Circulating tumor DNA (ctDNA) and RNAs and non-coding RNAs (ncRNAs) released into the bloodstream via microvescicles or tumor cell lysis represent, together with CTCs, different sides of the same coin: liquid biopsy. Liquid biopsy has emerged as a promising minimally invasive tool for precision medicine due to its ability to provide multiple global snapshots of primary and metastatic tumors at different times and more representative images of the spatial and temporal tumor heterogeneity [5] compared to tissue biopsy. In fact, even though tissue biopsy remains the gold standard for the histopatological definition and the molecular stratification of tumors, it is often difficult to perform, especially in relapsed and metastatic settings, and it does not support intratumoral heterogeneity and clonal evolutions related to driver mutations, which may occur during tumor development or treatment.

Among other elements potentially involved in cancer initiation, development, recurrence, and metastasis, one that only recently received its due attention is the host microbiota—and for CRC, especially the gut microbiota. The host microbiota is composed of bacteria (≈99%), viruses, and mycetes, existing in a condition of eubosis with the human body conferring important benefits related to physical and mental health, and the development of the individual [6]. In turn, this dynamic balance is affected by host genetics, lifestyle [7], and dietary habits [8] and gut microbiota dysbiosis may play a role in promoting inflammatory responses and alterations of the immunosurveillance, which can led to cancer initiation and/or progression [9].

In this review, we summarize the state of the art regarding the potential role and the future perspectives of liquid biopsy and host microbiome as "theragnostic" tools in CRC (Figure 1).

**Figure 1.** Potential clinical applications related to liquid biopsy and gut microbiota in colorectal cancer. Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), non-coding RNA (ncRNA), and exosomes are promising liquid biopsy markers for colorectal cancer with multiple potential advantages compared to tissue biopsy. CTCs from colorectal cancer (CRC) can be shed from the primary tumor into the bloodstream, which also contains ctDNA released from tumor tissue through apoptosis, necrosis, and secretion, as well as circulating normal DNA released from healthy tissue. NcRNAs (miRNAs and lncRNAs) encapsulated by exosomes can be actively secreted into the extracellular fluid by various types of cells in the tumor or passively released due to the apoptosis and necrosis of tumor cells and can eventually be found in the circulation. Besides liquid biopsy, several potential clinical applications for harnessing the gut microbiota in CRC include development of screening, prognostic and predictive biomarkers, and microbiota modulation for CRC prevention and treatment. FMT, fecal microbiota transplantation.

#### **2. Liquid Biopsy**

The term liquid biopsy refers to procedures of isolation of cancer-derived components such as CTCs, exosomes, ctDNA, ncRNAs, and proteins from peripheral blood or other body fluids, and their genomic or proteomic evaluation [10]. Assessment of such elements via non-invasive and low-risk blood-based detection tests could improve CRC screening, diagnosis, staging, and predict relapse and metastasis [11,12] and be effective in monitoring residual disease and drug resistance in CRC patients receiving systemic treatment [13,14].

#### *2.1. Circulating Tumor Cells (CTCs)*

CTCs are tumor cells released into the bloodstream from the primary tumor or metastases [15], which could escape from immune recognition and drug treatment, and subsequently form a niche in other tissues, promoting tumor recurrence and metastasis [16].

#### 2.1.1. Screening and Early Diagnosis

Since counting CTCs reflects the patient's tumor burden and the CTCs detection rate is positively correlated to the TNM stages, it is rather difficult and quite uncommon to detect CTCs in early-stage CRC, and therefore, their utility in CRC screening and early detection seems to be very poor [17]. However, a recent prospective study involving 667 patients (including healthy control subjects, patients with adenomas, and those with stage I–IV CRC) showed a significant association between CTC counts (performed using a novel CTC assay) and worsening disease status with respect to the adenoma-carcinoma sequence. Furthermore, the assay showed high specificity (86%) and sensitivity across all CRC stages (95%) and adenomatous lesions (79%) [18].

2.1.2. Prognostic and Predictive Factor, Staging Tool and Guide for Systemic Treatment, Resistance Evaluation, and MRD Assessment

CTCs could potentially play a role as a prognostic marker, in monitoring treatment outcomes and follow-up, for modulating the intensity of systemic therapies and for detecting resistance against these. A meta-analysis of 15 studies including 3129 non-metastatic and metastatic CRC (non-mCRC and mCRC) patients showed significantly worse progressionfree survival (PFS) and overall survival (OS) for CTC-positive with respect to CTC-negative CRC patients, regardless of sampling time (baseline or during treatment), detection methods (CellSearch, RT-PCR and others), and cut-off value of CTC (≥1, ≥2 and ≥3/7.5 mL blood), thus providing strong evidence for the presence of CTCs as an independent prognostic factor of poor survival [19]. A study conducted on 158 patients showed that rising of CTCs counts in 2 mL of peripheral blood (0 for healthy, 1 for benign, 5 for non-mCRC, and 36 for mCRC patients) was associated with tumor progression and poor prognosis at baseline. Notably, after 2 year follow-up on the non-mCRC patients, those who had ≥5 CTCs were eight times more likely to develop distant metastasis within one year after curable surgery than those who had <5 [20], therefore providing a support to the possible application of CTC detection during the follow-up of early CRC patients. Intensive firstline regimens with a triplet chemotherapy backbone plus the antiangiogenic bevacizumab provided better survival outcome if compared with doublet regimens, especially in *RAS*-*BRAF* mutated mCRC [21,22], paying the price of a major incidence of adverse events. Patient stratification by CTC detection could help modulate the intensity of the systemic treatment by reserving a more aggressive therapy to patients with a worse prognosis. In the randomized phase III VISNÚ-1 trial, a first-line systemic treatment with FOLFOXIRI (oxaliplatin, irinotecan, 5-fluorouracil (5-FU), and leucovorin) plus bevacizumab significantly improved PFS compared with FOLFOX (association of oxaliplatin, 5-FU, and leucovorin) plus bevacizumab in mCRC patients with ≥3 CTCs/7.5 mL blood at baseline [22]. In *RAS*-*BRAF* wild-type mCRC, a standard first-line regimen includes a doublet chemotherapy backbone in association with an anti-EGFR antibody (panitumumab or cetuximab), usually followed at disease progression by the alternative doublet regimen in association with an antiangiogenic drug [23,24]. As showed by a prospective study on 38 *RAS*-*BRAF* wild-type

mCRC patients who received a third-line treatment with irinotecan and cetuximab, early CTC-negative and CTC status changes assessment during treatment were significantly associated with tumor response and better PFS and OS, predicting treatment failure in advance compared to imaging-based tools [25].

#### *2.2. Circulating Tumor DNA (ctDNA)*

Circulating tumor DNA (CtDNA) is a kind of double-stranded DNA, a fragment of cell-free DNA (cfDNA), that originates from active, apoptotic, necrotic, or circulating tumor cells. CtDNA retains epigenetic characteristics and harbors tumor-specific mutations detectable in the bloodstream and other body fluids [10,26]. Importantly, ctDNA half-life varies from several minutes to a few hours, and as for CTCs, its plasma levels depend on tumor load, ranging from 50% to 90% in non-metastatic and metastatic cancer patients, respectively [4,10,25,27]. Furthermore, healthy people and cancer patients can be distinguished according to the fragment length distribution pattern of cfDNA [26]. These data suggest that ctDNA analysis may represent a real-time tumor burden assessment.

#### 2.2.1. Screening and Early Diagnosis

Even if a recent meta-analysis concerning quantitative analysis of ctDNA for CRC screening, including 1258 CRC patients and 803 healthy individuals from 14 studies, concluded that the diagnostic accuracy of ctDNA has unsatisfactory sensitivity but acceptable specificity for CRC diagnosis [28], there is growing evidence that ctDNA detection could be used along with the traditional screening methods (i.e., colonscopy, FOBT, digital rectal examination, and serum tumor marker) to improve the diagnosis of early CRC [16,29]. In particular, ctDNA, especially when combined with carcinoembryonic antigen (CEA), showed higher diagnostic capacity (area under the ROC curve (AUC) 0.92, with 84% sensitivity and 88% specificity) [30]. Furthermore, epigenetic changes as DNA methylation and histone modifications are early events in carcinogenesis and clinical data that suggest that ctDNA methylation shows better sensitivity than traditional serum tumor markers in early-stage CRC [31,32]. Particularly, a meta-analysis of 25 studies assessing the diagnostic role of methylated Septin 9 (mSEPT9) promoter in ctDNA for CRC screening highlighted the efficacy of Epi proColon 2.0 with 2/3 algorithm (Epigenomics). A positive ratio of mSEPT9 was higher in advanced CRC stages (45%, 70%, 76%, 79% in I, III, III, and IV, respectively) and low-grade tumors (31%, 73% and 90% in high, moderate, and low grade, respectively), with a sensitivity, specificity, and AUC of 0.71, 0.92, and 0.88, respectively. Previous results confirmed the poor ability of mSEPT9 to identify precancerous lesions [31]. On the other hand, a recent prospective cohort study conducted on a high-risk population of 1493 individuals demonstrated that a particular single ctDNA methylation marker, cg10673833, could reach high sensitivity (89.7%) and specificity (86.8%) for the detection of CRC and precancerous lesions [32].

2.2.2. Prognostic and Predictive Factor, Staging Tool, and Guide for Systemic Treatment, Resistance Evaluation, and MRD Assessment

A systematic review and metanalysis including 1076 mCRC patients treated with chemotherapy and/or targeted agents showed that lower baseline levels of cfDNA correlated with better OS [33]. A more recent systematic review and meta-analysis including 1779 non-mCRC and mCRC patients found that the presence or high concentration of ctDNA with KRAS mutation was associated with poor disease-free survival (DFS), PFS, and OS [34]. Moreover, as anti-EGFR therapy with cetuximab and panitumumab is approved for wild-type *RAS* mCRC and *KRAS* and *BRAF* are considered effective predictors of anti-EGFR therapy [35,36], ctDNA detection could represent an alternative tool for the selection of anti-EGFR treatment due to its correlation with *RAS* mutational status of tumor tissue [37]. In particular, *RAS* clones raised in blood during EGFR blockade decline after the withdrawal of anti-EGFR antibodies, therefore restoring the drug sensitivity of cancer cells and providing a rationale for anti-EGFR retreatment [37]. Moreover, ctDNA has a great potential to supplement Response Evaluation Criteria in Solid Tumors (RECIST) evaluation.

As already discussed, ctDNA is strictly dependent by tumor load, and tumor burden can be monitored in real-time due to the short half-life of ctDNA [26,27]. Compared to radiological approaches, serial monitoring of ctDNA is able to track treatment response weeks to months earlier, allowing anticipating disease progression and modifying treatment consequently [37]. A prospective phase II clinical trial of cetuximab in *RAS* wild-type mCRC patients combined the sequential profiling of ctDNA and matched tissue biopsies with imaging and mathematical modeling of cancer evolution, showing that liquid biopsies were able to detect spatial and temporal heterogeneity of resistance to anti-EGFR monoclonal antibodies [38]. In another phase II trial that tested the multikinase inhibitor regorafenib in *RAS* mutated mCRC patients, combining dynamic contrast-enhanced (DCE), MRI, and ctDNA predicts the duration of antiangiogenic response to regorafenib, improving patient management with potential health and economic implications [39]. As for CTCs, ctDNA concentration is positively correlated with tumor size, resulting lower in stage I with respect to stage IV CRC patients [15,16,40]. As a result of the strong link between CTCs, ctDNA, primary tumor, and metastasis, it has been suggested to integrate the blood-based liquid biopsy into the actual TNM staging system, and the concept of "TNMB" (B as blood) has been proposed to improve the existing cancer staging system [2,4,41]. In this regard, the ability to optimize systemic treatments, especially in the adjuvant setting in stage II-III CRC patients, has been historically limited by the use of clinicopathologic characteristics, which are not always able to properly prognosticate the risk of recurrence [42], and by conventional surveillance modalities (CEA, computed tomography (CT), and colonoscopy), which are not perfectly able to identify MRD and early recurrence [2,4,11,12]. In a prospective cohort of 230 stage II CRC patients, 7.9% were postoperative ctDNA positive, 79% of whom relapsed, while disease relapse occurred only in 9.8% of ctDNA-negative patients. The presence of ctDNA after the completion of chemotherapy was also associated with worse recurrence-free survival [43]. In a recent prospective cohort of 130 stage I–III CRC patients, ctDNA was quantified pre- and postoperatively, and after adjuvant chemotherapy. CtDNA-positive patients after surgery, adjuvant chemotherapy, and during follow-up were respectively 7, 17, and 40 times more likely to relapse with respect to ctDNA-negative patients [44]. Some authors proposed that monitoring ctDNA levels every 3-6 months after surgery can be used to supplement serum markers, CT, endoscopy, and other conventional monitoring tools, emphasizing that positive ctDNA preceded radiological and clinical evidence of recurrence by a median of 3 months, even if 6% of patients with positive ctDNA never relapsed [45]. A great effort is ongoing to validate the clinical utility of ctDNA, particularly in the adjuvant setting of CRC (Table 1).

**Table 1.** List of major ongoing prospective

 trials

investigating

 liquid biopsy in colorectal cancer.

follow up (ctDNA tested later)

*Biomedicines* **2021**, *9*, 140

 circulating DNA; Endp: study endpoints reported study description; planned enrollment; observational; interventional; chemotherapy; liquid biopsy; dPCR: digital PCR; ddPCR: digital droplet PCR; SOC: standard of care; ACT: adjuvant chemotherapy; NA: not available; Enco/Bini/Cet: Encorafenib/Binimetinib/Cetuximab; HR: high risk according to histopathological factors; LR: low risk according to hystopatological factors; ctDNA clear: ctDNA clearance or modification rate of every study arm and correlation with clinical outcome measures (according to the design of each study); OS: overall survival; DFS: disease-free survival; RFS: relapse-free survival; LR: local recurrence; DR: distant recurrence; QoL: quality of life; TT: treatment toxicity/treatment-related adverse events; pCR: pathologic complete response; TTF: time to treatment failure; TTR: time to recurrence.

