Colorectal cancer (CRC) is a heterogeneous disease that results from the interaction of multiple genetic modifications and exogenous factors such as diet, lifestyle, and microbiome [1]. Despite this heterogeneity, advances in genomic technologies have significantly improved the management of cancer patients. Identification of a driver gene mutation or other biomarkers can lead to specific targeted therapies, resulting in precision and personalized medicine that can improve the clinical outcomes of these patients [2]. The implementation of precision medicine and molecular genetic testing for cancer patients remains an ongoing educational process for physicians in both hospitals and educational centers ^[3][4][3,4]. The identification of potential new biomarker-based pharmacological treatments and therapeutic studies depends largely on the experience and knowledge of the medical team involved in the treatment of these patients.

Although numerous guidelines have been published on biomarker determination in CRC ^[1][5][6][1,5,6], the large amounts of rapidly evolving literature on precision oncology means that many of them are often outdated, challenging clinicians to be up to date in the biomarker field and critically examine all of this information to provide patients with the best possible molecular counseling ^[7][8][7,8]. In addition, genomic technologies generate massive amounts of information that can be difficult for the clinician to interpret and use adequately, resulting in a large disparity between clinical knowledge and genetic potential in cancer care [3].

Through several discussion meetings, a group of oncology experts from Spain and several Latin American countries reviewed the latest literature on the topic to provide practical recommendations on the determination of biomarkers in CRC based on their clinical experience.

There are several types of markers in CRC that have value in diagnosis, prognosis, and prediction of therapeutic response, disease monitoring, and recurrence. These markers include genetic or molecular alterations and even the anatomic location of the primary tumor.

CRC has been considered a successful model for the determination of genetic biomarkers in oncology such as KRAS, NRAS, BRAF mutations, HER2 amplification, and microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR) ^[1][9][1,9]. Genomic alterations influence treatment selection, and since targeted therapy for the treatment of advanced or metastatic CRC (mCRC) is essential, some international guidelines recommend assessing the mutational status of the genes involved in tumor development ^{[1][10][11][12]}[1,10,11,12]. Table 1 shows brief and practical recommendations for the determination of biomarkers involved in CRC. TableTable S1 S1 (can be downloaded at: https://www.mdpi.com/article/10.3390/cancers15174373/s1) shows the prognostic and predictive value of each biomarker ^{[1][2][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62]}[1,2,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62].

Along with these biomarkers, it is also important to highlight the role of homologous recombination deficiency (HRD) in CRC. HRD is caused by loss of the BRCA1/2 allele during carcinogenesis or by other genomic aberrations, which increases vulnerability to therapies such as platinum that crosslink DNA and cause DNA double-strand breaks, exceeding the ability to repair DNA damage. HRD-related somatic mutations are found in 13.8% of patients with CRC and are enriched in MSI-H, right-sided, and BRAF mutant cancers [63].

Although the anatomic location of a primary tumor across the colon is not a biomarker per se, it has been correlated with the prognosis of patients with CRC. Left-sided tumors may exhibit superior survival compared with those that are right-sided [64]. Moreover, it has been reported that right-sided tumors exhibit more CDX2-negative tumors compared with left-sided tumors. Lacking this transcription factor has been associated with several adverse prognostic variables and a high pathological grade. CDX2-negative CRCs are associated with a higher risk of recurrence and seem to benefit from adjuvant chemotherapy compared with CDX2-positive colorectal tumors [64].

Despite the fact that many of the biomarkers described are not routinely determined, it is important to mention them by emphasizing their predictive and prognostic value, even if only briefly, as they add value to the classical approach of biomarkers so far considered, which have been reduced classically only to RAS, BRAF, and MSI.

Histopathology has an important role in establishing an adequate molecular classification of CRC. Thanks to this classification, patients can be stratified according to their risk of metastasis or recurrence, which helps to select the most appropriate treatment in each case. Several classification systems and histopathological procedures are described below.

