1. Statistical, Computational, and Informatics Tools for Biomarker Analysis Methodology Development at the D ata M anagement and C oordinating C enter of the E arly D etection R esearch N etwork

2. 18 Laboratories 8 Centers CDCP 2 Laboratories NIST Chair: David Sidransky Chair: Bernard Levin EDRN ORGANIZATIONAL STRUCTURE An “infrastructure” for supporting collaborative research on molecular, genetic and other biomarkers in human cancer detection and risk assessment. Early Detection Research Network

3. Specimens with matching controls and epidemiological data Infrastructure to provide preneoplastic tissues: - Prostate - Lung - Ovarian - Colon - Breast BIOREPOSITORY Early Detection Research Network INFRASTRUCTURE

4. Early Detection Research Network INFRASTRUCTURE Capability in high-throughput molecular and biochemical assays Ability to respond to evolving technologies for EDRN needs Extensive experience and scale-up ability in proteomics and molecular assays Outstanding infrastructure for handling multiple assays and validation requests LABORATORY CAPACITY

5. Early Detection Research Network INFRASTRUCTURE Outstanding track record in biomarker research Statistical and data mining technology Statistical and predictive models for multiple biomarkers Novel statistical methods to interpret high-throughput data DATA STORAGE AND MINING

6. Early Detection Research Network INFRASTRUCTURE Improving informatics and information flow Network web sites public web site secure web site Early Detection Research Network Exchange (ERNE) Standardizing of Data Reporting: CDEs Developed DATA EXCHANGE AND SHARING

7. Early Detection Research Network (EDRN) INFORMATICS AND INFORMATION FLOW

8. Contact one of the EDRN Principal Investigators to serve as a sponsor for an application. Three types of collaborative opportunities are available: Type A: Novel research ideas complementing EDRN ongoing efforts; one year of funding at $100,000 Type B: Share tools, technology and resources, no time limit Type C: Allow to participate in the EDRN Meetings and Workshop For details on how to apply, see http://www.cancer.gov/edrn How To Become an Associate Member EARLY DETECTION RESEARCH NETWORK COLLABORATION

9. DMCC Statisticians Margaret Pepe, Lead of Methodology Group Ziding Feng, Principal Investigator Yinsheng Qu Mary Lou Thompson Mark Thornquist Yutaka Yasui

10. Biomarker Lab Collaborators at Eastern Virginia Medical School Bao-Ling Adam John Semmes George Wright

11. Focus of Presentation Design: Phase Structure for Biomarker Research Analysis: Statistical Methods for Biomarker Discovery from High-Dimensional Data Sets

12. Design: Phase Structure for Biomarker Research Three phase structure for therapeutic trials well-established Structure promotes coherent, thorough, efficient development Similar structure needs to be developed for biomarker research

13. Biomarker Development Categorize process into 5 phases Define objectives for each phase Define ideal study designs, evaluation and criteria for proceeding further Standardize the process to promote efficiency and rigor

15. The Details of Study Design Specific Aims Subject/Specimen Selection Outcome measures Evaluation of Results Sample Size Calculations Limitations / Pitfalls

16. Specific Aims Phase 1 Identify leads for potentially useful biomarkers Prioritize these leads Phase 2 Determine the sensitivity and specificity or ROC curve for the clinical biomarker assay in discriminating clinical cancer from controls

17. Specimen Selection -- Cases Phase 1 Cancers that are ultimately serious if not treated early, but treatable in early stage Spectrum of sub-types Collected at diagnosis Phase 2: same criteria as for phase 1 Wide spectrum of cases Clinical specimen at diagnosis From target screening population

18. Specimen Selection -- Controls Phase 1 Non-cancer tissue same organ same patient Normal tissue non-cancer patient Benign growth tissue non- cancer patient Phase 2 From potential target population for screening

19. Outcome Measures Phase 1 True positive and False positive rates (binary result) True positive rate at threshold yielding acceptable false positive rate ROC curve Phase 2 Results of clinical biomarker assay

20. Evaluation of Results Phase 1 Algorithms select and prioritize markers that best distinguish tumor from non-tumor tissue Initial exploratory studies need confirmation with new validation specimens Phase 2 ROC curves ROC regression to determine if characteristics of cases and/or characteristics of controls effect biomarker’s discriminatory capacity

21. Sample Size Phase 1 Should be large enough so that very promising biomarkers are likely to be selected for phase 2 development Phase 2 Based on a confidence intervals for the TPR or FPR, or confidence intervals for the ROC curve at selected critical points

22. Findings: Sample Size Estimation For phase 1 microarray experiments, use of ROC curves is more efficient than comparing means For phase 2 studies, equal numbers of cases and controls is often not optimally efficient Sample size calculations and look-up tables are now in EDRN website

23. 1.Pepe et al. Phases of biomarker development for early detection of cancer. Journal of the National Cancer Institute 93(14):1054–61, 2001. 2.Pepe et al. “Elements of Study Design for Biomarker Development” In Tumor Markers, Diamandis, Fritsche, Lilja, Chan, and Schwartz, eds. AAAC Press, Washington, DC. 2002. 3.Pepe. “Statistical Evaluation of Diagnostic Tests & Biomarkers” Oxford U. Press, 2003.

