Biomarkers that predict and cause resistance to checkpoint inhibitor treatments in solid cancers

ICIs offer a groundbreaking approach to treating various malignant solid tumors, contrasting with traditional cytotoxic therapies [1]. As immune cell surface receptors, ICIs have the capacity to either activate or inhibit the immune response. In the context of IT, they function as blockers of T-lymphocyte cell surface receptors, thereby enhancing the antitumor immune response [2].

For a long time, the progress in IT has been eclipsed by the rapid advancements in radiotherapy (RT) and chemotherapy [1,3]. However, recent research focused on cancer immunology and IT has enhanced our understanding of the antitumor response of T cells, as well as their interactions with cancer cells, antigen-presenting cells (APCs), immunosuppressive cells, and other immune mechanisms [1,3]. Studies by Bretcher and Cohn and Jenkins and Schwartz highlight that T-cell activation occurs via the T-cell receptor (TCR), which recognizes specific antigens, and through the costimulatory interaction between a T-cell costimulatory receptor and its ligand on an APC [4,5]. The inhibitory signals in T cells ensure tolerance to self-antigens, while the stimulatory signals promote an adaptive immune response to foreign antigens, collectively regulating immune activation [6]. Immune checkpoints allow inhibitory stimuli to modulate the inflammatory response following T-cell activation and protect normal cells from T-cell-mediated cytotoxicity [6]. This mechanism is crucial as it prevents the T-cell immune response from targeting cancer cells within the TME, serving as a protective measure [6].

Over the past decade, several types of ICIs have been studied and approved by the FDA. The first of these was the CTLA-4 inhibitor, Ipilimumab, approved in 2011 for metastatic malignant melanoma [2]. CTLA-4 exerts a negative regulatory effect on T cells, and its blockade can inhibit tumor formation, making it useful in cancer IT [2]. Additionally, the FDA has approved three anti-PD-1 antibodies—Pembrolizumab, Nivolumab, and Cemiplimab—and three anti-PD-L1 antibodies—Atezolizumab, Durvalumab, and Avelumab [1]. PD-1 and PD-L1 induce T-cell exhaustion, and blocking these pathways has been shown to enhance tumor-specific T-cell responses and promote tumor regression [2]. Ongoing research is exploring other potential ICI targets, including inhibitory receptors like TIM-3, V-domain Ig suppressor of T-cell activation (VISTA), and LAG-3, as well as activating molecules such as OX40 (CD134) and glucocorticoid-induced TNFR-related protein (GITR) [2]. However, studies have yielded mixed results, with some patients experiencing sustained therapeutic responses and others showing diminished responses over time [7]. This variability has highlighted the need to identify molecular biomarkers to predict treatment response and resistance mechanisms to checkpoint inhibitors, which will be discussed further in this article.

Malignant cells possess immunosuppressive properties, such as the expression of PD-L1 and the secretion of suppressive cytokines, which help them reduce their immunogenicity [8]. The resistance mechanisms of malignant cells can be categorized into two main strategies: evading immune recognition and creating an immunosuppressive TME [8]. These mechanisms rely on tumor cell-intrinsic and -extrinsic factors that interfere with antigen processing and presentation or enable tumors to recruit immune-suppressing cells that counteract the activity of effector cells [9]. Recent studies highlight the pivotal role of the TME in tumor-induced immunosuppression, as it is involved in downregulating effector T-cell activity and recruiting immunosuppressive cells [10].

Biomarkers can be categorized as either predictive or prognostic markers. Predictive markers, like the BRAF mutation status in melanoma, help determine the likelihood of a patient’s response to specific treatments, such as BRAF/MEK inhibitors [11,12]. Prognostic markers, such as the overall stage of disease in solid cancers and prostate-specific antigen levels at prostate cancer diagnosis, are independent of cancer interventions and can predict disease recurrence or patient survival [11,13]. The hormonal status of breast cancer serves a dual purpose as both a prognostic and predictive biomarker. Recent studies suggest that ongoing research and a deeper understanding of biological processes will enhance existing biomarkers and aid in developing new ones. PD-L1 expression and MSI are the two FDA-approved biomarkers used with ICIs. However, these biomarkers have shown limitations. Due to the genomic variability of humans and tumor cells, future approaches will likely require the use of multiple biomarkers to predict responses to ICIs accurately [11].

Recently, significant interest has emerged in studying the role of the gut microbiota in shaping responses to ICIs [11]. Several studies have identified bacterial species linked to favorable responses to ICIs, such as Akkermansia muciniphila, and others associated with resistance, like Ruminococcus obeum [11,14,15]. In patients with NSCLC or RCC, the use of antibiotics during ICI initiation has been associated with reduced microbiome diversity, leading to decreased overall survival and progression-free survival [14,16]. Additionally, a high-fiber diet has been shown to have a more beneficial effect on ICI response than a low-fiber diet [16].

A class of medications known as ICIs is used to treat cancer [17]. These medications stimulate the body’s immune system to target cancer cells more successfully. It is useful to have some understanding of the interactions between cancer cells and the immune system to comprehend how they function [17].

The immune system’s mission is to identify aberrant cells, including cancerous ones, and eliminate them. Immune cells, known as T cells, are essential to this process [18]. But to keep the immune system from attacking normal, healthy cells, it has “checkpoints” that function as brakes [18]. These are T-cell surface proteins, and they must be activated (or inactivated) to initiate an immunological response [18]. These checkpoints can be used by cancer cells to deter immune system attacks [19]. They possess the ability to express proteins that interact with these checkpoints and essentially shut down T cells, thus permitting the malignancy to spread unrestrained [19].

