Statistical Methods for Biotechnology Products

Statistical Methods for Biotechnology Products
Part II: Biochip Diagnostic Products Examples of Regulatory Evaluation of Biochip Products by Professor, Jen-pei Liu, PhD Division of Biometry, Department of Agronomy National Taiwan University, and Division of Biostatistics and Bioinformatics National Health Research Institutes 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Regulatory Evaluation of Biochip Products
HercepTest PATHWAYTM Her 2 (Clone CB11) Affymetrix GeneChip Microarray Instrumentation System Roche AmpliChip CYP450 Microarray 2C19 2D6 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Regulatory Evaluation of Biochip Products
References: US FDA Decision Summary – P (1998) US FDA Decision Summary – P (2000) US FDA Decision Summary – K042279 US FDA Decision Summary – K043576 US FDA Decision Summary – K042259 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Copyright by Jen-pei Liu, PhD; Products are only for illustration
HercepTest HER2 (the human epidermal growth factor receptor 2) is a member of the HER (erbB) family of transmembrane tyrosine kinase Enhanced level of HER2 is associated with mammary epithelial cell transformation and shorter survival in patients with breast cancer  25% of invasive breast cancers exhibit HER2 gene amplification The rate of HER2 gene amplification or protein in ductal carcinoma in situ (DCIS) is higher than invasive cancer  pathogenic role in the initiation of mammary carcinoma Treatment of Herceptin - requirement of screening the patients with over-expressed HER2 level 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

HercepTest HercepTest is an immunohistochemical (IHC) test intended to aid in the assessment of patients being considered for Herceptin treatment A Class III device – require clinical studies Interpret results Negative for HER2 overexpression – 0 or 1+ Positive for HER2 over expression – 2+ or 3+ 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

HercepTest Potential Adverse Effects False positive – the benefit of Herceptin to patients with normal or lower level of HER2 is unknown False negative – The risks of Herceptin is not negligible 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

HercepTest Device Stability Storage conditions: 20o and 37oC Experiment Three freeze-thaw cycles Heating to 37oC for 24 hours Stored at 10oC; three freeze-thaw cycles Stored at 10oC Three lots at 2, 4, and 8 weeks Stable up to 8 weeks at 37oC A projected shelf-life for the kit of 6 months at 2o to 8oC 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

HercepTest HER2 Antigen Stability Use freshly cut tissue sections stored in the dark at room temperature 20o Two paraffin embedded control cell line pellets and 2 well-characterized breast cancer tumors tested at days 1, 15, and 43 No change in epitope responsiveness during the 43-day holding period Freshly cut tissue sections can be stored in the dark at room temperature 20o up to 6 weeks 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Control Cell Lines 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Antibody Specificity 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Reproducibility Studies
Intra-run (one lab.) 5 specimens with different IHC intensity staining scores Three replicates for each specimen in a blinded random format Use of negative control to assess background staining Manual staining vs. automated staining reproducible + vs - results All +3 and +2 IHC scores were 100% reproducible 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Inter-run Three different laboratories Each of 5 specimens paired with negative control 5 pairs of slides were randomized and blinded from run to run Satisfactory reproducibility + vs – results No irreproducibility seen in distinguish 2+ from 3+ 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Inter-laboratory 6 labs but 3 labs without valid control results 3 labs (40 specimens each) The over percent agreement: 82% to 90% for + vs. – 15 discrepant results from 120 samples Additional 12 discrepant results between +2 and +3 staining intensity 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Lot-to-lot 3 lots The control cell line - identical results 2 different breast carcinomas expressing the HER2 receptor One lot is off by one IHC intensity score. One is positive and the other is between +2 and a negative result A normal HER2 negative tissue - identical results 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Clinical Studies Compared to an ICH clinical trial assay (CTA) used in the Herceptin trials Goal: at least 75% agreement with 95% confidence ( Ho P  0.75 vs. Ha: P>0.75) One central laboratory 274 + and 274 – (randomly selected) specimens 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Clinical Studies 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Clinical Studies (accuracy)
2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

PATHWAYTM Her 2 (Clone CB11)
A mouse monoclonal antibody Semi-quantitative detection of c-erbB-2 antigen Binding of an antibody to an antigen of interest Visualization of the bound primary antibody by an indirect biotin-avidin system coupled to an enzyme Interpret results Negative for HER2 overexpression – 0 or 1+ Positive for HER2 over expression – 2+ or 3+ 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

