Complete Response Associated With Combination Treatment Regorafenib and Sintilimab in a Sorafenib-refractory Hepatocellular Carcinoma Patient

Most of patients diagnosed with Hepatocellular carcinoma (HCC) have advanced diseases and many are not eligible for curative therapies. There is a growing evidence suggesting that combination treatment of PD-1/PD-L1 inhibitors and tyrosine kinase inhibitors (TKIs) becomes a prospective trend for advanced HCC. For those HCC patients with sorafenib resistance, the ecacy of regorafenib combined with PD-1/PD-L1 inhibitors remains unclear. function of HCC The published open-label, phase 3 (IMbrave150) trial atezolizumab combined with bevacizumab resulted in better overall and progression-free survival outcomes than sorafenib with a objective response rate (ORR) of 33.2% versus 13.3% in patients with advanced HCC, which was granted as a rst-line treatment for patients with advanced or metastatic HCC by the FDA Studies show that the anti-VEGF and PD-1/PD-L1 inhibitor combination improves antigen-specic T-cell migration. These results provide evidence for eciency of immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs) combination in advanced and metastatic HCC, indicating the combination therapy might become a prospective trend for advanced HCC. clinical ecacy of the combination rationale ICIs and TKIs, we case lung metastasis setting HBV-induced liver cirrhosis sorafenib-regorafenib sequential PD-1 after initial liver resection we hope explore further study for combination therapy of regorafenib and PD-1/PD-L1 inhibitor for HCC in the future.

Introduction Hepatocellular carcinoma (HCC) is the third leading cause of cancer death in the world and its incidence rate is rising in recent years [1]. In China, the number of HCCs accounts for nearly half of the world's new cases [2]. Both the incidence and mortality rates are 2 to 3 times higher in China than those in majority of other countries [3]. Liver resection, liver transplantation and local ablation are curative treatments for earlier stages of HCC, which achieve good surgical outcomes. However, HCC frequently relapses and most patients have locally advanced or extrahepatic metastasis when curative treatments are no longer available. For these patients, evidence for highly effective therapies on overall survival (OS) is still lacking.
Trans-arterial chemoembolization (TACE) can be used to treat HCC con ned to the liver and the therapeutic effect is limited.
Sorafenib, a small-molecule multi-kinase inhibitor, is the rst systemic therapeutic agent for patients with advanced HCC after a landmark study which revealed improvements in time to progression (TTP) and OS [4,5]. However, the therapeutic impact of sorafenib remains limited, and patients often acquire resistance soon after treatment. Thus, for HCCs who are refractory or intolerant to sorafenib, the second-line treatment for HCC is very important. Regorafenib is a multikinase inhibitor that blocks the activity of several protein kinases involved in angiogenesis, oncogenesis, metastasis, and tumour immunity [6]. It has a distinct molecular target pro le and had more potent pharmacological activity than sorafenib in previous studies [6,7]. Nevertheless, this therapy remains unsatisfactory. Therefore, there is an urgent need for more effective systemic therapies for HCC, particularly after treatment with sorafenib and regorafenib.
There is growing evidence suggesting that HCC was considered as an immunogenic tumor, stemming from an immunosuppressive environment. In the past few years, breakthroughs in immune therapy have offered new therapeutic options for many tumors [8]. The inhibitors of the programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) have emerged as a promising therapeutic strategy in a variety of cancers, such as melanoma, lung cancer and renal cell carcinoma [8,9]. The chronic in ammation, viral infection and liver cirrhosis underlying the formation of most HCC tumors highlight a complicated relationship between the immune biology and the development of HCC [10]. The liver is constitutively immunosuppressive as it promotes systemic tolerance to foreign antigens, which prevents excessive reactions to toxins and antigens absorbing from the enteric circulation [11]. HCC takes advantage of the immune tolerance to initiate and promote HCC carcinogenesis and progression. These characteristics of HCC may steer immunotherapeutic strategies to those that prevent immune suppressive mechanisms, rather than directly increase the immune function of HCC patients. The recently published open-label, phase 3 (IMbrave150) trial reported atezolizumab combined with bevacizumab resulted in better overall and progression-free survival outcomes than sorafenib with a objective response rate (ORR) of 33.2% versus 13.3% in patients with advanced HCC, which was granted as a rst-line treatment for patients with advanced or metastatic HCC by the FDA [12]. Studies show that the anti-VEGF and PD-1/PD-L1 inhibitor combination improves antigen-speci c T-cell migration. These results provide evidence for e ciency of immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs) combination in advanced and metastatic HCC, indicating the combination therapy might become a prospective trend for advanced HCC.
To evaluate clinical e cacy of the combination rationale including ICIs and TKIs, we represent a case of HCC with lung metastasis in the setting of HBV-induced liver cirrhosis responding dramatically to the combination of sorafenib-regorafenib sequential treatment and PD-1 inhibitor after initial liver resection and we hope to explore further study for combination therapy of regorafenib and PD-1/PD-L1 inhibitor for HCC in the future.