Checkpoint inhibitors have been approved for the treatment of a wide range of malignancies, particularly those that have proven to be resistant to conventional therapies like radiation or chemotherapy [20]. They are categorized based on the target molecule they each have, and they were first licensed for distinct malignancies in the European Union (EU) and the United States of America (USA) at different points in time, just as Table 1 describes below, but later on, it was discovered that they were also effective in other locations [20].

Numerous new immune checkpoint targets have been discovered recently, including VISTA, TIM-3, LAG-3, and T-cell Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) [32]. These studies suggested that inhibiting one immunological checkpoint could cause the TME to upregulate other checkpoint receptors in a compensatory manner [33]. Lung cancer was found to have a comparable compensatory mechanism between TIM-3 and PD-1 [34].

One of the earliest and most successful checkpoint inhibitor applications was in melanoma. ICIs like Pembrolizumab, Nivolumab, and Ipilimumab have significantly improved survival rates for patients with advanced melanoma [22,29].

Regarding other cutaneous malignancies, ICIs such as Avelumab and Pembrolizumab are used to treat Merkel cell carcinoma, a rare and aggressive skin cancer, particularly in advanced stages, while Cemiplimab is used in the treatment of cutaneous CSCC [23,28,35].

ICIs like Pembrolizumab, Nivolumab, Atezolizumab, or Durvalumab are also widely used in advanced NSCLC, either as a first-line treatment or after chemotherapy [36,37]. Some patients are tested for PD-L1 expression to determine the likelihood of response to treatment. Atezolizumab and Durvalumab are often used in combination with chemotherapy as a first-line treatment for extensive-stage small cell lung cancer (SCLC) [38].

In RCC, ICIs like Nivolumab, Pembrolizumab, and Atezolizumab are used both alone and in combination with other therapies like tyrosine kinase inhibitors (e.g., Axitinib) to treat advanced kidney cancer [39].

ICIs like Atezolizumab, Pembrolizumab, Nivolumab, Durvalumab, and Avelumab are often used in patients with advanced or metastatic bladder cancer (urothelial carcinoma) who are not eligible for chemotherapy or after chemotherapy has failed [39].

Another use for ICIs such as Nivolumab, Pembrolizumab, and Atezolizumab (in combination with Bevacizumab) is advanced HCC, especially when other treatments like surgery or local therapies are not an option [40].

Pembrolizumab and Atezolizumab are ICIs that are used in combination with chemotherapy for advanced triple-negative breast cancer (TNBC), especially in patients whose tumors express PD-L1 [41].

Pembrolizumab and Nivolumab are two ICIs particularly effective in patients with microsatellite instability-high (MSI-H) or dMMR tumors, which are subtypes of CRC [42]. They are also effective in advanced or metastatic gastric and esophageal cancers, often in patients whose tumors express PD-L1 [43].

Pembrolizumab is also approved for use in advanced or metastatic cervical cancer with PD-L1 expression [44]. Pembrolizumab and Dostarlimab are typically used in advanced or recurrent cases of endometrial cancer, particularly for tumors with MSI-H or dMMR [45].

ICIs can be used in lymphatic malignancies other than solid tumors [46]. In primary mediastinal large B-cell lymphoma (PMBCL), Pembrolizumab is approved for use in this rare type of non-Hodgkin lymphoma, especially after other treatments have failed [47]. In Hodgkin lymphoma, ICIs like Pembrolizumab and Nivolumab have been highly effective in treating it, particularly in cases where the disease has relapsed or is resistant to other treatments [46].

Research indicates that progression-free survival and duration of response to Nivolumab (an anti-PD-1 antibody), Ipilimumab (an anti-CTLA-4 antibody), or Pembrolizumab (another anti-PD-1 antibody) are greater in individuals with high tumor mutational burdens (TMBs) compared to those with lower mutational burdens [48].

ICIs can interact with tumor cells and have antitumoral effects through a variety of mechanisms, such as the following:

As discussed in the next subchapter, several predictive biomarkers have already received FDA approval and are utilized to monitor the therapeutic response to ICIs. Recent research is concentrating on evaluating the effectiveness of new biomarkers, including DNA damage response (DDR) gene alterations, MHC genotypes, beta-2-microglobulin (B2M) deficiency, polymerase epsilon (POLE) mutations, Janus kinase 1/2 (JAK1/2) mutations, tumor-infiltrating T cells (TILs), peripheral T cells, and the gut microbiome [98]. The latter will be discussed separately in a separate section of this article.

The DDR represents the cellular response to both endogenous and exogenous DNA damage, being implicated in repair processes through different pathways: dMMR, direct reversal, homologous recombination repair, nucleotide excision repair, base excision repair, and non-homologous end joining [98,99,100]. Recent studies have shown that immune checkpoint blockade holds great promise for NSCLCs. In a study by Chae et al., the authors examined a predefined list of 812 immune metagenes, derived from 813 microarrays across 36 studies, to accurately predict the presence of up to 28 intratumoral immune cell types in a large cohort of lung adenocarcinoma patients from The Cancer Genome Atlas. It was demonstrated that tumors with DNA repair deficiencies (n = 230), particularly in the dMMR pathway, which can accelerate mutation rates and impact prognosis, have significant potential to be used as biomarkers for TMB, neoantigen load, and tumor-infiltrating lymphocytes in patients undergoing anti-PD-1/PD-L1 therapy [98,101]. In urothelial cancer (UC), recent studies have indicated reduced response rates to IT, yet still an improvement overall compared to previous systemic therapies. In a study by Teo et al., the researchers hypothesized that DDR gene mutations could serve as biomarkers for anti-PD-1/PD-L1 therapy in patients with metastatic UC [102]. They analyzed sixty patients with metastatic UC treated with anti-PD-1/PD-L1 antibodies, enrolled in three separate prospective trials between April 2014 and December 2016, and identified seventy-four DDR gene alterations, including twenty-seven deleterious DDR gene alterations, before treatment initiation [102]. Patients with deleterious DDR gene alterations exhibited a lower incidence of hepatic involvement and were associated with a higher TMB [102]. Their findings showed that DDR gene alterations were linked to clinical benefits in patients receiving anti-PD-1/PD-L1 therapy, with more pronounced effects observed in those with deleterious DDR alterations (80%) and in those whose tumors had wild-type DDR genes (19%; p = 0.001) [102]. Additionally, Teo et al. emphasized the significance of deleterious DDR alterations beyond MMR deficiency, which constituted only 5% of their study cohort, in contrast to findings by Chae et al. [101,102].