#### *2.3. MicroRNAs (miRNAs) and Long Non-Coding RNAs (lncRNAs)*

MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are ncRNAs molecules involved in the regulation of protein-coding gene expression through mRNA degradation and silencing or activating and repressing genes via a variety of mechanisms at both transcriptional and translational levels. Both classes of ncRNAs regulate multiple cellular processes such as growth, development, and differentiation showing to be crucial for cancer initiation, progression, and dissemination and can be found in serum or other body fluids bound to protein or lipid complexes, or more frequently inside extracellular vescicles (i.e., exosomes) [46]. Furthermore, these elements seem to be strongly associated with the development of drug resistance in CRC [47–50]. For these reasons, miRNA and lncRNAs could have potential application in diagnosis, prognosis, and treatment of CRC.

#### 2.3.1. Diagnosis and Prognosis

MiR-150 appears upregulated in CRC and its downregulation together with elevated Gli1 (glioma-associated oncogene homolog 1) expression seems to be involved in the process of epithelial–mesenchymal transition (EMT), which is a necessary step in promoting invasion and metastasis in CRC [51]. The results of a recent metanalysis suggest that miR-150 could be effective as a diagnostic biomarker for CRC patients, while no significant evidence was found concerning its prognostic role [52]. Mir-181 seems to be involved in multiple signaling pathways such as FOXO, PI3K-Akt, VEGF, HIF-1, mTOR, and cAMP, therefore representing a promising biomarker with potential predictive and prognostic significance in CRC [53]. MiR-21, miR-200a, miR-543, miR-32, miR92a, miR-26a, miR-1061, and miR-181a act as oncogenes downregulating the oncosuppressor PTEN (phosphatase and tensin homolog), which is a diagnostic factor for CRC patients, therefore representing potential targets for CRC therapy [54]. The upregulation of miR21, miR215, miR143-5p, and miR106a is associated with worse prognosis in stage II CRC patients [55]. A panel of miR-21, miR29a, and miR125b is able to carefully distinguish between early CRC and healthy controls (AUC = 0.827) [56]. Serum miR-203 upregulation seems to be related to worse prognosis (HR = 2.1) and higher risk of liver (OR = 6.2) or peritoneum (OR = 7.2) metastasis [57]. In a population of 400 CRC patients, a four-miRNA panel (miR-142-5p, miR-23a-3p, miR376c-3p, and miR271-3p) showed good diagnostic performance (AUC = 0.922), while a two-miRNA signature (miR-23a-3p and miR-376c-3p) proved to be a prognostic tool for 3-year OS (HR = 2.30) [58].

A study focusing on circulating serum exosomes showed that the levels of lncRNA HOTTIP could predict OS in CRC patients and discriminate between CRC and healthy controls (AUC = 0.75) [59].

A recent systematic review and meta-analysis of 111 articles including 13,103 gastrointestinal cancer patients (3123 with esophageal cancer, 4972 with gastric cancer, and 5008 with CRC) showed that 74 lncRNAs were closely associated with poor prognosis in gastrointestinal cancer, including 58 significantly upregulated and 16 significantly downregulated lncRNA expression, and with a strong interaction with miRNAs for 12 of these lncRNAs [60].

#### 2.3.2. Drug Resistance

Several oncogenic miRNAs can promote platinum and fluoropyrimidine resistance. Complex interactions between miRNAs (miR-181a-5p, miR-136, miR-363-3p, miR20b-5p, miR-218, miR-145, Let-7a, miR141) and lncRNAs (CRNDE, LUCAT1, MALAT1, GIHCG, CASC15, ANRIL, MEG3, CCAL) in the context of Wnt/β-catenin and MDM2-P53 signaling pathways are ultimately involved in oxaliplatin resistance [47]. Moreover, mir-153, miR19b-3p, miR-203, and miR-625-3p upregulation in the context of FOXO3a, SMAD4, and ATM pathways, respectively, is associated with oxaliplatin resistance [48]. The upregulation of LncRNA NEAT1 acts as an oncogene in CRC through the regulation of CPSF4 expression, sponging miR-150-5p. The upregulation of NEAT1 ultimately results in 5-FU resistance, suppressed apoptosis, and enhanced invasion of CRC [49]. A recent systematic review and

meta-analysis of 39 studies including 2822 CRC patients consistently showed that multiple miRNAs (almost 60) could act as clinical predictors of chemoresistance and sensitivity for a combination of 14 drugs, including 5-FU and oxaliplatin. Particularly, 28 miRNAs were associated with chemosensitivity, 20 were associated with chemoresistance, 1 was associated with differential expression and radiosensitivity, while 10 were not associated with any impact on chemotherapy. These results outline the importance of almost 34 drugregulatory pathways of chemoresistance and chemosensitivity in CRC that are potentially targetable [61].

#### **3. Microbiota**

The study of microbiota started several years ago, and multiple definitions have been conceived to explain its meaning [62]. In general, the terms "microbiota" and "microbiome" refer to the complex of organisms found within a specific environment and their genomic pool, respectively [63,64]. Thus, the human gut microbiota consists of a multitude of microorganisms colonizing the gut and existing in that complex state of dynamic equilibrium (i.e., eubiosis), which is made of reciprocal interactions and multiple networks between themselves and the host cells. This is an equilibrium with specific spatial and temporal characteristics, whose deregulation might lead to dysbiosis [63].

The human gut microbiota—with its thousands of different bacterial taxa, eucaryotic microbes, and virus together with the intestinal barrier—is a very selective and important filter for the well-being of the whole organism, and as a neuroendocrine structure today considered as a "second brain", it is a component of the complex gut ecosystem [65,66]. The gastrointestinal microbiota varies according to the anatomical location and among individuals [11], and it plays different roles, from the supply of nutrients to the control of inflammation and carcinogenesis [63]. Commensal bacteria instruct the immune and physiological systems throughout life and are responsible for the presence of inflammatory and immune cells in the healthy intestine: the so-called "physiological" or "controlled" inflammation [67]. For this purpose, numerous evidence has demonstrated that a direct relationship between modification in the gut microbiota composition and some pathologies exist [68,69]. Among these diseases, obesity and metabolic alterations induced by some nutrients and diet, or autoimmune diseases such as type 1 diabetes and inflammatory bowel disease, are characterized by changes in the microbiome and gut dysbiosis [70].

#### *3.1. Microbiota and Cancer*

Gut microbiota emerged as a critical player also in the development of cancer. Several studies support the idea that a disturbance of the gut microbiota composition could lead to the onset of CRC [71]. Moreover, several studies reported a deep association between microbiota and CRC, demonstrating that microbiota dysbiosis can affect cancer susceptibility and progression through the modulation of several mechanisms such as inflammation, or inducing DNA damage, and producing metabolites involved in oncogenesis or tumor suppression [72]. For example, various bacterial pathogens are linked with the DNA damage response (DDR) pathway activation, which can be caused by both a direct effect of microbe produced genotoxins or an indirect effect of ROS produced in response to an excessive activation of immune cells stimulated by certain microbes or their metabolic end-products [73,74].

In particular, fecal metagenomic samples from CRC patients identified a CRC-enriched microbiota including *Enterobacteriaceae* [75], *Escherichia coli* [76,77], Enterotoxigenic *Bacteroides fragilis* (ETBF) [78], and *Fusobacterium nucleatum (Fn)* [79]. These bacteria seem to act as "pro-oncogenic" agents in different ways: promoting inflammation, impairing antitumor activity, inducing DNA damage, and tumor cell proliferation via the activation of β-catenin and other oncogenic pathways [75]. Several studies reported an association between an abundance of *Fusobacterium nucleatum*, carcinogenetic risk factors, and gene mutations in CRC [80]. In addition, a high abundance of *Fusobacterium nucleatum* was associated with CIMP status, wild-type p53, and MSI in colon tumor tissue [81].

On the other hand, the *Firmicutes* phylum (particularly the *Ruminococcaceae* and *Lachnospiraceae* families) [82] as well as *Bifidobacteria, Lactobacilli* [83,84] and non-enterotoxigenic *Bacteroides fragilis* (NTBF) [84] are substantially underrepresented in CRC patients [85] and have shown "anti-oncogenic" activities, such as a reduction of pro-inflammatory citokines, enhancement of antitumor immunity, epithelial cell renewal, regulation of intestinal barrier integrity, and short-chain fatty acid (SCFA) production [82–86]. SCFAs, by modulating histone deacetylase inhibitory activity, promote the accumulation and differentiation of Treg cells controlling tumor progression [86].

Similarly, a deeper review on the role of gut microbiota in the carcinogenesis of humans and animals observed that some bacteria appeared often augmented (including Fusobacteria, *Alistipes*, Porphyromonadaceae, Coriobacteridae, Staphylococcaceae, *Akkermansia* spp., and Methanobacteriales), whereas others decreased in CRC (*Bifidobacterium, Lactobacillus, Ruminococcus, Faecalibacterium* spp., *Roseburia*, and *Treponema* [87]. In addition, some microbial metabolites (such as nitrogenous compounds) were consistently elevated, whereas others (such as butyrate) were decreased throughout colonic carcinogenesis [87].

#### *3.2. Signaling Pathways Activated in Microbiota and Cancer*

The gut microenvironment homeostasis requires an intricate balance between cell proliferation, differentiation, and apoptosis processes in which several regulatory pathways are involved such as the Wnt, Notch, BMP, and Hedgehog signaling pathways [88,89]. Deregulation of these main signaling pathways can potentially determine a disruption of intestinal homeostasis and contribute to CRC development. For example, the Wnt/βcatenin signaling pathway is supposed to be closely connected with cancer biology [90]. In particular, the *adenomatous polyposis coli (APC)* gene truncating mutations that stabilize β-catenin are highly prevalent in CRC, making *APC* one of the most mutated genes in human cancers [91].

Among the canonical and non-canonical Wnt signaling pathways, the first is certainly the most critical for its function as regulator of the transcriptional co-activator β-catenin, in turn regulating inflammatory, proliferative, and differentiation pathways [92,93]. As reported in several studies, the Wnt pathway has been frequently considered together with the *RAS* pathway one of the major drivers of CSC expansion [93].

The gut microbiome can be the trigger of the (EMT), a transition taking place through the involvement of WNT and TGF-β signaling, as previously reported, causing the invasion and metastasis of CRC cells [94].

Recently, preclinical evidence demonstrated that defects in the colon barrier integrity associated with dysbiosis and with an increased expression of several inflammatory factors such as IL-17, *Cxcl2, Tnf-α,* and IL-1 can be responsible for the development of benign (e.g., hyperplastic polyp), pre-malignant (e.g., tubular adenoma), or malignant (e.g., colorectal adenocarcinoma) neoformations [71,95].

Taken together, these data suggest that alterations of gene expression or modifications of microbiota composition can trigger the development of cancer involving the deregulation of proliferative and inflammatory signaling pathways even though a clear cause–effect relationship between microbiota composition and changes in gene expression have not been well elucidated.

In conclusion, not only a genetic but also an epigenetic role has been highlighted in CRC progression and metastatization, as recently reported by Wu et al. [96].

#### *3.3. Microbiota and Efficacy of Anticancer Agents*

An emergent approach is taking into consideration the influence of the microbiota on the activity and efficacy of chemotherapy and immunotherapy drugs. For example, the hypothesis that gut microbiota can be strictly related to the pharmacological effects of chemotherapy agents, such as 5-FU, is supported by a pioneer study conducted with a CRC mouse model and high-throughput sequencing. The authors compared the tumor size and profiled the gut microbiota of mice treated with 5-FU, combined with probiotics or

ABX (an antibiotic cocktail of antibiotics), demonstrating the importance of pre-existing gut microbiota communities in the host response to 5-FU treatment. In particular, they found that antibiotics-induced dysbiosis during CRC treatment determined a dramatic increase of Proteobacteria, which may interact with the host inducing systemic inflammation and abolishing the therapeutic efficacy of the drug [97].

Regarding human studies, Zhang and colleagues investigated the relationship between *Fn* infection and efficacy of a systemic treatment with 5-FU in 94 CRC patients. They initially hypothesized a mechanism of reduced chemo-sensitivity of CRC cells to 5-FU linked to the upregulation of BIRC3, which is a member of the inhibitor of apoptosis proteins (IAPs). Next, they demonstrated that *Fn*-induced BIRC3 expression could be mediated by the TLR4/NFkB pathway. Indeed, other scientists had recently reported that *Fn* may mediate chemoresistance by activating the autophagic pathway in CRC [98].

Immunotherapy has revolutionized cancer treatment, and immune checkpoint inhibitors (ICIs) are now a standard of care in microsatellite-instable (MSI) CRC patients [99].

Recently, Lang et al. in their study showed that ileal microbiota can orchestrate the immunogenic cell death of ileal intestinal epithelial cells (IECs). They registered an accumulation of follicular T-helper (TFH) cells in CRC patients and mice and the suppression of IEC apoptosis. This effect could be linked to the impairment of the immunosurveillance mechanisms by chemotherapy directed against CRC in mice [100]. Protective immune responses in the ileum were associated with the colonization of specific bacteria such as *Bacteroides fragilis* and *Erysipelotrichaceae* that stimulate the production of programmed cell death (PD-1) molecules +TFH by secretion of interleukin 1R1 and interleukin 12. Moreover, the demonstration of apoptosis in the ileum can be considered a prognostic factor for CRC patients [100].

As for the relationship between bacteria species infection and efficacy of treatments, it has been postulated that the richness and diversity of species could be influenced by the different stages of gastric carcinogenesis and progression. In particular, more relevant changes seem to occur at the stage of precancerous lesions of gastric carcinoma (PLGC), suggesting that it is a turning point during GC progression. Moreover, the depletion of some bacteria such as *Akkermansia* and an enrichment of pathogenic bacteria such as *Escherichia Shigella* can overlap with the tumor progression stage [100].

Moreover, researchers have reported a reduction in the efficacy of immunotherapy regimens in metastatic renal cell carcinoma (mRCC) patients when treated with antibiotic drugs. In particular, worse clinical outcomes in terms of PFS and OS were found in mRCC patients who received antibiotics within four weeks of treatment initiation with respect to non-users [101].

#### *3.4. Recent Advances in Metagenomics Technology for Diagnosis and Prognosis*

Currently, the study of microbiomes, also named metagenomics, is based on two main approaches, which consider different aspects of the microbial community in a given environment. The structural metagenomics approach takes into consideration the structure, composition, and dynamics in a specific ecosystem of the uncultivated microbial population. Instead, functional genomics aims to study a specific gene coding for a function of interest. This approach requires the generation of expression libraries with thousands of metagenomics clones and its subsequent screening [102].

