Biomedical Engineering Reference
In-Depth Information
Our own laboratory has been focused on AML patients, linked to a large clinical
and translational research program testing new approaches to treatment. In a multi-
center phase I clinical trial conducted by the National Cancer Institute of Canada
Clinical Trials Group, patients with chemotherapy refractory AML were treated with
sorafenib, which at that time was believed to be a selective Raf kinase inhibitor, as a
single agent [27]. Blood samples were obtained for pharmacodynamic monitoring
predose and at weekly intervals during the first treatment cycle. The flow cytometry
protocol originally developed for pharmacodynamic monitoring of sorafenib in
peripheral blood lymphocytes was modified to include activation via the c-Kit
receptor using its ligand stem cell factor (SCF), in addition to phorbol ester as used
in the earlier version [18]. This was intended to provide a more physiological stimulus
to the ERK pathway, but unexpectedly it was found that although no pharmacody-
namic effect was detectable at any dose level using phorbol ester to drive the ERK
pathway, similar to our finding in the solid tumor trial, there was an obvious dose-
dependent inhibition of ERK activation when SCF was added to the blood samples,
with almost 100% inhibition after 1 week treatment at the highest dose level
(Figure 7.2). In patients who were randomized to sorafenib treatment on an inter-
mittent schedule, drug target inhibition was lost after 2 weeks off treatment.
Furthermore, the extent to which SCF activation of ERK signaling was inhibited
in patients during treatment was found to correlate with the plasma drug concentra-
tion [27]. The apparent paradox that a pharmacodynamic effect could be detected
using SCF to activate ERK, but not with phorbol ester, is explained by the fact that
sorafenib has multiple signaling targets in addition to Raf kinase. Using normal
donor-mobilized peripheral stem cells, we subsequently confirmed that ERK activa-
tion by SCF in the CD34 positive cells was inhibited at low micromolar concentra-
tions of sorafenib that are clinically achievable, whereas several hundred micromoles
were required to inhibit stimulation by phorbol ester [28].
Although this early sorafenib trial established the potential to monitor pharma-
codynamic effects in sequential blood samples obtained from leukemia patients,
several weaknesses in the flow cytometry protocol were identified, including poor
sensitivity and difficulty in identifying the blast cells in some samples due to the
degradation of light scatter. It was also felt that the technique was technically too
difficult for point of care application. A major overhaul of the method was therefore
undertaken, as described above, and applied to a new trial that also targeted the c-Kit
receptor in AML patients, this time using imatinib in combination with second-line
chemotherapy to treat patients whose disease was considered refractory to the first-
line drugs, but who were still candidates for intensive myelo-ablative chemotherapy.
An important feature of the optimized flow cytometry protocol is the ability to identify
leukemic blast cells at frequencies as low as 0.1%, an important consideration for
early-phase clinical trials inAML since patients enrolled onto these studies often have
fairly slow tempo, low count disease [16]. The study design included a 4-day lead in
period when the patients were treated with imatinib alone, and more intensive
pharmacodynamic monitoring during this period. Although results from this study
are still being analyzed, it is evident that when patients are treated with a fixed dose of
imatinib, there is considerable heterogeneity in the level of c-Kit inhibition achieved,
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