**4. Dynamic predictive and prognostic soluble biomarkers in cancer immunotherapy**

In the subsections below, we will focus on blood-based candidate biomarkers that can be utilized as predictive or prognostic markers in cancer immunotherapy.

#### **4.1 Blood cell counts/ratios, C-reactive protein and lactate dehydrogenase**

Changes in the blood cell counts and their ratios including neutrophils, lymphocytes, neutrophil to lymphocyte ratio as well as C-Reactive Protein (CRP) and Lactate Dehydrogenase (LDH) have been reported as prognostic/predictive outcome markers for immunotherapy [2]. Several studies have shown that low neutrophils and high lymphocytes are associated with overall survival (OS) in cancer patients [58, 115, 116]. For example, melanoma patients on nivolumab treatment having absolute lymphocyte counts of >1000/μL and absolute neutrophil count of <4000/μL were observed to have better overall survival [115]. On the other hand, pre-treatment neutrophil-to-lymphocyte ratio (NLR) and derived NLR (dNLR) can also serve as an index of the systemic inflammatory response and therefore considered as useful indicators of response in immunotherapy. Pre-treatment NLR/ dNLR levels and survival association studies in advanced cancers including melanoma, non-small-cell lung cancer (NSCLC) and genitourinary have reported that high pre-treatment NLR and dNLR levels are associated with poor progression free survival (PFS)/OS with increased risks of death in immunotherapy treated patients indicating their usefulness as predictive and prognostic biomarkers [117–120].

CRP is an inflammatory marker that induces the expression of acute-phase proteins such as neutrophils and has been correlated with poor prognosis in several cancers [121, 122]. With regards to immunotherapy, post treatment increased CRP levels have been associated with inflammation, disease progression and in some cases immune-related adverse events. On the other hand, low CRP levels post immunotherapies have been associated with better antitumor response/ survival [93, 123].

LDH is a final enzyme in the glycolysis pathway that catalyzes the interconversion of pyruvate and lactate. In cancers, high levels of LDH leads to increased utilization of glycolysis as their energy requirement in the microenvironment [124]. Studies have confirmed that LDH is a significant negative prognostic factor for immunotherapy treated stage 4 melanoma patients [125]. Elevated baseline LDH in melanoma and lung cancer patients treated with pembrolizumab/nivolumab is associated with poor OS and higher risk of death [126–128]. Similar results have been reported for advanced esophageal squamous cell carcinoma patients treated with the anti-PD-1 immune checkpoint inhibitor camrelizumab where elevated LDH levels were found to correlate with poor OS [129].

The fact that blood cell counts/ratios, CRP and LDH tests are performed as part of a routine diagnosis and also are highly assessable/measurable at various treatment timelines in patients making them attractive dynamic biomarkers.

*Advances in Precision Medicine Oncology*

regulator of mDCs-associated cytokine secretion and antigen-specific CD4<sup>+</sup>

favorably to the anti-tumoral immune response [108].

proliferation [105]. Moreover, subsets of intra-tumoral MDSCs that express PD-1 and CTLA-4 are found to display decreased arginase 1 expression and activity upon anti-CTLA-4 or anti-PD-1 treatment in mice [106]. In murine models, it has been shown that arginase 1 impairs T cells functions and contributes to immune evasion [107]. Furthermore, it has been recently reported that anti-PD-1 therapy was able to prevent the block in myeloid cell lineage maturation, thereby allowing the myeloid precursors to maturate into effector macrophages and dendritic cells contributing

A decline in circulating MDSCs under anti-CTLA-4 is found to correlate with patient outcome in several studies [37, 90, 109, 110], although this association is not universally reported [111]. Moreover, these studies showed discordant observations regarding the dynamics and predictive value of the major MDSCs subsets (monocytic MDSCs (mo-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) subsets) [112]. In addition, several studies showed that anti-PD-1 treatment had no effect on the level of circulating mo-MDSC and PMN-MDSC subsets [9, 113, 114]. Yet a particular study revealed a prominent restructuration of the myeloid compartment after initiation of anti-PD-1 therapy in metastatic melanoma patients when studied

T cell

**36**

**Figure 3.**

*Effect of PD-1 and CTLA-4 blockade on myeloid cells.*

#### **4.2 Soluble immune checkpoint inhibitors**

Soluble forms of immune checkpoints (sICs) are shed in the plasma/serum and have been associated with modulation of the immune system by affecting the binding capacity of immunotherapeutic drugs and thus influencing the efficiency of immune system. Studies have demonstrated that sICs can serve as markers for prognosis, response to treatment and overall response rate (ORR) in immunotherapy treated patients [130]. In addition to this, these markers can also be important for prediction of immune related adverse events which is an area poorly explored with respect to these biomarkers. Here, in this sub-section, we will discuss sICs evidenced in literature as prognostic and predictive markers in ICIs treatment.

