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Biomarkers and future prospects for immunotherapy in hepatocellular carcinoma.
Hepatocellular carcinoma is the sixth most common malignancy worldwide, causing hundreds of thousands of deaths in 2020, however, current treatment options for patients with advanced hepatocellular carcinoma remain limited.
Tumor immunotherapy has developed rapidly in recent years, especially in the field of immune checkpoint inhibitors ( ICIs ), which are designed to activate and enhance anti-tumor immunity, offering new prospects for the treatment of patients with advanced cancers, including liver cancer.
Currently, atezolizumab ( anti-PD-L1 blocker ) combined with bevacizumab ( VEGF antibody ) has shown encouraging clinical results in patients with liver cancer.
Nonetheless, only a small fraction of liver cancer patients currently benefit from ICI therapy, and the incidence of immune-related adverse events ( IRAEs ) is also an important issue, highlighting the need for a better understanding of ICI-tumor interactions, as well as the identification of immunotherapy response predictive biomarkers.
PD-L1 is widely expressed on the surface of tumor cells, antigen-presenting cells and other immune cells. It has been reported that PD-L1 expression is generally low in HCC ( about 10% of tumor cells ) and is associated with recurrence and shorter OS.
Several clinical trial results have been published in which PD-L1 was assessed as a predictive biomarker of ICI response.
In the CheckMate 459 phase III clinical trial, patients with PD-L1-positive tumors responded better to nivolumab than sorafenib, although nivolumab did not cause a significant improvement in OS.
However, the predictive value of PD-L1 is unclear, and in the CheckMate 040 and NCT02658019 trials, response rates were disappointing in all patients regardless of PD-L1 expression levels.
Even so, further analysis of patients in the CheckMate 040 trial showed that tumor PD-L1 expression was associated with improved OS.
The conflicting results of these clinical trials may be due in part to limitations in the detection of PD-L1 levels, including the lack of standard methods for assessing PD-L1 expression and its spatiotemporal heterogeneity, as well as the lack of standard thresholds that allow determination of ‘overexpression’.
There are two methods used to define PD-L1-positive expression, the ratio of PD-L1-positive tumor cells, the so-called tumor proportion score ( TPS ), and the ratio of PD-L1-stained tumor and immune cells, the so-called combined Positive Score ( CPS ).
The KEYNOTE-224 phase II trial assessed PD-L1 using two scoring methods, and CPS proved to be the more applicable biomarker.
Furthermore, the predictive value of PD-L1 expression may be underestimated because it is usually assessed at a single time point, whereas PD-L1 expression may be dynamic and inducible.
Tumor mutational burden/microsatellite instability
Tumor mutational burden ( TMB ) refers to the number of non-synonymous mutations found in a single tumor genome, including DNA damage response genes and alterations in genes encoding catalytic subunits of DNA polymerase epsilon ( POLE ) and delta ( POLD ), and has been assessed Its potential as a biomarker for multiple tumor types.
A study evaluating the frequency of genomic biomarkers of ICI response in 755 HCC patients found a median TMB of 4 mutations/Mb, with high TMB found in only 6 tumors ( 0.8% ).
Furthermore, in a small sample of cases ( N=17 ), TMB was not associated with ICI response.
However, as in the case of PD-L1 expression, TMB assays lack standardized thresholds and there are differences in quantification methods.
Therefore, the clinical value of TMB should be interpreted with caution.
DNA mismatch repair ( MMR ) is a key mechanism for maintaining genome integrity and stability. The lack of MMR activity results in a hypermutated phenotype called microsatellite instability ( MSI ).
A study evaluating the efficacy of pembrolizumab monotherapy in 86 patients with advanced MMR-deficient cancer showed ORR and complete response ( CR ) rates of 53% and 21%, respectively, and the results of this study support MSI as a solid tumor inhibitor to PD-1. predictors of clinical response to diagnosis.
Although MSI is considered an indicator for selecting responders to ICI therapy, data in HCC patients are still lacking. Ang et al assessed MSI in 542 HCC specimens and found only one case ( 0.2% ) with high MSI and high TMB, while several other studies reported generally low rates of MSI in HCC.
These findings highlight that MSI may not be an ideal biomarker for predicting response to ICIs in HCC patients.
Tumor infiltrating lymphocytes
Since T cell infiltration within the TME is a prerequisite for immune checkpoint blockade, baseline intratumoral T cell density and phenotype have been extensively studied.
Retrospective biomarker analysis conducted in the CheckMate 040 trial showed that, although not statistically significant, increased numbers of CD3+ or CD8+ tumor-infiltrating T cells in nivolumab-treated HCC patients were associated with a trend toward improved OS.
The study further demonstrated an association between increased frequency of CD3+ T cells and optimal overall response.
