Livin in Prognosis of Childhood ALL Livin- member of inhibitor of apoptosis proteins (IAP). – IAP- acts on effector and initiator caspases Function of Livin- antagonizes death receptor and mitochondria-based apoptotic pathways through the inhibition of caspases 3, 7, and 9, as well as the participation of JNK1 – Poor prognostic factor in cancers such as bladder cancer, neuroblastoma

Livin in Prognosis of Childhood ALL Choi et al. : expression of Livin in 222 patients diagnosed with ALL < 15 y.o. September March – Median age 65.5 months Mononuclear cells isolated from 2 mL bone marrow aspirate at diagnosis. DNA extraction→ complementary DNA→ mRNA Choi et al. Expression of Livin, an antiapoptotic protein, is an independent favorable prognostic factor in childhood acute lymphoblastic leukemia. BLOOD, 15 JANUARY 2007 VOLUME 109, NUMBER 2

Quantitative RT-PCR to measure mRNA expression levels of Livin and glyceraldehyde-3- phosphate dehydrogenase. Cytotoxicity assay to test susceptibility of leukemic blasts to apoptosis by chemotherapeutic agents Protein levels of Livin, monoclonal antibodies against Livin, caspase 3, poly (ADP-ribose) polymerase, anti-β tubulin Cleaved form of Livin isolated by Western blot Levels of Livin and anti-β tubulin

Livin mRNA in 57 of 222 patients Expression rate higher in females, patients age 1-9 years, patients with t(12;21), low-risk patients.

Expression associated with favorable response to induction chemotherapy. Higher levels of Livin expression in patients with favorable day 7 bone marrow response. Percentage of leukemic blasts after culture with methylprednisolone lower with expression of Livin.

L-asparaginase and Increase of Asparagine Synthetase Link b/w L-asparaginase resistance, high levels of asparagine synthetase. Depletion of asparagine in vitro-> amino acid dependent up-regulation of mRNA, protein, activity of asparaginase synthetase.

31 newly-diagnosed children with ALL. Baseline bone marrow, peripheral blood samples before administration of PEG-Asp Peripheral blood samples collected for 5 consecutive days until start of chemotherapy. Mononuclear cells extracted – All samples containing 90% leukemic cells – Total cellular DNA extracted – Reverse transcription forming cDNA

Real-time PCR to determine levels of asparagine synthetase and glyceraldehyde 3- phosphate dehydrogenase

Good response to PEG-Asp: blast number < 1×10^9/L at 5 days post PEG-Asp Intermediate response: blast number 1×10^9/L at 5 days post PEG-Asp Poor response: blast number 10 ×10^9 at 5 days post PEG-Asp

Continuous decrease of leukemic cells at 0, 5 days after PEG-Asp (median of 44.7 × 10^9/L at 0 days post PEG-Asp to 0.2× 10^9/L at 5 days post PEG-Asp). No difference between baseline expression of synthetase mRNA between good and intermediate responders, good and poor, or intermediate and poor.

No significant difference in up-regulated levels of asparagine synthetase 24 hours after PEG- Asp administration among the different groups of responders. Up-regulation of AS mRNA induced by L- asparaginase not related to reduction of lymphoblasts in childhood ALL.