#### *4.2.1 Soluble immune inhibitory markers*

#### *4.2.1.1 Soluble PD-1, PDL-1 and PDL-2*

sPD-1 has been documented to inhibit all three PD-L1/PD-1 interactions: PD-L1/ CD80, PD-L1/PD-1, and PDL2/PD-1 [131]. Researchers have demonstrated that expressed sPD-1 blocks PD-L1/PD-1 interactions that can lead to inhibition of tumor growth via various mechanisms including blockade of PD-L1 on tumor cells, upregulation of CD8<sup>+</sup> T cells, reduction in the expression of IL-10, increased production of inducible nitric oxide synthase, TNF-α and IFN-γ and enhancement of the immune response through interaction with immune cells [132–134]. sPD-1 has been reported as a modulator of immune response during ICIs treatment in serum of cancer patients. A study on 22 NSCLC patients observed that sPD-1 decreased significantly in clinically responding patients during Nivolumab treatment. In addition to this, patients with performance status of 0 had a decreased sPD-1 during treatment and these patients were found to have better immune fitness with low levels of immunosuppression [135]. Similarly, a study on 177 unresectable metastatic melanoma patients treated with anti-PD-1 showed interesting results. High pre-treatment serum concentrations of PD-1 and PD-L1 were correlated with poor prognosis and survival. The authors postulated that circulating serum PD-1 molecules might be directly targeted by therapeutic anti-PD-1 antibodies and this interaction might impair the effectiveness of anti-PD-1 therapy via neutralization. It is possible that this is a tumor escape mechanism that facilitates poor outcome. Thus, quantification of circulating PD-1 and PD-L1 molecules can translate into prognostic and predictive factors in immunotherapy treated patients [136].

sPD-L1 is produced by tumor cells/activated mature DCs and is known to have structural similarities to mPD-L1 [137]. It has been postulated that sPD-L1 has the capability to exert a competing effect against anti-PD-L1 drugs. A study by *Gong et al.* reported that NSCLC patients who were refractory to ICIs treatment secreted a sPD-L1 variant (without the transmembrane domain) in serum and this variant competed, bound and then inhibited the activity of immunotherapeutic drug in such patients [138]. This is a critical finding as it gives evidence on the dynamic role of sPD-L1 and its utility as a biomarker of response in immunotherapy. Furthermore, studies on various cancers have also reported on this aspect. For example, a study on ipilimumab treated melanoma patients showed that patients with high pre-treatment sPD-L1 levels showed poor prognosis and disease progression [139]. This is an interesting observation and sheds light on the dynamic nature of this biomarker. Similarly, two studies on NSCLC have documented that high levels of sPD-L1 correlated with poor prognosis, OS, and abdominal metastasis [140, 141]. However, to date no correlation of sPD-L1 with tissue PD-L1expression has been reported indicating the dynamic nature of secreted PD-L1 that is distinct from tissue PD-L1.

**39**

*Evolving Dynamic Biomarkers for Prediction of Immune Responses to Checkpoint Inhibitors…*

tool for patient stratification with regards to anti-PD-1 therapy benefit.

Several published studies evidence consistent data on its utility as a monitoring tool to test the efficacy of ICIs as a prognostic biomarker. Further studies with systematic uniform methodologies may allow better understanding of sPD-L1 as an effective

sPD-L2 is a splice variant protein product that lacks transmembrane domain and is secreted into the blood. Distinct expression pattern of PD-L2 variants in leukocytes of distinct cellular status have been observed suggesting that modulation of sPD-L2 expression may have an influence on the outcome of the immune response [142]. However, limited data on sPD-L2 as an immune related biomarker is available. A study by *Zizari et al.,* on NSCLC patients treated with Nivolumab showed that sPD-L2 was dynamically modulated during ICIs treatment. The concentrations of sPD-L2 were found to be significantly lower in responding patients. In addition to this, soluble mediators including low PD-L1, CD137, Tim-3 and BTLA-4 in combination with low sPD-L2 are associated with favorable clinical response indicating that sPD-L2 works in synergy with other molecules to modulate the immune response. This allows understanding on the dynamic interaction of soluble immune modulators as useful biomarkers of response [135]. In addition to response prediction, a study on NSCLC patients treated with Nivolumab reported interesting results with respect to immune related adverse events. Low sPD-L2 concentration at diagnosis as well as in pre-treatment samples was found to be associated with occurrence of immune grade 3–4 toxicity indicating that sPD-L2 can also serve as potential predictive biomarker for immune related adverse events in ICIs treated patients [143].