Case Presentation
A 56-year-old man with a history of chronic HBV infection for more than 30 years, presented to our department with abdominal malaise in September 2015. Enhanced abdominal magnetic resonance imaging (MRI) revealed a mass with the largest measuring up to 11.0*9.5 cm in size in segment 7 and 8 within the right lobe of liver ( Fig. 1A and 1B). The patient was diagnosed of HCC with the background of cirrhosis secondary to HBV infection based on imaging and con rmed with the clinical diagnosis of Barcelona Clinic Liver Cancer (BCLC) A and Child-Pugh class A. Entecavir treatment was routinely used once per day from then on. His alpha-fetoprotein (AFP) level was in the normal range. On September 23, 2015, he underwent segment 7 and 8 resection and cholecystectomy. The postoperative pathological examination showed hemorrhage with necrosis in the middle of the tumor, with moderate differentiation and vascular cancer embolus. The incisal edge was negative and not invaded the hepatic capsule ( Fig. 1C and 1D). He recovered well and discharge from hospital. With regular examination, unfortunately, in March, 2017, he was found to liver recurrence and had lung metastases (BCLC C). According to the guidelines, he started systemic treatment with sorafenib (400 mg, twice per day) and could tolerated this dose. Enhanced abdominal CT showed a heterogeneous irregular mass measuring up to 2.3*2.2 cm with arterial phase enhancement and venous phase washout in the left lobe of liver (Fig.S1). Chest CT revealed that multiple pulmonary nodules on both sides of the lung which were diagnosed as lung metastases, the largest up to 8-mm diameter ( Fig. 2A (Table 1). At his last follow-up, nearly 5 years have elapsed since the diagnosis of HCC and up to 40 months since lung metastases was diagnosed. He was observed to be in a very good condition, without evidence of disease progression. Written informed consent was obtained from the patient for publication of this case report and any accompanying images. Discussion Liver resection is recommended as the rst line of treatment for patients with early stage HCC with wellpreserved liver function [13]. However, resection of even very early tumors, less than 2 cm and without vascular invasion or satellites (BCLC stage 0), is associated with a recurrence rate of approximately 60% at 5 years [13,14]. HCC patients are often diagnosed at advanced stages when recurrence occurs, and curative therapy options are limited. The prognosis is extremely poor, leading to a 5-year overall survival rate of 2% [3].
The systemic treatment options available for HCC patients with advanced stages are limited. Overall, more than 50% of patients receive systemic therapies at some point during the disease process [15]. Sorafenib, a molecular kinase inhibitor, was thought to be a breakthrough in treating unresectable HCC although only 3 months longer OS was found in SHARP trial [4]. For a decade, sorafenib was the only FDA-approved therapy and the bene ts were limited for lack of either therapeutic alternative or second-line treatment for those who are intolerant or resistant. However, since 2017, treatment for patients with advanced HCC is dramatically changed by novel multi-target inhibitors approved, such as regorafenib, lenvatinib and cabozantinib, or immune checkpoint inhibitors, such as nivolumab and pembrolizumab. Regorafenib is an oral multikinase inhibitor that blocks the activity of protein kinases involved in angiogenesis, oncogenesis, metastasis, and tumour immunity [6,7]. Regorafenib is the only systemic treatment shown to provide survival bene t in advanced HCC patients progressing on sorafenib treatment. RESORCE trial indicated that regorafenib improved OS with a hazard ratio of 0.63 (95% CI: 0.50-0.79; one-sided p < 0.0001); median survival was 10.6 months (95% CI: 9.1-12.1) for regorafenib versus 7.8 months (6.3-8.8) for placebo [6].
Although regorafenib has been approved as a second-line treatment for patients with advanced HCC who show progression after sorafenib therapy, the treatment e cacy remains insu cient. With the development of immune therapy, the treatment effects on HCC need to be urgently explored.
PD-1 is expressed by activated T lymphocytes and is a pivotal immune checkpoint receptor that mediates immunosuppression upon binding to the PD-L1 expressed by tumor cells [9]. In recent years, immune checkpoint blockade has brought a paradigm shift in the treatment of a number of solid tumors. Various immune checkpoint blocking agents are being tested for their e cacy in HCC. Furthermore, PD-1 pathway offers a potential treatment strategy based on the encouraging results of KEYNOTE-224 [16] and Checkmate 040 trials [17]. Reccently, IMbrave150 trial showed better overall and progression free survival outcomes of atezolizumab plus bevacizumab as compared with sorafenib in patients with unresectable hepatocellular carcinoma who had not previously received systemic therapy, which inspired us to administrate this combination therapy in advanced or metastatic HCC [12]. Sintilimab (Innovent Biologics, Suzhou, China) is a highly selective, humanised, monoclonal antibody that blocks interactions between PD-1 and its ligands and has been tested regarding the safety and activity in patients with advanced-stage solid tumor and was approved for lymphoma by Chinese Center for Drug Evaluation in China in 2018 [18][19][20]. Findings from a phase 1 study of sintilimab in advanced solid tumours suggested 200 mg every 3 weeks as the recommended dose, and clinical bene t from the therapy was observed [19,21].