The link between TIL and response to ICI therapy has also been investigated in other immune cell types. Ng et al analyzed HCC samples from 49 patients treated with ICIs and reported that patients with high intratumoral CD38+CD68+ macrophages had better median OS compared with those with low CD38+CD68+ macrophages ( 34.43 vs 9.66 months ), possibly because CD38hi macrophages produced more IFN-γ and related cytokines.
Deep single-cell RNA sequencing was performed on 5063 T cells isolated from peripheral blood, tumor tissue, and adjacent normal tissue of 6 HCC patients.
The results showed that specific subsets, such as exhausted CD8+ T cells and regulatory T cells ( Treg ), were preferentially enriched in HCC.
Despite the importance of focusing on lymphocytes in the TME, its clinical application as a biomarker remains far away. How to select lymphocytes with specific characteristics and how to determine positive criteria are one of many unanswered questions.
Jointly assessing the number of multiple immune cells and PD-L1 expression to develop a TME score may be one answer.
Specific gene changes
In recent years, molecular maps associated with the emergence of NGS have provided information on actionable targets and identified specific genetic alterations associated with responses to ICIs.
Mutations in the CTNNB1 gene leading to activation of the WNT/β-catenin signaling pathway are characteristic of immune-rejecting HCC ( cold tumors ) and are associated with significantly lower enrichment scores for several immune signatures.
In a subgroup of 31 patients treated with different ICIs, altered activation of the WNT/β-catenin pathway was associated with lower disease control rates ( 0% vs 53% ), shorter intermediate median PFS ( 2.0 vs 7.4 months ) was associated with shorter median OS ( 9.1 vs 15.2 months ).
Furthermore, TP53 gene alterations are the most common mutations in HCC patients and are mutually exclusive with CTNNB1 mutations.
Studies have shown that changes in TP53 are closely related to the immune microenvironment of liver cancer, with less infiltration of CD8+ T cells and more infiltration of FOXP3+ Treg, resulting in down-regulation of immune responses.
Recent studies have shown that immune-related long noncoding RNAs ( lncRNAs ) can predict the response to ICI therapy in liver cancer. Peng et al. reported that the host gene of lncRNA MIR155 was strongly positively correlated with the expression of CTLA-4 and PD-L1 in liver cancer tissues, and had predictive value for the efficacy of ICI therapy.
Furthermore, researchers identified an immune-related lncRNA signature associated with poorer survival as an independent prognostic biomarker in HCC patients.
Several studies have shown that epigenetic alterations inherent in cancer cells are associated with carcinogenesis and tumor progression as well as changes in the TME, suggesting its potential as a predictor of immunotherapy.
Furthermore, given that DNA methylation can be measured in liquid biopsies, epigenetic biomarkers may offer additional advantages.
These observations suggest that the presence of specific genetic mutations, particularly those associated with CTNNB1, TP53, noncoding RNAs, and methylation, may influence response to ICI therapy by interacting with the immune microenvironment.
Although these studies are still preliminary, these alterations may represent new biomarkers of selection in patients with liver cancer.
Immune-related gene markers
A comprehensive analysis of tumor transcriptome profiling data was recently performed to characterize the responsiveness of the immune microenvironment to ICI treatment.
Ayers et al identified an 18-gene T-cell inflammatory expression profile and 6-gene IFN-γ signature that predicted response to pembrolizumab treatment in multiple solid tumors.
The retrospective analysis of the results of the CheckMate 040 trial also included an assessment of the predictive value of 4-gene inflammatory signatures ( PD-L1, CD8A, LAG3 and STAT1 ). This feature was found to be associated with improved responses and OS associated with nivolumab treatment during the dose escalation and dose expansion phases.
Recently, a 5-gene immune-related signature ( LDHA, PPAT, BFSP1, NR0B1, and PFKFB4 ) was identified and used to establish a prognostic model of HCC treatment response that could stratify immunotherapy-sensitive patients.
While these immune-related gene signatures show potential for predicting ICI response, these signatures require further testing for clinical application.
Peripheral blood biomarkers
Continued availability of tumor samples from ICI-treated patients is critical for biomarker research, however, this is difficult to achieve due to the invasive nature of biopsies.
In contrast, circulating biomarkers can be easily collected and repeatedly measured after treatment, making them more convenient for clinical use.
Studies have shown that TGF-β plays a central role in immunosuppression and tumor immune evasion within the TME.
In HCC, there is a strong correlation between TGF-β signaling and depleted immune signaling. In a phase II trial analyzing several representative circulating biomarkers in 29 patients with unresectable liver cancer treated with pembrolizumab, baseline plasma TGF-β levels <200 pg/mL were better Effective predictors of OS and PFS.
Some soluble immune checkpoint-related proteins have recently been shown to have good predictive value in various cancer types. Like PD-L1 exosomes, in one study, the magnitude of the increase in circulating exosomal PD-L1 levels could distinguish clinical responders from non-responders.