The major source of sCTLA-4 is Tregs, monocytes and immature DCs [144]. sCTLA-4 has been reported in several studies as a plausible marker for response in ICIs treatment. For example, in melanoma patients treated with ipilimumab, high pre-treatment expression of sCTLA-4 was associated with response to treatment and longer OS [145]. Another study on metastatic melanoma patients treated with ipilimumab showed similar results with high levels of sCTLA-4 at baseline associated with disease responsiveness and survival. Interestingly, the study observed that in responding patients, sCTLA-4 concentration increased with subsequent treatment cycles while in progressing patients, sCTLA-4 decreased subsequently indicating that sCTLA-4 can serve as a valuable dynamic marker for treatment monitoring. In addition to this, it was also observed that patients with high pre-treatment sCTLA-4 were at a higher risk of developing immune related adverse events providing further insight into its utility as a biomarker of response/adverse event monitoring [146]. However, in different cancers, its utility may be distinct based on its interaction with other molecules and subsequent immune modulation. For example, a recent study on NSCLC patients treated with nivolumab showed that lower concentration of sCTLA-4 at 3 months of clinical evaluation was associated with response. In addition to this, patients with performance status of 0 consistently maintained a lower expression of sCTLA-4 from the time of treatment initiation until 3 months of clinical evaluation indicating that sCTLA-4 can be an indicator of immune fitness in ICIs treated patients [135]. It is postulated that during ICIs treatment, sCTLA-4 might be involved in enhancing the ability of host cytotoxic T cells to attack tumor cells and

*DOI: http://dx.doi.org/10.5772/intechopen.96494*

*4.2.1.2 Soluble CTLA-4, TIM-3 and LAG-3*

thereby enhancing the antitumor effect of immunotherapy.

sTIM-3 secreted in blood lacks mucin and transmembrane domains. It is postulated that sTIM-3 is shed from the cell surface due to metalloproteinase-dependent cleavage and may serve as a decoy receptor for TIM-3 ligands thereby interfering with the inhibitory function of TIM-3 [147]. However, the exact function of sTIM-3 is still unknown. A study on plasma levels of sTIM-3 in ICIs treated patients observed that NSCLC

#### *Evolving Dynamic Biomarkers for Prediction of Immune Responses to Checkpoint Inhibitors… DOI: http://dx.doi.org/10.5772/intechopen.96494*

Several published studies evidence consistent data on its utility as a monitoring tool to test the efficacy of ICIs as a prognostic biomarker. Further studies with systematic uniform methodologies may allow better understanding of sPD-L1 as an effective tool for patient stratification with regards to anti-PD-1 therapy benefit.

sPD-L2 is a splice variant protein product that lacks transmembrane domain and is secreted into the blood. Distinct expression pattern of PD-L2 variants in leukocytes of distinct cellular status have been observed suggesting that modulation of sPD-L2 expression may have an influence on the outcome of the immune response [142]. However, limited data on sPD-L2 as an immune related biomarker is available. A study by *Zizari et al.,* on NSCLC patients treated with Nivolumab showed that sPD-L2 was dynamically modulated during ICIs treatment. The concentrations of sPD-L2 were found to be significantly lower in responding patients. In addition to this, soluble mediators including low PD-L1, CD137, Tim-3 and BTLA-4 in combination with low sPD-L2 are associated with favorable clinical response indicating that sPD-L2 works in synergy with other molecules to modulate the immune response. This allows understanding on the dynamic interaction of soluble immune modulators as useful biomarkers of response [135]. In addition to response prediction, a study on NSCLC patients treated with Nivolumab reported interesting results with respect to immune related adverse events. Low sPD-L2 concentration at diagnosis as well as in pre-treatment samples was found to be associated with occurrence of immune grade 3–4 toxicity indicating that sPD-L2 can also serve as potential predictive biomarker for immune related adverse events in ICIs treated patients [143].

