Multivariate analysis of various standard independent prognostic factors in ALL cases.

*<p>Cutoff value is 1.1 p-4EBP1 OD.</p><p>RR – relative risk, 95% CI –95% confidence interval.</p><p>na – not applicable.</p

The amount of mTOR activity dependent phospho-proteins (p-4EBP1 and p-S6) in lymphoma/leukemia cells (ELISA).

<p>(a.) P-4EBP1 ODs are shown for normal control peripheral blood mononuclear cells (N1, N2, N3), isolated peripheral normal B- and T-cells (B, T), isolated peripheral blood mononuclear cells and isolated bone marrow mononuclear cells from non-leukemic patients (NL) and isolated primary childhood ALL cells from representative patient samples, and from leukemia (Jurkat, CEM, Mn60, Nalm6) and lymphoma cell lines (KMH2). (b.) Relative p-4EBP1 and p-S6 expression was determined in samples from ALL patients (n = 10) and in leukemia/lymphoma cell lines (Jurkat, CEM, Mn60, Nalm6, KMH2); expression of normal lymphoid cells is considered to be 1. (OD: optical density; MNC: mononuclear cells. Equal cell numbers and equal protein concentrations were confirmed for comparisons.).</p

Summary of clinical data and mTOR activity related p-4EBP1 in 49 primary ALL patients.

§<p>cutoff equals 1.1 (OD p-4EBP1 ELISA).</p>*<p>based on cytogenetic and molecular genetic analysis; the hyperdiploid group also includes two cases with ETV6/RUNX1 fusion (good prognosis). High risk genetic lesions are: BCR/ABL fusion (two cases), ABL amplification (one case) and RUNX1 amplification (one case).</p>#<p>based on steroid response and current status; see methods: SR - standard, IR - intermediate, HR - high risk arm of the therapy; TX – transplantation, TRM - transplantation related mortality.</p>φ<p>– Fisher’s exact probability and,</p>χ<p>- chi² tests.</p>π<p>- significant correlation with mTOR activity level.</p

Survival analysis (Kaplan-Meier curves).

<p>Kaplan-Meier curves show overall and relapse free survivals for patients (n = 49) stratified by mTOR activity. Cutoff value between low and high mTOR activity groups (n = 37 and n = 12, respectively) was determined to be 1.1 for p-4EBP1 ODs (ELISA). We found that patients with low mTOR activity had significantly longer overall and relapse free survival (P<<0.05 with log-rank test; o: complete event, +: censored cases).</p

Changes in mTOR related p-4EBP1 expression in samples of ALL patients after <i>in vivo</i> chemotherapy and in cultured ALL cells treated with rapamycin <i>in vitro</i>.

<p>(a) P-4EBP1 OD is reduced by chemotherapy, but re-increases during relapse in ALL patient samples. Data is shown from 2 representative patients with good prognosis and 2 patients with poor prognosis at day 0, day 33, after three months of treatment, and at relapse (ELISA). (b) <i>In vitro</i> rapamycin sensitivity is variable in different human ALL samples (n = 4). P-4EBP1 levels variably decrease or increase after <i>in vitro</i> rapamycin treatment (50 ng/ml, 24 h) in isolated ALL cells (ELISA). A decrease in p-4EBP1 was associated with concomitant induction of apoptosis by rapamycin (>50% apoptotic cells in the first two samples; data not shown). (The first three samples were cultured from cells isolated from primary ALL cases and one at relapse.) *- P<0.05.</p

mTOR activity related p-4EBP1 expression in samples of ALL patients with different prognoses (ELISA) and detection of p-4EBP1 and p-S6 in ALL cells by flow cytometry.

<p>(a) P-4EBP1 OD values in 49 ALL samples (n = 37 for patients with good prognosis; n = 12 for poor prognosis, criteria for prognostic groups are described in the Methods section). P-4EBP1 OD levels were significantly different between patients with good and poor prognosis (p<0.05). The cutoff value between p-4EBP1 OD values representing high and low mTOR activity was determined to be 1.1 by ROC curve analysis. (b) p-S6 and p-4EBP1 overexpression is confirmed by flow cytometry in ALL cells. Representative histograms for p-4EBP1 and p-S6 flow cytometric analysis in normal PMNC and in ALL samples. Expression was calculated as the difference between MFIs of p-4EBP1 or p-S6 stained (p-4EBP1 and p-S6, respectively) and parallel unstained controls (-co). Differences between MFIs (ΔMFI) was 2.35 (pS6) and 1.56 (p-4EBP1) for normal peripheral mononuclear cells. However, ΔMFI was 42.6 and 32.4 for p-S6 and p-4EBP1, respectively, in the representative ALL case shown here.</p