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Battaglin et al. J Cancer Metastasis Treat 2018;4:12 I http://dx.doi.org/10.20517/2394-4722.2018.04 Page 11 of 25

Secondary resistance to anti-EGFRs is often dependent on clonal selection induced by targeted treatment
pressure. Emerging mutations in the RAS/RAF/MAPK signaling pathway can be detected after disease
progression in tumor biopsies from previously KRAS wild-type tumors and multiple mutations can coexist
at the same time in the same sample . This seems to be the result of the amplification of pre-existing
[140]
minor sub-clones, suggested by a significant overlap in the genetic events associated with primary and
acquired resistance . Moving from these data, several trials are currently exploring different approaches to
[141]
multiple targeted inhibition based on the emergence of selected resistance drivers, such as the combination
of anti-EGFRs with MEK or MET inhibitors. Mutations in the ectodomain of EGFR represent an additional
mechanism of resistance limited to the acquired setting [142,143] . Notably, a subset of mutations including EGFR
S492R as well as other acquired mutations recently identified (S464L, G465R and I491M) appears to confer
resistance to cetuximab but not panitumumab. The binding epitopes of cetuximab and panitumumab on
EGFR, in fact, overlap but are not identical [144,145] . Retrospective analyses from the ASPECCT trial, comparing
panitumumab to cetuximab in chemorefractory mCRC patients, revealed that EGFR S492R mutations
occurred in 1% vs. 16% of patients treated with panitumumab and cetuximab, respectively . The possible
[146]
rationale for using panitumumab after the detection of these mutations as a mechanism of resistance to
cetuximab still need further validation. Other strategies to overcome acquired resistance to anti-EGFRs
include treatment with novel antibodies targeting different epitopes of the EGFR ectodomain, which can
increase receptor internalization and degradation such as MM-151 and Sym004 .
[148]
[147]
VEGF pathway
Angiogenesis plays a key role in CRC development and progression, and VEGF is a key regulator in both
physiological and pathological angiogenesis. Therapeutic agents targeting VEGF/VEGFR signaling (i.e.
bevacizumab, aflibercept, ramucirumab and regorafenib) proved to be effective across different treatment
lines in mCRC and contributed greatly to improve patients’ survival in recent years [9,10] . However, despite
extensive efforts to identify predictive biomarkers for antiangiogenic therapies in the last decade, no predictive
marker is available in clinical practice yet . The complexity of the angiogenesis signaling network and the
[149]
overlap between various angiogenic factors, in fact, represent a challenge to pharmacogenomic biomarkers
discovery.

In 2012, Bates et al. retrospectively analyzed CRC tumor samples from the phase III bevacizumab E3200
[150]
trial to explore the predictive value on treatment outcomes of VEGF165b, a VEGF splice isoform. Despite
not reaching a statistical significance, patients with a lower level of VEGF165b appeared to benefit more from
bevacizumab treatment. Focusing on a different candidate marker, recently published data demonstrated
that patients treated with first-line bevacizumab-containing regimens had a significantly longer PFS when
affected by Homeobox B9 (HOXB9)-negative tumors compared with those with HOXB9-positive tumors (18.0
vs. 10.4 months, P = 0.048). HOXB9 is known as a highly conserved homeobox transcription factor gene which
drives neoplastic transformation and tumor progression exerting an anti-apoptotic effect and promoting
tumor cell invasion. The authors demonstrated, both with preclinical and clinical data, that transcription
factor HOXB9 mediates resistance of CRC to bevacizumab modulating a complex network of alternative
pro-angiogenic and pro-inflammatory secreted factors . A prospective validation of these promising
[151]
results is highly anticipated. In another interesting analysis, NOTCH1 expression has been proposed as a
detrimental prognostic factor in mCRC patients treated with chemotherapy plus bevacizumab . Of note,
[152]
a phase Ib trial is ongoing exploring safety and preliminary efficacy of a bispecific antibody targeting VEGF
and the NOTCH ligand DLL4 (OMP-305B83) in combination with FOLFIRI as second-line treatment in
mCRC . Finally, a novel emerging player in the angiogenesis regulatory pathways is the protein apelin
[153]
(APLN). APLN signaling takes part in multiple physiological functions including angiogenesis, and interacts
at different levels with key mechanisms regulating cell growth, survival and apoptosis. Recent preclinical
data based on the analysis of tumor-derived endothelial cells from patients receiving bevacizumab showed
that APLN mRNA levels are significantly associated with treatment response. In fact, APLN levels were high