#### *4.2.1.2 Soluble CTLA-4, TIM-3 and LAG-3*

The major source of sCTLA-4 is Tregs, monocytes and immature DCs [144]. sCTLA-4 has been reported in several studies as a plausible marker for response in ICIs treatment. For example, in melanoma patients treated with ipilimumab, high pre-treatment expression of sCTLA-4 was associated with response to treatment and longer OS [145]. Another study on metastatic melanoma patients treated with ipilimumab showed similar results with high levels of sCTLA-4 at baseline associated with disease responsiveness and survival. Interestingly, the study observed that in responding patients, sCTLA-4 concentration increased with subsequent treatment cycles while in progressing patients, sCTLA-4 decreased subsequently indicating that sCTLA-4 can serve as a valuable dynamic marker for treatment monitoring. In addition to this, it was also observed that patients with high pre-treatment sCTLA-4 were at a higher risk of developing immune related adverse events providing further insight into its utility as a biomarker of response/adverse event monitoring [146]. However, in different cancers, its utility may be distinct based on its interaction with other molecules and subsequent immune modulation. For example, a recent study on NSCLC patients treated with nivolumab showed that lower concentration of sCTLA-4 at 3 months of clinical evaluation was associated with response. In addition to this, patients with performance status of 0 consistently maintained a lower expression of sCTLA-4 from the time of treatment initiation until 3 months of clinical evaluation indicating that sCTLA-4 can be an indicator of immune fitness in ICIs treated patients [135]. It is postulated that during ICIs treatment, sCTLA-4 might be involved in enhancing the ability of host cytotoxic T cells to attack tumor cells and thereby enhancing the antitumor effect of immunotherapy.

sTIM-3 secreted in blood lacks mucin and transmembrane domains. It is postulated that sTIM-3 is shed from the cell surface due to metalloproteinase-dependent cleavage and may serve as a decoy receptor for TIM-3 ligands thereby interfering with the inhibitory function of TIM-3 [147]. However, the exact function of sTIM-3 is still unknown. A study on plasma levels of sTIM-3 in ICIs treated patients observed that NSCLC

*Advances in Precision Medicine Oncology*

**4.2 Soluble immune checkpoint inhibitors**

*4.2.1 Soluble immune inhibitory markers*

*4.2.1.1 Soluble PD-1, PDL-1 and PDL-2*

upregulation of CD8<sup>+</sup>

Soluble forms of immune checkpoints (sICs) are shed in the plasma/serum and have been associated with modulation of the immune system by affecting the binding capacity of immunotherapeutic drugs and thus influencing the efficiency of immune system. Studies have demonstrated that sICs can serve as markers for prognosis, response to treatment and overall response rate (ORR) in immunotherapy treated patients [130]. In addition to this, these markers can also be important for prediction of immune related adverse events which is an area poorly explored with respect to these biomarkers. Here, in this sub-section, we will discuss sICs evidenced in literature as prognostic and predictive markers in ICIs treatment.

sPD-1 has been documented to inhibit all three PD-L1/PD-1 interactions: PD-L1/ CD80, PD-L1/PD-1, and PDL2/PD-1 [131]. Researchers have demonstrated that expressed sPD-1 blocks PD-L1/PD-1 interactions that can lead to inhibition of tumor growth via various mechanisms including blockade of PD-L1 on tumor cells,

duction of inducible nitric oxide synthase, TNF-α and IFN-γ and enhancement of the immune response through interaction with immune cells [132–134]. sPD-1 has been reported as a modulator of immune response during ICIs treatment in serum of cancer patients. A study on 22 NSCLC patients observed that sPD-1 decreased significantly in clinically responding patients during Nivolumab treatment. In addition to this, patients with performance status of 0 had a decreased sPD-1 during treatment and these patients were found to have better immune fitness with low levels of immunosuppression [135]. Similarly, a study on 177 unresectable metastatic melanoma patients treated with anti-PD-1 showed interesting results. High pre-treatment serum concentrations of PD-1 and PD-L1 were correlated with poor prognosis and survival. The authors postulated that circulating serum PD-1 molecules might be directly targeted by therapeutic anti-PD-1 antibodies and this interaction might impair the effectiveness of anti-PD-1 therapy via neutralization. It is possible that this is a tumor escape mechanism that facilitates poor outcome. Thus, quantification of circulating PD-1 and PD-L1 molecules can translate into prognostic and predictive factors in immunotherapy treated patients [136].

