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CD8+ T cells, in order to reduce local tumour-infiltrating immune responses. Frequent mechanisms
include loss of MHC class I expression, dysregulation of antigen processing machinery, production of
immunosuppressive factors (TGF-beta; IL-10; VEGF; indoleamine 2,3 dehydrogenase), recruitment
[12]
of immunosuppressive cells (i.e T-reg) and activation of negative costimulatory signals in tumour
[13]
microenvironment like PD-L1 .
2. Tumour microenvironment consists of regulatory immune cells, extracellular matrix proteins, and
fibroblasts (cancer-associated fibroblasts, CAFs). They secrete tumour-promoting factors that contribute
[14]
to tumor invasion and neoangiogenesis . CAFs play a critical role in CRC immunosuppression,
particularly in CRC RAS mutated; in fact they lead to tumour progression by activating epithelial
[15]
mesenchymal transition and TGF-beta/SMAD signalling : high levels of CAFs markers are correlated
[16]
with poor prognosis in CRC .
3. The immune system includes innate and adaptive immune cells. The innate cells consist of macrophages,
mast cells, neutrophils, dendritic cells, and myeloid-derived suppressor cells (MSDCs) and natural
killer (NK) cells. The adaptive T cells include CD8+ cytotoxic T lymphocytes (CTLs), CD4+ Th1 cells,
[7]
CD4+ Th17 cells and regulatory T cells (Tregs) . A strong lymphocytic infiltration is associated with
better clinical outcome in many tumors, including colorectal cancer. Particularly, high densities of
CD3+ T cells, CD8+ cytotoxic T cells and CD45RO+ memory T cells are associated with a longer
[17]
disease free survival (DFS) and improved overall survival (OS) . Therefore in CRC also the infiltration
of M1 macrophages, DCs and NK cells is associated with good prognosis, while the presence of M2
[18]
macrophages, MDSCs, Th17 and B cells is related with poor outcome .

All these findings have been translated into the elaboration of the so called “immunoscore”. It is based on
the quantification of two lymphocytes populations (CD3/CD45RO, CD3/CD8 or CD8/CD45RO) in both
tumor core and invasive margin. It ranges from 0 (low density of both cells in both cancer regions) to 4 (high
[19]
density) and may predict DFS and OS in CRC . It may help identify patients with early stage disease who
[20]
might benefit from adjuvant chemotherapy or immunotherapies according to T-cell densities .

FROM MOLECULAR SUBTYPES TO STROMAL CLASSIFICATION
The development of CRC is supported by the accumulation of genetic and epigenetic alterations that
transform colonic epithelial cells into colon adenocarcinoma cells. The genomic instability occurs early in
carcinogenesis and it facilitates the acquisition of alterations in tumour suppressor genes and oncogenes in a
clone of cells, resulting in cancer [21,22] . Three fundamental pathways are implicated in this process:
1. Chromosomal instability (CIN): CIN is observed in the majority of sporadic CRCs (65%-75%) and is
characterized by a variety of chromosomal alterations leading to defects in chromosomal segregation,
telomere stability, and the DNA damage response as well as a loss of heterozygosity. CIN-high tumours
typically accumulate mutations in specific tumor-suppressor genes and oncogenes that activate critical
pathways for CRC pathogenesis, including KRAS, CTNNB1, PIK3CA, APC, TP53 and SMAD4.
2. CpG-island methylator phenotype (CIMP): it is defined by hypermethylation of the symmetrical
dinucleotide CpG and a global DNA hypomethylation. In many human genes there’s a CpG island
in their promoter region, and the methylation of the cysteines of the CpG-island implicates the
[23]
transcriptional silencing of the gene . When this happens in promoters of tumour suppressor genes, it
[24]
supports the possible development of cancer .
3. Microsatellite instability (MSI): it occurs in 15% of early-stage colorectal tumors. It is caused by a lack
of expression in the DNA mismatch repair (dMMR) proteins, which normally are involved in the
correction of DNA replication errors. This defect results in accumulation of mistakes in microsatellite
regions, which are short repetitive sequences of DNA, with unit length ranging from one to six bases.
They are scattered throughout the coding and noncoding regions of the genome. MSI can be due