Roche Molecular Systems, Inc. cobas® 4800 BRAF V600 Mutation Test Pleasanton, CA 94588-2722 Module 3: Labeling Draft Package Insert I cobas® 4800 BRAF V600 Mutation Test c(obase 2 FOR IN VITRO DIAGNOSTIC USE. 3 cobas® DNA Sample Preparation Kit 24 Tests P/N: 05985536190 4, 5 cobas® 4800 BRAF V600 Mutation Test IRgAF 1 24 Tests P/N: 05985579190 6 7 NOTICE: The purchase of this product allows the purchaser to use it for amplification and detection of nucleic acid 8 sequences by polymerase chain reaction (PCR) and related processes for human in vitro diagnostics for the specific intended 9 use stated in this Package Insert. No general patent or other license of any kind other than this specific right of use from 10 purchase isgranted hereby outside of the indications for use stated here. II TABLE OF CONTENTS 12 TABLE OF CONTENTS...... ................. .... .. ...... ... ..... ....... .... 13 INTENDED USE ....................... ................................. ..................... 14 SUMMARY AND EXPLANATION OF THE TEST........................... ....................... 3 I5 PRINCIPLES OF THE PROCEDURE...........................................3 16 Specimen Preparation . e o . .. .... .... .... ...... .. ........ ...3 17 PCR Amplification and Detectio ............. ............ 18 T arget Selection .................... .... ...... .... ...... ... ..3 19 Target Am plification .................................................. 20 Automated Real-time Detection........................ ............. ..... .. ... ..4 21 Selective Am plification . ... ... ..... .. .... ... ..... ...... ... .... .. .. ...4 22 REAG ENTTS HL . ............................U MS...... .. .............. ..4 23 WARNINGS AND PRECAUTIONS................ 7 24 STORAGE AND HANDLING REQUIREMENTS ............ ............ 8 25 MATERIALS ROVID PROVIDED...................... ............................................... 8 26 MATERIALS REQUIRED BUT NOT PRO VIDED.............................................. ....9 27 Instrumentation and Sofiware..............I......... ............ 1 28 SPECIMEN COLLECTION, TRANSPORT AND STORAGE ........................ ...................... 1 29 A. Specim en Collection ....................... ... .... .... 10 30 B. Specim en Transport ........................ ..... .. I .... ... ... .. 1 31 C. Specim en Storage ................................ .... ...... ... .. 1 32 INSTRUCTIONS FOR USE .............. .... ..................... .10I........ 33 Run Size ... ..... ......... ... .... .......... ....... ......... .0 34 Workflow ............. ........... 10 35 Reagent Preparation ............................ ....... ... ...... ....... .... ... ........ ... ....... .... .. 0 36 Deparaffinization of FFPET Sections Mounted on Slides ................................... ............... 11 37 Deparaffinization of FFPET Sections not Mounted on Slides....................... ...................... 11 38 DN A 1solation ........................ .... ....... ... ... ... .. 12 39 DN A Quantitation........................... ....................... 13 40 AM PLIFICATION AND DETECTION ................................................. 13 41 Instrum ent Set-U p ............................................... 13 42 Test Order Set-U p .................................................................... .............................. .............. .................................. I3 43 Dilution Calculation of Specimen DNA Stock at Concentrations from 5 ng/pL to 35 ng/pL ................................................ 14 44 Dilution Calculation of Specimen DNA Stock at Concentrations >35 ng/IL ......................... .4........................ 14 45 Specimen Dilution......................................... ..................................... .............14 46 Preparation of Working Master Mix (MM X) .......................... ..................... 15 47 Addition of Controls and Specimens ......................... ....................I5 PMA M100022 CONFIDENTIAL AND PROPRIETARY Draft Package Insert Print Date: August 29, 2011 Page 1 of 30 Roche Molecular Systems, Inc. cobase 4800 BRAF V600 Mutation Test Pleasanton, CA 94588-2722 Module 3: Labeling Draft Package Insert 48 Starting PC R ............................... I .6 49 INTERPRETATION OFRESULTS ............. ........... 16 50 Retesting of Specimens with Invalid Results.......................... ........................ 17 5 1 Q UALITY CONTROL ... 1........ ............. I7 52 BRAF MutantControl ....................... ............................................ ........................ 17 53 BRA F Wild-Type Control ............... ......... .... .....................17 54 PROCEDURAL PRECAUTIONS............ .......... 17 55 PROCEDURAL LI MITA TION ................................... ....................... 