1. Summit on Drug Discovery Tuberous Sclerosis Alliance July 7, 2011 Biomarkers in TSC and LAM

2. Definition of a biomarker A characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic response to a therapeutic intervention.”

3. Types of biomarkers Diagnostic/Screening Risk – Prognostic-risk of disease development or progression (e.g.-HER-2 overexpression in breast CA) – Predictive-likelihood of response to therapy (e.g.-HER-2 overexpression in breast CA) Pharmacodynamic- markers of drug effect – Proximal-e.g.-phosphorylation of target kinase – Distal-e.g.-FDG-PET Biological progression- markers of tumor progression – Tumor markers-PSA doubling time after treatment, CTC – Anatomical imaging markers-e.g-CT scanning Surrogate clinical endpoints – Response, disease free survival

4. The ideal biomarker Repeatable Reproducible Minimally invasive Little or no risk Inform audit trail Reflect biology of tumor

5. Clinical ‘manifestations’ in need of diagnostic, predictive, prognostic biomarkers in TSC and LAM CNS – Seizures – SEGAs – Hydrocephalus – Cognition – Autism Renal – Renal failure – AML growth – AML bleeding – Renal cancer Lung (LAM) –Respiratory insufficiency –Pneumothorax –Exercise tolerance –Heart –Rhabdomyomas

6. Discussion of selected diagnostic, predictive, and prognostic biomarkers in TSC and LAM CNS – Seizures serial EEGs to detect & preemptively treat epileptiform activity before the first seizure Renal – AML bleeding AML volume, aneurysm size and fat content on MRI imaging can be used to predict bleeding LAM -Diagnosis.Circulating tumor cells.VEGF-D-SLAM &TSCLAM.MMP-9 -Respiratory insufficiency.Prolactin levels.Bronchodilator response.Menopausal status.LAM histology score.DLCO – Pneumothorax Cyst size Collagen, MMP-1 Heart – Rhabdomyoma Intrauterine ultrasound is a useful biomarker for TSC

7. Serum VEGF-D levels distinguish women with Sporadic LAM from those with other cystic lung diseases. n=155 female subjects with definitive diagnosis by biopsy or genetics Serum VEGF-D evaluated by ELISA (R&D Systems)

8. Elevated serum VEGF-D levels are associated with the presence of LAM in women with TSC. Women with TSC only had a normal chest HRCT within 18 months of testing. Young et al, Chest 2010

9. In the MILES trial, VEGF-D declined on sirolimus

10. In the MILES cohort, baseline serum VEGF-D correlated with Baseline markers of airflow obstruction, hyperinflation, diffusing capacity and quality of life Lung function (FVC) response to sirolimus

11. Biomarker tool kit for TSC and LAM trials Need biomarkers that: – Optimize patient selection – Demonstrate treatment effect earlier (target modulation, cell/tissue effects) – Perform well as surrogate endpoints that correlate with response and survival

12. The Pharmacologic Audit Trail Effect of the drug on the target, pathway, cell, tissue Effect of body on the drug – PK and metabolism Therapeutic and toxicological effects of the drug Sarker D. Biomarker Med 2007 Sarker D. Adv Cancer Res 2007 Workman P. Mol Canc Ther 2003

13. Pharmacologic audit trail Sarker D. Biomarker Med 2007 Sarker D. Adv Cancer Res 2007 Workman P. Mol Canc Ther 2003

14. John Lennon’s playlist on Omni DC Stationary for 1 st US Concert at Washington Coliseum, Feb 11, 1964 Beethoven From Me to You I Saw Her Standin’ There This Boy All My Lovin’ I Wanna Be Your Man Please Please Me Till There Was You She Loves You Twist and Shout Long Tall Sally

15. Group B playlist for TSC and LAM biomarker development Incorporate proven and exploratory biomarker analyses early and often into all phases of trials Consider molecular context when designing selection criteria, audit trail and surrogate endpoints for trials Mine data and conduct high throughput biomarker screens of serum/tissue from completed RCTs Establish functional and efficient central repository for TSC data and tissue