The fibrosis-4 index is a prognostic factor for cholangiocarcinoma patients who received immunotherapy

Background Research of immunotherapy for cholangiocarcinoma has yielded some results, but more clinical data are needed to prove its efficacy and safety. Moreover, there is a need to identify accessible indexes for selecting patients who may benefit from such treatments. Methods The medical records of 66 cholangiocarcinoma patients who underwent immunotherapy were retrospectively collected. The effectiveness of immunotherapy was assessed by tumor response, progression-free survival (PFS), and overall survival (OS), while safety was evaluated by adverse events during treatment. Univariate and multivariate Cox regression analyses were performed to identify prognostic risk factors for PFS and OS, and Kaplan-Meier curves of potential prognostic factors were drawn. Results Overall, in this study, immunotherapy achieved an objective response rate of 24.2% and a disease control rate of 89.4% for the included patients. The median PFS was 445 days, and the median OS was 772.5 days. Of the 66 patients, 65 experienced adverse events during treatment, but none had severe consequences. Multivariate Cox analysis indicated that tumor number is a prognostic risk factor for disease progression following immunotherapy in cholangiocarcinoma patients, while tumor differentiation and the fibrosis-4 (FIB-4) index are independent risk factors for OS. Conclusion In general, immunotherapy for cholangiocarcinoma is safe, with adverse events remaining within manageable limits, and it can effectively control disease progression in most patients. The FIB-4 index may reflect the potential benefit of immunotherapy for patients with cholangiocarcinoma.


Introduction
Cholangiocarcinoma is a highly malignant tumor with heterogeneity and can be categorized based on the location of occurrence into intrahepatic, perihilar, and distal cholangiocarcinoma.There is a considerable variation in the incidence rates across the world, with higher rates in China and other Asian countries (1).Moreover, the incidence is continually rising globally (2).To date, surgery remains the only potentially curative treatment for this disease.However, cholangiocarcinoma is characterized by insidious onset, rapid progression, and very poor prognosis.Often, it is diagnosed at a late stage when the surgical options are no longer viable.There is also a high tendency for recurrence or metastasis after surgery, further reducing the chances for additional surgical interventions.Therefore, the treatment of cholangiocarcinoma should involve a multidisciplinary approach centered around surgery, combined with systemic treatments such as chemotherapy, targeted therapy, and immunotherapy (3,4).
The NCCN Guidelines for Biliary Tract Cancer recommend chemotherapy regimens with capecitabine, gemcitabine, oxaliplatin, cisplatin, and FOLFOX as systemic therapy (5).Compared to other types of cancer, such as non-small cell lung cancer, cholangiocarcinoma is relatively insensitive to immunotherapy, with research and application in this area progressing slowly.Fortunately, clinicians and researchers are striving to advance research on immunotherapy for bile duct cancer.Several studies have explored the effectiveness and safety of immune checkpoint inhibitors as monotherapy or in combination for patients with cholangiocarcinoma, from clinical trials to small-scale clinical applications.Studies on the application of pembrolizumab for incurable cholangiocarcinoma in Phase I b and II trials have found that regardless of PD-L1 expression, it can effectively control 6%-13% of advanced bile duct cancers, with manageable toxicity (6).A real-world study based on Phase III clinical trial TOPAZ-1, incorporating durvalumab into the first-line chemotherapy regimen for cholangiocarcinoma, demonstrated a 20% reduction in the risk of death (HR=0.80,95% CI: 0.66-0.97,P=0.021), confirming the efficacy and safety of durvalumab for cholangiocarcinoma patients (7).Subsequently, more studies and case reports on the application of other immune checkpoint inhibitors in cholangiocarcinoma have emerged, further bolstering the case for immunotherapy in cholangiocarcinoma (8)(9)(10)(11).Currently, the combination of durvalumab with gemcitabine and cisplatin is an option for first-line treatment of locally advanced or metastatic cholangiocarcinoma.However, further research is needed to verify the application of immune checkpoint inhibitors in cholangiocarcinoma and to identify prognostic risk factors for patients receiving immunotherapy.
The fibrosis-4 (FIB-4) index, incorporating age, ALT, AST, and platelet levels, is one of the most powerful indicators of liver fibrosis (12,13).Studies have shown that FIB-4 can be used to predict the progression of extrahepatic tumors (14).Another study indicated that liver fibrosis could promote immune escape in hepatocellular carcinoma (15).Therefore, we hypothesize that FIB-4 can be a prognostic predictor of cholangiocarcinoma patients receiving immunotherapy.Inflammatory biomarkers such as the systemic immune-inflammation index (SII), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) can reflect a patient's systemic inflammatory and immune status (16,17).Tumor-related inflammation is closely related to tumor occurrence, progression, immune responses, and patient prognosis (18)(19)(20)(21)(22).The calculation formula for SII is neutrophil count * platelet count/lymphocyte count.The prognostic nutritional index (PNI), which combines peripheral blood lymphocyte count and serum albumin level in a certain ratio, can be used to assess a patient's nutritional and immune status (23) and is related to prognosis after receiving immunotherapy (24).
Therefore, the aim of this study is to explore the effectiveness and safety of immune checkpoint inhibitors in the treatment of cholangiocarcinoma and to investigate the predictive ability of the FIB-4 and inflammatory markers for patients with cholangiocarcinoma who received immunotherapy, providing further reference for application of immunotherapy in cholangiocarcinoma.