MHC-I molecules present intracellular proteins to T cells, enabling them to identify mutated or foreign peptides, which influences antigenicity [98]. Recent research indicates that the insufficient presentation of driver mutation neoantigens by MHC-I may explain why certain tumors do not respond to ICIs [98]. MHC-II molecules, found specifically in APCs such as macrophages and DCs (DC), have also been studied. Recent findings suggest that human leukocyte antigen-DR isotype (HLA-DR), a component of MHC-II, is elevated in malignant cells and is associated with favorable prognosis and improved response to ICI treatment [103,104]. In a study conducted by Johnson et al., the authors analyzed two independent cohorts of melanoma patients treated with anti-PD-1 therapy, and they highlighted that malignant cells, particularly melanoma cells, with high levels of HLA-DR are more responsive to PD-1 antibodies due to their inflammatory signals, even in patients with poor prognosis, such as those with liver metastases [105]. In their study, 75% of HLA-DR-positive patients responded to anti-PD-1 therapy, compared to only 27% of HLA-DR-negative patients (p = 0.025), and they also demonstrated a significantly better survival rate (median not reached versus 27.5 months, log-rank p = 0.003) [104]. Additionally, a study by Jiang et al. evaluated the IT response in 85 NSCLC patients, using HLA typing, revealing that those expressing HLA-DRB4 had an improved overall survival of 26.3 months compared to 10.2 months in untreated patients. This finding was significant in both univariate (p = 0.022; 26.3 months vs. 10.2 months) and multivariate analysis (p = 0.011, HR 0.49, 95% CI = 0.29–0.85), emphasizing the significance of MHC-II in the antitumor immune response during ICI treatment [104]. However, Jiang et al. also noted an increased risk of endocrine adverse reactions in patients with HLA-DRB4 undergoing ICI treatment (11 patients, 81.8% had HLA-DRB4, p = 0.0139), although the cause remains unknown [104]. Importantly, Jiang et al. found that patients who developed adverse reactions to ICIs had increased overall survival rates, underscoring the complex relationship between treatment response and adverse reactions [104].

Recent studies have shown that B2M deficiency, a subunit of MHC-1, can downregulate MHC-1 antigen presentation, thereby increasing cancer immune evasion [98]. In a study by Sade-Feldman, the authors analyzed longitudinal tumor biopsies from 17 metastatic melanoma patients and observed that B2M deficiency was more prevalent in non-responder patients (29.4%) compared to responders (10%) treated with anti-CTLA-4 or anti-PD-1 therapy. This deficiency was associated with poorer survival rates, suggesting that B2M could be a potential biomarker for therapy resistance [106]. The study highlighted that one of the main reasons for this resistance is the absence of proteins that can substitute for B2M in HLA class I presentation [106].

POLE is a crucial eukaryotic DNA polymerase involved in DNA replication and repair. A study by Rousseau et al. discovered that POLE mutations can serve as predictive biomarkers for patients with CRC or endometrial carcinoma treated with PD-1 antibodies, as these mutations are associated with increased TMB and tumor-infiltrating lymphocytes [107]. The study highlighted that POLE mutations are primarily found in young male patients with CRC and young female patients with endometrial carcinoma, often indicating a favorable prognosis [107]. Their analysis revealed that tumors harboring POLE mutations exhibited higher levels of cytotoxic T helper (Th) 1 cell infiltrates and PD-1 expression, along with significant upregulation of IFN-γ, PD-L1, and CTLA-4, which are critical for patients undergoing ICI therapy [107]. Using Nivolumab, an anti-PD-1 antibody therapy, the study observed therapeutic responses exclusively in CRC and endometrial carcinoma patients with POLE mutations, reporting a survival rate of 5.4 months [107]. Remarkably, they noted a near-complete response in a 42-year-old patient with unresectable CRC and POLE mutations [107]. The study also emphasized the correlation between dMMR and POLE mutations, suggesting that DNA repair defects can be effective biomarkers for ICI treatment in certain cancer types [107]. Furthermore, they underscored the importance of associating POLE mutations with low-TMB tumors, which do not benefit from PD-1 blockade, in contrast to high-TMB tumors [107].

JAK1/2-inactivating mutations have been shown to increase tumor resistance to Pembrolizumab, an anti-PD-1 therapy, by disabling the IFN-γ responsive pathway [98]. In a study by Shin et al., these mutations were analyzed in patients with melanoma or CRC and high TMB that did not respond to anti-PD-1 therapy due to the diminished IFN-γ pathway and consequent lack of PD-L1 expression [108]. This finding is intriguing, as many studies have shown that patients with high TMB generally respond better to IT [91]. Biopsies from non-responder patients with advanced melanoma and high TMB revealed JAK1 domain mutations, along with low or absent CD8 infiltrates and PD-1/PD-L1 expression [108]. Conversely, in responder patients, JAK2 mutations were observed but lacked the same functional impact [108]. The results indicated that patients with JAK1 mutations did not respond to IFN-α, IFN-β, or IFN-γ, and consequently to anti-PD-1 therapy, whereas patients with JAK2 mutations were unresponsive only to IFN-γ but showed PD-L1 upregulation when exposed to IFN-α or IFN-β [108]. Nonetheless, survival rates for patients with tumors harboring JAK1/2 mutations were significantly lower [108]. The study concluded that JAK1/2-inactivating mutations are both acquired and primary causes of resistance to anti-PD-1 therapy, serving as effective predictive markers for this resistance [108].

