is also one of the important reasons why CAR-T has a good therapeutic effect in hematological malignancies but has not yet achieved a good effect in solid tumors.
Although CAR-T technology has many advantages, its application in solid tumors is still in the exploratory stage. The specificity and multi-target problems of solid mid-way targets are still the bottleneck of treatment. Target leakage, massive release of cytokines, and dissolution A series of problems such as syndrome need to be solved urgently .
5. Combination therapy related to radiotherapy
Radiotherapy for HCC started with wide-field low-dose radiotherapy for the whole liver. Before the 1990s, radiation liver disease and liver failure were often caused. With the application of three-dimensional conformal radiotherapy, intensity-modulated radiotherapy and SBRT, targeted The accuracy of lesions is significantly improved . Que et al.  retrospectively analyzed 54 patients with advanced HCC with portal vein tumor thrombus from January 2009 to June 2016.
The results showed that the ORR of SBRT combined with sorafenib group and SBRT alone was 77.77%. And 75.00% (P=0.43), mPFS was 6 months vs 3 months (P=0.24), and the 1-year and 2-year progression-free survival rates were 25.7% and 15.2% vs 11.1% and 8.3% (P=0.122) 5). The mOS was 12.5 months vs. 7 months (P=0.28). The SBRT combined with sorafenib group had higher efficacy, but the trend of various indicators did not reach statistical significance, and the test proved that the combination of sorafenib The rafenib group also increased the side effects of grade 3 or more.
PD-1/PD-L1 and CTLA-4 are important targets for immune checkpoint therapy in recent years. Preclinical studies  have confirmed that after radiotherapy, the PD-L1 of tumor cells mediated by γ-interferon produced by CD8+ T lymphocytes is up-regulated and induces local anti-tumor responses. Radiotherapy alone cannot produce long-term anti-tumor immunity, but blocking the PD-1/PD-L1 axis can alleviate the immune limitation of radiotherapy, and when radiotherapy is combined with anti-PD-1/PD-L1 treatment, it can activate systemic immunity to exert long-term immunity. End anti-tumor ability, there is a rare phenomenon that the tumor in the irradiated area and the tumor in the field regress together.
CTLA-4 and CD28 are homologous receptors co-expressed on the surface of CD4+ and CD8+ T lymphocytes, both of which can bind to CD80 and CD86 on the surface of APC, but CTLA-4 regulates the inactivation of T lymphocytes and has a higher affinity for ligands than CD28 . Radiotherapy combined with immune checkpoint inhibitors has the unique effect of eliminating primary lesions and distant metastases at the same time, but most of the related studies are in the preclinical stage. Only a few clinical studies retrospectively analyze the effects of radiotherapy combined with immunotherapy in melanoma, non-small cell lung cancer, etc.
Variable factors such as the long-term efficacy of malignant tumors, the optimal dose of radiotherapy, the optimal point of administration of the two treatment methods, and the relevant biomarkers of tumor prediction require a large number of clinical trials to explore. At the same time, new The development of immune targets and the matching of different immunosuppressive factors for radiotherapy to suit different types of cancer are also issues that need to be considered in the future.
5. Combination treatment related to local treatment
Studies  have shown that in the residual liver cancer tissue after TACE or ablation treatment, not only the local peripheral immune response will be induced, resulting in the suppression of the anti-tumor immune response, but also the number of VEGF-positive cells in the local residual tissue will increase significantly. The level of VEGF-A is significantly increased, which promotes the formation of local neovascularization, and becomes an important factor causing local recurrence after treatment. After local treatment, using immunosuppressive drugs to inhibit local immune escape or targeted drugs to inhibit local neovascularization, theoretically has a synergistic effect in controlling local recurrence.
The randomized, double-blind phase II clinical trial SPACE analyzed 307 mid-stage multinodular HCC patients with time to disease progression (TTP) as the primary endpoint. The results showed that although sorafenib combined with TACE is safe and effective, Compared with the control group, TTP was not prolonged in clinical significance (mTTP: 169 d vs 166 d, unilateral P=0.072).
Similar results were obtained in the other three clinical trials POST-TACE, trialTACE2, and BREISK-TA the result of. However, the phase II clinical trial TACTICS showed that the mPFS of the sorafenib+TACE group was significantly longer than that of the TACE-only group (25.2 months vs 13.5 months, P=0.006), and the median time to discontinue treatment in the sorafenib+TACE group It was also significantly longer (26.7 months vs 20.6 months, P=0.02) .