Study design and participants
This study included a consecutive cohort of cholangiocarcinoma patients at Tongji Hospital affiliated with Huazhong University of Science and Technology from June 2018 to May 2022, including intrahepatic, perihilar, and extrahepatic cholangiocarcinoma.Pathological specimens obtained through surgery or biopsy were confirmed by expert pathologists.Inclusion criteria were as follows: (1) undergoing more than one cycles of immunotherapy; (2) immune checkpoint inhibitors were not used in the perioperative period; (3) no history of other malignancies; (4) complete data.All data were collected retrospectively from medical records, including general data, laboratory tests, and imaging data within 7 days before the first immunotherapy.The end point of follow-up was the patient's death, loss to follow-up, or survival at the end of the follow-up period, which was December 31, 2023.This research was approved by the Research Ethics Committee of Tongji Hospital affiliated with Huazhong University of Science and Technology.

Response assessment
Tumor response was assessed 4-6 weeks after the second immunotherapy by CT or MRI, compared with imaging data within one week prior to the first immunotherapy and reported by expert radiologists.The criteria for assessment were based on RECIST 1.1 (25).The results included immune complete response (iCR), immune partial response (iPR), immune stable disease (iSD), and immune progressive disease (iPD).Additional evaluation metrics were the objective response rate (ORR) and disease control rate (DCR).ORR was the sum of iCR and iPR, and DCR was the sum of iCR, iPR, and iSD.

Safety evaluation
Patients' immune-related adverse events (irAEs) during treatment with immune checkpoint inhibitors were evaluated for the safety of immunotherapy through retrospective review of medical records, including blood routine, liver function, renal function, thyroid function, and recorded symptoms such as weight loss, fatigue, decreased appetite, rash, etc.

Statistical analysis
The presentation of data is described in the tables.Continuous variables were presented as median (interquartile range).Categorical variables were presented as numbers (percentages).The cutoff values for continuous variables were the critical values of the test items or obtained through the R package surv_cutpoint based on the time and status of overall survival (OS).Risk factors for progression-free survival (PFS) or OS were analyzed using a Cox regression model, and a P-value of less than 0.05 was considered statistically significant.Survival curves were generated using the Kaplan-Meier (K-M) method through the survminer package in R, with p-values calculated via the log-rank test.The K-M curves and adverse events were visualized using the ggplot2 package.Analysis involving the specified R packages was conducted using R 4.3.2,while sections not mentioned to use R packages were analyzed using SPSS 26.