TILs, especially tumor-reactive cytotoxic CD8+ T cells, have been linked to better responses in patients with various tumor types treated with ICIs [98,109,110]. Tissue-resident memory T cells (TRM), a recently discovered subtype of CD8+ memory T cells, have shown promise in improving outcomes for patients with NSCLC, oral, or breast cancer treated with anti-PD-1 therapy [98,109,110]. These cells enhance responses by blocking PD-1 or CTLA-4 expression, making them potential biomarkers for ICI treatment [98,109,110].

Corgnac et al. conducted a retrospective study on a cohort of 111 patients with NSCLC, discovering that those with higher pre-IT levels of CD8+ TRM cells, characterized by the CD103+CD49a+CD69+ phenotype, had increased TRM levels during anti-PD-1 treatment. This was associated with improved outcomes compared to non-responders who had low CD8+ levels (HR = 0.40, 95% confidence interval [CI], 0.20–0.77, p = 0.006). The median immunotherapy progression-free survival for these patients was 30 months (95% CI, 2.73, not reached) [109]. This improvement was attributed to the higher presence of activated tumor antigen-reactive T cells. Thus, TRM cells were suggested as potential biomarkers for ICI treatment in NSCLC patients [109].

Another study by Lee et al. demonstrated that TRM cells with the CD39+ phenotype contributed to favorable responses in patients with triple-negative breast cancer treated with ICIs and predicted patient survival, primarily due to their functional restoration capability [110]. Importantly, CD39+ TRM cells influenced both local and systemic antitumor immunity. Their study also showed that combining anti-CTLA-4 and anti-PD-1 therapy yielded better outcomes than anti-PD-1 therapy alone in these patients [110].

Chronic antigen stimulation leads to the exhaustion of tumor-reactive cytotoxic CD8+ T cells, giving rise to exhausted T cells and their precursors (TPEX) [111]. These TPEX cells have been recently studied in melanoma and NSCLC patients undergoing anti-PD-1 therapy, being correlated with enhanced response duration, suggesting their potential as biomarkers for ICI treatment [98,111].

Miller et al. found that TPEX cells were more effective than terminally exhausted T cells in controlling tumor growth [111]. In their study of melanoma patients who responded to ICI therapy, they observed higher expression of TPEX cells compared to terminally exhausted T cells, concluding that TPEX cells could be biomarkers for longer response duration to ICIs in melanoma patients [111]. Similarly, Liu et al. observed higher TPEX cell levels and lower terminally exhausted T-cell levels in NSCLC responders compared to non-responders following anti-PD-1 therapy, reinforcing the potential of TPEX cells as biomarkers [112].

Immuno-oncology has undergone significant changes over time due to rapid advancements in research. Today, thousands of potential therapies are available in immuno-oncology, thanks to robust drug development pipelines. However, despite promising preclinical data, some studies have observed hyperprogressive disease in patients during later clinical trials when treated with a combination of immune checkpoint inhibitors (ICIs) and other immuno-oncology therapies [48].

Chen et al. conducted a comprehensive pan-cancer analysis, examining the prevalence of Kelch-like ECH-associated protein 1 (KEAP1) mutations in a cohort of 40,167 patients with various cancer types (including lung cancer, endometrial cancer, hepatocellular carcinoma, head and neck cancer, bladder cancer, colorectal cancer, and esophagogastric cancer) using data from the cBioPortal online database [113]. They found that the overall prevalence of KEAP1 mutations was 2.7%, with the highest prevalence observed in patients with NSCLC (15.8%) [113]. The study also revealed that patients with KEAP1 missense mutations exhibited the highest TMB levels, indicating that KEAP1 mutations could serve as negative predictive biomarkers for immunotherapy outcomes [113]. The authors demonstrated the prognostic significance of KEAP1 mutations in both early-stage (p = 0.0099) and advanced-stage cancers (p < 0.0001) [113]. Moreover, when correlating KEAP1 mutations with overall survival, they found that patients with these mutations had a significantly reduced overall survival rate (39 months) compared to those without KEAP1 mutations (109 months) (p < 0.0001) and shorter disease-free survival (97 vs. 158 months, p = 0.0677) [113]. A contributing factor to these outcomes was the significantly lower infiltration of CD8+ T cells in patients with KEAP1 mutations, particularly in endometrial cancer, breast cancer, bladder cancer, colorectal cancer, lung adenocarcinoma, and lung squamous cell carcinoma, with the lowest levels observed in lung adenocarcinoma [113].

In a retrospective study by Papillon-Cavanagh et al., the researchers analyzed mutations in KEAP1 and serine/threonine kinase 11 (STK11) among 2276 patients with stage IIIB, IIIC, IVA, or IVB NCSLC, including 574 patients who received anti-PD-1/anti-PD-L1 inhibitor treatment. The study found that both STK11 and KEAP1 mutations were associated with a poor response across multiple therapeutic classes [114]. However, this poor response was not specifically observed in the cohort treated with ICIs, nor was there a significant impact on progression-free survival or overall survival. For STK11 mutations, the hazard ratios were HR 1.05 (95% CI 0.76 to 1.44, p = 0.785) for progression-free survival and HR 1.13 (95% CI 0.76 to 1.67, p = 0.540) for overall survival [114]. For KEAP1 mutations, the hazard ratios were HR 0.93 (95% CI 0.67 to 1.28, p = 0.653) for progression-free survival and HR 0.98 (95% CI 0.66 to 1.45, p = 0.913) for overall survival [114]. The authors concluded that these mutations serve more as prognostic biomarkers rather than predictive ones for anti-PD-1/anti-PD-L1 therapy [114].

