**5. Correct treatment sequence after the implementation of ICI in CRC armamentarium**

Nowadays, most CRC patients (75%) are diagnosed with an early stage (I–III) due to performant screening programs providing a chance for cure. However, 25% of them have metastatic disease at presentation and, therefore, poor prognosis [61].

For early-stage CRC, the standard of care consists of upfront surgery of the primary tumor and regional lymph nodes, followed by adjuvant chemotherapy in selected patients [62]. Following surgical resection, the 5-year DFS is 95% for stage I, 82–88% for stage II, and 45–50% for stage III CRC [63]. The primary role of adjuvant chemotherapy is to eradicate the micrometastatic residual disease after surgery. Identifying micrometastatic residual disease is unreliable; therefore, the gold standard used to confirm the clinical benefit of adjuvant chemotherapy is the 5-year OS [64]. Since the most challenging issue of the existing treatment parading in early-stage CRC is the incapacity to detect micrometastatic disease, the available guidelines recommend adjuvant chemotherapy for all stage III CRC patients. For stage II CRC, the benefit of adjuvant chemotherapy is still debatable. To date, it is recommended only for patients with high-risk clinicopathologic features (positive resection margins, <12 examined lymph nodes, T4, perineural invasion, lymphovascular emboli, perforation, and obstruction). The preferred chemotherapy regimens for this setting are a combination of fluoropyrimidine and oxaliplatin (FOLFOX or CAPOX) [65]. The addition of oxaliplatin led to OS improvement, and the risk of death was further reduced by 16%, 17%, and 12% in the MOSAIC, XELODA, and NSABP C-07 trials [66–68].

### *Immunotherapy for Colorectal Cancer in the Era of Precision Medicine DOI: http://dx.doi.org/10.5772/intechopen.105377*

In the last 20 years, the prognosis of mCRC patients has significantly improved due to remarkable progress made in precision medicine. The currently available guidelines recommend resectioning metastasis performed either upfront or after previous downsizing treatment in selected patients [69]. In a recent meta-analysis, the 5-year survival rate was approximately 38% in patients who underwent resection of the liver metastasis [70]. If, however, this goal is not realistic, systemic therapy has shown significant survival benefits for mCRC patients. The fundamental development in mCRC treatment was the addition of oxaliplatin (a platinum derivate) and irinotecan (a topoisomerase I inhibitor) to 5-FU-based chemotherapy. Therefore, FOLFOX (5-FU, folinic acid, and oxaliplatin) and FOLFIRI (5-FU, folinic acid, and irinotecan) demonstrated better response rates and DFS compared to 5-FU alone, representing the mainstay of first-line chemotherapy [71, 72].

Further, after decades of clinical and translational research, an important step toward precision medicine was discovering treatment options based on the tumor's molecular characteristics. The first biologic therapy included in the mCRC treatment strategy was bevacizumab, a mAb targeting vascular endothelial growth factor-A (VEGF-A) [73]. Bevacizumab is recommended for RAS-mutated mCRC either as first-line or second-line in combination with chemotherapy [74]. Similarly, cetuximab and panitumumab are anti-EGFR mAbs associated with chemotherapy in the first and second lines of treatment but for restricted patients harboring RAS/BRAF-WT (wild-type) tumors [75]. Moreover, aflibercept (a synthetic receptor for VEGF-B, VEGF-A, and PIGF) and ramucirumab (anti-VEGFR-2) demonstrated clinical benefit in the second-line therapy while combined with chemotherapy [76, 77]. In further line, regorafenib (a multikinase inhibitor) also showed clinical efficacy [78]. Owing to improved surgical procedures and expanded therapeutic options, most mCRC patients experience an improved survival between 24 and 36 months, allowing a continuum of care [79].

Even if MSI-H/dMMR tumors represent a small subset of mCRC (5% or all cases), the discovery and introduction of ICIs into the continuum of care has been a significant step forward in precision medicine. Based on the clinical benefit observed in clinical trials, the current guidelines recommend nivolumab ± ipilimumab and pembrolizumab as first-line and non-first-line therapy for MSI-H/dMMR mCRC patients [33, 34, 40, 80]. Surprisingly, the phase III KEYNOTE-177 trial, which compared pembrolizumab with standard first-line therapy in MSI-H/dMMR mCRC, demonstrated a doubling PFS in pembrolizumab-treated patients (16.5 months). This outcome is the longest PFS ever reported by phase III trials for any first-line therapeutic options in mCRC [34]. Additionally, pembrolizumab and nivolumab ± ipilimumab are also recommended in the neoadjuvant setting for resectable MSI-H/dMMR mCRC patients [81].