17 56 NON-CLINICAL PERFORMANCE EVALUATION........... ...................................... ..I.8 57 Analytical Sensitivity - Limit of Blank (LoB)......................... .I........................ I7 58 Analytical Sensitivity- Limit of Detection (LoD) ............................................... 18 59 Analytical Sensitivity Using Specimen Blends................................................19 60 Analytical Sensitivity Using FFPET Specimens........................ 9..................... 19 61 Analytical Sensitivity UsingCell Line Blend ....................... ........................ 19 62 Minim al Tum or Content..... ......... .......... .................... ....... 20 63 C ross-Reactivity ........ ................ ...... ......... .... ......... ... ....... ........... ...... 2 1 64 BRAF Non-V600E Melanoma FFPET Specimens ......................... 2....................... 22 65 BRAF Non-V600E Plasmids .................................................. 22 66 Plasm ids of BRA F Homologs ......................... .......................... 23 67 Skin-related Microorganism s.. ... ..-. ... ......... .. ..... ........ ... ..... ... .23 68 Interference .......................... ...... ...... ......... 23 69 Reproducibility .2...................... ....... .... .... .25 70 Vemurafenib Clinical Efficacy.. ... ...... .......... .. .. .. .............. ..... 28 71 REFERENCES.................................................................. ................... 30 72 PMA M100022 CONFIDENTIAL AND PROPRIETARY Draft Package Insert Print Date: August 29, 2011 Page 2 of 30 Roche Molecular Systems, Inc. cobaso 4800 BRAF V600 Mutation Test Pleasanton, CA 94588-2722 Module 3: Labeling Draft Package Insert 73 INTENDED USE 74 The cobaso 4800 BRAF V600 Mutation Test is an in vitro diagnostic device intended for the qualitative detection of the BRAF V600E 75 mutation in DNA extracted from formalin-fixed, paraffin-embedded human melanoma tissue. The cobas® 4800 BRAF V600 Mutation 76 Test is a real-time PCR test on the cobas 4800 system, and is intended to be used as an aid in selecting melanoma patients whose tumors 77 carry the BRAF V600E mutation for treatment with vemurafenib. 78 SUMMARY AND EXPLANATION OF THE TEST 79 Activating mutations of the proto-oncogene BRAF occur in many human cancers, including malignant melanoma, colorectal cancer, 3 80 ovarian cancer, and thyroid cancer. 1,2 BRAF mutations have been identified in 40%-60% of malignant melanomas. Mutations are also 81 common in benign nevi,4 suggesting that such mutations are a very early event. The discovery of such somatic mutations in the BRAF 82 gene in melanoma and other human tumors has helped to elucidate the central role of the BRAT kinase in signaling pathways that 83 control cellular proliferation, differentiation and cell death. In normal cells, BRAF is part of a highly regulated signaling pathway that 84 mediates the effects of growth factor receptors (such as EGFR) through RAS. RAF, MEK and ERK. Oncogenic mutations in BRAF 85 result in a gain of kinase function, rendering the RAF-MEK-ERK pathway constitutively active in the absence of the typical growth 86 factors. 87 The majority of BRAF mutations in melanoma and other human tumors occur in codon 600.' The predominant mutation at codon 600 is 88 the V600E mutation (GTG>GAG). A number of dinucleotide mutations affecting codon 600 [V600K (GTG>AAG), V600R 89 (GTG>AGG), V600E2 (GTG>GAA), and V600D (GTG>GAT)] have also been observed less commonly, primarily in melanoma and 90 rarely in other tumors, such as colorectal cancer. The cobas' 4800 BRAF V600 Mutation Test is a real-time PCR assay designed to 91 detect the presence of the V600E (T1799A) mutation. The cobas" 4800 BRAF V600 Test is used as a companion diagnostic test for 92 vemurafenib, a compound which inhibits the mutant V600E version of BRAF. Clinical trials of vemurafenib in patients with advanced 93 melanoma have shown that patients with a V600E-mutant tumor are likely-to experience clinical benefit from the compound67. 94 PRINCIPLES OF THE PROCEDURE 95 The cobas® 4800 BRAF V600 Mutation Test is based on two processes: (1) manual specimen preparation to obtain genomic DNA from 96 formalin-fixed, paraffin-embedded tissue (FFPET); (2) PCR amplification and detection of target DNA using a complementary primer 97 pair and two oligonucleotide probes labeled with different fluorescent dyes. One probe is designed to detect the wild-type BRAF V600 98 sequence and one is designed to detect the V600E mutation sequence. Two external run controls are provided and the wild-type allele 99 serves as an internal, full process control. 100 Specimen Preparation 101 FFPET specimens are processed and genomic DNA isolated using the cobas®