sPD-L1 is produced by tumor cells/activated mature DCs and is known to have structural similarities to mPD-L1 [137]. It has been postulated that sPD-L1 has the capability to exert a competing effect against anti-PD-L1 drugs. A study by *Gong et al.* reported that NSCLC patients who were refractory to ICIs treatment secreted a sPD-L1 variant (without the transmembrane domain) in serum and this variant competed, bound and then inhibited the activity of immunotherapeutic drug in such patients [138]. This is a critical finding as it gives evidence on the dynamic role of sPD-L1 and its utility as a biomarker of response in immunotherapy. Furthermore, studies on various cancers have also reported on this aspect. For example, a study on ipilimumab treated melanoma patients showed that patients with high pre-treatment sPD-L1 levels showed poor prognosis and disease progression [139]. This is an interesting observation and sheds light on the dynamic nature of this biomarker. Similarly, two studies on NSCLC have documented that high levels of sPD-L1 correlated with poor prognosis, OS, and abdominal metastasis [140, 141]. However, to date no correlation of sPD-L1 with tissue PD-L1expression has been reported indicating the dynamic nature of secreted PD-L1 that is distinct from tissue PD-L1.

T cells, reduction in the expression of IL-10, increased pro-

**38**

patients treated with nivolumab had a lower level of sTIM-3 at three months of clinical evaluation and this correlated with the response and longer survival of the patient [135]. Though, the study does give an indication of sTIM-3 as biomarker of response, further studies are needed to understand its dynamic nature in ICIs treatment.

sLAG-3 plays a role in immune pathways and has been associated as a Th1 activity marker in serum. sLAG-3 has been reported to bind to MHC class II and induce maturation of dendritic cells thereby facilitating attack on tumor cells [148, 149]. This makes sLAG-3 an attractive biomarker in ICIs treatment. Though several studies on LAG-3 and cellular response has been documented, there is paucity of data on the utility of sLAG-3 in serum of ICIs treated patients. A study on nivolumab treated NSCLC patients reported that sLAG-3 was significantly increased during treatment and this increase was retained in non-responding patients. In addition to sLAG-3, other soluble mediators including sPD-1 and sPDL-2 were also increased in these patients indicating the dynamic interactive nature of LAG-3 and its role as predictive marker [135].

#### *4.2.1.3 Soluble IDO, CD163 and NKG2DL*

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that acts as an immune checkpoint and inhibits T-cell proliferation by starving the cells from tryptophan (trp) in order to sensitize them to apoptosis [150]. IDO facilitates tumor immune escape and several preclinical studies have associated IDO activity with immunotherapeutic resistance [151]. A study on nivolumab treated NSCLC patients found that lower baseline level of IDO activity in serum was significantly associated with better PFS/ OS while higher levels were associated with early progression indicating that high serum levels can serve as early marker of response/indicator of resistance to anti-PD-1 treatment [152]. On the other hand, a larger study on 27 NSCLC patients treated with nivolumab indicated the dynamic nature of sIDO at baseline, 2 months and at disease progression. The study aimed to evaluate the dynamic nature of IDO as a predictor of response. Interestingly, the authors observed that at baseline, IDO activity was higher in responding patients than non-responding patients. However, in patients who did not benefit from immunotherapy, a statistically significant increase was observed between baseline sample and sample taken at the time to disease progression indicating the potential utility of IDO as therapeutic resistance marker to anti-PD-1 treatment [153].

CD163 is the hemoglobin/haptoglobin complex scavenger receptor expressed exclusively on circulating monocytes/tissue macrophages. It is involved in antiinflammatory functions associated with macrophages and therefore plays an important role in suppressing anti-tumor immune responses [154]. sCD163 is secreted in plasma via proteolytic shedding and is considered a specific marker for TAM. To demonstrate the utility of sCD163 as a marker of response in immunotherapy, *Fujimura et al.* conducted a study on 59 cases of advanced cutaneous melanoma and 16 cases of advanced mucosal melanoma treated with nivolumab. It was observed that in advanced cutaneous melanoma group, sCD163 was significantly increased at 6 weeks of treatment in the response group as compared to non-response group indicating that sCD163 is an early marker of response in nivolumab treated patients [155]. On the other hand, another study by the same group determined the utility of sCD163 as predictor of immune related adverse events (irAE) in nivolumab treated advanced melanoma patients. It was observed that at day 42 of treatment, the absolute value of sCD163 significantly increased in patients with adverse nivolumabinduced, immune-related events indicating that sCD163 can also serve as a valuable predictor of irAEs in immunotherapy [155]. However, due to limited published data, further studies will provide a better understanding on this marker.