In contrast, many studies have highlighted the poor prognosis of patients with STK11 gene codes for liver kinase B1 (STK11/LKB1) and KEAP1 mutations treated with ICIs. However, recent research evaluating their impact in KRAS wild-type patients, or independent of KRAS mutations, suggests that the poor prognosis is primarily linked to concurrent KRAS mutations [115]. These contradictory findings largely stem from study limitations, such as the inclusion of other gene mutations and the lack of comparison arms involving patients who received alternative treatments [115].

Recent studies have emphasized the significance of targeting the nuclear factor erythroid 2-related factor 2 (NRF2)–KEAP1 axis in various diseases, including cancer, due to the cytoprotective functions of proteins encoded by NRF2-target genes, such as those involved in antioxidant defense, detoxification, and anti-inflammatory processes [116]. However, targeting the NRF2-KEAP1 axis was not previously considered a priority, largely because of its extensive range of biochemical activities that extend beyond the regulation of antioxidant systems, many of which are governed either directly or indirectly by NRF2, leading to nonspecific effects [116]. The benefits of targeting the NRF2-KEAP1 axis have only recently gained attention, including the maintenance of redox signaling, enhanced biotransformation of xenobiotics, regulation and resolution of inflammation, suppression of gluconeogenesis and hepatic lipogenesis, support of proteostasis, and inhibition of fibrosis [116]. Recent research has identified numerous effective drugs targeting the NRF2-KEAP1 axis for both neoplastic and non-neoplastic diseases, with many already approved and in use, and several others still under investigation [116]. Table 2 summarizes drugs targeting the NRF2-KEAP1 axis that have shown efficacy in certain types of cancer.

In a comprehensive pan-cancer analysis by Lee et al., researchers examined the genomic landscape of Kirsten rat sarcoma viral oncogene homolog (KRAS)-altered solid and hematologic malignancies in 426,706 patients treated with chemotherapy, chemotherapy combined with ICIs, or ICI monotherapy [119]. They found that KRAS alterations were present in 23% of the cases, with 88% being mutations, most commonly observed in CRC with KRAS G12D and G12V mutations, followed by NSCLC with KRAS G12C mutations [119]. The study also evaluated co-altered genes related to immunotherapy response, including TMB and PD-L1 expression, as well as co-mutations in STK11 and KEAP1, which were associated with low (28% and 16%) or negative (53% and 27%) PD-L1 expression, respectively [119]. The researchers observed similar overall survival rates in NSCLC patients with KRAS G12C and KRAS G12V mutations (11 vs. 10 months, HR 1.0, 95% CI 0.86–1.20, p = 0.88) and KRAS G12D mutations (11 vs. 12 months, HR 0.91, 95% CI 0.75–1.11, p = 0.36) [119]. Notably, patients with KRAS alterations had decreased overall survival compared to those without KRAS alterations but with other oncogenic mutations (11 vs. 27 months, HR 0.55, 95% CI 0.49–0.63, p < 0.001) [119]. Similar findings were reported in CRC and other cancer types, emphasizing the significant implications for developing KRAS inhibitors, particularly targeting G12C, G12D, and G12V mutations [119]. The authors acknowledged study limitations, including the lack of detailed clinical and treatment information for patients in the database and the selection bias introduced by using comprehensive genomic profiling [119].

Selective KRAS G12C inhibitors are the only treatments that have shown clinical benefits in patients with NSCLC and KRAS G12C mutations who have undergone at least one prior systemic therapy [117]. Sotorasib, an approved selective KRAS G12C inhibitor, has demonstrated clinical efficacy and a tolerable safety profile in these patients [117]. Similarly, Adagrasib, another approved KRAS G12C inhibitor, has proven effective in pretreated NSCLC patients [117]. However, some newer inhibitors are still under investigation and face limitations due to intrinsic and acquired resistance mechanisms. KRAS mutations are also present in pancreatic ductal adenocarcinoma and CRC [117].

Sotorasib is a KRAS G12C inhibitor that binds irreversibly to the S-IIP pocket, locking KRAS in its inactive GDP-bound state and inhibiting KRAS oncogenic signaling [117]. Additionally, Sotorasib enhances its interaction with KRAS G12C and increases potency by binding to a surface groove created by an alternative orientation of histidine at position 95, surpassing the effectiveness of AMG-1620, another KRAS G12C inhibitor [117]. Studies using xenograft models have demonstrated that Sotorasib can inhibit ERK phosphorylation and tumor cell growth in KRAS G12C-mutant cells, leading to durable tumor regression as a monotherapy [117]. It can also be used in combination with chemotherapy [117].

Adagrasib is another KRAS G12C inhibitor with a mechanism similar to that of Sotorasib, irreversibly binding to the mutant protein and locking it in its inactive GDP-bound form [117]. However, Adagrasib offers some advantages over Sotorasib, including high oral bioavailability, a long half-life (approximately 24 h), extensive tissue distribution, and the ability to penetrate the central nervous system [117]. Notably, studies have shown that Adagrasib exhibits synergistic effects when combined with inhibitors of the EGFR family, SHP2, mTOR, or CDK4/6 [117]. Table 2 provides a summary of the key drugs used to inhibit the KRAS G12C mutation across various cancer types.

