Introduction Review of Molecular Diagnostics 2017). Companion diagnostics (CDx)


Nowadays, pharmacotherapy
is mostly characterized by “trial and error”, with a consequently low success
rate after treatment, resulting in profound consequences not only for the
patient, but also for the healthcare system and the society. Early diagnosis
and early intervention are crucial elements for severe chronic diseases and
especially for cancers, as an incorrect or delayed treatment decision will
often result in disease dissemination with very little or no chances of cure. Optimally,
any pharmacological treatment decision should be timely and rely on a deep insight
into disease’s pathobiology and the mechanism of the drug’s action. Over the
last few years, the advances in molecular medicine and molecular diagnostics
have given a sufficient insight into the practice of a more sensible drug
development process and pharmacotherapy, leading to the development of companion diagnostics assays (Jørgensen, Expert Review
of Molecular Diagnostics 2017).

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Companion diagnostics (CDx) assays

diagnostics (CDx) assays are predictive biomarker tests associated to an explicit
drug. An expanding number of drugs are being developed together with a prognostic
biomarker assay that support the development progress and the use of the drugs as
an important treatment decision mechanism (Hersom, The Drug Monit. 2017).
Companion diagnostic assays have as a point of reference a single patient,
moving towards a more precise and individualized pharmacotherapy (Jørgensen,
Expert Review of Molecular Diagnostics, 2017). The success of this type of
targeted therapy is widely contingent on the performance of those assays (Jørgensen,
Ann Transl Med 2016 /Agarwal, Pharmacogenomics and Personalized Medicine, 2015),
which measure the condition of the drug target using common technologies such
as PCR, DNA sequencing, or immunohistochemistry (IHC) (Dracopoli et al.,
CellPress 2016).

As reported
by the US FDA guidance document issued in August 2014, a companion diagnostics test
is defined as “an in vitro diagnostic
device that provides information that is essential for the safe and effective
use of a corresponding therapeutic product”. Such an assay could be imperative
for better monitoring the therapy of a patient, while adapting patient’s
eligibility to a unique treatment to improve safety and efficacy. (Jørgensen,
Expert Review of Mol Diagn 2017/ Jørgensen, Ann Transl Med 2016).

substantial majority of CDx assays are applied for drugs that suppress signal
transduction pathways, either by inhibiting the activity of an intracellular
kinase using a small molecule or by blocking the activation of a receptor
induced by a ligand with a monoclonal antibody (Dracopoli et al., CellPress

alterations specific for tumors have been identified that allow the development
of agents against these targets, emerging the necessity of developing a potent
and proper diagnostic test to mark tumors harboring these modifications. The
past few years there has been a significant increase in new approved anticancer
drugs with the associated companion diagnostics. Those CDx tests are applied to
patients who, by harboring the molecular modification, have more chances to
react to the pharmacologic blocking of the target. The selecting of patients
using a CDx assay brings various potential assets, including greater rates of
response and conceivably reduced toxicity. Moreover, the development of
companion diagnostics provides challenges, such as the identi?cation of
clinically compatible biomarkers, their evaluation and validation, and
acknowledgement of the optimal clinical setting for their handling (Tibau et
al., Mol Diagn Ther. 2017). 

Current CDx markers indicated for Oncology

HER2 against transtuzumab-pertuzumab in breast cancer

The theory
of having a predictive test in conjunction with a drug was first introduced in
clinic regarding the development of trastuzumab (Herceptin, Roche/Genentech)
for the treatment of patients with advanced breast cancer (Jørgensen, Expert
Rev. Mol. Diagn 2015). This monoclonal antibody that targets HER2, binds to the subdomain IV of HER2,
triggering HER2 degradation and a
cell-mediated antibody-dependent cytotoxicity (Dracopoli et al., CellPress 2016),
and has revolutionized the treatment of patients with HER2-positive breast cancer (Richard S et al., An. Acad. Bras. Ciênc. 2016).

this accomplishment, most patients with HER2-positive
metastatic breast cancer continue to demonstrate progression of their disease,
highlighting the urgency for different therapies. The ongoing concern in new
targeted agents drove to the development of a novel recombinant humanized
antibody, namely pertuzumab, which is directed against the extracellular domain
II of HER2 protein. This protein is
important for the HER2
heterodimerization with other HER
receptors, contributing to the activation of downstream signaling pathways.
Pertuzumab, in combination with trastuzumab and docetaxel, was approved for the
first-line treatment for patients that have HER2-positive
metastatic breast cancer and it is currently being indicated as a standard of
care. Pertuzumab, when administered with trastuzumab, completely blocks the HER2/3 axis, and as a result these two therapeutic
antibodies have synergistic activity, due to their complementary mode of action
(Richard S et al., An. Acad. Bras. Ciênc. 2016).