According to the CMS classification, mCRCs with MSI-H/dMMR phenotype are considered immune-activated and belong to the CMS1 subgroup. Conversely, MSS/ pMMR tumors, representing 95% of all mCRCs, display a low immune infiltrate, do not respond to ICIs, and are a serious challenge for clinical management [19]. It has been revealed that radiotherapy, chemotherapy, and targeted agents can induce immunogenic cell death (ICD), releasing tumor neoantigens and increasing the immune infiltrate in the tumor microenvironment (TME). Based on this hypothesis, many clinical trials are currently investigating the combination of ICIs with other anticancer therapies in MSS/pMMR mCRC to overcome the primary resistance to immunotherapy [82, 83].

### **6. Biomarkers**

### **6.1 Microsatellite instability (MSI)**

"Short tandem repeats" or microsatellites are repeated noncoding DNA sequences with a length from one to six base pairs. DNA polymerases are more predisposed to make errors either by removing or by inserting additional bases in these particular regions, leading to mismatched DNA strands [84]. Therefore, the MSI molecular phenotype is a consequence of deficient MMR proteins. The most directed genes from the MMR family associated with genome instability are MLH1, MLH2, PMS2, and MSH6. It is estimated that only 15% of CRCs are microsatellite unstable (MSI-H) [85]. Germline MMR gene mutation is the hallmark of Lynch syndrome, an autosomal dominant condition associated with an increased risk of colorectal (80%), endometrial (60%), stomach, small intestine, kidney, bladder, and brain tumors [86]. However, the MSI phenotype appears due to somatic mutations in most cases, usually caused by epigenetic silencing of the MLH1 promoter. Less commonly, the inactivation of MMR proteins can occur due to somatic biallelic MMR gene mutations. It is worth mentioning that a subset of MSI-H tumors has no detected alterations in the MMR genes [87]. These tumors were shown to overexpress various micro-RNAs (miRNAs), like miRNA-21 and 122, that might silence MMR genes [88].

Considering that the human genome comprises hundreds of thousands of microsatellites, the MSI assay evaluates only five of them via polymerase chain reaction (PRC) for practical reasons. Therefore, a tumor is defined as MSI-H if at least two microsatellites have a shift in size, and a size shift in only one locus represents an MSI-L tumor. By contrast, tumors with no unstable microsatellites are defined as microsatellite stable (MSS). The immunohistochemistry (IHC) assay of key MMR proteins has a high concordance rate and similar performance characteristics to the MSI assay via PRC. Hence, loss of protein expression defines a tumor as dMMR, while the presence of all MMR proteins labels the tumors as pMMR (MMR proficient) [89]. Besides IHC and PCR, next-generation sequencing (NGS) is a novel approach for detecting MSI status with high sensitivity (95%) and specificity (98%) [90]. To further clarify the notions, MSI-H and dMMR are considered the same types of tumor, and MSS and pMMR tumors are also mostly overlapping.

Regardless of the origin (sporadic or hereditary), all the MSI-H/dMMR CRCs have some characteristic histologic features. The high mutational load resulting from the deficiency of MMR proteins leads to the accumulation of a robust number of tumors neoantigens with great immunological potential [91]. MSI-H/ dMMR tumors are frequently located in the right colon, have mucinous histology, are poorly differentiated, and, more importantly, have increased TILs. Moreover, MSI-H/dMMR tumors were reported to highly express immune checkpoints (CTLA4, PD-1, and PD-L1) [92].

