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Page 266 Franca et al. Cancer Drug Resist 2019;2:256-70 I http://dx.doi.org/10.20517/cdr.2019.004

particular, hsa-miR-34a-5p and has-miR-125b-5p showed the largest significant negative correlation with
TPMT expression. Interestingly, TPMT activity measured in erythrocytes is negatively associated with age
[23]
in children with ALL and age has been shown to modulate thiopurine pharmacokinetics in pediatric
[87]
patients . The molecular mechanisms by which age influences TPMT activity could involve epigenetic
factors, however more research is needed to clarify this phenomenon.

While the contribution of epigenetic factors to mercaptopurine induced adverse effects in pediatric ALL is
still uncertain and speculative, further studies on this topic may bring important insights to improve therapy
of patients taking mercaptopurine.

Genotyping vs. phenotyping
Many genes involved in thiopurine pharmacogenetics (i.e., TPMT, NUDT15, ITPA) encode for enzymes
whose activity can be measured in patients, for examples in erythrocytes. For TPMT there are many studies
considering both phenotyping and genotyping, while for the other enzymes the information available is still
limited.

Both TPMT genotyping or phenotyping before starting thiopurines treatment allows to identify patients with
reduced enzymatic activity and increased risk of drug induced adverse effects and to adjust starting dose
accordingly or to use an alternative therapy. There is a risk of TPMT misclassification when only genotyping
[22]
or phenotyping is used. Genotype-phenotype concordances are usually set between 76% and 99% . Indeed,
the conduction of both tests may give clinicians a more complete picture about patient’s risks during therapy,
but there is no consensus on the evaluation of both in all patients, also for the associated costs. The Food and
Drug Administration (FDA) still indicates genotyping or phenotyping before drug administration to identify
[88]
TPMT status , while the American College of Gastroenterology indicates phenotyping as the first choice
for patients using thiopurines for IBD because phenotyping reports a quantitative level of TPMT enzyme
[89]
activity . Genotyping certainly detects variations useful to predict the enzyme activity and the possible
development of adverse effects directly associated with enzyme deficiency. One advantage of genetic tests is
that they can be performed before starting therapy and the information obtained will be unchanged for the
entire patient lifespan. Despite the relatively simple and rapid procedure, genetic tests provide data about
key variants generally detecting the most common genetic variants associated with enzyme decrease but is
not able to give a full prediction because they do not verify the presence of rare or previously undiscovered
[90]
variants that will not be detected by variant-specific genotyping methods . The advent of genome wide and
next-generation sequencing (NGS) approaches and the continued reduction in the costs of these technologies
will partly overcome this problem. Moreover, the genotype is not solely responsible for patient’s susceptibility
of a drug. Genotyping tests will always provide a partial point of view since they do not consider variability
of enzyme activity due to the presence of drug interactions, presence of others pathologies or other
epigenetic factors. Phenotyping consists in a more direct observation of how the drug is metabolized and
considers also inter-individual differences between patients with the same genetic arrangement. For this
purpose, the activity of TPMT in lysed erythrocytes can be directly and successfully determined using
reversed-phase high performance liquid chromatography (HPLC) . It is important to measure TPMT
[91]
activity on a blood sample taken before starting thiopurine administration and during therapy, because its
activity can increase during treatment and thus can interfere with test results . Moreover, phenotyping test
[36]
[92]
shall be performed at least 3 months after a transfusion of erythrocytes : during ALL chemotherapy many
patients receive transfusions therefore the utility of phenotyping in this setting may be limited.
In conclusion, TPMT genotyping and phenotyping provide complementary information. Despite the usually
quite high levels of concordance between the analysis, the combination of both genotype and phenotype
tests insures a higher precision in the treatment personalization.