Additionally, other mutations, such as mesenchymal–epithelial transition (MET), human epidermal growth factor receptor 2 (HER2), rearranged during transfection (RET), and neurotrophic tyrosine receptor kinase (NTRK), are still under study and have shown promising clinical potential for the development of specific inhibitors [117].

Despite the remarkable efficacy of ICIs, many patients (~70%) do not respond well or develop resistance to these drugs [120]. The response rate to ICIs varies between 15% and 30% in most solid tumors and 45% and 60% in MSI-H cancers and malignant melanoma [121].

Currently, the FDA has only approved three predictive biomarkers for ICI therapy in malignancies [122]. These biomarkers are PD-L1, MSI or dMMR, and TMB [122].

PD-L1, also known as programmed death ligand 1, has gained recognition as a significant biomarker in breast cancer in recent times [123]. There is a correlation between this factor and poorer outcomes in patients with hormone receptor-positive breast cancer. However, it has a predictive function in guiding the response to systemic treatment specifically in the TNBC subtype, particularly in cases where the cancer has spread [123]. ICIs are now being incorporated into the treatment regimen for a growing number of patients with TNBC [123].

Monoclonal antibody IT that targets PD-1 andvPD-L1 has become a widely accepted treatment for patients with lung cancer (NSCLC) [124]. It is used in both initial and subsequent treatment stages, and has shown long-lasting positive responses in around 10-20% of treated patients [124].

In 1992, T. Honjo and his colleagues at Kyoto University discovered that PD-1 is a gene related to apoptosis [125]. However, the process of apoptosis does not necessitate the overexpression of PD-1 [125].

PD-1 function requires tyrosine in T and B lymphocytes [126]. The orderliness in the cytoplasmic tail of PD-1 can be bound by protein tyrosine phosphatase 1 and 2 (SHP-1, SHP-2) [126]. However, it is unknown whether PD-1 takes SHP-1 and/or SHP-2 in a physiological setting. Cysteine is responsible for the homodimerization of CD28, CTLA-4, and inducible costimulatory molecules [127]. On the other hand, PD-1 is monomeric on the cell surface and is unable to form covalent homodimers because it lacks the extracellular surface [128].

Subsequent research conducted in 1996 by the same group revealed that PD-1 expression was stimulated by antigen receptors on T and B cells [129] and had a role in suppressing immunological responses, since PD-1-deficient mice displayed autoimmune symptoms [130,131].

Nobel prize laureate Tasuku Honjo and his team have used J43 mAb immunoprecipitation and flow cytometry to determine the PD-1 gene product, which is a 50–55 kDa membrane protein produced on the cell surface of 2B4.11 cells, and various PD-1 cDNA transfectants [132].

The PD-1 antigen is present in specific subsets of thymocytes and spleen T cells, which may cause T-cell activation or death [129]. In a similar manner, in vitro treatment with an anti-Ig M antibody caused PD-1 expression on spleen B cells [129]. PD-1, on the other hand, was not substantially expressed on lymphocytes after treatment with lipopolysaccharide, dexamethasone, or growth factor deprivation [129]. The findings indicate that PD-1 antigen expression is closely controlled and stimulated by signal transduction via the antigen receptor [129]. While PD-1 expression is not necessary for the common process of apoptosis, it remains possible that the PD-1 antigen contributes to lymphocyte clonal selection [133].

PD-1 functions as PD-L1’s T-cell-inhibitory receptor, and both PD-L1 (CD274) and PD-L2 (CD273; also known as B7-DC) are expressed in cancer cells and APCs in the TME [134,135,136].

The goal of targeting PD-1 and its ligands is to attack tumors with the help of the body’s immune system [137,138]. The development and use of such blocking antibodies fundamentally altered the course of cancer IT [137,138].

PD-1 is activated in several peripheral hematopoietic cells by cytokines and antigen receptor signaling; its expression occurs throughout thymic development [127]. During TCR β rearrangement, immature CD4−CD8− (double negative) thymocytes express PD-1 [139]. Upon their activation, peripheral CD4+ and CD8+ T cells, B cells, monocytes, NK T cells, and certain subsets of DCs express PD-1 through induction [140].

PD-1 on T cells and APCs can be induced by estrogen [141]. As a result of being activated, peripheral T cells (CD4+ and CD8+), B cells, NK T cells, monocytes, and certain types of DCs (DC) show PD-1 expression. BCR or TCR activation makes PD-1 function [142].

Macrophages, memory B cells, and mast cells exhibit inducible PD-L2; PD-L2 is expressed in fewer cell types compared with PD-L1 [126,143]. Hematopoietic and non-hematopoietic cells generally express PD-L1 [144]. Although produced constitutively in T cells, PD-L1 levels are raised after the activation of B cells, DCs, macrophages, and mast cells originating from bone marrow [127].

Mice naturally express more PD-L1 than humans [127]. Neurons, pancreatic islet cells, endothelial cells, epithelial cells, fibroblastic reticular cells, and cells identified in immune-privileged regions, such as retinal pigment epithelial cells and the placenta, all contain PD-L1 [127].

Due to the widespread expression of PD-L1 on both hematological and non-hematopoietic cells, and the expression of PD-1 on B cells, T cells, some DCs, and macrophages, there are several potential interactions in both directions between PD-1 and PD-L1 [145]. T cells excel in facilitating the function of PD-1 [145].

PD-1 interacts with one of its ligands during TCR signaling, reducing T-cell survival and inhibiting T-cell proliferation, cytokine generation, and cytolytic activity [146,147].

PD-Ls have been shown in several studies to have the ability to inhibit T-cell cytokine production and cell proliferation. However, they have also been seen to stimulate T-cell activation. The origins of these divergent results are now unclear [148,149].

