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 1998- March 2006.– 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.