Metagenomics, investigating the wide populations of microbial communities and analyzing all the DNA present within a sample, can provide comprehensive and useful data regarding the state of the microenvironment of CRC patients. In metagenomics, datasets acquired from recent studies of the taxonomic clades related to CRC have been discovered [103].

Moreover, Meyerson et al. by using whole-genome sequences established the configuration of microbiota in healthy and CRC patients [104]. By the way, thanks to the multi-omics approach based on the plethora of recent technologies including genomics, transcriptomics, proteomics, and metabolomics respectively able to analyze DNA markers, RNA transcript, protein, and metabolites produced inside the colon, researchers have a remarkable opportunity for the discovery of novel prognostic, diagnostic, and therapeutic biomarkers [104], even though the question of whether the microbiota and its metabolites could be considered replicable and useful biomarkers across cohorts and populations remains unclear. So, the aim of this interesting approach tries to examine the differences in patients and healthy individuals for identifying biomarker patterns to work toward a personalized medicine therapeutic approach [105].

For example, in a large cohort study conducted on 616 participants undergoing colonoscopy, the presence of distinct patterns of the microbiome in cases of multiple polypoid adenomas has been demonstrated. *Fn* appeared significantly elevated from intramucosal carcinoma to more advanced stages. Moreover, *Atopobium parvulum* and *Actinomyces odontolyticus*, which co-occurred in intramucosal carcinomas, were significantly increased only in multiple polypoid adenomas and/or intramucosal carcinomas. In addition, metabolome analyses indicated a significant increase of metabolites such as branched-chain amino acids and bile acids in intramucosal carcinomas. Futhermore, the authors suggested that the shift in the microbiome and metabolome seemed to occur from the very early stages of CRC development, confirming the potential diagnostic and etiological role of multi-omics data. Therefore, the authors proposed metagenomic and metabolomic markers to discriminate cases of intramucosal carcinoma from the healthy controls, highlighting the possible etiological and diagnostic importance of large-cohort multi-omics data [106]. Indeed, the application of metagenomics to explore the gut microbiota profile has also been prospectively investigated in 60 CRC patients and 30 healthy controls. This study revealed the importance of data from the gut microbiome in association with known clinical risk factors of CRC to discriminate between adenoma and carcinoma clinical groups [107]. On the other hand, a similar conclusion has been reported by a European study based on fecal samples metagenomic sequencing and taxonomic classification of a mixed group of CRC, adenomas patients, and healthy subjects. Indeed, this study indicated that observed gene pool differences may reveal tumor-related host–microbe interactions [108].

An emerging approach to study the intersection of the gut–microbial communities and human health is based on the study of microbe-derived extracellular vesicles (EVs). EVs, separated into three different types, outer membrane vesicles (OMVs), shedding vesicles, and apoptotic bodies, are composed of different macromolecules including lipids, proteins, nucleic acids, and metabolites [109,110].

For example, via metagenomic and metabolomics analysis of gut EVs of CRC and healthy subjects, Kim et al. found an alteration of compositional bacteria and metabolites profile in CRC patients, suggesting a potential diagnostic role of EVs metabolites profiles in the identification of cooperation between microbiome and cancer development [109].

#### *3.5. Organoids Engineering*

Organoid engineering has become an important tool for cancer assessment but also in modeling host–microbe interactions. New insights are rapidly being gained on the role of the microbiome in CRC development, and it is clear that CRC patients have an altered gut microbe population compared to healthy ones. However, whether they play a direct or indirect role in cancer development is a topic of great discussion [111].

Research suggests a key role for microbes in developing an inflammatory environment in which cancer cells can grow; they can also influence cancer development by producing metabolites that influence the host metabolism [112].

From a practical point of view, microbes can be administered to cell culture media, allow basolateral exposure, or be microinjected into the lumen of the organoid to faithfully reproduce the microbial activity [113].

For example, Pleguezuelos-Manzano et al. focused on the abundance, in stool samples of CRC patients, of some bacteria including *E. coli* and pks + *E. coli*, which are capable of producing the genotoxin colibactin. This toxin has been shown to damage DNA and create a non-physiological base pairing in epithelial cells [77].

By the use of organoids constituted with *E. coli* pKs + obtained from the colon of CRC patients co-cultured with the epithelial cells, these researchers reproduced in vivo the intestinal situation and demonstrated that exposure to *E. coli* pKs + would appear to be a risk factor in the development of CRC [114]. Therefore, in conclusion, the specificity of colibactin-induced mutations supports the need for further investigations relating to its link with cellular DNA as well as representing a valid support in the identification of a preventive biomarker [77].

New knowledge is also rapidly gaining in the field of "nutrition and gut microbiota". Several studies have established that after the ingestion of phytochemicals and fibers, the intestinal microbiota initiates complex catabolism that releases important metabolites of the intestinal microbiome (GMMs). Moreover, thanks to the use of organoids derived from colorectal lesions, the impact of diet and metabolites on tumorigenesis has been also investigated [115].

Recently, Toden and colleagues identified evidence that metabolites produced by the microbial catabolism of flavan-3-ols in the distal gastrointestinal tract could induce programmed cell death, inhibiting cancer and promoting gut health [116]. They used intestinal organoids as a preclinical model system and noted that flavan-3-ols suppressed the formation and growth of both intestinal organoids—those derived from APCM in mouse models and those from human CRC tumors—by inhibiting the cell cycle and inducing apoptosis. The gene expression profile revealed the suppression of survival and self-renewal pathways in organoids treated with flavan-3-ols. Flavan-3-ols is a commercial grape seed extract, consisting of monomers, dimers, and trimers. These compounds include proanthocyanidins (PACs); they can reach the distal gastrointestinal tract almost intact and are effectively transformed into low molecular weight phenolic compounds by the colonic microbiota [117–119]. The flavan-3-ols monomers, dimers, and trimers that reach the colon become available for the gut microbiota. Then, microbial catabolism begins, producing hydroxy-phenyl-γ-valerolactones (PVLs) and, to a lesser extent, their derivative hydroxy-phenylvaleric acids (PVAs), with only a small percentage of non-metabolized PACs remaining [120].

#### *3.6. Therapeutic Use of Antibiotics, Probiotics, and Fecal Microbiota Transplantation*

Several approaches, which include dietary interventions, antibiotic treatments, preand probiotics, and fecal microbiota transplantation (FMT), have been explored to modulate gut microbiota composition, including its physiology and metabolites involved in CRC occurrence, progression, or drug resistance.

Diet plays a significant role in the modulation of the microbiome. A normal gut microbiota depends upon the fermentation of the indigestible fiber component of our diet for its energy supplement. The symbiotic gut microbiota ferments dietary fibers into shortchain fatty acids (SCFAs) such as propionate, acetate and, most importantly, butyrate [67]. In a prospective cohort study, a diet rich in whole grains and dietary fiber was associated with a lower risk to develop *F. nucleatum*-positive CRC but not *F. nucleatum*-negative CRC, supporting a potential role for intestinal microbiota in mediating the association between diet and colorectal neoplasms [121]. As no clear guideline regarding the type of nutrition and cancer incidence has been established, different forms of reduced caloric intake, such as fasting, demonstrated a wide range of beneficial effects in cancer prevention and anticancer drug efficacy [122], at least in part mediated by gut microbiota. Indeed, every-other-day fasting leads to an increase in fermentation products such as acetate and lactate altering gut microbiota composition, with enriched levels of Firmicutes, the production of SCFAs, and reduction in Bacteroides, Actinobacteria, and Tenericutes [123]. Since tumors are not able to metabolize ketone bodies due to deficiencies in key mitochondrial enzymes, a ketogenic diet with low-carbohydrate and high-fat intake, mimicking the metabolic state of fasting by inducing a physiological increase in acetoacetate and beta-hydroxybutyrate, might be a

reliable therapeutic strategy to inhibit cancer progression [124]. Omega-3 polyunsatured fatty acids (PUFAs) are widely used as nutritional supplements and multiple benefits have been claimed, included anticancer activity. PUFAs seem to increase "anti-oncogenic" bacteria, including *Bifidobacterium* and *Lactobacillus* other than SCFA-producing genera such as *Blautia*, *Bacterioides*, *Roseburia,* and *Coprococcus* [125]. A randomized trial showed that omega-3 PUFA supplementation induces a reversible increase in several SCFA-producing bacteria [126].

Since antibiotic administration represents an aggressive and non-selective means of manipulation of gut microbiota composition, its role in CRC management seems to be controversial. Although preclinical evidence showed that gut microbiome depletion seems to inhibit cancer progression [127], multiple lines of evidence highlight how antibiotics can undermine immunotherapy efficacy or promote disease progression emphasizing microbial dysbiosis [128,129].

Of course, a potential strategy of CRC prevention and management is represented by probiotics and fecal microbiota transplantation (FMT). Probiotics are living microorganisms with the potential to positively influence resident microbiota, intestinal epithelium cells, and the immune system, and they are generally considered safe and well tolerated in healthy subjects [130]. A randomized trial with *Lactobacillus* and *Bifidobacterium* strains significantly reduced the levels of proinflammatory cytokines such as TNF-α, IL-6, IL-10, IL-12, IL-17, and IL-22 and prevented post-surgical complications.

FMT consists of the transplantation of gut microbiota from healthy donors to patients to restore intestinal dysbiosis and reduce the activation of inflammatory, proliferative, and procarcinogenic pathways. These specimens are prepared according to well-established protocols to avoid potential risk factors such as viruses and parasites and stored in banks of donated feces [131]. Treatment with chemotherapy and ICIs can result in adverse events including colitis. FMT treatment has been shown to improve ICI-induced colitis in cancer patients [132]. Additionally, FMT reduced the severity of intestinal mucositis and diarrhea following FOLFOX treatment in preclinical models by suppressing IL-6 levels, increasing the number of goblet cells and zonula occludens-1, decreasing apoptotic and NFkB-positive cells as well as the expression of Toll-like receptors and MYD88, leading to a restoration of gut microbiota composition without complications such as bacteremia [133]. Another study conducted in a mouse model to assess the efficacy of FMT to reverse antibiotic- and chemotherapy-induced gut dysbiosis suggests that FMT may effectively help in preventing acute intestinal inflammation and mucosal barrier dysfunction. In particular, the administration of FMT reduced the proportions of pathogenic species and an increase of the relative distribution of Clostridium scindens and Faecalibacterium prausnitzii, which are species that exhibited anti-inflammatory properties [134].

Finally, as demonstrated by Hefazi et al. in cancer patients treated with cytotoxic chemotherapy, FMT treatment determined a reduction of multiply recurrent Clostridium difficile infection (CDI) and diarrhea episodes remarking its highly therapeutic efficacy [135].

#### **4. Future Perspectives and Conclusions**

Despite the recent advances in the systemic treatment of molecularly selected CRC patients with advanced disease (i.e., pembrolizumab in MSI [99] or the association of the anti-BRAF encorafenib, the anti-MEK binimetinib and the anti-EGFR cetuximab in BRAF V600E mutated [136] tumors), the survival benefit is limited to a small percentage (10–20%) of patients harboring these alterations.

The use of CTCs, ctDNA, miRNAs, and lncRNA could help find new potentially targetable biomarkers for the management of CRC. Furthermore, as a minimally invasive and repeatable procedure, liquid biopsy can improve CRC screening, early diagnosis, clinical staging, and prognostic stratification, allowing a higher rate of cure. Moreover, liquid biopsy might be useful to monitor minimal residual disease after surgical treatment, possibly allowing a finer modulation of the adjuvant systemic therapy, integrating clinicopathological risk factors and ctDNA or CTC detection. Finally, if properly integrated with clinical and instrumental assessment, liquid biopsy might help monitor disease progression, treatment efficacy, and acquired resistance to chemotherapy and targeted agents in CRC.

Of course, there is urgent need to optimize pre-analytical and analytical processing for clinical validity, to standardize laboratory methods in ensuring the reproducibility of the results and to properly assess the cost-effectiveness [137]. Indeed, the lack of clinical applicability is currently due to the large quantity of liquid biopsy assays, with different detection limits, sensitivity, and specificity [138]. To solve the pitfalls for liquid biopsies due to the difficulty of CTC detection, the application of various microfluidic platforms based on CTC characteristics has been explored [139]. Recently, for the selection of CTC, a "negative depletion" microfluidic chip has been developed [140]. In this system, named leukapheresis, the leukocyte depletion strategy can enrich for untagged CTCs in a "tumorindependent" manner applicable to all tumor types, as demonstrated in several tumor types [141–144].

CTC analyses performed on leukapheresis products should improve the reach of liquid biopsies in metastatic cancer, and combined with CTC detection, they may play a critical role in screening high-risk patients for early cancer, identifying the tissue of origin, and reducing the need for invasive biopsies.

Therefore, once the multiple ongoing randomized phase II–III trials will define and validate the role of liquid biopsy especially in the adjuvant setting of early CRC (Table 1), a process of harmonization of procedures and data will be necessary to transfer from bench to bedside this important tool of personalized medicine.

On the other hand, it is clear that CRC carcinogenesis is also defined by gut microbiota metabolic activity and its dysbiotic composition. Therefore, the integrated analysis of the gut microbiome and its interactions with the host, anticancer drugs, and other exogenous factors [7,139] is essential to improve the outcomes of CRC patients. Recent findings support the potential of microbial markers in cancer diagnosis and prognosis and the potential of FMT or pre-probiotics in remodeling the tumor microenvironment or in potentiating antitumor immunity. Continuous monitoring of changes in microbiota profiles and biomarkers may help in the identification of dysplasia. In addition, in this context, an important collaborative effort is required to elucidate the role of the gut microbiota in modulating responses to cancer treatment, and this aspect is particularly clear in several ongoing clinical trials investigating the effect of FMT in patients with cancer who are refractory to ICI. These trials, along with further validations, will determine whether the selective modulation of gut microbiota, either by FMT, probiotic treatment, or other means, enables CRC patients to overcome resistance to chemotherapy or immunotherapy (Table 2). Of course, a more complete and holistic approach toward cancer treatment should include host–microbiota interactions as important screening and treatment factors.

