Hepatocellular carcinoma (HCC) accounts for approximately 90% of primary liver cancers, and it is one of the leading causes of cancer-related death worldwide (1). Immunotherapy plays an increasingly critical role in the systemic treatment of HCC (2, 3). Previous clinical trials have confirmed the definite antitumor efficacy of programmed death-1(PD-1) inhibitors on HCC (4, 5). The newly released results of RATIONALE-301 (Tislelizumab versus Sorafenib) and LEAP-002 (Lenvatinib plus pembrolizumab versus Lenvatinib) on the European Society for Medical Oncology in 2022 highlighted the key role of PD-1 inhibitors monotherapy in the treatment of HCC (NCT03412773, NCT03713593). PD-1 inhibitors are emerging as a critical part of guideline-recommended first-line therapy for advanced hepatocellular carcinoma (NCT03298451) (6).

Preserved liver function is the premise of various treatments for HCC, and it is an important predictor of superior prognosis (7). Previous research confirmed that better liver function was positively associated with the prognosis of patients after hepatectomy (8), local therapy (9, 10) and systemic therapy (11, 12). Child−Pugh grade is extensively used to evaluate patient liver function when systemic therapy is recommended, and most patients are in Child−Pugh grade A, so it is necessary to develop other liver function evaluation systems to further stratify them into different groups according to their prognosis.

Alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) are readily available as part of serum liver function tests, and they are elevated in the presence of cholestasis or liver parenchymal damage. Previous studies suggested that elevated serum ALP and GGT were independent risk factors for the prognosis of HCC patients (13, 14). Other scholars have found that ALP and GGT have the potential to be used as biomarkers to predict the cancer risk and mortality of all comers (15, 16). Few studies have explored the role of ALP and GGT in the prognosis of HCC patients receiving programmed death 1(PD-1) inhibitors.

In this retrospective study, we analyzed the data of patients with HCC who received PD-1 inhibitors to explore the prognostic effect of ALP and GGT in HCC.

Our study confirmed that elevated baseline serum ALP and GGT levels, as indicators of liver parenchymal damage, were prognostic risk factors of HCC patients receiving PD-1 inhibitors, and the interference of molecularly targeted drugs was excluded according to the inclusion criteria. We also reported that ALG grade, a new combination of ALP and GGT, was a superior predictor of prognosis for the first time, and ALG grade can further stratify patients with Child−Pugh grade A into subgroups with different prognoses.

PD-1 inhibitor therapy for HCC is an emerging systemic therapy, and liver function at baseline is a confirmed prognostic factor. Child−Pugh grade is one of the most common clinical indicators for evaluating liver reserve capacity, and studies have confirmed that the prognosis of patients with Child−Pugh Grade B were worse than that of patients with Child−Pugh Grade A (19), and Albumin-Bilirubin (ALBI) grade is another common liver function scoring method that can replace Child−Pugh score (20). Our study found that Child-Pugh grade, rather than ALBI grade was associated with patient prognosis. Since most patients receiving systemic therapy have good liver function reserve in the early stage, Child−Pugh grade and ALBI grade cannot accurately stratify the prognosis of such patients, we explored other indicators in serum liver function tests. In our study, both PFS and OS of patients with normal ALP and GGT levels at baseline were superior than those of patients with elevated ALP and GGT receiving PD-1 inhibitors. Previous studies supported that elevated ALP and GGT were associated with poor survival of patients receiving liver resection (21, 22), liver transplantation (23, 24), local therapy (25, 26) and molecular targeted therapy (27, 28). This was consistent with our findings in anti-PD-1 therapy, and it is noteworthy that serum ALP and GGT levels could stratify patients with Child−Pugh grade A, enhancing the accuracy of prediction in the current study. Cox regression further confirmed the results of survival analysis. Liver function is also a critical factor in the long-term survival of patients, and this is similar to our findings. We further found that ALG grade 3 was also a prognostic risk factor for patients with Child−Pugh grade A, and there was no significant correlation between Child−Pugh grade and ALG grade. ALG grade is a reliable prognostic factor independent of Child−Pugh grade, but ALG grade 3 was an independent risk factor for PFS but not for OS in our study. The reason of this result may be: 1. most of the patients enrolled have not yet reached the clinical endpoint, only 23 patients died during follow-up; 2. the follow-up time (18 months) was relatively short; 3. Statistical differences should not be the only factor in the validity of prognostic indicators, ALG grade 3 was risk factor for PFS which has confirmed the prognostic effect of ALG grade to a certain extent.

Serum ALP and GGT are common diagnostic indicators for liver diseases. As early as 1985, elevated GGT was systematically reported to be associated with the occurrence of liver cancer (29). Drug-induced liver injury is common in patients with advanced HCC, elevated serum ALP and GGT levels hinder recovery from drug-induced liver injury (30, 31), and the concentrations of ALP and GGT in the bile duct can be used as indicators to quantify bile duct injury (32). GGT increases the level of intracellular glutathione, which can promote tumor cells to maintain balance in reactive oxygen species and avoid cell death through redox pathways during pro-oxidant therapy (33), GGT-positive tumors possess stronger proliferative ability and drug resistance (34, 35). Researchers have demonstrated that granulocyte colony-stimulating factor (G-CSF) upregulates GGT1 and enhances the immunosuppressive function of myeloid-derived suppressor cells, and GGT inhibitors can alleviate tumor immunosuppression and the tumor-promoting effect of G-CSF (36). Alkaline phosphatase is ubiquitous expressed in human body, which has four types: intestinal ALP, placental ALP, germ cell ALP and single tissue non-specific ALP (37). Previous studies have found that placental ALP is highly expressed in HCC cell lines, and this type of ALP can transform cells with specific survival advantages to malignant transformation (38). Tumor cells with ALP overexpression are more likely to form immunogenic cold tumors, and targeted killing of ALP-overexpressing tumor cells can convert immunogenic cold tumors to hot (39). This further confirms the plausibility of our finding that GGT and ALP are adverse long-term prognostic factors for HCC patients receiving PD-1 inhibitors.

There are still some limitations in the current study: First, this is a single-center, retrospective clinical study, and the level of evidence for the findings is not strong enough; Second, the number of participants in the study was relatively small, sufficient sample size and follow-up time can more realistically analyze the prognosis of patients; Third, systemic treatments are diverse for HCC patients, and our study only excluded the interference of molecular targeted therapy on risk factor analysis. Mutation and amplification of oncogenes and tumor suppressor genes also affect the long-term prognosis of patients with HCC (40). Multicenter, prospective clinical trials are still needed to further explore the role of ALG grade in the prognosis of patients with HCC receiving PD-1 inhibitors.

Our study indicated that ALG grade 3 at baseline predicted poor prognosis of patients with HCC receiving PD-1 inhibitors. ALG grade, combining ALP and GGT, is a novel and readily available prognostic marker and the predictive effect of ALG grade on patient prognosis is independent of Child−Pugh grade.

The original contributions presented in the study are included in the article/supplementary materials. Further inquiries can be directed to the corresponding authors.

Our study was approved by the Medical Ethics Committee of Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology (TJ-IRB20220936). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

LX and CL wrote the paper; LX, BinZ, and ZL collected and analyzed the clinical data;QL and HL added part of clinical information; XC revised the manuscript; CL and BixZ designed this subject. All authors contributed to the article and approved the submitted version.