Biomedical Engineering Reference
In-Depth Information
antibodies to Akt, Bowers et al. devised an ingenious assay that makes use of the PI3
kinase dependence of platelet activation in response to fibrinogen. This assay uses the
expression of the platelet activation marker CD41a in response to a synthetic peptide
as a readout for PI3 kinase inhibition in peripheral blood samples [22]. When applied
to blood samples obtained from tumor-bearingmice treated with the experimental PI3
kinase inhibitor wortmannin, the pharmacodynamic effects detected with this assay
were found to correlate closely with the extent of Akt phosphorylation seen in tumor
tissue, suggesting that this will prove to be a useful assay applied to cancer patients
during early-phase clinical trials.
7.2.4.2 Leukemia
Inmost leukemia patients, themalignant cells are present in the
peripheral blood and are therefore readily sampled. However, in the case of acute
myeloid leukemia (AML), which is the common form of aggressive leukemia in
adults, cell proliferation takes place predominantly in the bone marrow where it is
regulated by complex interactions with the stroma. The leukemic stem cell population
is also believed to reside in the bone marrow. Bone marrow aspiration samples,
therefore, probably give a better appreciation of signaling traffic in the leukemic cell
population, and are also needed in cases where the leukemic blasts do not enter the
circulation. However, they are less readily obtained than peripheral blood.
In contrast to normal blood cells, constitutively activated signaling proteins can
be detected in the peripheral blast cells in the majority of AML patients, indicating
some degree of autonomy from external sources of pathway activation. For
example, STAT5 is activated via bcr/abl in cases of chronic myeloid leukemia,
and by the Flt-3 receptor tyrosine kinase in cases of AML carrying the Flt-3 internal
tandem duplex mutation (Figure 7.1a). The S6 ribosomal protein, which is involved
in the regulation of protein translation, can be activated by several upstream
signaling pathways, including ERK and Akt/mTOR, and we have shown that
peripheral AML blast cells can show great heterogeneity in the extent to which
S6 is constitutively activated by these pathways (Figure 7.1b) [23]. Using isolated
peripheral blast cells from AML patients, Irish and Nolan observed significant
differences in the extent to which signaling pathways could be acutely activated by
different growth factors, with some of these patterns carrying important prognostic
information [24]. Based on the above observations, it seems likely that complex cell
signaling assays could be adapted for individual leukemia patient treatment selec-
tion by identifying the critical aberrant pathways in the blast population. However,
that is outside the scope of this chapter.
The application of flow cytometry to the pharmacodynamic monitoring of signal
transduction inhibitors in leukemia patients is still in its infancy, but appears to have
major potential. Chronic myeloid leukemia, which is driven by the bcr/abl fusion
protein, is the most obvious candidate for this since there is a consistent pattern of
activated signaling elements present in nearly all cases. Flow cytometry has been used
to measure dose-dependent loss of constitutively activated STAT5 and CrkL in CML
cell lines treated with the bcr/abl inhibitor imatinib, and subsequently demonstrated
in CML patients, although to date this has not developed into a routine clinical
test [25, 26].
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