Obs: observational; RR: response rate; TT: treatment toxicity/treatment-related adverse events; SOC: standard of care; T: clinical trial; LM: luminal microbiota; MAM: mucosal associatedmicrobiota; CT: chemotherapy; IT: immunotherapy; NSCLC: non-small cell lung cancer; TNBC: triple negative breast cancer; PC: pancreatic cancer; HR: high-risk; MO3UFA: marineomega-3 polyunsaturated fatty acid; FMT: fecal microbiota transplant.

**Author Contributions:** A.P. and S.D.M. conceptualized and organized the manuscript; A.P., S.D.M., F.P. have been involved in collection of bibliographic materials and writing—original draft preparation; A.P., S.D.M., F.P, V.M., K.C., G.P. have been involved in writing—review and editing, A.P., S.D.M., G.P and C.F. provided supervision and proofread the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by ALCLI "Giorgio e Silvia" ONLUS, a non-profit association.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

#### *Article*

## **Clinical Significance of Preoperative Inflammatory Markers in Prediction of Prognosis in Node-Negative Colon Cancer: Correlation between Neutrophil-to-Lymphocyte Ratio and Poorly Differentiated Clusters**

**Giulia Turri 1, Valeria Barresi 2, Alessandro Valdegamberi 1, Gabriele Gecchele 1, Cristian Conti 1, Serena Ammendola 2, Alfredo Guglielmi 1, Aldo Scarpa <sup>2</sup> and Corrado Pedrazzani 1,\*,†**

**Abstract:** Although stage I and II colon cancers (CC) generally show a very good prognosis, a small proportion of these patients dies from recurrent disease. The identification of high-risk patients, who may benefit from adjuvant chemotherapy, becomes therefore essential. We retrospectively evaluated 107 cases of stage I (*n* = 28, 26.2%) and II (*n* = 79, 73.8%) CC for correlations among preoperative inflammatory markers, histopathological factors and long-term prognosis. A neutrophilto-lymphocyte ratio greater than 3 (H-NLR) and a platelet-to-lymphocyte ratio greater than 150 (H-PLR) were significantly associated with the presence of poorly differentiated clusters (PDC) (*p* = 0.007 and *p* = 0.039, respectively). In addition, H-NLR and PDC proved to be significant and independent survival prognosticators for overall survival (OS; *p* = 0.007 and *p* < 0.001, respectively), while PDC was the only significant prognostic factor for cancer-specific survival (CSS; *p* < 0.001,). Finally, the combination of H-NLR and PDC allowed an optimal stratification of OS and CSS in our cohort, suggesting a potential role in clinical practice for the identification of high-risk patients with stage I and II CC.

**Keywords:** colon cancer; poorly differentiated clusters; prognostic factors; inflammatory markers; histopathological markers; immune system

#### **1. Introduction**

Colorectal cancer (CRC) is one of the most frequent malignancies in the Western population [1]. Prognosis is mainly influenced by the completeness of surgical resection and pathological stage [2–4]. However, some histopathological and molecular features may play a relevant role in the definition of long-term outcomes [5] in patients affected by this neoplasia. The identification of additional prognostic factors, able to distinguish high-risk from low-risk patients, is particularly relevant in case of node-negative disease. Indeed, the use of adjuvant chemotherapy in these patients is controversial in view of the overall good prognosis [6].

In addition to tumor stage, the host immune system may play an important role in tumor development and progression [7–9]. Some inflammatory markers, expression of an imbalanced immune reaction, have been evaluated as prognostic factors in cancer patients [8,10,11]. In particular, neutrophil-to-lymphocyte ratio (NLR) has been tested in

Valdegamberi, A.; Gecchele, G.; Conti, C.; Ammendola, S.; Guglielmi, A.; Scarpa, A.; Pedrazzani, C. Clinical Significance of Preoperative Inflammatory Markers in Prediction of Prognosis in Node-Negative Colon Cancer: Correlation between Neutrophilto-Lymphocyte Ratio and Poorly Differentiated Clusters. *Biomedicines* **2021**, *9*, 94. https://doi.org/10.3390/ biomedicines9010094

**Citation:** Turri, G.; Barresi, V.;

Received: 17 December 2020 Accepted: 16 January 2021 Published: 19 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

oncological patients [12–14], and its prognostic value was also suggested in patients with CRC. Various studies investigated the correlation between preoperative NLR and overall as well as disease-free survival in CRC, suggesting different threshold values and results. Most of the studies concluded that elevated NLR was associated with worse outcomes in patients with both localized and metastatic CRC [10,15–23]. Similarly, platelet-to-lymphocyte ratio (PLT) [23–25] and platelet count (PC) could predict long-term outcomes in patients with CRC [11,26,27].

Among the histopathological factors, the presence of lymphatic or vascular invasion, poor differentiation according to the World Health Organization (WHO) grading system and tumor budding are currently considered indicators of worse prognosis in stage II CRC [28–30]. More recently, the presence of poorly differentiated clusters (PDCs) [31] has gained attention in view of its significant correlation with higher recurrence risk and shorter overall survival in patients with stage II CRC [32–34]. Nevertheless, the assessment of PDC is rarely adopted for prognostic stratification in routine clinical practice, as conventional tumor grading system is still preferred in the AJCC guidelines [35].

This study aimed at evaluating the potential correlations between preoperative inflammatory biomarkers and histopathological characteristics in node negative colon cancer, as well as their impact on long-term prognosis.

#### **2. Materials and Methods**

#### *2.1. Inclusion Criteria and Population under Study*

The original population under study consisted of all patients undergoing surgery for CRC (*n* = 1418) at the Division of General and Hepatobiliary Surgery, University of Verona Hospital, between January 2005 and December 2015 The inclusion criteria were: age ≥ 18 years; histology-proven colon cancer; absence of nodal or distant metastasis (AJCC/UICC TNM Stage I and II); availability of histological slides or paraffin block of the primary tumor; data on preoperative NLR, PLR and PC; and absence of residual disease after surgery (R0 resection). Patients with rectal cancer were excluded from the analysis.

#### *2.2. Assessment of Inflammatory Markers*

Neutrophil count, lymphocyte count and PC were obtained from venous blood within 2 weeks before the date of surgery. NLR and PLR were calculated by dividing the absolute number of neutrophils or platelets by the absolute number of lymphocytes, respectively. Blood samples were drawn by an expert phlebotomist in vacuum blood tubes containing K2-EDTA (Terumo Europe NV, Leuven, Belgium). The complete blood cell count (CBC) was performed using Advia 2120 (Siemens Healthcare Diagnostics, Tarrytown NY, USA). The local reference ranges are 150–400 × 109/L for platelets, 4.3–10.0 × <sup>10</sup>9/L for total white blood cells (WBC), 2.0–7.0 × 109/L for neutrophils and 0.95–4.5 × <sup>10</sup>9/L for lymphocytes. The same analyzer was used throughout the study period. The quality and comparability of test results were validated by data of both internal quality control (IQC) and external quality assessment (EQA) [36].

#### *2.3. Histological Evaluation*

All cases included underwent histopathological revision as previously described in detail [32]. Briefly, hematoxylin–eosin-stained histological slides were revised to assess the depth of infiltration (pT1, pT2, pT3 and pT4) and histological grading according to the WHO criteria, lympho-vascular invasion (LVI), perineural invasion (PNI), tumor budding, presence of inflammation and PDC count. PDC were defined as clusters of at least 5 tumor cells lacking a glandular structure, at the invasive front or in the tumor stroma and counted in one hot spot under the microscopic field of ×20 objective lens (i.e., a microscopic field with a major axis of 1 mm). The 8th Edition of the American Joint Committee on Cancer (AJCC) and the Union International Contre Le Cancer (UICC) criteria were used for reporting the pathology specimens [35].

#### *2.4. Preoperative Work-Up and Surgical Technique*

All patients were staged with preoperative colonoscopy, chest-abdomen-pelvis computed tomography (CT) and carcinoembryonic antigen (CEA) measurement. In the case of dubious hepatic lesions, magnetic resonance was used to clarify the preoperative staging.

The main goal of surgery was the complete excision of the cancer burden in order to obtain an R0 resection. The extent of the resection was planned according to cancer location, disease stage and patient's general conditions. Anatomical resections with ligation of vessels at their origin were the procedures of choice in order to achieve an adequate lymphadenectomy [37].

#### *2.5. Data Collection and Statistical Analysis*

Data were extracted from a prospectively maintained database. Demographic, clinical, surgical, hematological and histopathological variables were analyzed. All methods used in this study were performed in accordance with the relevant ethical guidelines and regulations of the University Hospital of Verona, where the investigation was carried out. The study was approved by the Verona University Hospital Ethics Committee (09/07/2016, ID number: 42763-CRINF-1034 CESC). Informed consent was obtained from all patients enrolled in the study. On preliminary analysis, preoperative NLR, PLR and PC were found to be normally distributed. The optimal cut-off values for NLR (≥3) [19,20], PLR (≥150) [25] and PC (≥<sup>350</sup> × 109/L) [10] as dichotomous predictors of survival were chosen based on previously published literature. The correlation between preoperative inflammatory markers and pathological features was investigated using independent t test or Mann–Whitney U test for continuous variables and chi-squared test or Fisher's exact test for categorical variables, as appropriate. Continuous data were reported as mean (+SD) or median (range) as appropriate according to distribution, while categorical data were reported as numbers and percentages.

Survival and follow-up data were obtained by collecting outpatient clinical records or by directly contacting the patient or their relatives. The median length (range) of follow up was 104 (3–160) months considering the whole population and 113 (76–160) months considering surviving patients only. At the time of analysis, 75 patients had completed their follow-up and 32 have died.

Survival analysis was computed using the Kaplan–Meier method and compared by the log-rank test, with time of overall survival (OS) measured from the date of surgery to the date of death from any cause or most recent follow-up and cancer-specific survival (CSS) as months from the date of surgery to the date of death from cancer. Multivariate analysis was performed by Cox regression model taking into account clinical and pathological characteristics and inflammatory markers that were found to significantly influence longterm survival on univariate analysis.

All statistical tests were two-sided, and association were considered statistically significant at a nominal level of 0.05 (*p* < 0.05). Statistical analysis was performed using SPSS (version 23, SPSS, Chicago, IL, USA).

#### **3. Results**

In total, 107 patients fulfilled the inclusion criteria and were included in the analysis (Figure 1). Twenty-eight (28) tumors (26.2%) were classified as TNM stage I and 79 (73.8%) as stage II. None of the patients received adjuvant chemotherapy.

Table 1 reports the correlation between inflammatory markers and clinical-pathological variables. Forty-five patients (42.1%) had an NLR value greater than 3 (H-NLR). H-NLR was significantly associated with serosal invasion (31% vs 11.3%; *p* = 0.036) and presence of PDC (51.1% vs 24.2%; *p* = 0.007). A PLR value greater than 150 (H-PLR) was associated with a significantly higher rate of mucinous histotype (18.8% vs 4.6%; *p* = 0.042) and presence of PDC (43.7% vs 23.2%; *<sup>p</sup>* = 0.039). Finally, a PC greater than 350 × 109/L (H-PC) was associated with a higher rate of right-sided CC (76.7% vs 39%; *p* = 0.001), mucinous histotype (30% vs 6.5%; *p* = 0.003) and poorly differentiated (G3) tumors (23.3% vs 2.6%; *p* = 0.002). No significant associations were demonstrated between NLR, PLR and PC values and the amount of inflammatory reaction, nor with lympho-vascular, perineural invasion or tumor budding.

**Figure 1.** CONSORT diagram for patient inclusion.

**Table 1.** Correlations between NLR, PLR and PC and main clinical and pathological variables for the 107 patients under study.

SD, standard deviation; CACI, Charlson Adjusted Comorbidity Index; LVI, Lymphovascular Invasion; PNI, Perineural Invasion; PDC, Poorly Differentiated Clusters. Number in parentheses are percentages, unless specified otherwise.

> At survival analysis, NLR, among inflammatory markers and PDC, among histopathological factors, demonstrated to significantly and independently influence OS and CSS (Figure 2 and Table 2).

Accordingly, the combined effect on long-term outcomes of NLR and PDC was evaluated. As shown in Figure 3, excellent long-term outcomes were observed in PDC negative cases almost independently from NLR values. Conversely, long-term survival demonstrated to be negatively influenced by the presence of PDC, with a significantly worse prognosis in H-NLR cases, both considering OS (*p* < 0.001) and CSS (*p* < 0.001).

In the Cox regression multivariate analysis, age above the median (*p* < 0.001), TNM stage II (*p* = 0.035), H-NLR (*p* = 0.007) and the presence of PDC (*p* < 0.001) were independent predictors of shorter OS. Presence of PDC was the only independent prognostic factor for shorter CSS (*p* < 0.001), although H-NLR was nearly significantly associated (*p* = 0.072) (Table 3).

**Figure 2.** Kaplan–Meier estimates of overall survival (OS) and cancer-specific survival (CSS) according to PDC and NLR status: (**a**) OS according to PDC status (*p* < 0.001); (**b**) CSS according to PDC status (*p* < 0.001); (**c**) OS according to NLR status (*p* < 0.001); and (**d**) CSS according to NLR status (*p* = 0.011).

**Table 2.** Kaplan–Meier estimates of survival probability at 5 years according to main clinical–pathological variables for the 107 patients under study.

**Figure 3.** (**a**) Kaplan–Meier estimates of OS (*p* < 0.001) according to the combined PDC/NLR variable; and (**b**) Kaplan–Meier estimates of CSS (*p* < 0.001) according to the combined PDC/NLR variable. <sup>100</sup>

**Table 3.** Multivariable analysis for overall- and cancer-specific survival.

The Cox regression multivariate analysis for OS and CSS was also conducted using the combination of PDC and NLR. The presence of both negative prognostic factors showed an additive effect; the HR for PDC-present/L-NLR was 19.91 (2.14–185.11) compared to an HR of 56.67 (95% CI 6.63–483.94) for PDC-present/H-NLR (*p* < 0.001) (Table 4).

**Table 4.** Multivariable analysis for overall- and cancer-specific survival conducted with the combination variable.

#### **4. Discussion**

In the current study, we analyzed the association and the prognostic role of preoperative inflammatory markers and the main histopathological features in surgically resected CCs in the absence of lymph node metastases. The main results of this study are: (1) H-NLR values are significantly associated with the presence of PDCs; (2) both H-NLR and

PDC confirmed to be significant and independent survival prognosticators; and (3) the combination of NLR and PDC allows a better stratification of OS and CSS in TNM Stage I and II colon cancer. To the best of our knowledge, this is the first study demonstrating a correlation between preoperative inflammatory markers and the presence of PDCs in patients with CC.