### **6.2 PD-L1 expression**

The detection of PD-L1 using immunohistochemical staining is one of the most explored predictive biomarkers for the response to ICIs. Studies reported that upregulation of PD-L1 is correlated with high infiltration of effector T cells. Moreover, these tumors have a high likelihood of responding to ICI. In contrast to other tumor types

*Immunotherapy for Colorectal Cancer in the Era of Precision Medicine DOI: http://dx.doi.org/10.5772/intechopen.105377*

like non-small-cell lung cancer (NSCLC), melanoma, and gastric cancer, the PD-L1 expression predicted no response to ICIs in mCRC patients [93]. An update from the CheckMate-142 trial investigating nivolumab +/− ipilimumab in MSI-H/dMMR CRC demonstrated that the ORR was irrespective of PD-L1 expression [39]. Moreover, the KEYNOTE-016 trial investigating the clinical benefit of pembrolizumab in mCRC with both MSS and MSI-H phenotypes showed no statistically significant correlation between PD-L1 expression and OR or PFS [31].

The reported disparities among tumors could be explained by the dynamic nature of this surface protein, which is influenced by the TME and treatment options. Furthermore, the lack of standardization for PD-L1 expression assay limits its clinical significance [94].

### **6.3 POLE/POLD1**

POLE (DNA polymerase epsilon) and POLD1 (DNA polymerase delta) are two enzymes responsible for the correct genome replication during the cell cycle. Somatic mutation of either POLE or POLD genes affects their proofreading function, increasing the predisposition to numerous cancer types, including CRC [95]. Similar to the MSI-H/dMMR, these tumors have an ultramutated phenotype [96]. POLE-mutated CRCs express an upregulation of immune checkpoint molecules and also have a high level of TILs. Moreover, these tumors seem to be a rare finding (1% of CRCs), appear more frequently in young male patients, and have an early stage at presentation [97].

To date, limited evidence is available regarding the clinical benefit of ICIs in POLE/POLD1-mutated tumors. An excellent response to pembrolizumab was seen in a patient suffering from endometrial cancer who had a POLE mutation seen at genomic profiling. Since MSI-H/dMMR CRCs have similar characteristics (hypermutated phenotype, upregulated immune checkpoints, and inflamed TME), it was supposed that POLE/POLD1-mutated CRCs might be better suited for ICIs [98]. Further data are, however, needed to support this hypothesis.

### **6.4 Immunoscore**

The immunoscore represents an immunohistochemical and digital pathologybased assay derived from the immune contexture. It quantifies two lymphocyte populations, CD8+ and CD3+, both in the tumor core (TC) and invasive margins (IM). The purpose of immunoscore was to translate the immune contexture into a viable biomarker for CRC [99]. The immunoscore ranks from I0 (immunoscore 0), characterized by a low density of CD8+ and CD3+ in both TC and IM, to I4 (immunoscore 4), with a high density of both lymphocyte populations in both regions. The advantage of immunoscore appears to be dual. First, this score is reported to be a prognostic factor for DFS and OS in early CRC. Moreover, it also seems to be an important tool for novel therapeutic approaches, including immunotherapy [100].

The prognostic value of immunoscore is supported by several studies. According to the phase III NCCTG N0147 trial, a high immunoscore was statistically significantly associated with a longer 3-year DFS than a low immunoscore in stage III CRC patients [101]. An international consortium including 14 centers from 13 countries assessed the prognostic value of immunoscore in stage I–III CRC patients (samples from 2681 patients). Patients with high immunoscore had a statistically significant lower risk of recurrence at 5 years compared to low immunoscore (HR = 0.20, 95%

CI 0.10–0.38; p < 0.0001). In the multivariant analysis, the association between immunoscore and the time to recurrence (TTR) was independent of T stage, N stage, patient's age, sex, microsatellite instability, or other existing prognostic factors (p < 0.0001) [102]. Besides its prognostic value, immunoscore holds great potential as a predictive biomarker. An international study conducted by the Society for Immunotherapy of Cancer analyzed the association of immunoscore with the effect of adjuvant chemotherapy in time to recurrence (TTR) in stage III CRC patients. A high immunoscore was associated with the lowest risk of recurrence, and it showed a significant correlation with prolonged TTR, DFS, and OS in this subset of patients (all p < 0.001) [103]. The immune context might also predict the clinical response to ICIs. CD8+ T cells were reportedly a good predictor of response to CTLA4 blockade in melanoma patients. Moreover, CD8+ lymphocytes were associated with response to anti-PD-1 molecules [100, 104].

To date, immunoscore was introduced among the "Essential and Desirable Diagnostic Criteria" for CRC in the fifth edition of the World Health Organization (WHO) classification of digestive tumors. This detail brings us closer to the notion of TNM-I classification ("I" from "immune") [105].