Immune cells that target particular antigens and do not have PD-1 receptors respond more strongly to antigen-containing resting DCs, mostly via CD8+ T cells. It is the antigen itself that causes this reaction [150]. A negative impact on effector T-cell reactivation, proliferation, and function may also be exerted via the PD-1/PD-L pathway [148]. Some tumor-penetrant cancer cells and APCs may also display PD-1/PD-L1 interaction in cis when these cells express PD-1 and PD-L1, according to recent studies [151]. This interaction is called cis expression [151]. Researchers found that when PD-1 and PD-L1 are produced on the same cancer or antigen-presenting cell (APC), they touch each other directly on the cell surface. Hence, this contact, called cis interaction, reduces the capacity of PD-L1 to bind to PD-1 on T cells in a trans manner [152,153]. This goal was achieved by the creation of new methods for re-creating lipid bilayer systems in vitro and by conducting experiments in cell cultures [154].

The microbiota, alongside PD-L1 expression, TMB, dMMR, MSI, and TME, has emerged as a crucial biomarker under continuous investigation for its role in the therapeutic response of solid cancers. It significantly contributes to the development of the intestinal immune system [2,196].

Recent studies have demonstrated a strong association between the microbiota and the efficacy of therapeutic responses in solid tumors, including ICIs [197]. The microbiota induces an immune response against malignant cells by activating T cells and increasing the levels of human IL-2 and IFN-γ [198]. Figure 3 depicts the enhancement of treatment with ICIs through the influence of the intestinal microbiota.

The role of IFN-γ in producing a favorable response to ICI therapy is achieved through its regulation of 30 genes, stimulation of NK cell activity, enhancement of antigen presentation, and ability to increase macrophage lysosomal activity, thereby exerting antitumor and immunoregulatory effects [196,198]. Similarly, IL-2 contributes to a favorable response to ICI therapy by regulating leukocyte activity, interacting with antibodies, promoting hematopoiesis and tumor surveillance, and inducing NK cells to produce cytokines such as IFN-γ and tumor necrosis factor-α (TNF-α) [196,198].

Dysbiosis, an altered state of the gut microbiota, has been linked to chronic health conditions and cancer, but also a poor response to ICIs [16,198,199]. Dysbiosis caused by antibiotics and various diets can influence antitumor immune responses by reducing the production of deoxycholate and short-chain fatty acids [199]. Bacterial species most associated with effective ICI responses include Bifidobacterium, Faecalibacterium, Lactobacillus, and Akkermansia (HR = 2.92, 95% CI = 1.08–7.89), particularly in cases of melanoma or HCC, modulating tumor responses to checkpoint blockade IT [16,199].

A study by Gopalakrishnan et al. found significant differences in the gut microbiota of patients with melanoma. Responders to ICI treatment (CTLA-4 inhibitors and PD-1 antibodies) exhibited higher alpha diversity and an abundance of Ruminococcaceae bacteria (p < 0.01), along with enrichment of anabolic pathways and enhanced systemic and antitumor immunity [16]. The gut microbiota of responder patients was enriched with Clostridiales, Faecalibacterium, and Ruminococcaceae (p < 0.01), whereas non-responders had increased amounts of Bacteroidales, Escherichia coli, and Anaerotruncus colihominis (p < 0.01) [16].

The study also observed that responder patients had elevated levels of CD4+ and CD8+ T cells, with preserved cytokine response and a higher density of immune cells and markers of antigen processing and presentation, which can modulate antitumor immune responses [16]. In contrast, non-responders exhibited higher levels of regulatory T cells and MDSCs, along with a reduced cytokine response [16].

Unfortunately, some studies have highlighted the risk of immune-related adverse events in patients treated with ICIs. Despite Faecalibacterium being associated with a positive effect on ICI efficacy, an increased risk of immune-related adverse events has been observed [199]. The gut microbiome can influence the local immune system and the trafficking of bacterial peptide-primed T cells distally, potentially leading to increased toxicity from ICI treatment [199].

In a study by Xu X et al., the efficacy of PD-1 antibody IT was evaluated in CT26 tumor-bearing mice with CRC, treated with different types of antibiotics (vancomycin and colistin) and different gut microbiota compositions, compared to a control group given non-antibiotic sterile drinking water [198]. Initially, the antibiotic-treated mice showed reduced tumor growth, but this trend reversed significantly after introducing PD-1 antibody treatment [198]. Tumor growth progressed more in the vancomycin group (moderately) and the colistin group (significantly), compared to the control group, which responded favorably to the PD-1 antibody [198]. The bacterial species observed were Bacteroides spp. (18.76%) and Bacteroidales (4.6%) in the control group, Bacteroides spp. (20.83%), Prevotella spp. (7.75%), and Bacteroidales in the colistin-treated group, and Prevotella spp. and Akkermansia muciniphila (3.85%) in the vancomycin-treated group [198]. Prevotella and Akkermansia muciniphila were associated with a favorable response to IT due to their roles in the biosynthesis of steroid hormones, mannose, phenylpropane, beet red, and carotene [198]. In contrast, Bacteroides and Bacteroidales were linked to a poor therapeutic response through tumor signaling pathways, antigen recognition and presentation, Th17 cell differentiation, IL-17 secretion, estrogen signaling pathways, pyrimidine metabolism, and nucleic acid shearing and repair [198].

Routy et al. [14] also highlighted the positive effects of Akkermansia muciniphila in epithelial tumors (p = 0.004). Studies have shown that Akkermansia muciniphila enhances gut barrier function, normalizes metabolic endotoxemia and adipose tissue metabolism, improves immune responses, and decreases serum ALT, AST, pro-inflammatory cytokines, and chemokines [200,201,202].