Baseline characteristics of patients
Between June 1, 2018, and May 31, 2022, a total of 225 cholangiocarcinoma patients receiving immunotherapy were registered at our center.These patients all underwent surgical resection or percutaneous biopsy to obtain tumor tissue and were diagnosed with cholangiocarcinoma following pathological examination.After excluding patients who only received one cycle of immunotherapy, those for whom immunotherapy was used as neoadjuvant therapy, those with a history of other malignancies, those with incomplete case data or who were lost to follow-up, 66 patients were ultimately included in this study.The number of patients excluded for each criterion is shown in the flowchart (Figure 1).
The study follow-up was conducted up until December 31, 2023, with the shortest follow-up duration being 18 months for the included patients.All patients had an ECOG score of either 0 or 1.The study population comprised 37 (56.1%)males and 29 (43.9%)females, with an age distribution of 29-79 years and a median age of 57.5 years.Based on the originating site within the biliary system, there were 57 cases (86.4%) of intrahepatic cholangiocarcinoma, 7 cases (10.6%) of perihilar cholangiocarcinoma, and 2 cases (3.0%) of distal cholangiocarcinoma.Among them, 21 patients had a history of hepatitis B virus infection, accounting for 31.8% of the total.At the time of immunotherapy, more than half of the patients (59.1%) had carbohydrate antigen 19-9 (CA19-9) levels beyond the normal range, and a small portion of patients (26.9%) had abnormal carcinoembryonic antigen (CEA) levels.Approximately half of the patients (45.5%) had abnormal liver enzymes with or without increased bilirubin levels.The vast majority of patients had adenocarcinoma pathology (95.5%).Among all patients, only one case was highly differentiated, with the rest being poorly differentiated (25.8%) or moderately differentiated (72.7%).Immunohistochemistry results showed a median Ki67 value of 40, with quartiles of 15 and 69.Those included in this study for immunotherapy were all at an advanced stage of the disease, with a median tumor largest diameter of 4.8cm.39 cases (59.1%) had multiple lesions, 10 cases (15.2%) had major vascular invasion, 33 cases (50%) local lymph node metastasis, 36 cases (54.5%) liver The flowchart of this study.

Treatment strategy for immunotherapy
The treatment strategy is displayed in Table 2.In this study, the median number of continuous immunotherapy sessions was 4.5, with one individual undergoing up to 27 sessions.Most patients had previously undergone various treatments before receiving immunotherapy, such as surgery, TACE, microwave ablation, radiotherapy, chemotherapy, or combinations of these treatments.21 patients used immunotherapy as a first-line treatment; among them, a few employed only immunotherapy as the first-line treatment, often combined with chemotherapy or targeted therapy or both simultaneously.Among all cases included in this study, during the immunotherapy phase, 13 individuals (19.7%) applied immunotherapy alone, around half of the patients (n=34, 51.5%) combined with chemotherapy, and 11 patients (12.1) combined with targeted therapy, with 8 patients receiving a combination of chemotherapy, immunotherapy, and targeted therapy.

Efficacy
In this study, a total of eight different immune checkpoint inhibitors were used, with Camrelizumab being the most frequently employed.No cases were rated as having achieved iCR.Overall, the ORR post-immunotherapy was 24.2%, and the DCR was 89.4%.Specific to each inhibitor, Durvalumab had the highest DCR of 100%.Following up were Camrelizumab and Tislelizumab with DCRs of 91.3% and 90.9%, respectively.Toripalimab had the lowest ORR (12.5%) and DCR (75.0%).In the entire cohort, the median overall survival was 772.5 days, and the median progression-free survival was 445 days.Information was detailed in Table 3.
For potential risk factors identified by univariate analysis, K-M curves were plotted according to cut-off values, and P-values were calculated between different groups using the log-rank test.As shown in Figure 3, patients with higher levels of CA19-9 at the start of immunotherapy had a higher risk of tumor progression and shorter survival, with median PFS (mPFS) of 1007 vs. 348 days, and median OS (mOS) of 1509 vs. 669 days.Patients with multiple tumors had significantly shorter progression times than those with a single tumor (mOS: 1007 vs. 383 days), and notably shorter survival times, with half of single-tumor patients surviving at the conclusion of follow-up.Figure 4 shows the impact of FIB-4, SII, PLR, and PNI on OS.Patients with higher levels of FIB-4, SII, and PLR had shorter OS, while those with higher PNI levels had longer OS.K-M curves of NLR and TBil are shown in Supplementary Figure S1.Values are presented as n (%).TACE, transarterial chemoembolization.