In addition, soluble PD-L1 ( sPD-L1 ) is associated with immunotherapy response in NSCLC patients, whereas data on HCC patients are lacking and further studies are needed.
Circulating immune cells in peripheral blood have been widely evaluated as predictive biomarkers.
In a trial of tremelimumab in combination with locoregional therapy in patients with HCC, the frequency of CD4+PD-1+ cells in peripheral blood mononuclear cells at baseline was found to be higher in responders than in nonresponders.
In addition, in 16 patients with liver cancer treated with nivolumab, lower baseline levels of PD-1+ B cell positivity and PD-L1+ monocyte positivity in peripheral blood were observed to correlate with disease control.
Circulating tumor DNA ( ctDNA ) can predict the response of several tumors to ICI therapy.
However, a study using ctDNA to analyze the mutational status of advanced HCC reported that WNT pathway-related mutations were not associated with clinical outcomes after ICI therapy, suggesting that further studies are needed to determine whether ctDNA can indeed serve as a predictive biomarker for HCC.
High alpha-fetoprotein ( AFP ) levels are considered to be a prognostic marker of poor clinical prognosis in patients with liver cancer.
Recently, it has been reported that decreased serum AFP levels after treatment is associated with better ICI treatment outcomes in advanced HCC.
However, results from the checkmate040 clinical trial showed that while baseline AFP <400 µg/L was associated with longer OS than AFP ≥ 400 µg/L, ORR and DCR were similar regardless of baseline AFP level.
Given that AFP levels are closely related to patients’ baseline characteristics, their predictive effects should be interpreted with caution.
Biomarkers in peripheral blood have the great advantage of being less invasive, and numerous studies have shown their potential as predictors of immunotherapy efficacy.
However, supportive data from large clinical trials are still lacking.
Furthermore, there is currently no evidence that blood samples have better predictive accuracy than tissue samples for any given biomarker.
Growing evidence suggests that the gut microbiota plays a critical role in the development and regulation of innate and adaptive immunity, and research has found that there appears to be a relationship between the gut microbiota and response to ICI treatment .
Zheng et al. recently reported dynamic changes in gut microbiome composition during anti-PD-1 immunotherapy for liver cancer by metagenomic sequencing.
They observed that fecal samples from immunotherapy responders showed higher taxonomic richness and higher gene counts compared to non-responders, the microbial composition of the responder group remained relatively stable, while the number of Proteobacteria in the non-responder group remained relatively stable.
Significant increase from week 3 and predominance at week 12.
Furthermore, antibiotic use at the start of ICI treatment has been reported to be associated with worse outcomes, suggesting the impact of gut microbiota on HCC treatment.
A clinical trial ( NCT03785210 ) is currently underway to evaluate the efficacy of a combination antibiotic ( vancomycin ) and ICI therapy.
Potential biomarkers for IRAE
Most studies have focused on identifying biomarkers that predict immunotherapy efficacy, while relatively few studies have investigated biomarkers associated with IRAEs.
In addition, most of the findings were from patients with melanoma. However, these studies can serve as a reference for identifying biomarkers that predict IRAE in HCC.
In patients receiving ipilimumab, baseline serum IL-6 and IL-17 levels were significantly associated with an increased risk of severe toxicity, with IL-17 being associated with severe diarrhea/colitis.
In a retrospective study involving 167 adult patients with solid tumors, increased baseline lymphocyte counts were associated with an increased risk of IRAEs in patients treated with nivolumab or pembrolizumab.
Future studies should explore the underlying biological mechanisms associated with the efficacy of ICIs, and how to balance IRAE incidence and immunotherapy response deserves serious consideration.
In HCC, although several studies have been conducted to identify predictive biomarkers, HCC-related immunotherapy is still in its infancy, and basic research and clinical trials exploring immunotherapy biomarkers to predict efficacy are still limited and currently impossible Identify which biomarkers are effective in predicting the efficacy of immunotherapy.
Of particular note is that biomarkers undergo dynamic changes in patient populations.
Therefore, it is crucial to obtain samples from patients before and during treatment to assess these dynamic changes and correctly determine the predictive value of assessing biomarkers, with due consideration for their application in clinical practice.
Advances in research techniques such as NGS, single-cell RNA sequencing, and artificial intelligence will allow us to gain a more complete understanding of the various components of the TME and their interactions, and allow extensive screening of potential biomarkers at the genomic scale to determine the determinants of therapeutic efficacy. predictor.
1. Biomarkers and Future Perspectives for Hepatocellular Carcinoma Immunotherapy. Front Oncol. 2021; 11: 716844.
Biomarkers and prospects for immunotherapy in hepatocellular carcinoma
(source:internet, reference only)