According to current guidelines [28–30], adjuvant chemotherapy in stage II CC is considered only for patients with specific risk factors, namely serosal infiltration (pT4), presence of lymphatic or vascular invasion and fewer than 12 analyzed nodes. However, among node-negative CCs, 5% of stage I and 12% of stage II tumors will develop a recurrence within five years from surgery [6]. Some molecular parameters, such as microsatellite instability and KRAS/BRAF mutations, have been associated with survival outcomes [5,38,39], however their assessment requires sophisticated and expensive techniques. Therefore, great interest has been directed towards the identification of some readily available and inexpensive markers which could be useful for the detection of patients who may benefit from systemic chemotherapy. Since neutrophils, platelets and lymphocytes are routinely measured as part of the preoperative work-up of patients undergoing surgery, their possible prognostic value could be very relevant in clinical practice.

The clinical impact of inflammatory markers has been partially confirmed in our study. Although NLR showed a significant association with increased risk of death from cancer (Table 2), and it was an independent prognostic variable for shorter OS at the multivariate analysis (Table 3), PLT and PC did not demonstrate any relevant association with long-term outcomes. This is in accordance with previously published studies [10,22,23,40]. Although other studies concluded that NLR is an important inflammatory biomarker in CRC, several issues should be remarked. In the study by Li and colleagues [21] on 5336 patients with CRC, which is largest published series, H-NLR was an independent prognostic factor for OS at multivariate analysis. However, the significance of NLR and other inflammatory markers in patients who did not undergo adjuvant chemotherapy was not demonstrated; this is in line with our results. Likewise, Haram at al. [22] conducted a systematic review to assess the prognostic role of NLR in metastatic and non-metastatic CRC. They concluded that preoperative NLR > 5 was associated with poorer overall survival in patients with CRC, but no association was found with the other chosen cut-offs. Malietzis et al. [20] did not identify an independent prognostic role of H-NLR (>3) in 506 patients with non-metastatic CRC who did not receive adjuvant chemotherapy. Finally, the systematic review and metaanalysis by Zhang et al. [23] found a significant association between NLR > 5, PLR > 150, PC > 400 and overall survival. However, none of the study evaluated cancer-specific survival. Furthermore, most of the studies included colon as well as rectal cancer [41,42], therefore producing results that may be biased because of the difference in treatment and prognosis of the two locations.

With regards to histopathological markers of poor prognosis, the presence of PDCs has recently gained attention as a promising prognostic factor in patients with CRC [33,34]. They reflect tumor de-differentiation, and their evaluation on hematoxylin and eosinstained slides is more reproducible than WHO grading [32,43]. No previous study evaluated the association between PDC and inflammation-based scores in CC. This study is the first to show a significant association between H-NLR and PDC, and their cumulative negative effect on OS and CSS. The presence of an imbalanced inflammatory response measured on peripheral blood may reflect the presence of a more de-differentiated and aggressive disease. A previous study reported a significant correlation of tumor budding with preoperative neutrophil count, but not with NLR [44]. Although tumor budding and PDC have morphological and immunohistochemical similarities and might both represent tumor de-differentiation [45,46], there is not a clear evidence that they biologically overlap.

In patients with TNM stage I and II CC, inflammatory markers may permit preoperative identification of high-risk patients, whereas pathological markers lead to postoperative stratification of patients with a reduced survival probability and a higher risk of recurrence. Namely, patients with cT4 and one of these risk factors may be considered for neoadjuvant

treatment, or patients with H-NLR and PDCs may receive adjuvant chemotherapy even in absence of node metastases and other risk factors. In fact, it should be noted that the association between NLR and PDC resulted to be a better prognosticator of CSS than TNM stage itself, suggesting than even TNM stage I patients with PDC (14 cases, 50%) or H-NLR (11 cases, 39.2%) may benefit from adjuvant chemotherapy. Similarly, PDC and NLR may assist in selecting endoscopically resected "early cancers" that should merit to undergo surgical resection.

The main limitation of this study relates to its retrospective nature and the limited sample size. Although we considered an initial large population, many patients were excluded due to the unavailability of all histological slides and/or hematological parameters. However, our study also has many strengths. First, the population includes homogeneous cases of node-negative CC who did not receive adjuvant chemotherapy. In fact, at their time of surgery, no adjuvant chemotherapy was indicated for TNM stage II cancers, even in the presence of pathological risk factors. This allows us to abolish the potential bias related to the administration of adjuvant chemotherapy. Second, the consistent follow-up time assured the identification of some late recurrences that may characterize the postoperative course of early stages CC.

#### **5. Conclusions**

Our study suggests that both NLR and PDC significantly affect survival even when limiting the analysis to stage I and II CCs. Noteworthy, we observed an increased rate of PDC positivity in patients with high values of NLR. In addition, NLR significantly and independently stratified OS and CSS in cases with PDC positivity. Further studies with a higher number of cases are required in order to confirm our observations and identify the effective clinical value of the association of H-NLR and PDC.

**Author Contributions:** Conceptualization, G.T., V.B., S.A. and C.P.; Data curation, G.T., A.V., G.G., C.C. and S.A.; Formal analysis, G.T., G.G. and C.C.; Investigation, G.T., V.B., A.V., G.G., C.C., S.A. and C.P.; Methodology, C.P.; Resources, V.B.; Software, G.G.; Supervision, V.B., A.V., A.G., A.S. and C.P.; Validation, V.B., A.V., G.G., C.C., A.G., A.S. and C.P.; Writing—original draft, G.T. and C.P.; and Writing—review and editing, G.T., V.B., C.C., A.G., A.S. and C.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** FUR 2019: University of Verona, Italy, to VB.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the local Ethics Committee (09/07/2016, ID number: 42763—CRINF-1034 CESC).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy reasons.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Review* **Recent Advances in Monoclonal Antibody Therapy for Colorectal Cancers**

**Kyusang Hwang †, Jin Hwan Yoon †, Ji Hyun Lee † and Sukmook Lee \***

Biopharmaceutical Chemistry Major, School of Applied Chemistry, Kookmin University, Seoul 02707, Korea<EMAIL_ADDRESS>(K.H<EMAIL_ADDRESS>(J.H.Y<EMAIL_ADDRESS>(J.H.L.) **\*** Correspondence<EMAIL_ADDRESS>Tel.: +82-2-910-6763

† These authors contributed equally to this work.

**Abstract:** Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide. Recent advances in recombinant DNA technology have led to the development of numerous therapeutic antibodies as major sources of blockbuster drugs for CRC therapy. Simultaneously, increasing numbers of therapeutic targets in CRC have been identified. In this review, we first highlight the physiological and pathophysiological roles and signaling mechanisms of currently known and emerging therapeutic targets, including growth factors and their receptors as well as immune checkpoint proteins, in CRC. Additionally, we discuss the current status of monoclonal antibodies in clinical development and approved by US Food and Drug Administration for CRC therapy.

**Keywords:** colorectal cancer; monoclonal antibody; therapeutic target; therapy

#### **1. Introduction**

Monoclonal antibody therapy is an effective therapeutic intervention to treat patients with chronic (e.g., cancers and immunological disorders) as well as acute infectious diseases [1]. Since the discovery of hybridoma technology by Kohler and Milstein in 1975 [2], OKT3, the first US Food and Drug Administration (FDA)-approved mouse monoclonal antibody specific to CD3, was developed to prevent or reverse graft rejection by blocking T-cell activation [3]. However, it is not widely used in clinics because of the immunogenicity issue observed in OKT3 treatment [4]. Consequently, the remarkable development of recombinant DNA technology created many cutting-edge technologies for the development of therapeutic antibodies, including antibody library construction, phage display, high-throughput-based antibody selection, affinity maturation, humanization, and overproduction [5–10]. For example, phage display is the most common and practical technology for peptide or antibody selection that was initially developed by. Smith and Winter, the 2018 Nobel laureates in chemistry. Antibodies, such as antigen-binding fragments (Fab) or single-chain variable fragment (scFv), are displayed on a phage that confers antibodies with the key properties of replicability and mutability [11]. Furthermore, Gregory P. Winter and his team pioneered the humanization techniques to lower the immunogenicity elicited by nonhuman monoclonal antibodies [12]. Moreover, antibody humanization is a state-of-the-art technique to humanize the variable region of the antibodies obtained from nonhuman species including mice, rabbits, and chickens. Among several approaches, the complementarity-determining region (CDR) grafting method has been mostly used in antibody humanization for the development of therapeutic antibodies including cetuximab, rituximab, and infliximab. Humanized antibodies by CDR grafting are rendered by transferring the CDRs of a variable region to a human antibody scaffold [10,13–15]. As of December 2019, 79 therapeutic monoclonal antibodies have been approved by the US FDA. Furthermore, the global therapeutic monoclonal antibody market is the fastest-growing pharmaceutical industrial market and is expected to generate revenue of \$300 billion by 2025 [14]. In addition, several antibody fragments such as Fab and scFv have also entered

**Citation:** Hwang, K.; Yoon, J.H.; Lee, J.H.; Lee, S. Recent Advances in Monoclonal Antibody Therapy for Colorectal Cancers. *Biomedicines* **2021**, *9*, 39. https://doi.org/10.3390/ biomedicines9010039

Received: 30 November 2020 Accepted: 31 December 2020 Published: 5 January 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

clinical trials [16]. Antibody fragments retain the targeting specificity of whole monoclonal antibodies and can be more economically produced. Furthermore, these fragments are smaller and easily penetrate tissues and tumors more rapidly and deeply than monoclonal antibodies having a higher molecular weight of 150 kDa. Antibody fragments have been also forged into multivalent and multispecific reagents, linked to therapeutic payloads (e.g., radionuclides, toxins, enzymes, liposomes, and viruses), and engineered for enhanced therapeutic efficacy [16–19].

Colorectal cancer (CRC) is the third most commonly occurring malignancy and second leading cause of cancer death worldwide. The increasing prevalence of CRC across the globe is one of the key factors driving the growth of the market [20]. According to statistics, the global CRC therapeutic market is expected to reach \$18.5 billion by 2023 from \$13.7 billion in 2018, at a compound annual growth rate of 6.1% from 2018 to 2023 [21]. Traditionally, 5-fluorouracil (5-FU)-based chemotherapeutic regimens, such as FOLFIRI (irinotecan-containing regimen) and FOLFOX (oxaliplatin-containing regimen), have been used clinically as standard therapies for treating CRC patients [22]. Recently, monoclonal antibodies have been used in combination with standard chemotherapy to improve the clinical outcomes of CRC patients. Compared with traditionally used chemotherapeutic agents, monoclonal antibodies have fewer side-effects because their target specificity and versatility are also being applied to next-generation antibody-based therapeutics, including bispecific/multispecific antibodies, antibody-drug conjugates, and chimeric antigen receptor T cells or natural killer cells [23–25].

Over several decades, extensive in vitro and in vivo biochemical and molecular biology studies have suggested many key signaling molecules closely associated with CRC progression and metastasis. Among them, several growth factors, including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) and their cognate receptors, are proven therapeutic targets in CRCs for monoclonal antibody therapy [26,27]. Further, human EGF receptor type 2 (HER2) is also known as a monoclonal antibody target in CRCs [28]. More recently, Alison and Honjo, the 2018 Nobel prize winners in physiology, discovered immune checkpoint proteins, such as programmed cell death protein-1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4), which are key negative regulators of the immune system and cancer growth [27]. Presently, these immune checkpoint proteins are drawing attention as the most promising therapeutic targets in other types of cancers as well as CRCs for monoclonal antibody therapy. In addition, with infrastructural and technical advancement in monoclonal antibody development, blockbuster humanized and fully human monoclonal antibodies, including cetuximab, bevacizumab, and pembrolizumab, have received FDA approval and are widely used to treat CRC patients [29].

In this review, we first highlight recent studies of the roles and relevance of therapeutic targets in CRCs for monoclonal antibody therapy to understand the pathological mechanism of CRCs governed by the target molecules. Simultaneously, presenting the current status of FDA approved monoclonal antibodies in clinical development for CRC therapy will provide insight into unmet medical needs in CRCs for monoclonal antibodybased therapy.

#### **2. Physiological and Pathophysiological Roles of Therapeutic Targets in CRCs** *2.1. EGF/EGFR*

EGF is a 6 kDa growth factor with 53 amino acid residues; it binds to epidermal growth factor receptor (EGFR) that is a single transmembrane glycoprotein with 1186 amino acids. ErbB family members comprise ErbB1 (EGFR, HER1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). EGF binding to EGFR occurs within the 622-amino acid extracellular domain (ECD), which is divided into four distinct domains: I–IV. Especially, domains I and III are responsible for ligand binding, whereas domains II and IV have two cysteine-rich regions that form disulfide bonds. Further, EGFR also has a 23-amino acid residue α-helical

transmembrane domain, 250-amino acid tyrosine kinase domain, and 229-amino acid C-terminal tail with regulatory tyrosine residues [30–32].

Under physiological conditions, EGFR activation proceeds sequentially by two steps. First, prior to ligand binding, domain II is folded into domain IV via disulfide bonds in a tethered conformation. Second, once EGF binds to domains I and III of EGFR monomers, EGFRs promote domain rearrangement to expose dimerization arms in domain II, which leads to receptor dimerization via domain II [31,33]. In turn, the EGFR dimers induce trans-autophosphorylation by tyrosine kinase domains within the cytosolic parts of each EGFR, resulting in activation of their downstream signaling cascades, such as the rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt pathways [33–35]. The RAS–RAF– MAPK pathway is a major downstream signaling route of the ErbB family. EGF binding to EGFR and consecutive tyrosine phosphorylation in EGFRs leads to activation of RAS, a small GTP-binding protein, with the help of growth factor-bound protein 2 (GRB2), an adaptor protein, and son of sevenless (SOS), a guanine nucleotide exchange factor. In turn, activated RAS then activates downstream signaling molecules, including RAF and MAPK [36]. Activated MAPKs phosphorylate specific transcription factors and participate in regulation of cell migration and proliferation. EGFR activation also stimulates PI3K composed of separate regulatory (p85) and catalytic (p110) subunits. The p85 regulatory subunit directly binds to EGFR through the interaction of its Src homology domain 2 (SH2) with phosphotyrosine residues in activated EGFR. At the same time, the p110 catalytic subunit catalyzes phosphorylation of phosphatidylinositol 4,5-diphosphate to generate phosphatidylinositol 3,4,5-triphosphate (PIP3), which in turn activates the protein serine/threonine kinase, Akt [37–40] (Figure 1).