Discussion
Recent years have seen a burgeoning interest in clinical and basic research on immune checkpoint inhibitors, which have shown promising results in a variety of cancers, including some progress in cholangiocarcinoma.The combination of durvalumab with gemcitabine and cisplatin as a first-line treatment for cholangiocarcinoma has been approved as an indication for durvalumab.Additional clinical studies using immune checkpoint inhibitors as a first-line treatment for cholangiocarcinoma are underway (26,27).At present, before immunotherapy, the most effective method for assessing possible tumor response and predicting the efficacy of immunotherapy is to biopsy the tumor tissue for PD-L1 immunohistochemistry staining.The tumor proportion score (TPS), immune cell score (IC), and tumor burden score (TBS) can be calculated based on the staining results to assess PD-L1 expression (28).However, PD-L1 testing is not routinely available in many hospitals, and patient acceptance is not high.For those diagnosed with a needle or aspiration biopsy, the sample volume is limited and may not be sufficient for PD-L1 staining.Therefore, we need simple and effective indicators to predict the prognosis of cholangiocarcinoma patients undergoing immunotherapy.This study explores the potential of comprehensive indicators such as FIB-4, SII, NLR, PLR, PNI based on various simple blood test results to predict patient prognosis.
A total of 66 patients with pathological results and who had received immunotherapy were included in this study.Most were in the advanced stages of the disease at the time of immunotherapy, with multiple lesions or macrovascular invasion, or distant metastasis.The mOS in the cohort was 772.5 days, and the mPFS was 445 days, with an DCR of 89.4%, congruent with previous studies (29).Among the 66 patients, only one did not experience any adverse events, with decreased weight being the most commonly reported side effect.In patients who developed irAEs, only eight individuals (12.1%) experienced a single adverse event, while the remainder had at least two types of irAEs.Adverse reactions affected the immunotherapy regimen in 33 patients, accounting for 50.7% of the cohort, with renal damage being the most common severe (grade 3 or higher) adverse event.Notably, one patient receiving sintilimab discontinued treatment due to severe bone marrow suppression (grade 4).According to a multicenter study that included 53 patients, researchers reported that all patients experienced irAEs (30).Fatigue, both in any grade and grade 3 or higher, was the most common adverse event, with 41.5% of patients experiencing severe (grade 3 or higher) reactions, and one case of grade 4 bone marrow suppression, findings similar to those of our study.Another study (31) divided 59 cholangiocarcinoma patients who underwent immunotherapy into two groups based on the occurrence of irAEs (32 in the irAE group, accounting for 54.2%) and compared differences between them.They observed statistical differences in total bilirubin levels and relapse between the two groups.Through Cox regression analysis, an interesting conclusion was drawn-whether irAEs occurred was an independent risk factor for the OS and PFS of patients with advanced cholangiocarcinoma.Our research, along    Occurrence rate of adverse events in this study.with these studies, underscores the necessity of investigating irAEs in cholangiocarcinoma patients, as it is meaningful for assessing the safety and efficacy of immunotherapy.
To explore prognostic risk factors, univariate and multivariate Cox regression analyses were conducted with PFS and OS as outcome variables, and the results of the univariate analysis showing statistical significance were subjected to log-rank testing.We found that patients with multiple tumors before immunotherapy had a worse prognosis, more likely to show progression after immunotherapy treatment (mOS: 1007 vs. 383 days), with a significantly shorter overall survival.A meta-analysis based on 47 research cohorts suggested tumor number as a predictive factor in hepatocellular carcinoma OS and PFS predictive models (32).Kaplan-Meier analyses revealed that, in this study, the traditional tumor marker CA19-9 has good shortterm predictive power for OS and PFS in patients with cholangiocarcinoma.A Chinese study on pancreatic cancer patients undergoing immunotherapy found that higher baseline levels of CA19-9 were associated with accelerated tumor growth during immunotherapy and worse prognosis (33).Furthermore,   poorly differentiated tumors were found to grow rapidly, to be more invasive, more likely to metastasize, and had significantly worse prognosis (mOS: 669 vs. 1509 days), which was an independent risk factor predicting the outcome of immunotherapy for cholangiocarcinoma.Obstruction often occurs in biliary diseases, leading to elevated serum liver enzyme and bilirubin levels.In our study, bilirubin levels within a week before receiving immunotherapy were related to the total survival time of the patients (mOS: 1508 vs. 263 days), with higher levels indicating a worse prognosis.Bilirubin levels affect patients with cholangiocarcinoma's prognosis in several ways: Patients with hilar cholangiocarcinoma with higher bilirubin levels at diagnosis may not benefit from surgery (34); The albumin bilirubin (ALBI) grade, which combines bilirubin and albumin levels, is an independent prognostic factor for OS and PFS in