Efficacy of lenvatinib for unresectable hepatocellular carcinoma based on background liver disease etiology: multi-center retrospective study

It was recently reported that hepatocellular carcinoma (HCC) patients with non-alcoholic steatohepatitis (NASH) are not responsive to immune-checkpoint inhibitor (ICI) treatment. The present study aimed to evaluate the therapeutic efficacy of lenvatinib in patients with non-alcoholic fatty liver disease (NAFLD)/NASH-related unresectable-HCC (u-HCC). Five hundred thirty u-HCC patients with Child–Pugh A were enrolled, and divided into the NAFLD/NASH (n = 103) and Viral/Alcohol (n = 427) groups. Clinical features were compared in a retrospective manner. Progression-free survival (PFS) was better in the NAFLD/NASH than the Viral/Alcohol group (median 9.3 vs. 7.5 months, P = 0.012), while there was no significant difference in overall survival (OS) (20.5 vs. 16.9 months, P = 0.057). In Cox-hazard analysis of prognostic factors for PFS, elevated ALT (≥ 30 U/L) (HR 1.247, P = 0.029), modified ALBI grade 2b (HR 1.236, P = 0.047), elevated AFP (≥ 400 ng/mL) (HR 1.294, P = 0.014), and NAFLD/NASH etiology (HR 0.763, P = 0.036) were significant prognostic factors. NAFLD/NASH etiology was not a significant prognostic factor in Cox-hazard analysis for OS (HR0.758, P = 0.092), whereas AFP (≥ 400 ng/mL) (HR 1.402, P = 0.009), BCLC C stage (HR 1.297, P = 0.035), later line use (HR 0.737, P = 0.014), and modified ALBI grade 2b (HR 1.875, P < 0.001) were significant. Lenvatinib can improve the prognosis of patients affected by u-HCC irrespective of HCC etiology or its line of treatment.


Scientific Reports
| (2021) 11:16663 | https://doi.org/10.1038/s41598-021-96089-x www.nature.com/scientificreports/ males, < 20 g/day in females)] of fatty liver patients and/or based on pathological findings 8 . Burned-out NASH liver cirrhosis was diagnosed clinically based on the clinical course (e.g., no history of alcohol abuse, history of obesity and/or fatty liver, or past pathological diagnosis) by each institution. Patients without autoimmune liver disease (AIH or PBC) other than the above, or those in whom hepatic fibrosis was not observed pathologically were classified as cryptogenic liver disease. Positive for severe fibrosis was defined based on elevated FIB-4 index (≥ 3.25) 9 .
The therapeutic effects of lenvatinib in all 557 patients with Child-Pugh class A were examined as Study-1. Furthermore, therapeutic responses were compared between patients with NAFLD/NASH (n = 103), and those with chronic hepatic viral infection or alcohol abuse (Viral/Alcohol group) (n = 427), after exclusion of cryptogenic patients (n = 27), as Study-2 ( Fig. 1). HCC diagnosis. HCC was diagnosed based on an increasing trend of alpha-fetoprotein (AFP), as well as typical findings obtained in dynamic CT 10 , MRI 11,12 , and contrast enhanced ultrasonography (CEUS) with perflubutane (Sonazoid®, Daiichi Sankyo Co., Ltd., Tokyo, Japan) examinations 13,14 , and/or pathological findings. To evaluate tumor progression, Barcelona Clinic Liver Cancer (BCLC) stage 15 and tumor node metastasis (TNM) stage were used, and determined as previously reported in a study for TNM staging of HCC conducted by the Liver Cancer Study Group of Japan (LCSGJ) 6th edition (TNM-LCSGJ) 16 .
Assessment methods for hepatic reserve function and therapeutic response. Child-Pugh classification 17 and albumin-bilirubin (ALBI) grade were used for assessment of hepatic reserve function [18][19][20] .
To perform more detailed evaluations of patients with the middle ALBI grade of 2, a revised grading system was used that consisted of four levels, with sub-grading for the middle grade of 2 (2a and 2b) based on an ALBI score of − 2.27 as the cut-off (modified ALBI, mALBI grade), which was previously reported to result in a predictive value for indocyanine green retention after 15 min (ICG-R15) of 30% 21,22 . Progression-free survival (PFS) was analyzed according to the modified Response Evaluation Criteria In Solid Tumors (mRECIST) criteria 23,24 , based on results of dynamic CT examinations performed at intervals of 8-12 weeks.
Lenvatinib treatment and assessment of adverse events. After obtaining written informed consent from each patient, lenvatinib treatment was started. The drug was orally administered at 8 mg/day in patients weighing < 60 kg or 12 mg/day in those ≥ 60 kg, and discontinued when any unacceptable or serious adverse event (AE) occurred (any grade 3 or more severe AE, or any unacceptable grade 2 drug-related AE), or radiological tumor progression was observed, according to the guidelines for administration of lenvatinib. AEs were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0 25 . When a drug-related AE was noted, dose reduction or temporary interruption was maintained until the symptom was resolved to grade 1 or 2, according to the guidelines provided by the manufacturer. AEs of grade 3 or more were defined as severe, and the worst grade for each AE during the present observation period was recorded.