Significant differences in metabolic and immune processes were observed when comparing the two antibiotic-treated groups. In the colistin-treated mice, abnormal T-cell glucose metabolism and chronic inflammation induced by type I diabetes mellitus, antigen processing and presentation, cancer pathways, Th 17 cell differentiation, and IL-17 signaling pathways were noted [198,202,203]. In contrast, vancomycin-treated mice showed increased glycerolipid metabolism and glycosphingolipid biosynthesis, which were associated with a more favorable response to PD-1 antibody treatment [198].

An important aspect analyzed by Xu et al. was the similarity of bacterial species before and after PD-1 antibody treatment in the three groups, indicating that the microbiota did not change post-IT [198]. The authors also noted changes in IFN-γ and IL-2 cytokines in the TME of antibiotic-treated mice, secondary to changes in gut microbiota, leading to different efficacies of PD-1 antibody treatment [198].

Recent studies have demonstrated that anti-PD-1 antibodies exhibit some efficacy in treating HCC [204,205]. Zheng et al. conducted a study analyzing eight patients with stage C HCC, according to the Barcelona Clinic Liver Cancer classification, who were treated with anti-PD-1 antibodies without any concurrent antibiotic treatment [206]. At the beginning of ICI treatment, the patients exhibited no dysbiosis, with an enrichment of Gram-positive Firmicutes, Gram-negative Bacteroidetes, and Gram-negative Proteobacteria in their fecal samples [206]. Throughout the treatment, patients who responded did not show significant changes in gut microbiota composition [206]. After 12 weeks, Proteobacteria, particularly Escherichia coli, became dominant in non-responding patients, while Klebsiella pneumoniae increased in responding patients [206]. Additionally, responding patients exhibited elevated levels of Lactobacillus species, Bifidobacterium dentium, and Streptococcus thermophilus, which inhibit pathogenic microorganisms [206]. They also had higher levels of Lachnospiraceae, Ruminococcaceae species, and Akkermansia muciniphila, which help maintain normal intestinal permeability and systemic immunosuppression [206]. In conclusion, Zheng et al. highlighted that the gut microbiome could serve as a biomarker for early detection of the 6-month prognosis of patients treated with PD-1 antibodies, as early as 3-6 weeks into the treatment [206].

Some studies emphasize the importance of probiotics in the intestinal flora, noting that they can increase the concentration of bacterial metabolites such as propionate and butyrate, which in turn enhance the activity of immune cells [198,207,208]. Other studies have demonstrated that the oral administration of Bifidobacterium can enhance the efficacy of PD-L1 antibody therapy and that oral administration of Akkermansia muciniphila can increase the effectiveness of PD-1 antibodies [14,209]. Table 3 summarizes the types of bacteria and their effects on the efficacy of ICI treatment responses.

Many recent studies highlight the critical importance of controlling the gut microbiota before and during treatment with PD-1 antibodies, emphasizing that maintaining a normal gut microbiota is crucial for a favorable therapeutic response [198]. Therefore, all factors that can alter the taxonomic composition, functional metagenomics, and metabolic profiles of the microbiota must be considered before starting treatment with ICIs, as these factors can significantly influence the therapeutic response to ICIs [2].

ICIs have significantly transformed cancer IT, particularly in metastatic melanoma, by improving overall survival rates [210]. These therapies target immune checkpoints like CTLA-4 and PD-1, which play essential roles in T-cell activation and maintaining immunological tolerance [211]. Despite their success, ICIs present challenges, including treatment resistance, immune-related adverse events (irAEs), and limited safety data in pediatric patients [212]. Adverse events are notably more severe with CTLA-4 inhibitors, especially at higher doses, while PD-1/PD-L1 inhibitors generally have milder toxicities, with fatigue being the most common side effect [213]. The potential for life-threatening off-target effects in children is particularly concerning [214,215]. Ongoing research is exploring novel combinations of ICIs with other therapies—such as chemotherapy, targeted agents, RT, and T-cell-based treatments—to enhance outcomes, especially in patients who do not respond favorably to ICI monotherapy [51]. Additionally, the TME plays a crucial role in treatment response, posing further complexities that require continued investigation to optimize therapeutic strategies across various cancer types [20]. Combining RT and IT may enhance their effectiveness through synergistic mechanisms, offering hope against cancers resistant to conventional treatments [216]. Additionally, photodynamic therapy (PDT), which disrupts tumor vasculature and activates the immune system, holds promise but requires carefully designed photosensitizers to work effectively in the challenging tumor microenvironment, particularly under hypoxic conditions [217].

In conclusion, the gut microbiota has emerged as a significant biomarker influencing the therapeutic response to ICIs in solid cancers. Research shows that the microbiota can enhance immune responses against tumors by activating T cells and increasing cytokine levels, such as IL-2 and IFN-γ, thereby improving ICI efficacy. However, dysbiosis—an imbalance in the gut microbiota—can lead to poor therapeutic outcomes [218]. Specific bacterial species, like Akkermansia muciniphila and Bifidobacterium, are associated with favorable responses to ICIs, while others are linked to negative effects. The gut microbiome’s composition can also predict patient prognosis early in treatment [218]. Probiotics have shown the potential to enhance the efficacy of ICIs by modulating the gut microbiota, highlighting the importance of microbiota in cancer IT.

One limitation of our study is the heterogeneity of the included trials, particularly in the definition of microbiome diversity, which may limit the generalizability of these findings. Additionally, most studies did not control for antibiotic use, which is a significant confounder in microbiome research.

Therefore, many promising future studies should consider these variables regarding anti-PD-1 and anti-CTLA-4 treatments and the improvement of their efficacy.