24. Selecting Differentially Expressed Genes from Microarray Experiments Lead: Margaret Pepe Context gene expression arrays for n D tumor tissues and n C normal tissues Y ig = logarithm relative intensity at gene g for tissue i. for which genes are Y ig different in some/most cases from the normals? how many tissues, n D and n C, should be evaluated in these experiments? illustrated with ovarian cancer data

25. Statistical Measures for Gene Selection — typically use a two sample t-test for each gene — we argue that sensitivity and specificity are more directly relevant for cancer biomarker research. — focus attention on high specificity (or high sensitivity) — use the partial area under the ROC curve to rank genes, instead of the t-test

26. Example Gene Rank (among 100 genes) gene #5gene #97 t-test104 partial AUC331

28. traditional calculations based on statistical hypothesis testing These are exploratory studies, need new methods Propose to base calculations on the probability that a differentially expressed gene will rank high among all genes Use computer simulation for sample size calculations Sample Sizes for Gene Discovery Studies

29. with 50 tumor and 50 normal tissues we can be 83.6% sure that the top 30 genes will rank in the top 100 in the experiment.

30. Pepe et al. Selecting differentially expressed genes from microarray experiments. Biometrics (in press)

31. Summary The method we developed for selecting genes and calculating sample sizes are more appropriate for the purpose of diagnosis and early detection

32. Analysis: Statistical Methods for Biomarker Discovery from High-Dimensional Data Sets Method development motivated by SELDI data from John Semmes/George Wright at Eastern Virginia Medical School Data consist of protein intensities at tens of thousands of mass/charge points on each of 297 individuals Developed three approaches to biomarker discovery: wavelets, boosting decision tree, and automated peak identification

33. The EVMS prostate cancer biomarker project Prostate cancer patients:N=99 early-stage N=98 late-stage Normal controlsN=96 Serum samples for proteomic analysis by Surface Enhanced Laser Desorption/Ionization (SELDI) Goal: To discover protein signals that distinguish cancers from normals

34. An example of SELDI output 48,000 mass/charge points (  200K Da)

35. Normal The design of the biomarker analysis PCa- early PCa-late N=96N=99N=98 Training Data 167 PCa (84 early, 83 late) vs. 81 Normal Test Data 30 PCa 15 Normal (Blinded)

36. Wavelet Analysis Lead: Yinsheng Qu Steps in the wavelet analysis: Represent original data plot with a set of wavelets (dimension reduction) Determine those wavelets that distinguish between subgroups (information criterion) Define discriminating functions based on the distinguishing wavelets (Fisher discrimination)

39. Three Group Classification: Normal, Cancer, BPH 12,352 mass spectrum data points, reduced to 3,420 Haar wavelet coefficients, of which 17 coefficients distinguish between the three cases. 2 classification functions generated. Truth: Predicted:NormalCancerBPH Normal 14 0 0 Cancer 1 27 7 BPH 0 3 8

41. Qu Y et al. Data reduction using discrete wavelet transform in discriminant analysis with very high dimension. Biometrics, in press.

42. Boosted Decision Tree Method. Lead: Yinsheng Qu/Yutaka Yasui This method combines multiple weak learners into a very accurate classifier It can be used in cancer detection It can also be used in identification of tumor markers Using this method we can separate controls, BPH, and PCA without error in test set

43. Outline of boosting decision tree The combined classifier is a committee with the decision stumps, the base classifiers, as its members. It makes decisions by majority vote. The base classifiers are constructed on weighted examples: the examples misclassified will increase their weights on next round. The 2 nd stump’s specialty is to correct the 1st stump’s mistakes, and the 3 rd stump’s specialty is to correct the 2 nd stump’s mistakes, and so on. The combined classifier with dozens and even hundreds of decision stumps will be accurate. Boosting technique is resistant to over fitting.

44. Classifier 2: A boosted decision stump classifier with 21 peaks (potential markers)

45. The Boosting procedure Y i ={cancer, normal}={1, -1}, f m (x i )={1, -1} Initial weights (m=1), w i = 1 (i = 1,...,N). Choose first peak and threshold c. For m =1 to M: w i = w i exp{  m  (incorrect)} –where  m = ln(1-err)/err) and err is the classification error rate at the current stage –normalize the weights so they sum to N. –choose a peak and c (i-th subject with weight w i ) Final classifier: f(x) = sum(  m f m (x)) over m=1 to M. f(x i )> 0  i-th subject classified as cancer

46. When to stop iteration? minimal margin: minimum of y i f(x i ) over all N subjects The minimal margin in the training sample measures how well the two classes are separated by classifier. Even classifier reaches zero error on training sample, if iteration still increases the minimal margin --> improve prediction in future samples.

48. Qu et al. 2002. Boosted Decision Tree Analysis of SELDI Mass Spectral Serum Profiles Discriminates Prostate Cancer from Non-Cancer Patients. Clinical Chemistry. In press. Adam et al. 2002. Serum Protein Fingerprinting Coupled with a Pattern Matching Algorithm that Distinguishes Prostate Cancer from Benign Prostate Hyperplasia and Healthy Men. Cancer Research. 62:3609-3614.

49. Summary Wavelets approach: Does not require peak identification (black-box classification) Boosting decision tree: Requires peak identification first. Useful for both classification and protein mass identification

50. Final Summary The methods developed in the past two years are mainly for Phase 1&2 studies, reflecting the current needs of EDRN. EDRN DMCC statisticians are working on key design and analysis issues in early detection research. More work remains to be done (e.g., In classification, consider the mislabeling of Prostate cancer by BPH; exam gene by environmental interactions).