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Page 14 of 25 Battaglin et al. J Cancer Metastasis Treat 2018;4:12 I http://dx.doi.org/10.20517/2394-4722.2018.04

tumors and to be strongly correlated with shorter patients’ PFS and OS (PFS 2.4 vs. 7.4 months, P < 0.0001;
OS 6.1 vs. 17.8 months, P < 0.0001) . On the other hand, Khambata-Ford et al. discovered that patients
[177]
[178]
with overexpression of epiregulin (EREG) and amphiregulin (AREG), two EGFR ligands, are more likely
to achieve disease control when treated with cetuximab and show a significantly longer PFS. These data
[179]
have been confirmed by Jacobs et al. showing a significant association between cetuximab response and
AREG/EREG expression. In a recent work, EREG and AREG expression has been found to have a strong inverse
correlation with methylation and to be inversely associated with right-sided tumor location, CIMP-H status
and BRAF mutation . Additionally, the authors reported that treatment with hypomethylating agents (i.e.
[180]
azacitidine) increased EREG expression, and that a CIMP-H status was associated with shorter PFS outcomes,
also in BRAF/NRAS WT patients. Based on these data, promoter DNA methylation may be the main regulatory
mechanism of AREG/EREG expression, which may explain, at least in part, the association between right-
sided tumor location, CIMP-status and anti-EGFR treatment response in mCRC. DNA methylation may, then,
partially account for primary anti-EGFRs resistance, supporting the rationale to explore the possible synergistic
treatment effect of demethylating agents in combination with anti-EGFR drugs.

Despite promising evidence, the complexity and heterogeneity of epigenetic alterations in CRC still represent
a considerable challenge, which needs to be further addressed in order to identify reliable biomarkers and
translate current knowledge into actionable therapeutic strategies.

FUTURE PERSPECTIVES
CRC consensus molecular subtypes
In recent years, great advances have been made in understanding the complexity of tumor biology and genetic
landscape underlying tumor development and response to treatment. In 2015 an international consortium
developed the Consensus Molecular Subtypes, which classifies CRC into four distinct biological groups,
based on gene expression signatures and correlated with distinct genetic, epigenomic, transcriptomic,
microenvironmental, prognostic and clinical features . CMS1 (microsatellite instability immune, 14%)
[181]
tumors are associated with high tumor mutational load (TML), microsatellite instability, hypermethylation
status (CIMP+), BRAF mutation, and strong immune activation. The CMS2 (canonical, 37%) subtype is
characterized by an epithelial signature, marked WNT-β-catenin pathway and MYC signaling activation.
CMS3 (metabolic, 13%) tumors feature metabolic dysregulation; and CMS4 (mesenchymal, 23%) a prominent
transforming growth factor (TGF)-β activation, stromal invasion and angiogenesis. Samples with mixed
features (13%) are considered to represent a transition phenotype or intratumoral heterogeneity. CMS
subgroups show a strong prognostic value independent of tumor stage, with CMS4 associated with worse
survival. Moreover, retrospective analyses of clinical trials have suggested a potential predictive value for
[182]
CMS subtypes, including a better outcome following bevacizumab treatment for CMS1 , and a lack of
benefit from oxaliplatin and anti-EGFRs (irrespective of RAS mutational status) for the mesenchymal-
[183]
[184]
like phenotype. Although not yet implemented in clinical practice, this classification system has the
potential to better inform clinicians of prognosis and therapeutic response, and to guide novel therapeutic
strategies with subtype-based targeted interventions . In fact, data have been published from very recent
[6]
preclinical studies exploring models of CMS in large panels of CRC cell lines, primary cultures and patient-
derived xenografts (PDX), with the aim of developing “adapted” classifiers optimized for pre-clinical
research and investigate specific drug sensitivity of individual CMS [185,186] . Results from these studies show
interesting initial findings highlighting subtype-dependent response profiles, with a different sensitivity to
chemotherapy (either 5-FU or oxaliplatin)-induced apoptosis between CMS2 and CMS4, which relates to
the in vivo efficacy of chemotherapy in PDX models where a delay in outgrowth of CMS2, but not CMS4
xenografts, was observed. Additionally, a strong response to anti-EGFRs and HER2 inhibitors was observed
in the CMS2 subtype. Indeed, a deeper understanding of the unique drug-sensitivity profile of each CMS
subtype and the possibility of performing high-throughput in vitro and in vivo drug screening using PDX
technology have the potential to greatly advance precision medicine in CRC.