For those HCC patients with sorafenib resistance, the e cacy of regorafenib combined with PD-1/PD-L1 inhibitors remains unclear. Regorafenib was proved to be an immunomodulator in tumor microenvironment while PD-1 antibody blocks the co-inhibitory signals and unlocks the negative regulation of the immune response [22,23]. Therefore, further investigations for the combination treatment of regorafenib and PD-1/PD-L1 inhibitors are warranted to provide its e cacy data in clinical trials. Unfortunately, there are no published data from randomized controlled trials in HCC which were registered on clinicaltrial.gov currently ( Table 2). Standard combination and sequencing of the therapy need to be established with deeper insight into the rationale of combined action and further RCTs. What's more, for most of the patients enrolled in, present with preserved liver function, while the advanced HCC patients in real clinical phase may have a much worse performance. Whether they can tolerate the combination treatment is still unknown and the clinical trials won't take the risk to enroll these patients. Joerger et al. [24] presented the case of a sorafenibrefractory patient probably experiencing progressive disease during immune checkpoint inhibitor combination treatment with the anti-PD-1 monoclonal antibody nivolumab and the anti-GITR monoclonal antibody BMS-986156 within a clinical phase-1 trial followed by a prolonged tumor response according to RECIST v 1.1 during third-line treatment with regorafenib. In this case, sorafenib-immunotherapyregarofenib sequential treatment was applied and the last evaluation was documented as partial response and the patient started taking regorafenib at a reduced dose of 80 mg and later further reduced to 40 mg per day. The patient in our report was evaluated as complete response after combination therapy of regorafenib with standard dose and sintilimab. No life-threatening adverse events were found in our patient. The combination of regorafenib and PD-1/PD-L1 inhibitor will be a novel therapy method for the future gold standard in the systemic treatment of sorafenib-refractory HCC. The precise mechanism of synergistic effect between regorafenib and PD-1/PD-L1 inhibitors remains unclear. Previous study indicated that inhibition of JAK1/2-STAT1 signaling pathway blocked MHC-I expression while suppressing the MAPK pathway increased MHC-I expression [25]. Through inhibiting both the RET-Src axis/JAK1/2-STAT1 and MAPK pathway, regorafenib had little in uence on MHC-I expression.
Taken together, through blocking the IFN-γ induced PD-L1 and IDO1 expression with little in uence on MHC-I expression, regorafenib unleashed the IFN-γ side effect without compromising the antigen presentation processes. What's more, tumor-associated macrophage (TAM) is major population of immune cells that affects tumor development. One of the mechanisms described for the failure of anti-angiogenic therapy and tumor evasion is the strong in ltration of TAMs. TAM serves as an important driving factor in immunosuppressive tumor microenvironment (TME) and, for instance, the secreted TGF-β inhibits CD8 + Tcell responses to kill the cancer cells [26,27]. Regorafenib modulates TAM re-polarization and relieved the immunosuppressive effect of TAM via binding to CSF-1R [27]. Terme et al. [28] demonstrated that regorafenib reduces the proportion of Tregs and inhibits tumor-induced Treg proliferation via targeting the vascular endothelial growth factor A (VEGFA)/VEGF receptor 2 (VEGFR2) signaling pathway. Regorafenib targets a variety of kinases involved in angiogenic, tumor growth-promoting and tumor microenvironmental signalling pathways [7]. The probable mechanism of synergistic effects of regorafenib and PD-1/PD-L1 inhibitors are showed in Fig. 5.
In summary, this study provided the rst case of complete tumor response to the combination of regorafenib and sintilimab as second line treatment for a patient with advanced, pulmonary metastatic HCC which was refractory to rst-line sorafenib. Data from this clinical case report support future exploration of combination treatment of the oral multi-kinase inhibitor regorafenib with PD-1/PD-L1 inhibitors in sorafenib-refractory HCC patients. Regorafenib plus PD-1 or PD-L1 inhibitor may present a new potential treatment option for the advanced HCC patient with sorafenib resistance. However, their synergistic effects and safety need further investigation in a randomized phase 3 study.

Declarations
Acknowledgements None Authors' contributions ZYH was the chief physician who provided the case. ELZ providedfunding and was a major contributor in writing the manuscriptand performing the analysis and interpretation of data. ZYZ andLZ collected clinical data. SX and XPC revised the manuscript. All the authors read and approved the nal manuscript.

Funding
This work was supported by the grants from National Natural Science Foundation of China (81902839) and National Science and Technology Major Project of China (2017ZX10203207-002)

Availability of data and materials
Not applicable as all information and data is presented in manuscript.

Ethics approval and consent to participate
Ethical approval is not required as patient consent for publication wasobtained.
Consent for publication Figure 1 MRI showed a mass with the largest measuring up to 11.0*9.5cm in size in segment 7 and 8 within the right lobe of liver before surgical resection (A and B). Postoperative pathology revealed moderate differentiation and vascular cancer embolus (C and D).