Many previous reports have suggested the importance of EGFR as a therapeutic target in CRC. For example, immunohistochemical studies have shown that EGFR was highly overexpressed in 118 (80%) of 150 CRC patients, with a median follow-up of 40 months. In addition, *Balb/c* athymic nude mice subcutaneously injected with HCT116 or EGFR-knockout CT116 cells also revealed that depletion of EGFR in HCT116 cells was associated with reduced tumor growth [41]. Osimertinib (Tagrisso®, AstraZeneca, Cambridge, UK), a tyrosine kinase inhibitor of EGFR, was found to remarkably decrease the tumor size and growth rate in a DLD-1, a colorectal adenocarcinoma cell line, xenograft mouse model [42]. Administration of cetuximab, a human/mouse chimeric antibody, exhibited significant growth suppression and inhibited the EGFR/MAPK pathway in a HT29 xenograft mouse model [43]. Furthermore, another anti-EGFR antibody also showed similar growth inhibition of CRCs to cetuximab in preclinical settings. GC1118, a novel fully human anti-EGFR IgG1 antibody, exhibited potent inhibitory effects on EGFR signaling, enhanced antibody-mediated cytotoxicity, and significantly inhibited tumor growth in a CRC patient-derived xenograft (PDX) model [44]. Further, Ame55, an anti-EGFR IgG1 antibody, also inhibited tumor growth in a LoVo xenograft mouse model [45].

In addition, some reports show the interrelationship of EGFR with other biomarkers. Consequently, HER2 amplification has been implicated in therapeutic resistance to anti-EGFR antibody therapy in preclinical studies in metastatic CRC (mCRC) [46]. HER2 amplification is seen in a small subset of mCRC, predominantly in KRAS wild-type tumors, for which anti-EGFR antibodies are used as targeted therapies [47,48]. In these tumors, aberrant HER2 signaling results in the bypass of the activation of the RAS/MEK/MAPK signaling pathway, thereby blunting the effect of EGFR blockade [49,50]. Furthermore, the EGFR-MET interaction induced by transforming growth factor-α, a specific EGFR ligand, overexpression and concomitant phosphorylation of MET, and activation of MET downstream effectors have been proposed to be closely associated with the acquired resistance to cetuximab in CRC cells [51,52]. Thus, these pieces of evidence demonstrate that the combined inhibition of EGFR and other biomarkers can represent an effective strategy for overcoming cetuximab resistance in patients with CRCs [53].

**Figure 1.** Schematic representation of the physiological roles and signaling pathways of EGF and VEGF and their cognate receptors and the effect of antibodies targeting these signaling molecules on CRC progression and metastasis. pY means phosphotyrosine residues.

#### *2.2. VEGF/VEGFR*

VEGF is a disulfide-bonded dimeric glycoprotein of 45 kDa. The VEGF family has five isotypes, including VEGF-A, placental growth factor, VEGF-B, VEGF-C, and VEGF-D [54]. Among them, VEGF-A is a glycosylated mitogen protein that is closely associated with regulation of numerous pro-angiogenic functions, including endothelial cell growth and migration, and vascular permeability in angiogenesis [55]. However, less is known about the function and regulation of VEGF-B, -C, and -D. VEGFR receptor (VEGFR) is a member of the receptor tyrosine kinase (RTK) family having multiple immunoglobulin-like ECDs and tyrosine kinase activity. VEGFRs are divided into three types: VEGFR1 (Flt-1), VEGFR2 (KDR or Flk-1), and VEGFR3 (Flt-4). Especially, VEGFR2, a 200–230 kDa protein, mostly interacts with VEGF-A and has a key role in angiogenesis at early embryogenesis and mostly at lymph angiogenesis [54,56].

Similar to that of other RTKs, the binding of VEGF to VEGFR forms a receptor dimer and induces trans-autophosphorylation. Specifically, signaling of VEGFRs is initiated upon binding of a covalently linked ligand dimer to the ECD of the receptor. This signaling promotes receptor homodimerization followed by phosphorylation of specific tyrosine residues located in the intracellular juxtamembrane domain, the kinase insert domain, and the carboxy terminal tail of the receptor. Subsequently, a variety of signaling molecules are recruited to VEGFR dimers [57,58]. These interactions next activate phospholipase C (PLC)γ and protein kinase C (PKC) to induce transcription of genes necessary for angiogenesis and cell proliferation [59]. Simultaneously, VEGFR-induced activation of PI3K results in accumulation of PIP3, which induces phosphorylation of Akt to increase endothelial cell survival and also induces systemic destruction of the entire basement membrane to increase vascular permeability [60,61]. Ligand binding to VEGFR-2 also triggers the activation of the RAS pathway, initiating signaling through the RAF–MEK– MAPK pathway known to be important in VEGF-induced cell proliferation [62]. In addition, VEGF stimulates p38 MAPK to regulate the rearrangements of the actin cytoskeleton in cell migration [63] (Figure 1).

It has been reported that 70% of patients with stage IV CRCs had positive VEGF expression, whereas 50% and 47% of patients with stage II and III CRCs, respectively, had positive VEGF expression. A statistically significant correlation was found between VEGF and 10-year disease-specific survival: VEGF-expressing tumors were more frequent in patients who died of the disease than in those who survived for 10 years [64]. Further, other reports have shown that the VEGF level also increased in CRC patients. In a prospective study by Anastasios et al. that included 67 consecutive colorectal patients, VEGF was detectable in all control subjects. Their median serum VEGF level was 186 pg/mL. Additionally, serum VEGF levels were higher in 67 patients with newly diagnosed and histologically confirmed primary CRC (492 pg/mL) than in the control subjects (186 pg/mL) [65].

An increasing number of reports have suggested VEGF/VEGFR signaling as a promising therapeutic target in CRCs. First, Foersch et al. generated a conditional knockout for VEGFR2 to investigate the functional role and underlying molecular mechanisms of the signaling. Specific deletion of VEGFR2 was confirmed by qPCR of cDNA. Immunofluorescence staining revealed a lack of receptor expression relative to that of VEGFR2-expressing control mice. Consequently, significantly fewer tumors developed in VEGFR2-knockout mice than in control mice [66]. Second, regorafenib, a novel small-molecule multi-kinase inhibitor, markedly slowed tumor growth in five of seven PDX models. The antitumor effects of regorafenib were evaluated in seven PD CRC xenografts [67]. Third, bevacizumab, a humanized antibody to VEGF, showed a significant delay in CRC tumor growth relative to that of the non-treated animals [68]. Fourth, intraperitoneal injection of DC101, an anti-VEGFR mouse monoclonal antibody, inhibited tumor growth and induced apoptosis in CRC in a KM12L4 xenograft model. Further, treatment with DC101 decreased tumor vascularity, growth, proliferation, and increased apoptosis [69]. Taken together, this large number of studies shows the importance of VEGF/VEGFR signaling for developing pharmaceutical anti-angiogenic drugs.

#### *2.3. HGF/c-MET*

HGF is synthesized as an inactive precursor (pro-HGF) that undergoes site-specific proteolytic cleavage by extracellular serine proteinases into an active 90-kDa heterodimer containing α and β chains. HGF is predominantly secreted by stromal cells and activates mesenchymal-epithelial transition factor (c-MET) on adjacent epithelial cells [70]. HGF contains two c-MET binding sites; a high affinity site in the N-terminal and first kringle regions that binds to the immunoglobulin-like fold shared by plexins and transcriptional factors (IPT) 3 and IPT4 domains in c-MET and a low-affinity site in the serine protease homology domain that interacts with the semaphorin domain in c-MET [71]. As a tyrosineprotein kinase Met or HGR receptor, c-MET is synthesized as a 170-kDa single-chain precursor protein (pro-c-MET) that undergoes furin-mediated post-translational cleavage, yielding a disulfide-linked heterodimer composed of an extracellular α-subunit and a single transmembrane β-subunit [70,72].

In the physiological state, HGF binding to c-MET induces c-MET dimerization and trans-autophosphorylation of the tyrosine residues Y1003 in the juxtamembrane domain and Y1234 and Y1235 within the kinase activation loop, resulting in phosphorylation of two tyrosine residues, Y1349 and Y1356, in the C-terminus. Tyrosine phosphorylation of Y1349 and Y1356 residues creates docking sites for recruitment of key intracellular adaptor proteins and signaling molecules through SH2-mediated interactions, including GRB2, GRB2-associated binding protein 1 (GAB1), signal transducer and activator of transcription 3 (STAT3), the p85 subunit of PI3K, SRC, PLC-γ, and Shc [73,74]. The potency, duration, and versatility of HGF/c-MET signaling are modulated by signaling amplifiers and co-receptors. Recruitment and sustained phosphorylation of the multi-adaptor protein GAB1 is an important hallmark of sustained c-MET signaling that can either bind directly to c-MET through its unique 13-amino-acid c-MET binding site or indirectly by association with GRB2. The tyrosine-phosphorylated GAB1 protein serves as an auxiliary signal transduction platform through recruitment of various effector proteins, including PI3K. Cell survival response is related to the PI3K/Akt pathway activated by c-MET signaling [75,76]. Furthermore, HGF-induced c-MET-dependent RAS/RAF/MAPK activation has been found to require the co-receptor CD44v6, thus providing a platform for SOS recruitment to the complex and subsequently triggering proficient activation of RAS [32]. Invasion, branching morphogenesis, and tumorigenesis are mediated by STAT3 activation in a tissue-dependent manner. Typically, STAT3 activation is induced by phosphorylation on a critical tyrosine residue (Y705) that triggers STAT3 dimerization owing to reciprocal phosphotyrosine-SH2 domain interactions. In addition to tyrosine 705 phosphorylation, STAT3 is also activated through serine (S727) phosphorylation. Finally, the reversible acetylation of STAT3 by histone acetyltransferase on a single lysine residue (K685) represents a third mechanism of STAT3 activation. Acetylated STAT3 enhances the stability of STAT3 dimers, which are required for DNA-binding and transcriptional activity. Cellular migration and adhesion are mediated by a c-MET–SRC–focal adhesion kinase (FAK) interaction. Within the complex, Src phosphorylates Y576 and Y577 within the kinase domain activation loop and Y861 and Y925 within the C-terminal domain of FAK. The FAK–Src complex further binds to and phosphorylates various adaptor proteins, such as p130Cas and paxillin [77–79] (Figure 2).

**Figure 2.** The schematic representation of the physiological roles and signaling pathways of HGF/c-MET and HER2 and effect of antibodies targeting these signaling molecules on CRC progression and metastasis. pY means phosphotyrosine residues.

c-MET is overexpressed in CRCs [80]. Specifically, immunohistochemistry with 23 cases of colorectal adenoma and 102 cases of primary colorectal carcinoma as well their corresponding metastases (44 lymph nodes, 21 peritoneal deposits, and 16 liver metastases) showed that normal tissues had a negative or weak c-MET expression, whereas c-MET was highly overexpressed in adenomas and primary CRC. Moreover, c-MET expression in metastatic tissues was significantly higher compared with the primary tumor [81].

Currently, HGF/c-MET signaling is one of the key therapeutic targets in CRC therapy. Small hairpin RNA-mediated c-MET knockdown dramatically suppressed tumor growth in a SW480 xenograft mouse model as well as SW480 cell proliferation in vitro [82]. SU11274, an ATP-competitive inhibitor of c-MET, significantly inhibited tumor growth in a LoVo xenograft mouse model. ARQ 197 (tivantinib), a non-ATP-competitive inhibitor of c-MET, decreased tumor growth in a HT29 xenograft mouse model [83]. Antibody-based targeting of c-MET also gave results similar to those of pre-existing chemical inhibitors. For example, YYB-101, a humanized neutralizing antibody specifically binding to HGF, inhibits c-MET activation and cell scattering in vitro and suppresses tumor growth in HCT116 xenograft mouse models [84]. R13 and R28, two fully human antibodies against c-MET, synergistically inhibit HGF binding to c-MET and elicit antibody-dependent cellular cytotoxicity. The combination of R13/28 significantly inhibited tumor growth in xenograft models of various colon tumors, including OMP-C12, 27, and 28. Inhibition of tumor growth was associated with induction of hypoxia. Moreover, in an experimental metastasis model, R13/28 increased survival by preventing recurrence of otherwise lethal lung metastases [85].

#### *2.4. HER2*

HER2 is an ErbB family member with a molecular weight of 185 kDa comprising a 632 amino acid ECD, 22-amino acid α-helical transmembrane domain, and a 580-amino acid tyrosine kinase domain. Despite the many intensive studies on HER2, a ligand of HER2 has not been clearly identified yet. It is known that HER2 forms complexes with HER2 or other ErbB family members, including EGFR, ErbB3, and ErbB4, to activate downstream signaling pathways [86,87].

Similar to EGF/EGFR signaling pathways, it has been suggested that in normal cells, the HER2 complex formation, such as through homodimerization or heterodimerization, leads to continuous trans-autophosphorylation on tyrosine residues of HER2 and activates downstream signaling pathways, including the RAS/RAF/MAPK pathway, PI3K/Akt pathway, and PLC/PKC pathway. HER2 activation ultimately promotes cell growth, proliferation, and survival. The RAS/RAF/MAPK and PI3K/Akt pathways are the two most important and extensively studied downstream signaling pathways upon activation of HER2 receptors. A third key signaling in the network is the PLC-γ/PKC pathway. Binding of PLC-γ to phosphorylated HER2 stimulates PLC-γ activity and results in hydrolytic cleavage of phosphatidylinositol-4,5-bisphosphate (PIP2) to yield inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol. These second messengers are important for intracellular calcium release and activation of PKC. As a result of these signaling pathways, different nuclear factors are recruited and modulate the transcription of different genes involved in cell-cycle progression, proliferation, and survival [32,33,88,89] (Figure 2).