assays have already been introduced into clinical routines and various
molecular profiling tests have been developed for therapy prediction and prognosis
in early stage breast cancer patients. These assays do not rely on NGS
technologies, but mostly on RT-qPCR and DNA microarrays and cannot be strictly
considered as CDxs, since they are not associated with a particular drug.
Nevertheless, these tests provide valuable information regarding disease
prognosis for the individual patient, contributing to the determination of the
initiation, or not, of chemotherapy (Jørgensen, Expert Rev. Mol. Diagn 2015).

PD-1 against pembrolizumab in NSCLC 

Until recently, the common used therapy for
patients with non-small cell lung cancer (NSCLC) at advanced stage was
chemotherapy, with modest survival regarding the stage of the disease.
Nevertheless, within the last few years, several new target drugs have been
developed for certain subgroups of NSCLC patients with tumors harboring
specific driver mutations. These drugs are small-molecule tyrosine kinase
inhibitors and are guided by CDx assays to ensure that they will be received
only by patients with the specific molecular abberations.

The immune checkpoint inhibitors have made
available an alternative treatment for NSCLC patients with unknown drug-driver
mutations, besides chemotherapy. Until today, three immune checkpoint
inhibitors have been accepted for the treatment of metastatic NSCLC, the PD-1
inhibitors, pembrolizumab and nivolumab and the PD-L1 inhibitor, atezolizumab.
However, out of these, only the IHC PD-L1 assay of pembrolizumab is approved as
a CDx, whereas similar assays linked to nivolumab and atezolizumab were
certified as complementary diagnostics.

In metastatic NSCLC, pembrolizumab, nivolumab,
and atezolizumab have all shown clinical superiority over docetaxel-chemotherapy
as second-line treatment, with improved survival rate. However, pembrolizumab
is the only immune checkpoint inhibitor that has exhibited superiority over
platinum-based chemotherapy as first-line treatment, with a significant longer overall
survival and its PDL1 IHC 22C3 pharmDx assay (Dako, Glostrup, Denmark) has a regulatory
status as CDx assay (Reck et al., N Engl J Med 2016). It is a qualitative IHC
assay, which uses the monoclonal mouse anti-PD-L1 clone 22C3, blocking the
PD-1/PD-L1 interaction and promoting an anti-tumor response (Hersom et al., Ther Drug Monit. 2017). 

KRAS against cetuximab or
panitumumab in colorectal cancer

In contrast
to the previously mentioned biomarkers that select patients who can benefit
from the use of molecular-targeted applications, the KRAS mutation
in colorectal cancer selects against patients that are not going to benefit
from anti-EGFR receptor therapy, with the use of cetuximab or panitumumab (Ong et al., Expert Rev Mol Diagn 2012).

They are
two monoclonal antibody drugs that target the extracellular domain of epidermal
growth factor receptor (EGFR), inhibiting the proliferation, infiltration and metastasis of tumor cells and induce
apoptosis. They are both certified for first-line treatment of metastatic colorectal cancer (mCRC) (Ong et al., Expert Rev Mol Diagn. 2012/ Wang al., Cell Physiol Biochem 2017). These drugs may be administered
in combination with chemotherapy, or as monotherapy after the failure of chemotherapy.
Both strategies have improved survival rate of mCRC, but their benefit is
limited to patients with RAS wild-type
tumors (Adriana Camargo Carvalho et al., PLOS ONE 2017), and are ineffective
towards patients whose tumors occupy KRAS
mutations (Dracopoli et al., CellPress 2016).

RAS is a proto-oncogene encoding a monomeric
GTPase that can be “captured” in a constitutively activated condition by
mutations that restrain it from hydrolyzing GTP to GDP. Three RAS genes are observed in the human
genome, HRAS, KRAS, and NRAS, out of
which KRAS is one of the most
regularly mutated genes in human cancers (Dracopoli et al., CellPress 2016).
Mutations in the KRAS oncogene are overexpressed in colorectal
cancer but are prevalent in many other types of cancers as well. Mutations in
the gene contribute to aberrant cellular growth, proliferation and
differentiation because of the cell signaling activation. Most of the
procedures that are used to identify mutations engage PCR to amplify specific
exons of the gene, and distinguish wild-type from mutant sequences in explicit
codons (Ong et al., Expert Rev Mol Diagn. 2012).