Ethical approval. Written informed consent for lenvatinib treatment was obtained from each patient. This was a retrospective analysis of records stored in a database and official approval was received based on the Guidelines for Clinical Research issued by the Ministry of Health and Welfare of Japan. All procedures complied with the declaration of Helsinki. The study protocol was granted approval by the Institutional Ethics Committee of Ehime Prefectural Central Hospital (IRB No. 30-66) (UMIN000043219). Statistical analysis. Continuous variables are expressed as median values (first-third quartile). Statistical analyses were performed using Welch's t-test, Student's t-test, Fischer's exact test, or Mann-Whitney's U test, as appropriate. Cox hazard analysis (stepwise regression method), the Kaplan-Meyer method, and a log-rank test were used to analyze prognosis factors.

Discussion
The present analyses of u-HCC patients who received lenvatinib showed that PFS and OS in those in the NAFLD/ NASH group were favorable as compared with those in the Viral/Alcohol group (median PFS: 9.3 vs. 7.5 months, P = 0.012) (median OS: 20.5 vs. 16.9 months, P = 0.057). Also, when cryptogenic HCC was included in the NAFLD/NASH group, both PFS and OS were better in those patients (median PFS: 9.3 vs. 7.5 months, P = 0.012) (median OS: 21.0 vs. 16.9 months, P < 0.001). An interesting meta-analysis article recently reported by Pfister showed that patients with a viral etiology demonstrated therapeutic benefits with ICI treatment [HR 0.64], whereas those with nonviral etiology HCC did not [HR 0.92] (P = 0.03) 5 . That report also presented results of two different validation studies of ICI treatment for HCC, in which NAFLD-HCC cases showed significantly worse OS than cases of HCC with another etiology (HR 2.6, 95% CI 1.2-5.6, P = 0.017; median 8.8 vs. 17.7 months, P = 0.034). In patients undergoing ICI treatment, background liver disease etiology might be a biomarker of efficacy. As for a reason for that phenomenon, it has been reported that CD8 + T cells in HCC patients with NASH are increased and activated by IL-15-induced Fas-ligand dependent apoptosis through tumor necrotic factor (TNF) and acetate in the tumor, unlike MHC class-I dependent CD8 + T cell activation 27 , thus immune response to tumor antigens is impaired 28,29 . In contrast, the effectiveness of MTA is not related with mode of CD8 + T cell activation, but rather inhibition of multi-tyrosine kinase activity, thus MTA should be effective irrespective of HCC etiology including in NAFLD/NASH-related HCC cases.
Patients in the IMbrave150 study who underwent treatment with Atezo + Bev, a newly developed ICI and anti-VEGF-antibody combination, showed an overwhelmingly superior therapeutic efficacy as compared with those who received sorafenib (median OS: 19.2 vs. 13.4 months, HR 0.66, 95%CI 0.52-0.85) (ORR/CR by mRECIST: 35%/12% vs. 14%/3%) 30 . Although pooled analysis of the SHARP and Asia-Pacific trials found that positive for HCV was a predictive factor for therapeutic response to sorafenib [HR 0.47, 95% CI 0.32-0.69, P = 0.035] 31 , the IMbrave150 study showed superiority for the therapeutic effect (both OS and PFS) of Atezo + Bev as compared with sorafenib in HCV-HCC cases (HR 0.43, 95% CI 0.25-0.73 and HR 0.68, 95% CI 0.42-1.10, respectively) 30 . On the other hand, that study did not demonstrate superior findings for Atezo + Bev in regard to OS in HCC with nonviral etiology (HR 1.05, 95% CI 0.68-1.63 and HR 0.80, 95% CI 0.55-1.17) as compared to viral HCC cases. However, these results do not indicate that Atezo + Bev is not effective for non-viral HCC, as the OS in patients who received that treatment was 17.0 months, similar to that in patients with HBV HCC (19.0 months). Rather, the worse OS HR can be attributed to better efficacy of sorafenib even in non-viral HCC (18.1 months) as     www.nature.com/scientificreports/ compared with HBV-HCC (12.4 months) and HCV-HCC (12.6 months) cases, though the reasons are unknown. The present results suggested a similar phenomenon. Although liver fibrosis in the background of HCC patients with NAFLD/NASH may be milder as compared to that in those with Viral/Alcohol, elevated FIB-4 index was not significant prognostic factor both in PFS and OS. An explanation for these findings is not clear. Despite the retrospective nature of this analysis, it is possible to speculate that cryptogenic HCC is a subgroup of NAFLD/NASH HCC. In the present Study-1, patients with u-HCC due to alcohol abuse had PFS and OS similar to those with viral HCC, thus viral and alcoholic HCC were treated as a single group in comparisons of PFS and OS with those of NAFLD/NASH/cryptogenic HCC patients. In Study-2, after excluding cryptogenic HCC, the NAFLD/NASH and other etiology (Viral/Alcohol) groups were compared to confirm