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to germline mutations of MMR enzymes, as MLH1, MSH2, MSH6, and PMS2, which causes the so
called Lynch syndrome [25-27] . There are also sporadic dMMR CRCs, which arise mainly from epigenetic
silencing of MLH1 promoter, and they are associated with CIMP phenotype and BRAF V600E
mutations.

To define MSI, five microsatellites are evaluated trough PCR based assay: if ≥ 2/5 are unstable, the sample
is defined as MSI-high (MSI-H), while 1/5 or 0/5 are MSI-low (MSI-L) and MSS, respectively, which have a
[28]
similar behavior .

The progressive findings in the molecular characterization of CRC along with the identification of specific
gene alterations as prognostic and predictive factors in this cancer, led to the elaboration of various CRC
classifications, essentially based on gene expression [29-33] . However, there were many differences among
these classifications, so in 2015 the CRC Subtyping Consortium (CRCSC) developed a new classification,
identifying four consensus molecular subtypes (CMS), analyzing the results of six CRC subtyping
[34]
algorithms . Each CMS group had a specific pattern:
• CMS1 (MSI Immune, 14%): CMS1 samples were hypermutated, with low prevalence of somatic copy
number alterations (SCNAs), enriched of MSI and CIMP tumours with hypermethylation status. A
particular characteristic of this group was a more frequent presence of BRAF mutations, compared to
the other CMS. This subtype was defined as immune, because of the rich immune infiltrate (especially
Th1, cytotoxic T cells and NK cells) and the strong activation of immune evasion pathways, as we
[35]
typically see in MSI CRC .
• CMS2 (Canonical, 37%): this group exhibited the typical CIN pattern; it also showed more frequent copy
number gains in oncogenes and losses in tumour suppressor genes. It was characterized by epithelial
differentiation, with WNT and MYC activation, higher expression of the oncogenes EGFR, ERBB2
(also known as HER2), insulin-like growth factor 2 (IGF2), insulin receptor substrate 2 (IRS2) and
transcription factor hepatocyte nuclear factor 4α (HNF4A), as well as cyclins2.
• CMS3 (Metabolic, 13%): CMS3 samples were characterized by few SCNAs, a 30% significant
hypermutation with a mixed MSI status, a higher prevalence of CIMP low cluster and an intermediate
hypermethylation status. This subtype was defined as “metabolic” according to the common metabolic
alterations and the higher expression of KRAS mutations, which made this group of cancers similar to a
[12]
recently identified gastric cancer subtype .
• CMS4 (Mesenchymal, 23%): similarly to CMS2, this group had a high prevalence of SCNAs. It showed
the typical mesenchymal pattern, as the upregulation of genes involved in epithelial mesenchymal
transition, the TGFβ activation, angiogenesis, matrix remodeling, with a consequent stromal infiltration,
particularly CAFs.

This classification reflects also significant clinical and prognostic differences among the various subtypes:
CMS1 cancers are frequent in females with right-sided tumours and have a higher histopathological
grade, while CMS2 cancers are more frequently left-sided. Moreover, CMS4 cancers are often diagnosed at
advanced stages and they show worse overall survival (OS) and relapse-free survival (RFS). Patients with the
typical CMS1 pattern have poor survival after relapse, consistently with the known bad prognosis of patients
[36]
with MSI and BRAF mutated CRC after relapse , differently from CMS2 population, which has the best
survival after relapse of these CMS groups [37,38] .

IMMUNOTHERAPY IN MSI mCRC
Mismatch repair deficient CRCs represent 15% to 20% of stage II and III CRCs and are associated with better
prognosis than proficient (pMMR) tumors. In the metastatic setting, dMMR CRCs represent only around 5%
[39]
and are associated with a poor prognosis , as confirmed in the recent results presented at the 2017 ASCO