Although the tumor, node, and metastasis (TNM) staging system remains the gold standard for the stratification of patients with CRC, the heterogeneity of CRC points to the need for additional prognostic biomarkers ^[12][65][12,65]. The CMS classification of CRC is based on a combined analysis of RNA sequencing data from multiple international cohorts and includes molecular factors, tumor stroma, and signaling pathways for personalized systemic therapy ^[66][67][66,67]. However, this classification does not yet have a translation to clinical practice.

TB is an adverse histologic feature associated with poor prognosis that arises because of the loss of adhesion of the neoplastic cells of the tumor, which leads to the initiation of the metastatic process in units of 1 to ≤4 cells called buds ^[10][11][10,11]. According to the International Tumor Budding Consensus Conference (ITBCC), there may be BD1 (0–4 buds, low budding), BD2 (5–9 buds, intermediate budding), or BD3 (≥10 buds, high budding) [65]. Both BD2 and BD3 are risk factors for nodal metastases in patients with pT1 (stage I) CRC, whereas only BD3 is associated with an increased risk of recurrence and death in those with stage II CRC. In particular, BD3 tumors could be candidates for adjuvant therapy [68]. TB has been included in major staging systems/guidelines as an additional prognostic factor ^[68][69][68,69]. TB could potentially be considered relevant in the following scenarios: (1) to determine the risk of node metastasis in patients with early-stage CRC and thus report the need for radical surgery; (2) to identify patients with high-risk stage II colon cancer, a potential indication for adjuvant therapy; and (3) as an indicator of metastasis and lack of response to neoadjuvant therapy if detected in pretreatment biopsies [15]. According to this expert consensus, TB reporting is recommended but is not a required element.

ImmunoScore is a scoring system reported as percentiles of CD3+ and CD8+ immune cell densities in predefined regions of the tumor samples using software, with the aim of assessing the prognostic value of patients with stage III colon cancer, as well as its predictive value for response to adjuvant chemotherapy in these patients [70]. Patients with a high ImmunoScore will benefit more from chemotherapy in terms of risk of recurrence. Those with a high ImmunoScore have a low risk of recurrence and prolonged time to recurrence, overall survival, and disease-free survival ^[10][11][70][10,11,70]. According to experts’ consensus, there are no studies that suggest applying this scoring system routinely in clinical practice, although it is an auxiliary tool for predicting response to adjuvant therapy. It is available in some cases but is not approved.

The CRM should be recorded in all non-peritonealized resections, as poor prognosis is associated with compromised CRM [71]. A positive margin is defined as (1) a tumor < 1 mm from the sectioned margin, (2) a tumor < 2 mm from the sectioned margin, and (3) cells that are in contact with the electrocautery-affected area ^[10][11][10,11].

The mucinous component is defined as a tumor in which more than 50% of the lesion consists of extracellular pools of mucin, besides being phenotypically distinct from adenocarcinoma not otherwise specified [72]. Mucinous differentiation accounts for 5–15% of colorectal adenocarcinomas. This subtype of CRC responds poorly to chemoradiotherapy and has a poor prognosis. The genetic origins of mucinous colorectal adenocarcinoma are predominantly associated with BRAF, MSI, and CIMP pathways [73]. According to experts’ consensus, in pathology reports it is important to indicate presence of this component, as well as the corresponding percentage. Mucinous adenocarcinoma (MAC) with a signet ring cell component > 50% should be classified as signet ring cell carcinoma [74]. This is a rare histologic subtype of adenocarcinomas with a poor prognosis, usually due to the molecular alterations described in mucinous CRC and diagnosis in advanced stages ^[75][76][75,76]. The experts suggest reporting the presence of the signet ring cell component separately from the mucinous component.

Checkpoint inhibitors have changed the treatment paradigm in several types of solid tumors. The selection of patients potentially susceptible to responding to these treatments requires robust predictive biomarkers. Some of the most important biomarkers related to immunotherapy in CRC are described below.

