Genome-Wide Gene Expression Profiling of Patients With ITP Receiving Thrombopoietin Mimetics

1. Identification and validation of response-predictive genes. The normalized
pre-treatment microarray data of the training set is retrieved from SMD for statistical
analysis. The supervised analysis SAM (Significance Analysis of Microarrays, two class
unpaired) is performed to identify genes whose expression is significantly different
between responders and non-responders. Then a Leave-one-out cross-validated
gene-expression predictor for the 2 response classes is devised by the PAM (Predication
Analysis of Microarrays) method based on nearest shrunken centroids. The unsupervised
clustering of the independent test set is performed using the predictive genes and the
prediction accuracy is calculated. Quantitative real-time PCR is performed as further
validation using the un-amplified RNA samples and Taqman gene expression assays
(Applied Biosciences).

2. Gene expression changes correlated with TPO mimetic treatment and pathway analysis.

2.1. Hypothesis: The transcriptional profile of patients who respond to TPO agonists is
different than those who do not respond.

Plan: The expression data of pre-treatment as well as the 1-week and 1-month after
initiation of treatment samples is retrieved from SMD. The two class paired SAM analysis is
performed to compare pre-treatment samples with samples collected at either 1-week or
1-month after initiation of treatment in responders and non-responders. The two class
unpaired SAM analysis is also used to compare post-treatment samples of responders and
non-responders at the same time point. The significant genes (q value<0.05, fold change>2.5)
are subsequently analyzed by IPA (Ingenuity Pathway Analysis) system to be transformed into
a set of relevant networks based on the extensive records maintained in the Ingenuity
Pathway Knowledge Base. The statistically significant networks, molecular and cellular
functions, top canonical pathways and toxicity lists associated with each pair of dataset
will be recognized through this analysis. Hypothesis on non-response to TPO mimetics can be
generated based on the different functional subsets of significant genes. Genes involved in
important pathways identified by IPA analysis will be validated by QRT-PCR as in our recent
publication on oxidative stress pathways in ITP4. Our goal is to develop biomarkers which
predict likelihood of response to therapy and identify pathways associated with resistance
to therapy which could be targeted.

2.2 Hypothesis: Since available TPO agonists have different mechanisms of action, there may
be differences in responders and non-responders between the different drugs.

Plan: We recognize that TPO agonists have different mechanisms of action which could affect
downstream signaling pathways and transcriptional responses. For this reason in addition to
evaluating the TPO agonists as a group in 2.1 above, patients will also be analyzed by type
of agonist. The conclusions of this type of analysis will be limited by the numbers of
individuals treated with a particular drug but could be useful for hypothesis generation and
confirmation in a larger cohort.

Inclusion Criteria:

- clinical diagnosis of ITP TPO treatment

Exclusion Criteria:

- thrombocytopenia not due to ITP