In CRC, HER2 expression is varied because of many factors that influence the determination of HER2 expression, especially of the intracellular fraction of HER2. One report stated that HER2 overexpression was observed in 136 (11.4%) of 1195 CRC patients with moderately to poorly differentiated tubular adenocarcinomas. Further, HER2 overexpression correlated with shorter mean overall survival (OS) [90]. Other studies have also reported that membranous overexpression of HER2 occurs in only 5% of all CRC patients, whereas cytoplasmic HER2 overexpression is observed in a significant proportion (30%) of patients [91].

Several lines of evidence also support the idea that HER2 is a therapeutic target in CRCs. Tucatinib, a reversible inhibitor that binds to the ATP pocket of the internal domain of the HER2 receptor, prevents activation of HER2 signaling pathways [92]. Further, administration of tucatinib in a CRC PDX model significantly reduced tumor volume. Both H2Mab-19 and H2Mab-41, novel anti-HER2 IgG2 antibodies, significantly reduced tumor development in Caco-2 xenograft mouse models [93,94]. Treatment with Herceptin® (Genentech, San Francisco, CA, USA) caused a decrease in HER-2 protein levels in DLD-1, HT-29, Caco-2, and HCA-7 colon cancer cells in vitro. Treatment of athymic mice engrafted

with EGFR-dependent colon cancers, including HCA-7, DLD-1, and HT-29 with Herceptin® showed tumor regression and decreased EGFR tyrosine phosphorylation in tumor cells [95].

#### *2.5. Immune Checkpoint*

2.5.1. CTLA-4

CTLA-4, also designated CD152, is a type I transmembrane T-cell inhibitory molecule that functions as an immune checkpoint, downregulates immune responses and is found as a covalent homodimer of 41–43 kDa [96]. CTLA-4 is a member of the IgG superfamily that is expressed by activated T cells. CTLA-4 contains an ECD with one Ig-like V-type domain, a transmembrane domain, and cytoplasmic tail. CTLA-4 is homologous to the T-cell costimulatory protein, CD28, and both molecules bind to B7-1/B7-2 on antigen-presenting cells [97]. CTLA-4 binds CD80 and CD86 with greater affinity and avidity than CD28, thus enabling it to outcompete CD28 for its ligands. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal [98,99].

CTLA-4 is upregulated in a manner dependent on TCR stimulation. At the cell membrane, CTLA-4 undergoes dimerization, and each CTLA-4 dimer can bind two independent B7-1/B7-2 homodimers, forming a linear zipper-like structure between B7-1/B7-2 and CTLA-4 homodimers [100]. Activated CTLA-4 binds to PI3K, the tyrosine phosphatases (SHP1 and SHP2) and the serine/threonine phosphatase PP2A. SHP1 and SHP2 dephosphorylate TCR-signaling proteins, whereas PP2A targets phosphoserine/threonine residues and is known to interfere with the activation of Akt [101].

CTLA-4 can inhibit T-cell responses by several mechanisms. One mechanism involves antagonism of B7-CD28–mediated co-stimulatory signals by CTLA-4. The fact that CTLA-4 has a much higher affinity for B7 than CD28 supports the notion that the CTLA-4–mediated sequestration of B7 is closely associated with negative regulation of T-cell signaling [102]. Another mechanism for the inhibitory activity of CTLA-4 is related to direct interaction with the TCR–CD3 complex at the immunological synapse for negative regulation of downstream signaling after TCR activation [103]. When CTLA-4 interacts with the ITAMs present on the TCR–CD3 complex, the activated CTLA-4 binds to tyrosine phosphatases, including SHP1, SHP2, and PP2A, and eventually deactivates various downstream signaling molecules of activated T cells, including zeta-chain-associated protein kinase 70, spleen tyrosine kinase, and proto-oncogene tyrosine-protein kinase [104–106] (Figure 3).

Many previous reports have shown that CTLA-4 is a key therapeutic target in CRCs [107]. First, Long et al. used the CRISPR-Cas9 system to generate CTLA-4 knockout cytotoxic T lymphocytes (CTLs) and evaluated the effect on the antitumor activity of the CTLs [107]. The HCT-116 xenografted mice treated with CTLA-4 KO CTLs demonstrated repressed tumor growth and prolonged survival relative to those in the control group. All of the mice in the control group died from progressive tumors within 62 days. In contrast, only 10% of CTLA-4 KO CTLs treated mice died within that time [108]. Second, Fu et al. validated the efficacy of anti-CTLA-4 mouse monoclonal antibodies on tumor size in mice inoculated with CT26 cells. The tumor volumes were 2106 ± 205 mm3 on day 17 in the control group treated with vehicle only but were 23 ± 4 mm<sup>3</sup> in the group treated with the anti-CTLA-4 antibodies on day 5, which indicated a statistically significant difference in antitumor activity between the treated and vehicle groups [109]. Third, Lute et al. also reported that anti-human CTLA-4 human monoclonal antibodies-treated mice survived longer than the control Ig-treated mice in a human peripheral blood leukocytes-SCID mouse model. Fourth, administration of 9H10, an anti-murine CTLA monoclonal antibody, as monotherapy moderately inhibited growth and metastatic spread of the colon cancer cells in an orthotropically implanted CT26 xenograft mouse model [110]. Further, the sole CTLA-4 inhibition significantly increased intratumoral CD8<sup>+</sup> and CD4+ T cells and reduced FOXP3+/CD4+ Treg cells, which was associated with increased expression levels of the pro-inflammatory Th1/M1-related cytokines IFN-γ, IL-1α, IL-2, and IL-12 [111].

In summary, the evidence from the studies above shows that CTLA-4 blockade exerts inhibitory effects on growth and metastasis of CRCs.

**Figure 3.** Schematic representation of the physiological roles and signaling pathways of immune checkpoint proteins of PD-1, PD-L1, CTLA-4, and B7-1/2 and effect of antibodies targeting these signaling molecules on CRC progression and metastasis.

#### 2.5.2. PD-1/PD-L1

PD-1, also designated CD279, is a 55 kDa membrane protein consisting of an ECD followed by a transmembrane region and an intracellular tail containing two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Programmed cell death-ligand 1 (PD-L1, designated CD274, B7-H1) is a 44 kDa transmembrane protein expressed on T cells, B cells, macrophages, and dendritic cells as well as on tumor cells, including CRCs [112,113].

Under physiological conditions, when T cells recognize antigens on major histocompatibility complex of the target cell, inflammatory cytokines, such as tumor necrosis factor alpha and interferon gamma (IFNγ) are produced to initiate inflammatory processes. These cytokines upregulate the expression of PD-L1 on tissues and PD-1 on T cells. In turn, PD-1 directly interacts with PD-L1 to negatively regulate T-cell receptor (TCR) signaling, inhibits interleukin-2 (IL-2) production in T cells, and increases T-cell apoptosis [114,115]. Specifically, this PD-1/PD-L1 interaction induces lymphocyte-specific protein tyrosine kinase-induced phosphorylation of two tyrosine-based motifs within ITIM and ITSM of the cytoplasmic tail of PD-1. The recruitment of Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP1) and SHP-2 phosphatase then induces dephosphorylation of the TCR signalosome, including CD3ζ, ZAP70, and PI3K kinases, resulting in the deactivation of downstream signaling targets [116]. Moreover, the PD-1/PD-L1 interaction also downregulates the protein (casein) kinase 2 expression that phosphorylates the regulatory domain of phosphatase and tensin homolog (PTEN) and inhibits phosphatase activity to remove PIP3 produced by PI3K [112,117,118]. Thus, PTEN can terminate PI3K activities by dephosphorylating PIP3, which eventually leads to immune tolerance, a phenomenon

in which the immune system loses the control to mount an inflammatory response even in the presence of actionable antigens [117,119] (Figure 3).

PD-L1 has been reported to be overexpressed in CRC. More specifically, among the 80 tumor specimens, 22 (27.5%) showed high PD-L1 expression, 24 (30.0%) showed moderate expression, and 34 (42.5%) showed weak or no PD-L1 staining. Furthermore, the high PD-L1 expression in normal tissues was observed in four (6.3%) cases [120].

Many studies have suggested PD-1 and PD-L1 as promising therapeutic targets in CRCs. First, in *BALB/c Rag2*−*/*−*γc*−*/*− mice engrafted with PD-L1-overexpressing and PD-L1-knockout CT26 murine colon cancer cells, Gordon et al. found that after 3 weeks, tumors were significantly smaller in the PD-L1-knockout group than in the PD-L1 overexpression group [121]. Second, Cai et al. examined the efficacy of anti-mouse PD-1 rat immunoglobulin (Ig) G2 antibodies on tumor growth in a CT26 colon cancer xenograft mouse model. The antibody treatment showed significant inhibition of transplanted-tumor growth in mice [122]. Third, in a humanized CRC PDX model established by Capasso et al., treatment with nivolumab, a fully human IgG4 (S228P) monoclonal antibody to PD-1, led to significant tumor growth inhibition coupled with increased numbers of IFNγ-producing human CD8+ tumor-infiltrating lymphocytes [123]. Fourth, Stewart et al. reported that MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1, significantly inhibited the growth of human tumors in a novel CT26 xenograft model containing co-implanted human T cells. This activity is entirely dependent on the presence of transplanted T cells. Further, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 CRC cells [124]. The antitumor activity of anti-PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of high-motility group box 1 within CT26 tumors [125].

#### **3. Current Status of Monoclonal Antibodies for CRC Therapy**

#### *3.1. Cetuximab*

Cetuximab (Erbitux®), developed jointly by Merck KGaA (Darmstadt, Germany) and Imclone Systems (New York City, NY, USA), is a monoclonal antibody that binds to the ECD of the EGFR. It is a human/mouse chimeric IgG1 antibody that consists of the variable fragments (Fvs) of a murine anti-EGFR antibody and human constant heavy and kappa light chains [126,127].

Cetuximab was originally known as a blockade for inhibiting interactions between EGFR and all known EGFR ligands by specifically binding to domain III of the EGFR ECD [128]. Furthermore, its binding to EGFR is also able to promote receptor internalization and concomitantly downregulate EGFR protein levels expressed on the cell surface, resulting in suppression of EGFR-dependent downstream signaling pathways and transcription. In addition to these specific modes of action, cetuximab indirectly attacks cancer cells through antibody-dependent cell-mediated cytotoxicity (ADCC). After its binding to EGFR, the IgG1 portion of cetuximab may be recognized by Fcγ receptors (FcγR) on immune effector cells, such as natural killer cells and T cells, and participates in cancer cell death. In general, FcγRs bind effectively to IgG1 and IgG3 antibodies. Thus, cetuximab is speculated to more likely stimulate ADCC than panitumumab having IgG2. Consequently, cetuximab reduces tumor angiogenesis, invasiveness, and metastatic spread [129–132].

In 2004, cetuximab received FDA approval for metastatic CRCs and head and neck cancers, and its use is recommended in combination with standard chemotherapy for treatment of patients with metastatic CRCs having EGFR-positive and wild-type KRAS (Table 1). According to the CRYSTAL clinical trial (NCT00154102), compared with FOLFIRI alone, cetuximab plus FOLFIRI improved progression-free survival (PFS) from 8.0 to 9.0 months and OS from 20 to 23.5 months for treatment of patients with KRAS wild-type [133].

Antibody information was obtained from the FDA Label database and the Drug Approval and Databases site maintained by the US FDA (https://www.fda.gov/drugs/development-approval-process-drugs/drug-approvals-and-databases) or Clinical Trial Information Site (https://clinicaltrials.gov).

#### *3.2. Panitumumab*

Panitumumab (Vectibix™) originally developed by Abgenix Inc. (Freemont, CA, USA) is a fully human IgG2 monoclonal antibody that specifically binds to the ECD of EGFR. Especially and different from cetuximab, it can also bind to a single-point mutation in domain III of EGFR (S468R) that confers acquired or secondary resistance only to cetuximabtreated patients [134].

In 2006, panitumumab was approved by the US FDA for treatment of patients with EGFR-expressing metastatic CRCs with disease progression or following fluoropyrimidine-, oxaliplatin-, and irinotecan-containing regimens (Table 1). Later, it was also approved for treatment of patients with refractory metastatic CRCs having EGFR-positive and wild-type KRAS. Currently, panitumumab was the first monoclonal antibody to use KRAS as a predictive biomarker [135].

Panitumumab is being used in combination with chemotherapy in the first- and second-line treatment of metastatic CRCs [136]. In the PRIME (NCT00364013) clinical trial, compared with chemotherapy alone, a first-line treatment of panitumumab plus FOLFOX improved PFS from 8.0 to 9.6 months and OS from 12 to 14 months. Further, in the 20,050,181 clinical trial (NCT00339183), compared with chemotherapy, a second-line treatment of panitumumab plus FOLFIRI improved PFS from 4 to 6 months and OS from 19 to 24 months [137,138].

#### *3.3. Bevacizumab*

Bevacizumab (Avastin®) developed by Genentech (South San Francisco, CA, USA) is a humanized IgG1 monoclonal antibody that binds to segment β5–β6 of VEGF165, known as VEGF-A; its binding to VEGF-A inhibits angiogenesis by specifically inhibiting the interaction between VEGF-A and VEGFR2. Thus, bevacizumab inhibits angiogenic signaling caused by the interaction of VEGF-A and VEGFR2 [139–141].

In 2004, bevacizumab received FDA approval for first- or second-line treatment with 5- FU-based therapy for patients with mCRCs (Table 1). As the first anti-angiogenic antibody drug, bevacizumab is currently being used in clinics for treatment of patients with NSCLC, mRCC, epithelial ovarian cancer, and recurrent glioblastoma as well as metastatic CRCs. In the clinical trial ECOG3200 (NCT00069095) for treatment of patients with mCRCs, compared with FOLFOX4 alone, bevacizumab plus FOLFOX4 improved OS from 10.8 to 12.9 months and PFS from 4.7 to 7.3 months [142–144].

#### *3.4. Ramucirumab*

Ramucirumab (Cyramza®) developed jointly by ImClone Systems (New York City, NY, USA) and Dyax (Cambridge, MA, USA) is a fully human monoclonal IgG1 antibody that binds to the ECD of human VEGFR2, which is a key receptor that mediates angiogenesis and is highly expressed in not only tumor microvessels but also malignant tumors. Bevacizumab strongly neutralizes VEGF and blocks binding to VEGFR1/VEGFR2, whereas ramucirumab specifically blocks the VEGF/VEGFR2 interaction by binding to VEGFR2 [145–147].