HLA-I plays a critical role in antigen presentation to T lymphocytes, including tumor antigens. These molecules are frequently lost in CRC, resulting in immune escape to cytotoxic T lymphocytes during the natural history of cancer development [77]. This phenomenon has important implications when T-cell-mediated immunotherapy is applied in cancer patients [77]. The absence of HLA-I expression allows tumor cells to avoid recognition by cytotoxic T lymphocytes, whereas natural killer (NK) cells are activated [78].

Tumors comprise epithelial cells and stroma, both of which configure the TME made up of distinct and interacting cell populations ^[79][80][79,80]. Abnormalities of the extracellular matrix relieve the behavioral regulation of stromal cells and promote angiogenesis and tumor inflammation, resulting in resistance to immunotherapy in the TME [81]. Cancer-associated fibroblasts (CAFs) or myofibroblasts are responsible for the production and regulation of stroma in tumor tissue, depositing the same matrix components that make up tumor connective tissue, a process that is termed desmoplastic reaction (DR). CAFs modulate cancer cells through the production of growth factors [79]. Histological classification of DR provides important prognostic information that could contribute to the selection of patients with stage II colon cancer who would benefit from postoperative adjuvant therapy [82].

TGF-β is considered a tumor suppressor cytokine. However, TGF-β may transform from an inhibitor of tumor cell growth to a stimulator of growth and invasion in advanced stages of CRC [83]. An extensive meta-analysis concluded that high expression of TGF-β was a prognostic indicator in CRC patients undergoing surgery. The mortality rate of patients with a high expression of TGF-β was higher than that of patients with a low expression [83]. Thus, TGF-β could be a valuable prognostic biomarker in CRC. Inhibition of TGF-β signaling prevents metastasis or further development of certain advanced tumors such as CRC [84].

IFN-γ plays a dual and opposing role in cancer development. On the one hand, IFN-γ signaling inhibits tumor growth, and on the other hand IFN-γ contributes to tumor growth through the promotion of tumorigenesis and angiogenesis [85]. Several publications have shown that CTLA-4 and PD-1 inhibitors, as well as other immune checkpoint inhibitors, result in increased IFN-γ production, which in turn leads to the killing of cancer cells [85]. Resistance to immunotherapy is attributed to defects in IFN-γ signaling [86].

TMB is defined as the number of somatic mutations detected per megabase of tumor DNA. Its detection is of great relevance to increase the population benefiting from clinical immunotherapy [87]. A high TMB increases the probability of neoantigen generation; neoepitopes produced from mutated genes, when bound to major histocompatibility complex, are not recognized by T cells, leading to an effective antitumor immune response [88]. Higher TMB is associated with stronger immunogenicity, which could probably enhance the antitumor activity of immunotherapies. Of note is that high TMB overlaps with other biomarkers like MSI and TILs [89].

TILs are lymphocytes located in the inflammatory infiltrates present in tumor islets and in the peritumoral stroma of solid tumors and are composed of cytotoxic T lymphocytes (TCD8), NK cells, and T helper lymphocytes (TCD4). In CRC, several studies support the prognostic value of the density of infiltration by TILs, depending on the specific subtype of lymphocytes that compose them [90]. Thus, the higher frequency of TCD8 and NK effector cells in tumor islets and peritumoral tissue seems to be associated with better long-term survival [89]. In addition to prognostic information, evaluation of TILs in locally advanced rectal cancer may help predict the degree of response to neoadjuvant chemoradiotherapy [91].

TAMs have an impact on the prognosis and efficacy of chemotherapy and immunotherapy [84]; they are mainly recruited from the periphery by chemokines released from tumor tissues. Such factors bind to corresponding receptors for monocyte/macrophage recruitment [81]. TAMs play an important role in promoting tumor angiogenesis and express a variety of membrane-bound molecules [81]. TAMs can be divided into M1-like and M2-like, which are shown to have antitumor and protumor activity in TME, respectively [92].