The analysis found that trials such as SPACE received or terminated TACE treatment at a fixed time node regardless of the tumor control and liver function damage during the treatment process, resulting in transient liver function damage, and the liver function Child-Pugh Class B was the termination of TACE. The standard of treatment progress caused patients with transient liver injury to be terminated prematurely, and the above reasons for the difference in test results were further confirmed in the later follow-up.
In the clinical trial of anti-CTLA-4 antibody trimelimumab combined with ablation for the treatment of advanced HCC, 32 HCC patients with Barcelona staging C had mOS of 12.3 months, mTTP of 7.4 months, 6 months and 12 months. The monthly progression-free survival rates were 57.1% and 33.1%, respectively . Compared with the phase II clinical trial of trimelimumab monotherapy for HCC, in the case of monotherapy selected patients with better prognosis (43% Barcelona stage B), the combination therapy still achieved a longer mTTP (7.4 months vs 6.48 months) and longer mOS (10.1 months vs 8.2 months) .
7. Combined surgery
Translational therapy means that patients who are inoperable but have potential surgery can be transformed into an operable treatment mode through comprehensive measures to control tumor progression and even reduce clinical stage, improve liver function . Two clinical studies on translational therapy were reported at the ASCO meeting in 2020, namely “Lenvatinib combined with PD-1 monoclonal antibody first-line/second-line treatment of unresectable/advanced HCC real-world studies”  and “TKIs combined A clinical study of PD-1 monoclonal antibody conversion therapy for initial unresectable HCC” .
The former included 77 patients with advanced HCC, lenvatinib combined with PD-1 antibody for first-line and second-line treatment, achieved 60.5% and 43.8% ORR (mRECIST criteria), of which 6 patients were due to tumor shrinkage after treatment Surgery was performed; the latter included a total of 60 patients. After lenvatinib combined with PD-1 monoclonal antibody treatment, 11 patients were converted to resectable, of which 9 patients completed the operation. As of the last follow-up, 7 patients remained No tumor state.
A phase II clinical study on postoperative application of apatinib in patients with resectable stage IIIa HCC reported at the ASCO meeting in 2020 included 30 HCC patients with portal vein tumor thrombi, according to the Japanese Liver Cancer Research Group According to the classification, 1 case was Vp1, 11 cases were Vp2, and 12 cases were Vp3. The median treatment time was 4.8 months, and the median follow-up time was 14.3 months.
The test results showed that the median recurrence-free survival time was 7.6 months, the 1-year recurrence-free survival rate was 36.1%, the mOS was not reached, and the 1-year OS rate was 93.3%. All patients had treatment-related adverse events. There were 14 cases of grade 3 or 4 adverse events. The most common adverse events were thrombocytopenia and neutropenia.
The test confirmed that apatinib is tolerable to most patients after HCC resection with portal vein tumor thrombi. As for the efficacy of postoperative application of apatinib on tumor recurrence, a controlled study needs to be further proved. Another phase II clinical trial evaluating the perioperative efficacy of nivolumab or nivolumab combined with ipilimumab in patients with resectable HCC showed that the CR rate of 21 patients undergoing scheduled surgery Up to 24%, 6 cases of grade 3 treatment-related adverse events, no grade 4 or above adverse events occurred, reaching the main safety endpoint.
Combination therapy shows good therapeutic effects and controllable safety in the middle and advanced stages of HCC. However, the development of combination therapy requires clinicians to consider some issues:
(1) There are various combinations of combination therapy, which combination has the better effect, or whether patients of different stages have the best combination corresponding to the effect;
(2) The side effects of systemic treatment are relatively large, such as leukopenia or neutropenia, hypertension, diarrhea, skin rash, etc., whether the combined treatment will increase toxic and side effects, how to reduce the occurrence of toxic side effects, and how to give appropriate treatment after occurrence;
(3) There are dominant populations in the immune-targeted combination program, and exploring the biomarkers of combined therapy and screening the dominant populations of immune-targeted therapy is an urgent problem to be solved;
(4) HCC patients are often accompanied by HBV or HCV infection, and the use of immunosuppressants can cause fulminant liver function damage related to virus reactivation . Will the combination therapy based on immune checkpoint inhibitors increase the probability of virus reactivation? , And the corresponding treatment measures when activated are still to be explored;
(5) Early diagnosis and early treatment and prevention of metastasis and recurrence are critical in the treatment of HCC. Multidisciplinary diagnosis and treatment will be the only way. In the future diagnosis and treatment process, precision treatment for combined therapy biomarkers will be multidisciplinary. Comprehensive diagnosis and treatment will always be the latest and highest requirement for the treatment of middle and late stage HCC.
(source:internet, reference only)