Ramucirumab was isolated from a Dyax's phage antibody library and developed as a therapeutic antibody for treatment of solid tumors. In 2014, ramucirumab first received FDA approval as a single-agent treatment for advanced gastric or gastro-esophageal junction adenocarcinoma after prior treatment with fluoropyrimidine- or platinum-containing chemotherapy. In 2015, ramucirumab in combination with FOLFIRI was also approved by the US FDA for the treatment of patients with mCRC with disease progression on or after prior therapy with bevacizumab, oxaliplatin, and fluoropyrimidine (Table 1). In the RAISE clinical trial (NCT01183780) for treatment of mCRC patients, compared with FOLFIRI alone, ramucirumab plus FOLFIRI improved OS from 11.7 to 13.3 months and PFS from 4.5 to 5.7 months [148,149].

#### *3.5. Rilotumumab*

Rilotumumab (AMG-102) developed by Amgen Inc. (Thousand Oaks, CA, USA) is a fully human monoclonal IgG2 antibody that binds to the beta chain of HGF. HGF, a scattering factor, influences cancer cell proliferation, survival, invasion, and metastasis through interaction with c-MET. Therefore, specific binding of rilotumumab to HGF neutralizes the interaction between HGF and c-MET and inhibits c-MET phosphorylation and downstream signaling, resulting in inhibition of cancer cell proliferation, survival, and invasion through partial antagonism of c-MET phosphorylation [150,151].

It has been reported that in the phase II clinical trial (NCT00788957) for treatment of patients with wild-type KRAS metastatic CRCs, compared with panitumumab alone, rilotumumab plus panitumumab combination therapy improved OS from 8.6 to 9.6 months and PFS from 3.7 to 5.2 months [152] (Table 1).

#### *3.6. Onartuzumab*

Onartuzumab (MetMAb) developed by Genentech (South San Francisco, CA, USA) is a humanized monoclonal antibody that binds to the semaphorin domain of c-MET; it is a human IgG1 with a monovalent arm. c-MET is an RTK through which downstream signals are transduced after dimerization or oligomerization by HGF binding. HGF/c-MET signaling has also been implicated in the metastatic growth of multiple cancers, making it an attractive target for various therapeutic agents [153]. When onartuzumab binds to c-MET, it inhibits cell proliferation and survival, cell motility, migration, and invasion [154]. In a clinical trial (NCT01418222) of the MET inhibitor onartuzumab in combination with mFOLFOX-6 plus bevacizumab for treatment of mCRC patients, no significant differences existed in PFS or OS between onartuzumab combination therapy with mFOLFOX-6 plus bevacizumab and bevacizumab plus mFOLPOX-6 therapy. Therefore, the phase II clinical trial of onartuzumab failed because it did not show any clinical improvement [155] (Table 1).

#### *3.7. Trastuzumab*

Trastuzumab (Herceptin®) developed by Genentech (South San Francisco, CA, USA) is a humanized IgG1 monoclonal antibody that binds to domain IV in the ECD of the HER2. This antibody is composed of humanized Fvs of murine anti-HER2 antibody and human constant heavy and kappa light chains. The antibody suppresses cancer growth and proliferation [156].

Trastzumab binds to HER2 on the cell surface of CRCs and inhibits downstream signaling pathways, such as RAS/RAF/MEK and PI3K/Akt pathways, thereby inhibiting the proliferation and survival of CRCs. This demonstrates that trastuzumab can be used as a therapeutic agent in HER2-overexpressed CRCs. In addition, trastuzumab indirectly attacks cancer cells through Fc effect functions, such as ADCC and complement-dependent cytotoxicity (CDC) [157,158].

In 1998, trastuzumab first received US FDA approval for the treatment of patients with metastatic HER2-overexpressing breast cancers, and its indications for numerous cancer treatments have expanded [159,160]. Despite the low-expression pattern observed in CRC patients, intriguingly, recent preclinical trials have shown that trastuzumab or pertuzumab in combination with lapatinib significantly suppressed tumor growth in HER2 amplified CRC tumor xenograft animal models. According to the HERACLES clinical trial (NCT03365882), compared with chemotherapy alone, trastuzumab plus lapatinib therapy in patients with HER2-positive, wild-type KRAS metastatic CRC improved PFS by 2.9 months and OS by 11.5 months [161] (Table 1).

#### *3.8. Pertuzumab*

Pertuzumab (Perjeta®) developed by Genentech (South San Francisco, CA, USA) is a humanized IgG1 monoclonal antibody that binds to HER2. The antibodies act as a blockade to inhibit dimerization of HER2 with other HER receptors, especially HER3, by specifically binding to domain II in the ECD of HER2, a different epitope for trastuzumab, resulting in inhibition of cell growth and initiation of apoptosis [162]. In 2012, the US FDA approved pertuzumab use in combination with trastuzumab and docetaxel for treatment of patients with metastatic HER2-positive metastatic breast cancers. According to the HERACLES study (NCT03365882), the randomized phase II trial studies is ongoing by evaluating the efficacy of pertuzumab and tratuzumab in patients with HER-amplified mCRCs, compared with cetuximab and irinotecan hydrochloride [163] (Table 1).

#### *3.9. Ipilimumab*

Ipilimumab (Yervoy®) developed by Medarex (Princeton, NJ, USA), is a fully humanized monoclonal antibody that binds to the ECD of CTLA-4, specifically blocks the interaction between CTLA-4 and B7-1 or B7-2, and eventually maintains T-cell cytotoxicity to attack cancer cells [164,165].

In 2011, ipilimumab first received FDA approval for melanoma treatment. Furthermore, it underwent clinical trials for the treatment of NSCLC, SCLC, and bladder cancer. Moreover, it was approved by the FDA for the treatment of mCRC in 2018 [166] (Table 1). This antibody is currently being evaluated in the CheckMate-142 clinical trial (NCT02060188) for treatment of patients with dMMR/MSI-H mCRC. The CheckMate-142 trial that is evaluating the combination therapy of ipilimumab plus nivolumab is the same clinical trial that is evaluating nivolumab [167].

#### *3.10. Tremelimumab*

Tremelimumab (CP-675) developed by AstraZeneca (Cambridge, UK) is a fully human monoclonal IgG2-kappa antibody that binds to the ECD of CTLA-4 and has an epitope similar to that for tremelimumab and ipilimumab. Its binding to CTLA-4 specifically blocks the interaction between CTLA-4 and B7-H1 and B7-H2 and downregulates the immune system. The function of tremelimumab is similar to that of ipilimumab, which maintains T-cell cytotoxicity against cancer cells [166,168].

In 2015, tremelimumab first received US FDA approval as an orphan drug for the treatment of patients with malignant mesothelioma. Currently, the indications that can be treated through clinical trials are expanding. In clinical trials, tremelimumab has been administered in various combination therapies with durvalumab and anti-cancer drugs. In a phase II clinical trial (NCT02870920) that compared the combination therapy of tremelizumab plus durvalumab with best supportive care for treatment of patients with advanced mCRC, the PFS was estimated to have improved to 1.8 months and the OS to have improved to 6.6 months after a median follow-up of 15.2 months (Table 1). This clinical trial is ongoing until December, 2020 [169,170].

#### *3.11. Pembrolizumab*

Pembrolizumab (Keytruda®) developed by LifeArc (London, UK) is a humanized monoclonal antibody that binds to PD-1. This antibody was generated by grafting the variable region sequences of a high affinity mouse anti-human PD-1 antibody onto a human IgG4-kappa isotype framework containing a stabilizing S228P Fc mutation for preventing Fab-arm exchange. The antibody specifically binds the PD-1 C'D-loop and antagonizes the interaction between PD-1 and its known ligands, PD-L1 and PD-L2 [171,172].

In 2014, pembrolizumab received FDA approval as the first PD-1/PD-L1 blockade drug for the treatment of metastatic melanoma [173]. In 2020, the FDA also approved pembrolizumab as the first-line treatment for patients with unresectable or metastatic microsatellite high-instability or mismatch repair-deficient CRCs [174] (Table 1). According to the KEYNOTE-177 clinical trial (NCT02563002) for treatment of MSI-H/dMMR CRCs, pembrolizumab improved PFS from 8.2 to 16.5 months compared with the standard chemotherapy [175].

#### *3.12. Nivolumab*

Nivolumab (Opdivo®) developed by Medarex (Princeton, NJ, USA) is a fully human monoclonal antibody that binds to the IgV domain of PD-1. The antibody has a variable region grafted into the human kappa and IgG4-constant region containing an S228P mutation in the hinge region. Especially, nivolumab specifically binds to the N-terminal loop of PD-1 different from an epitope for pembrolizumab. Nivolumab is also an immune checkpoint inhibitor that potentiates the cytotoxicity of T cells to kill malignant tumors [176,177].

In 2014, nivolumab first received US FDA approval for treatment of patients with advanced melanoma and was then approved to treat lung cancer in 2015. Currently, immunotherapy using nivolumab is widely used to treat mCRC [178]. In the CheckMate-142 phase II clinical trial (NCT02060188) for treatment of patients with dMMR/MSI-H mCRC, the PFS was estimated to be 12 months and the OS rate improved from 73% to 85% compared with that of nivolumab monotherapy as the primary endpoint after a median follow-up of 13.4 months (Table 1). This CheckMate-142 clinical trial is ongoing until primary completion in July 2022 [179,180].

#### *3.13. Camrelizumab*

Camrelizumab (AiRuiKa) developed by Jiangsu HengRui Medicine Co. Ltd. (Lianyungang, Jiangsu, China) is a humanized monoclonal antibody that binds to the flexible Nand C'D-loops of PD-1 and specifically blocks the interaction between PD-L1 and PD-1. It has epitopes partly overlapped with epitopes that bind to nivolumab and pembrolizumab. The cancer cell-killing mechanism of camrelizumab in immunotherapy is similar to that of pembrolizumab and nivolumab [181,182].

In 2019, camrelizumab first received China Food & Drug Administration approval for treatment of patients with classical Hodgkin's lymphoma. The indications for camrelizumab are expanding through multiple ongoing clinical trials. A phase II clinical trial (NCT03912857) for treatment of patients with advanced mCRC, the combination of camrelizumab plus apatinib, a VEGFR2 inhibitor, is expected to increase the overall response rate of advanced mCRC after standard chemotherapy [183,184] (Table 1).

#### *3.14. Atezolizumab*

Atezolizumab (Tecentriq®) developed by Genentech (South San Francisco, CA, USA) is a monoclonal antibody that binds to the IgV domain of PD-L1, a ligand of the PD-1 receptor expressed on the surface of T cells. It is a humanized antibody in which the variable region of atezolizumab is changed to a human germline sequence and Fc region of atezolizumab engineered to reduce the Fc-mediated effector functions. PD-L1 acts as a

T-cell suppressor through interaction with PD-1. Atezolizumab specifically binds to PD-L1 and blocks the interaction between PD-1 and PD-L1, thereby maintaining the anti-cancer effect of T cells [185,186].

In 2016, atezolizumab received FDA approval as an immune checkpoint PD-L1 inhibitor for the treatment of patients with locally advanced or metastatic urothelial carcinoma [10,16]. In 2019, the FDA also approved atezolizumab for the first-line treatment of adult patients with extensive-stage small-cell lung cancer in combination with carboplatin and etoposide, (NCT02763579) [19]. According to a clinical trial (NCT02873195) for treatment of refractory mCRC, compared with bevacizumab plus capecitabine therapy, bevacizumab and capecitabine combination therapy improved PFS from 3.3 to 4.3 months. Additionally, OS was maintained for 20 months, a result similar to that for bevacizumab and capecitabine combination therapy [155] (Table 1).

#### *3.15. Avelumab*

Avelumab (Bavencio®) developed by Merck KGaA (Darmstadt, Germany) is a fully human IgG1 antibody that binds to the IgV domain of PD-L1 in a manner similar to atezolizumab and blocks the interaction between PD-L1 and PD-1. Avelumab consists of human antibody sequences in both the variable and constant lambda light-chain and IgG1 heavy-chain regions [187].

In 2017, avelumab first received US FDA approval for the treatment of metastatic Merkel cell carcinoma [17]. Clinical trials for treatment of CRC patients ongoing. Specifically, avelumab was administered in clinical trials for CRC patients with POLE mutation observed in 3% of total CRC patients. One trial is a phase II clinical trial of avelumab monotherapy (NCT03150706) being conducted at Asan Medical Center (AMC) in South Korea. The other trial is a phase III clinical trial of avelumab plus 5-FU combination therapy (NCT03827044) being conducted at Royal Marsden Hospital in the UK that is expected to end in 2024 [188] (Table 1).

#### *3.16. Durvalumab*

Durvalumab (Imfinzi®) developed by Medimmune (Gaithersburg, MD, USA) is a fully human monoclonal antibody that binds to the ECD of PD-L1 and blocks the interaction between PD-L1 and PD-1. However, it has been reported that an epitope of PD-L1 for durvalumab is different from that of avelumab. It was engineered to prevent cytotoxic effector functions (ADCC or CDC) against PD-L1-positive immune cells [189]. Durvalumab is an immune checkpoint inhibitor used to promote immune responses for cancer therapy [189,190].

In 2017, durvalumab first received FDA approval for the treatment of metastatic urothelial carcinoma and is currently expanding indications through combination therapy [191]. The clinical trials using durvalumab are for refractory mCRC or mutated mCRC. The ILOC-EORTC phase II clinical trial (NCT030101475) for treatment of patients with refractory mCRC is ongoing with duvalumab plus tremelimumab combination therapy after radiation therapy as a primary completion in August 2021. Another phase II clinical trial (NCT03435107) for treatment of patients with POLE-mutated mCRC is ongoing to compare duvalumab monotherapy with chemotherapy. That trial has a primary completion date of March 2022 [192,193] (Table 1).

#### **4. Conclusions**

CRC is a highly complex and molecularly heterogeneous disease harboring frequent mutations that are resistant to common treatment. Despite recent advances in identification of therapeutic targets in CRCs and concomitant development of their therapeutic antibodies, several medical needs remain unmet in CRC therapy. As indicated by cetuximab treatment, antibody-drug resistance is one of the major hurdles for treating CRC patients. Cetuximab is only responsive to wild-type EGFR- and KRAS-expressing CRC patients who represent 10–20% of all CRC patients, whereas cetuximab is not responsive to approxi-