NKG2D is an activating immunoreceptor of cytotoxic lymphocytes and is expressed on T, NK, and NKT cells. NKG2D has eight ligands including MIC (MICA

**41**

**5. Conclusion**

*Evolving Dynamic Biomarkers for Prediction of Immune Responses to Checkpoint Inhibitors…*

and MICB) or ULBP (ULBP1, ULBP2, ULBP3, ULBP4, RAET1G, and RAET1L) family. These NKG2D ligands are absent on normal cells but are usually overexpressed on tumor cells. Soluble NKG2D ligands (sNKG2DLs) are generated by proteolytic shedding of tumor cells which boosts tumor immune escape by binding and subsequent endocytosis/degradation of NKG2D receptor on NK/T cells thus suppressing antitumor immune responses [156, 157]. Various sNKG2DLs have been studied as predictive biomarkers of response in immunotherapy. A study in melanoma patients by *Maccalli et al.,* showed that absence of soluble sMICB, sULBP-1 and sULBP-2 in baseline serum of anti-PD-1 treated patients correlated with improved survival while detectable levels of these molecules was correlated with poor survival [158]. Similarly, another study by the same group on melanoma patients showed that elevated sULBP2 in early-stage patients on ICIs treatment was a strong indicator of poor prognosis indicating the clinical usefulness of sULBP2 as a distinguishing marker for classifying

prognosis in early- and late-stage melanoma patients on treatment [156].

CD27 is expressed in lymphocytes and is activated by its ligand, CD70, which is a member of the tumor necrosis factor receptor superfamily. Upon binding of CD70 to CD27, soluble CD27 (sCD27) is cleaved off by metalloproteinases and is secreted in serum, plasma, and urine samples. Studies have suggested that changes in sCD27 levels reflect the activity of systemic immunity [159]. In various hematological malignancies increased levels of sCD27 have been reported to correlate with poor prognosis [160]. A study on 16 advanced lung cancer patients on anti-PD-1 treatment were tested for their pretreatment sCD27 levels and correlated with their response patterns. It was observed that a sCD27 level was higher in patients with longer survival and in such patients the duration of treatment was shorter. The authors suggested that sCD27 levels can serve as prognostic marker for predicting

CD28 is a second messenger of T cell activation and is a critical immune checkpoint for recognition of dendritic cells by T cells. Previous studies have suggested that PD-1 antibodies rely on the activation of the CD28/B7 pathway to rescue the depletion CD8+ T cells and then achieve anti-tumor effects [162]. Soluble sCD28 has been reported as a modulator of T cells for proliferation and is therefore considered an attractive biomarker of response to ICIs treatment [163]. Recently, a study on 44 patients with various cancers (lung, tongue, esophageal and nasopharyngeal, colorectal, cholangiocarcinoma, gastric, duodenal adenocarcinoma, renal cell carcinoma, hepatocellular carcinoma, and malignant melanoma) on anti-PD-1 treatment were tested for serum CD28 along with other soluble markers. It was observed that patients with higher baseline sCD28 expression had a longer PFS and responded better to treatment than non-responsive patients [164]. This dynamic change in sCD28 during treatment gives a credible index in terms of its predictive efficiency as a promising response marker. However, larger studies on this aspect are needed to understand the role of this marker.

Using ICIs have shown promising effect in treating cancers. However, only small group of patients are responsive to this treatment strategy. Tumor resistance to the immune response can be mediated by the involvement of several immunological pathways. In this chapter, we reviewed the different immunological pathways that

*DOI: http://dx.doi.org/10.5772/intechopen.96494*

*4.2.2 Soluble immune stimulatory markers*

effectiveness of ICIs in advanced lung cancer [161].

