Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 340

is able to predict patients' drug susceptibilities and detect the existence of
drug-resistant cells by examination prior to starting treatment. The exami-
nation uses peripheral blood or bone marrow samples obtained by blood spec-
imen collection or bone marrow aspiration; although these sampling
techniques are invasive, they are included in conventional tests for CML,
so our analyses result in no additional patient burdens. For each sample, after
the separation of mononuclear cells (including the tumor cells) by density gra-
dient centrifugation, cDNA encoding Pickles is introduced into the cells by
electroporation. After confirming fluorescence emission at 24 h, drug treat-
ment is begun. The FRET efficiency in individual cells is then calculated from
the image data obtained using fluorescence microscopy ( Fig. 8.5E ).
Given that tumor cells in the chronic phase of CML contain cells at each
step of differentiation, it is not necessarily the case that BCR-ABL activity is
high in all leukemic cells. This phenomenon is reflected in the range of
FRET efficiencies observed. For this reason, a threshold value (D-FRET)
was used to identify cells in which BCR-ABL activity is high. This value
was mathematically derived from the mean and the standard deviation of
the FRET efficiency in cells that did not express BCR-ABL. Given that
the probability for cells with low BCR-ABL activity to exceed D-FRET
is less than 0.003, the BCR-ABL activity of cells that exhibit higher FRET
efficiencies than this value is significantly high.
We are currently engaged in drug efficacy evaluation using the afore-
mentioned processes and criteria. If cells from both peripheral blood and
bone marrow with values above D-FRET disappear upon drug treatment,
the patient is considered sensitive, whereas if they remain above this value,
we conclude that the patient is resistant ( Fig. 8.5E ). In addition, when
the distribution pattern of FRET efficiency was carefully compared with
the clinical course, we saw that we were able to anticipate the patients' re-
sponses to drugs, albeit only partially, using this analysis, which was per-
formed at the initial stage of the disease. For instance, we experienced a
case in which drug-resistant cells existed only in the bone marrow
sample, while the peripheral blood cells were susceptible to drug treat-
ment. At the beginning of treatment, a decrease in the number of
tumorcellswasobserved;however,the patient had achieved neither a
complete cytogenetic response nor a major molecular response after
12 months of therapy (a suboptimal response) and had to be put on a dif-
ferent dosage and ultimately different drugs. One can speculate that the
resistant cells that had been detected during the initial examination
remained and caused relapse.