1. Genetic Variations in the PI3K/PTEN/AKT/mTOR Pathway are Associated with Clinical Outcomes in Esophageal Cancer Patients Treated with Chemoradiotherapy Michelle Hildebrandt, Ph.D. Instructor Department of Epidemiology

2. Outline Pathway Experimental Details Results Conclusions Future Directions

3. PI3K/PTEN/AKT/mTOR Pathway

4. Downstream Processes Manning and Cantley, Cell (2007) mTOR TERT

5. Major Effectors PI3K PTEN AKT mTOR

6. PI3K and PTEN “Sensor” of cellular environment – PI3K activated by receptors at membrane RTK and GPCR – Transmitted through PIP2/3 PIP3 – on PIP2 – off – PTEN removes activation signal

7. AKT Major adaptor protein 3 isoforms (AKT1/2/3) AKT1 and AKT2 most important for this pathway in cancer

8. mTOR mammalian Target Of Rapamycin (FRAP1) Involved in regulation of translation Y Mamane, et al, Oncogene (2006)

9. Importance in Cancer Balance between cell survival and apoptosis – Activated  Survival This pathway is often constitutively active in MANY cancers – Gene amplifications – Activating mutations – Silencing of PTEN  no regulation

10. Chemotherapy Response In lung and ovarian cell lines: – Overexpression of PIK3CA and reduced PTEN activity results in cisplatin resistance – Cisplatin resistance associated with AKT overexpression – Inhibition of AKT increases efficacy of paclitaxel – Inhibition of mTOR increases cisplatin efficacy – Increased AKT activity found to increase response to 5-FU

11. Esophageal Cancer Estimated >16,000 new EC cases each year in the US Rate of adenocarcinoma increasing rapidly – Mirror rates of obesity  gastroreflux Surgery is standard treatment Multimodal approaches are often used – 5-year survival rate of 25-28%

12. Esophageal Cancer Phosphorylation of AKT increased during progression from Barrett’s to EC PI3K/PTEN/AKT/mTOR Pathway is activated in EC

13. The Question For EC patients treated with a taxane, fluoropyrimidine and/or platinum agent… Is genetic variation within the PI3K/PTEN/AKT/mTOR pathway associated with variation in clinical outcomes?

14. Experimental Details Patient Characteristics 186 Caucasian patients with resectable adenocarcinoma or squamous cell carcinoma Recruited between 1985 and 2003 at MDACC Concurrent chemoradiotherapy followed by surgery, or induction chemotherapy followed by concurrent chemoradiotherapy and then surgery

15. Experimental Details Patient Characteristics

16. Study endpoints were: – Overall Survival – Recurrence – Pathologic Response to Therapy Adjusted all analyses for: – Age at diagnosis, gender, smoking status, alcohol consumption, clinical stage, histological tumor type, tumor location, pathological stage and histological viability, radiation dosage, chemoradiotherapy sequence, and chemotherapy regimens

17. Experimental Details Treatment Information Stratified analyses by chemotherapeutic regimen – Any of the three – Fluoropyrimidine + any – Platinum compound + any – Taxane + any

18. Experimental Details SNP Selection tagged SNP Selection – Based on HapMap data from CEPH population Gene + 5 Kb flanking – LD tagged SNPs: r 2 > 0.8, MAF > 0.1 – tagger and LDselect

19. Experimental Details SNP Selection

20. Results 16 SNPs, 4 treatment groups, 3 clinical outcomes…

21. Results Recurrence Risk in Patients Treated with Any of the Three Three SNPs associated with recurrence – AKT1:rs2498804 – AKT2:rs892119 Increased risk of recurrence All treatment groups – PTEN:rs12357281 Decreased risk of recurrence Except for platinum compound + any

22. Results Recurrence Risk in Patients Treated with Any of the Three

23. Results Recurrence-free Survival by AKT2:rs892119 A: Any Three B: 5-FU C: Platinum agent D: Taxane

24. p-value95% CIHR* No Recurrence / Recurrence < 0.0012.34 - 18.186.5215 / 17 2 0.0241.15 - 7.192.8769 / 27 1 1 (reference)36 / 130 # of Unfavorable Genotypes Results Recurrence Risk by Unfavorable Genotype AKT1:rs2498804 and AKT2:rs892119 Consistent across all strata: Fluoropyrimidine - HR*: 6.36 Platinum Agent - HR*: 10.73 Taxane - HR*: 83.37

25. Results Gene-Gene Interactions in Patients Treated with a Taxane 7 SNPs associated with Recurrence 0.0080.006 - 0.4580.05CG + GG PTEN:rs12357281 0.0311.20 - 42.367.13CC PIK3CA:rs6443624 0.0151.60 - 80.8811.38TT PIK3CA:rs7621329 0.0351.19 -128.3812.35TT FRAP1:rs2295080 0.0061.62 - 16.885.23AG + GG AKT2:rs892119 0.0161.55 - 76.9810.94TT AKT1:rs1130214 0.0032.40 - 83.0214.10GT + TT AKT1:rs2498804 p-value95% CIHR*

26. Results Survival Tree Analysis for Recurrence

27. Results Survival in Patients Treated with Any of the Three Both FRAP1 (mTOR) SNPs significant – Consistent except for platinum compound group Taxane Platinum CompoundFluoropyrimidine Any of the Three 0.0038.280.0732.660.00044.660.0014.19TT FRAP1:rs2295080 0.0057.030.0912.770.0033.820.0043.53TT FRAP1:rs11121704 p-valueHR*p-valueHR*p-valueHR*p-valueHR*

28. Results Survival in Patients Treated with a Taxane AKT1 and AKT2 SNPs 0.0061.43 - 8.783.54AG + GG 0.0650.89 - 43.886.25GG 0.0141.27 - 8.403.27AG 1(reference)AA AKT2:rs892119 0.1570.79 - 4.191.82GT + TT 0.0141.56 - 51.178.92TT 0.2220.73 - 3.941.69GT 1(reference)GG AKT1:rs1130214 p-value95% CIHR*

29. Results Pathologic Response to Therapy AKT2:rs892119 p-valueOR*p-valueOR*p-valueOR*p-valueOR* TaxanePlatinum CompoundFluoropyrimidineAny of the Three 0.0264.120.0592.460.0082.980.0102.81 AG + GG GG 0.0423.680.1052.180.0182.680.0232.54 AG 1 (reference) AA

30. Results Pathologic Response to Therapy AKT1:rs3803304 in patients treated with Any of the Three – HR*: 0.50 (0.25 – 0.99), p-value: 0.049 FRAP1:rs11121704 in taxane group only – HR*: 2.76 (1.04 – 7.37), p-value: 0.042

31. Conclusions Genetic variation within this important pathway is important… AKT2:rs892119 in particular – Tags 5 SNPs – Results suggest increased signaling – Functional SNP?

32. Conclusions PTEN variation had expected opposite effect – Counteracts other effects Differences between treatment groups – Many more in taxane group  sample size? – Less in platinum compound group

33. Future Directions Analyze SNPs in a control group undergoing surgery alone Same in other cancers treated with these agents? Functional studies  mechanism

34. Thank You! Xifeng Wu Jafer Ajani Julie Izzo Mien-Chie Huang Esophageal Study Participants Wu Laboratory