*4.2.2.1 Soluble CD27 and CD28*

*Evolving Dynamic Biomarkers for Prediction of Immune Responses to Checkpoint Inhibitors… DOI: http://dx.doi.org/10.5772/intechopen.96494*

and MICB) or ULBP (ULBP1, ULBP2, ULBP3, ULBP4, RAET1G, and RAET1L) family. These NKG2D ligands are absent on normal cells but are usually overexpressed on tumor cells. Soluble NKG2D ligands (sNKG2DLs) are generated by proteolytic shedding of tumor cells which boosts tumor immune escape by binding and subsequent endocytosis/degradation of NKG2D receptor on NK/T cells thus suppressing antitumor immune responses [156, 157]. Various sNKG2DLs have been studied as predictive biomarkers of response in immunotherapy. A study in melanoma patients by *Maccalli et al.,* showed that absence of soluble sMICB, sULBP-1 and sULBP-2 in baseline serum of anti-PD-1 treated patients correlated with improved survival while detectable levels of these molecules was correlated with poor survival [158]. Similarly, another study by the same group on melanoma patients showed that elevated sULBP2 in early-stage patients on ICIs treatment was a strong indicator of poor prognosis indicating the clinical usefulness of sULBP2 as a distinguishing marker for classifying prognosis in early- and late-stage melanoma patients on treatment [156].

#### *4.2.2 Soluble immune stimulatory markers*

### *4.2.2.1 Soluble CD27 and CD28*

*Advances in Precision Medicine Oncology*

*4.2.1.3 Soluble IDO, CD163 and NKG2DL*

patients treated with nivolumab had a lower level of sTIM-3 at three months of clinical evaluation and this correlated with the response and longer survival of the patient [135]. Though, the study does give an indication of sTIM-3 as biomarker of response, further

sLAG-3 plays a role in immune pathways and has been associated as a Th1 activity marker in serum. sLAG-3 has been reported to bind to MHC class II and induce maturation of dendritic cells thereby facilitating attack on tumor cells [148, 149]. This makes sLAG-3 an attractive biomarker in ICIs treatment. Though several studies on LAG-3 and cellular response has been documented, there is paucity of data on the utility of sLAG-3 in serum of ICIs treated patients. A study on nivolumab treated NSCLC patients reported that sLAG-3 was significantly increased during treatment and this increase was retained in non-responding patients. In addition to sLAG-3, other soluble mediators including sPD-1 and sPDL-2 were also increased in these patients indicating the dynamic interactive nature of LAG-3 and its role as predictive marker [135].

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that acts as an immune checkpoint and inhibits T-cell proliferation by starving the cells from tryptophan (trp) in order to sensitize them to apoptosis [150]. IDO facilitates tumor immune escape and several preclinical studies have associated IDO activity with immunotherapeutic resistance [151]. A study on nivolumab treated NSCLC patients found that lower baseline level of IDO activity in serum was significantly associated with better PFS/ OS while higher levels were associated with early progression indicating that high serum levels can serve as early marker of response/indicator of resistance to anti-PD-1 treatment [152]. On the other hand, a larger study on 27 NSCLC patients treated with nivolumab indicated the dynamic nature of sIDO at baseline, 2 months and at disease progression. The study aimed to evaluate the dynamic nature of IDO as a predictor of response. Interestingly, the authors observed that at baseline, IDO activity was higher in responding patients than non-responding patients. However, in patients who did not benefit from immunotherapy, a statistically significant increase was observed between baseline sample and sample taken at the time to disease progression indicating the potential utility of IDO as therapeutic resistance marker to anti-PD-1 treatment [153]. CD163 is the hemoglobin/haptoglobin complex scavenger receptor expressed exclusively on circulating monocytes/tissue macrophages. It is involved in antiinflammatory functions associated with macrophages and therefore plays an important role in suppressing anti-tumor immune responses [154]. sCD163 is secreted in plasma via proteolytic shedding and is considered a specific marker for TAM. To demonstrate the utility of sCD163 as a marker of response in immunotherapy, *Fujimura et al.* conducted a study on 59 cases of advanced cutaneous melanoma and 16 cases of advanced mucosal melanoma treated with nivolumab. It was observed that in advanced cutaneous melanoma group, sCD163 was significantly increased at 6 weeks of treatment in the response group as compared to non-response group indicating that sCD163 is an early marker of response in nivolumab treated patients [155]. On the other hand, another study by the same group determined the utility of sCD163 as predictor of immune related adverse events (irAE) in nivolumab treated advanced melanoma patients. It was observed that at day 42 of treatment, the absolute value of sCD163 significantly increased in patients with adverse nivolumabinduced, immune-related events indicating that sCD163 can also serve as a valuable predictor of irAEs in immunotherapy [155]. However, due to limited published data, further studies will provide a better understanding on this marker. NKG2D is an activating immunoreceptor of cytotoxic lymphocytes and is expressed on T, NK, and NKT cells. NKG2D has eight ligands including MIC (MICA

studies are needed to understand its dynamic nature in ICIs treatment.