intrahepatic cholangiocarcinoma (35,36).The analysis of FIB-4, SII, NLR, PLR, and PNI showed that FIB-4 is an independent risk factor for OS in patients with cholangiocarcinoma undergoing immunotherapy.Initially identified as a non-invasive marker for predicting and diagnosing liver fibrosis, FIB-4 quickly gained widespread application and holds significant value in various diseases (37,38).Its most extensively studied aspect is the correlation between FIB-4 and the incidence, progression, and prognosis of liver cancer, as well as the response to treatment.Studies have shown that FIB-4 can predict the recurrence of hepatocellular carcinoma following curative hepatectomy (39).Large sample size researches in multiple countries have explored FIB-4 in relation to various diseases.For instance, a study in Germany found that elevated FIB-4 levels are associated with an increased risk of developing liver cancer (40,41).In the United States, a prospective study using FIB-4 as one of the markers for predicting liver fibrosis found that higher fibrosis levels were linked to an increased overall mortality rate from liver diseases (42).A retrospective study in Korea revealed that individuals with high FIB-4 levels had a significantly increased risk of dying from liver cancer (HR: 629.10, 95% CI: 228.74-1730.20)(43).Moreover, FIB-4's predictive value for the likelihood of hepatocellular carcinoma in moderate to heavy drinkers even surpasses that of ultrasound-detected liver cirrhosis indices (44).For individuals with HCV infection, FIB-4 levels are closely associated with the risk of developing liver cancer after achieving HCV clearance (45), and therapeutic interventions to reduce FIB-4 levels can decrease the risk of hepatocellular carcinoma in these patients (46,47).FIB-4 is also closely related to the prognosis of various extrahepatic tumors (48).A large cohort study in Korea involving 25,947 individuals explored the relationship between non-alcoholic fatty liver disease and various types of cancer, finding a strong correlation between high FIB-4 levels and the incidence of all types of tumors (49).In colorectal cancer patients, high pre-operative or pre-chemotherapy FIB-4 levels could reliably predict poorer long-term outcomes postsurgery (50), and patients with high FIB-4 levels had a greater likelihood of synchronous liver metastasis from colorectal cancer (51).Additionally, FIB-4 can predict the long-term prognosis after gastric cancer resection (52).FIB-4's correlation extends to mortality from non-tumorous diseases as well.Another large study in Korea found that an FIB-4 level >2.67 was associated with increased all-cause mortality, cardiovascular mortality, and liver-related mortality (53).This index could even predict the mortality rate in COVID-19 patients (54), with an FIB-4 score >2.53 being related to an increased risk of death (OR: 4.53, 95% CI: 2.83-7.25,P<0.001) (55).In this study, higher levels of FIB-4 were associated with a worse prognosis after treatment with immune checkpoint inhibitors.Research suggests that liver fibrosis can facilitate the immune escape of hepatocellular carcinoma (15).
Given the proximity of cholangiocarcinoma and hepatocellular carcinoma development sites, this might partially explain the poorer immune treatment responses in patients with elevated FIB-4 levels in our study.However, NLR levels were not significantly correlated with recurrence or prognosis.Higher Our study has limitations as PD-L1 expression was not routinely measured at our center.Consequently, we could not discuss the relationship between PD-L1 expression and immune response, progression, or prognosis in cholangiocarcinoma patients.Moreover, as this is a single-center, small sample, retrospective study, information bias cannot be completely avoided, and larger and prospective studies are needed to confirm the causal relationship between FIB-4 levels and prognosis in cholangiocarcinoma patients undergoing immunotherapy.Besides, this study did not include a comparison between patients who only received chemotherapy and those who underwent immunotherapy or a combination of immunotherapy and chemotherapy.Future research should aim to expand the cohort to include patients treated with chemotherapy alone, analyzing whether the FIB-4 levels or liver fibrosis can specifically predict the efficacy of immunotherapy in cholangiocarcinoma.Furthermore, the tumor's immune microenvironment plays a crucial role in influencing immune responses.Tertiary lymphoid structures can indicate the local immune infiltration status and their presence in various tumors has been associated with better immune responses (60,61).Thus, they hold significant potential for predicting immune reactions in cholangiocarcinoma (62) and warrant further exploration in subsequent studies.
Future research should focus on finding clinically simple and accessible indicators to guide treatment and predict whether there is a benefit from treatment modalities, providing support for clinical decision-making and maximizing patient benefits with minimal cost.

Conclusion
Our study indicates that immune checkpoint inhibitors can control the progression of most cholangiocarcinoma, with side effects within a safe range.FIB-4 may serve as a prognostic predictor for patients with cholangiocarcinoma receiving immunotherapy.

TABLE 2
Treatment strategy.

TABLE 3
Tumor response, PFS and OS.

TABLE 4
Univariate and multivariate analysis of OS.