1. Diagnostic Assays to Plan Specific Drug Treatment Elizabeth Hammond MD

2. Criteria for Development of Diagnostic assays Defined assay characteristics for total test Standardized procedures for assay, reference materials and training plan to assure initial and ongoing competence Acceptable interlaboratory variability evaluated in standard manner, possibly using TMA’s Plan for new laboratory validation Plan for ongoing laboratory competence assessment

3. Assay Characteristics Specimen adequacy: definition of unacceptable samples Specimen handling: time from surgery to processing, time in fixative, temperature Standardized Reference Materials Assay conditions: precise, clearly reported Interpretation and reporting criteria: precise, including how reference material is used in the evaluation and acceptable limits of interobserver variation

4. CAP FISH Survey Results FISH amplified 2000: 35 participants 35/35 = amplified 2001: 63 participants 58/63= amplified (others= no response) FISH non amplified 2000: 35 participants 35/35= non amplified 2001: 63 participants 49/63= non amplified (others= no response) CAP CYH survey results, 2000-2001

5. CAP IHC (MK) Survey FISH amplified case 357 participants IHC scores: 0 19 (3.7%) 1+ 4 2 + 52 3 + 296 FISH unamplified case 380 participants IHC scores: 0 275 1+ 69 2+ 27 (7%) 3+ 10 (3%) Source: 2000 CYH 01, 02 and MK Surveys, CAP

6. CAP MK B Survey Image Scoring PhotoScore01+2+3+Total 011+25352800457 022+06037620436 033+803445456 041+92350130455 062+713328233455 Score not provided for all images by all participants

7. FISH vs IHC Specimen handling, adequacy not specified for either assay type; DNA less affected than protein FISH for Her2 has internal standard (Chr 17) Standard procedures and training are mandatory for laboratories performing FISH, but not required for all IHC assays Interpretation criteria are quantitative and clearly specified for FISH, ambiguous for IHC

8. Conclusions Use of central laboratory during pivotal trial is necessary if there is not assay method standardization, standardized reference materials and training plans for assay and interpretation Interlaboratory comparison of assay and interpretation criteria should be conducted concurrently with pivotal trial Validated assay with standardized reference materials, training plan, and interpretation criteria is required prior to Phase III trial

9. Treatment Guidelines for Patients with Negative Assay Results Assay should not be used to assign treatment if: –Relationship between analyte and drug efficacy is unclear –Interpretation guidelines of assay do not clearly specify cut point of positive and negative –Assay method is non validated –Standardized reference materials are not available

10. Package Insert Information Specific information about specimen adequacy and handling parameters Assay performance characteristics with assay comparisons, where appropriate Assay interpretation guidelines Acceptable methods of assay validation in a lab Acceptable levels of interlaboratory variability Requirement of laboratory participation in ongoing competency assessment

11. Assay Utility Assessment Phase III clinical trials should be used to assess assay utility Tissue banking on such trials should be mandatory so that new assays can be validated on the same specimens TMAs can be created to promote new assay utility assessment while optimizing tissue bank specimen use.

12. TMA Concordance with Slides ArraysHead/NeckGliomaProstate One array90%86% Two arrays95%93% Three arrays99%95%93% Four arrays100%97% RTOG Study, presented USCAP, 2001; N= 150 tumors