**40**

CD27 is expressed in lymphocytes and is activated by its ligand, CD70, which is a member of the tumor necrosis factor receptor superfamily. Upon binding of CD70 to CD27, soluble CD27 (sCD27) is cleaved off by metalloproteinases and is secreted in serum, plasma, and urine samples. Studies have suggested that changes in sCD27 levels reflect the activity of systemic immunity [159]. In various hematological malignancies increased levels of sCD27 have been reported to correlate with poor prognosis [160]. A study on 16 advanced lung cancer patients on anti-PD-1 treatment were tested for their pretreatment sCD27 levels and correlated with their response patterns. It was observed that a sCD27 level was higher in patients with longer survival and in such patients the duration of treatment was shorter. The authors suggested that sCD27 levels can serve as prognostic marker for predicting effectiveness of ICIs in advanced lung cancer [161].

CD28 is a second messenger of T cell activation and is a critical immune checkpoint for recognition of dendritic cells by T cells. Previous studies have suggested that PD-1 antibodies rely on the activation of the CD28/B7 pathway to rescue the depletion CD8+ T cells and then achieve anti-tumor effects [162]. Soluble sCD28 has been reported as a modulator of T cells for proliferation and is therefore considered an attractive biomarker of response to ICIs treatment [163]. Recently, a study on 44 patients with various cancers (lung, tongue, esophageal and nasopharyngeal, colorectal, cholangiocarcinoma, gastric, duodenal adenocarcinoma, renal cell carcinoma, hepatocellular carcinoma, and malignant melanoma) on anti-PD-1 treatment were tested for serum CD28 along with other soluble markers. It was observed that patients with higher baseline sCD28 expression had a longer PFS and responded better to treatment than non-responsive patients [164]. This dynamic change in sCD28 during treatment gives a credible index in terms of its predictive efficiency as a promising response marker. However, larger studies on this aspect are needed to understand the role of this marker.

#### **5. Conclusion**

Using ICIs have shown promising effect in treating cancers. However, only small group of patients are responsive to this treatment strategy. Tumor resistance to the immune response can be mediated by the involvement of several immunological pathways. In this chapter, we reviewed the different immunological pathways that

can be modulated by immune checkpoint blockade and more specifically PD-1 and CTLA-4 inhibitors. We have summarized all the findings obtained in pre-clinical and clinical trials reporting an impact of anti-PD-1 and anti-CTLA-4 on intra-tumoral and peripheral immune response. Interestingly, the study of the dynamics of the immune system under CTLA-4 and PD-1 inhibitors shows a noticeable distinction in their regulatory mode of action on the anti-tumoral and peripheral immune response. Moreover, the findings discussed in this chapter show that CTLA-4 and PD-1 inhibitors do not only restore intra-tumoral effector T cells activity upon exhaustion but are also able to induce a consequential remodeling of the tumor microenvironment as well as the systemic immune response. Indeed, the field of immunological liquid biomarkers is fast evolving with many novel predictive and prognostics markers gaining attention. Though, liquid biopsies have many advantages including minimal invasiveness, longitudinal monitoring and simultaneous parallel testing with highly sensitive/ specific high throughput applications. Although several studies state the utility of soluble ICIs markers, it is observed that the characteristic feature of these markers is to modulate the immune response in synergy with each other. This makes them attractive candidates as up and down regulation of a combination of markers can allow better understanding of the immune modulatory and dynamic nature of soluble immune molecules involved in ICIs treatment. However, there are several limitations that need to be addressed for these markers. Mainly, standardization of sampling/measurement techniques as well as larger validation studies are required to verify the utility of these markers as promising tools to guide and monitor treatment decisions in ICIs treated patients. Finally, identification of dynamic biomarkers for prediction of ICIs tumor control and for monitoring of patient response under treatment is gaining considerable knowledge through recent technologies including proteomics and transcriptomics. Progress along this approach is critical to build reasoning for novel therapeutic combinations and to set forth a more personalized cancer immunotherapeutic strategy.