response to lenvatinib in clinically diagnosed NAFLD/NASH patients. The NAFLD/NASH group showed better PFS (P = 0.012). Although there was no significant difference in OS (P = 0.057), OS in the NAFLD/NASH-HCC patients treated with lenvatinib was very favorable (20.5 months) and tended to be better than that in the Viral/Alcohol HCC cases (16.9 months). It was recently proposed by Hessheier et al. that metabolic factors may be risk factors for development of liver diseases and cirrhosis 32 , while Eguchi et al. found "lean-NASH" (non-obese NASH, body mass index: BMI < 25 kg/m 2 ) existing in 20% to > 35% in patients in Japan 33 . Of the present cryptogenic HCC patients (n = 26), diabetes was observed in 44.4% (n = 12), hypertension in 51.9% (n = 14), and overweight (BMI ≥ 25 kg/m 2 ) in 25.9% (n = 7), while 70.4% (n = 19) had at least one of those co-factors (Supplemental Table S2). Thus, cryptogenic HCC might be categorized as NAFLD/NASH HCC. When cryptogenic HCC cases are included with NAFLD/NASH, in other words, without hepatitis viral infection or alcohol abuse history, such patients may receive benefit from lenvatinib treatment (Fig. 4e,f).
Since 2004, the number of adults with NASH awaiting liver transplantation in the United States has nearly tripled and NASH has become the second leading etiology of liver disease among such cases 34 . In meta-analysis results, the NAFLD incidence rate was reported to be 25.24% (all regions, 95% CI 22.1-28.65) and pooled overall NASH prevalence among biopsied NAFLD patients was estimated to be 59.10% (95%CI 47.55-69.73), while the annual rate of liver carcinogenesis from NAFLD was estimated to be approximately 0.04% (95% CI 0.29-0.66) 35 . Similarly in Japan, a rapidly increasing rate of HCC patients without hepatitis viruses has been reported 36 , with most cases of non-B, non-C HCC shown to be related to lifestyle/metabolic factors, such as obesity or diabetes, including cryptogenic HCC 37 . Recently, liver-related diseases, such as cirrhosis and HCC, have been reported to be the third leading cause of death in patients in Japan with type 2 diabetes mellitus, which is associated with NAFLD 38 . Furthermore, a recent review article of cases of HCC related to NAFLD mentioned that the impact of metabolic syndrome and its relevance in those patients is not clear 39 . Nevertheless, establishment of an effective treatment strategy for u-HCC related with NAFLD/NASH is considered to be a critical clinical issue. It is anticipated that the number and percentage of NAFLD-HCC cases will continue to increase, though liver cirrhosis is not present in all of those. However, HCC is often detected in an advanced stage because no surveillance program for NAFLD-HCC patients has been established. As a result, it is important to confirm which systemic treatment (e.g. MTAs or ICI combination) is a more effective therapeutic option for patients with NAFLD HCC as well as those with viral hepatitis-related HCC. Moreover, some favorable results regarding OS in u-HCC patients receiving lenvatinib as post-progression treatment following ICI have been reported. Aoki noted that the median OS of lenvatinib was 15.8 months (95% CI 8.49-23.17) after ICI failure 40 , while Yoo reported that patients who received lenvatinib as post-progression treatment after Atez/Bev failure showed good OS (median   41 . In the present analysis, lenvatinib showed a good therapeutic effect with both first and later line administration. Thus, not only for NAFLD/NASH u-HCC cases but also those with ICI treatment failure, lenvatinib can be selected for administration as an effective subsequent therapeutic option at any time, especially in patients with good hepatic function. The present study has some limitations, including its design as a retrospective multicenter study. Furthermore, the pathological diagnosis of disease etiology for the present patients without viral hepatitis was not adequately assessed. A future study in which prospective comparisons between lenvatinib and ICI treatment in NASH/ NAFLD HCC patients is needed.
In conclusion, lenvatinib was found to be effective for improving the prognosis of u-HCC patients irrespective of HCC etiology or line of treatment.

Data availability
The datasets generated and/or analyzed for the current study are not publicly available because of privacy reasons. Table 3. Prognostic factors for progression-free survival and overall survival in Study-2. ALT alanine aminotransferase, NAFLD non-alcoholic fatty liver disease, NASH non-alcoholic steatohepatitis, ECOG PS Eastern Cooperative Oncology Group performance status, ALBI score: albumin-bilirubin score, mALBI grade modified ALBI grade, AFP alpha-fetoprotein, TNM LCSGJ 6th tumor node metastasis stage by Liver Cancer Study Group of Japan 6th edition, BCLC stage Barcelona Clinic Liver Cancer stage.