PATHWAYTM Her 2 (Clone CB11)
Potential Adverse Effects False Positive the benefit of Herceptin to patients with normal or lower level of HER2 is unknown False Negative The risks of Herceptin include infusion toxicity (chills, fever, pain, asthenia, nausea, vomiting and headache) and cardiotoxicity 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Control Cell Lines 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Specificity 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Reproducibility Intra and inter-run – reproducible results Inter-laboratory – 3 labs No cases varies from clinically + to clinically – between sites All slides had staining variability no larger than 1 intensity level No invalid runs 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Reproducibility Inter-laboratory – 3 labs Variation between 3+ and 2+ intensity occurred in 4 out of 22 staining events (18%) Potential difficulty distinguishing a 2+ from 3+ 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Reproducibility Intra-laboratory (technician A vs. technician B) Varied by no greater than 1 intensity level No cases from clinically + to clinically – Variation between 3+ and 2+ 1/16 (6%) Intra-technician (technician A vs. technician A, one week apart) 3/14 (21%) 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Reproducibility Intra- and Inter-investigator Intra-investigator (4 weeks apart) Clinically + vs. Clinically - :72/74 (97%) 3+ and 2+ variation: 5/34 (15%) Inter-investigator Clinically + vs. Clinically -: Round 1: 71/74 (96%); Round 2: 70/75 (93%) 3+ and 2+ variation: Round 1: 4/34 (12%); Round 2: 1/35 (3%) 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Clinical Studies Compared to DAKO HercepTest Goal: at least 75% agreement with 95% confidence ( Ho P  0.75 vs. Ha: P>0.75) One central laboratory Multi-center: 3 sites 50+ and 100- specimens by HercepTest for each site + > 10% of cells staining scores of 2+ or 3+ 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Clinical Studies Observed overall agreement = 92.4 (416/450) with an exact 95% CI (89.6% to 94.7%) P-value for testing Ho: P0.75 is < The observe kappa statistic = 0.83 with a p-value for testing no agreement < P-value for McNemar test for equal proportion of clinically + is 1.00 Assume that HercepTest is gold standard Sensitivity: 88.7% (134/151); 95%CI: 82.6% % Specificity: 94.3% (282/299); 95%CI: 91.1% % 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Clinical Studies Observed overall agreement = 86.4 (389/450) with an exact 95% CI (82.9% to 89.5%) P-value for testing Ho: P0.75 is < The observe kappa statistic = 0.73 with a p-value for testing no agreement < P-value for test for marginal homogeneity is 1.00 Assume that HercepTest is gold standard Sensitivity for intermediate category: 46.2% (24/52); 95%CI: 32.3% % Sensitivity (+): 83.8% (83/99); 95%CI: 75.1% % Specificity (-) : 94.3% (282/299); 95%CI: 91.1% % 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Kappa Statistic  = (p0 – pe)/(1 – pe) Where p0 = pii and p0 = pi. p.i pij is the proportion of (i,j) entry of a rxr contingency table Pi. (p.j) is the sum of pij over category i, i=1,…,r SE = {1/[1- pe]sqrt(n)}{sqrt[C]} C = pe – p2e - pi. p.i(pi.+p.i) 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Kappa Statistic Example HercepTest PATHWAY Margin (0.6267) (0.0378) 299 (0.6644) (0.0378) 134 (0.2978) 151 (0.3356) Margin (0.6644) 151 (0.3356) 450 (1.0000) p0 = = pe = * * =  = ( – )/(1 – ) = 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Affymetrix GeneChip Microarray Instrumentation System
FS450Dx Fluidics Station 4 modules Each containing a single GeneChip microarray Perform functions required for hybridization, washing, and staining Up to 9 stations communicate to a workstation GCS3000Dx Scanner A wild-field, epifluorescent, confocal, scanning laser microscope Autoloader loads arrays from array cartridges GCOSDx (GenChip Operating Software) 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

GCOSDx Software Interface between the user and instrument system Instrument control Application of processing array Data collection Algorithms and reporting functions to produce a clinical result 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

GCOSDx Software Alignment algorithm to superimpose a grid on the image to delineate probe cells by using a checkboard image of control probes, located at the corner of the probe array to superimpose the grid on the scanned image Generate cell intensity data from the image data The cell analysis algorithm analyzes the image data and computes a single intensity value for each probe cell on the array and saved as a “cel” file 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Operational Environment Programmed in C++ MS Window 2000 SP3 or SP4 (will move to MS XP) Internet Explorer 6.0 Office XP MDAC 2.7 SP1 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Indications for use To measure fluorescence signals of labeled DNA targets hybridized to GeneChip arrays Special Conditions for Use Statements For use with separately cleared GeneChip microarray assays 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Affymetrix GeneChip Microarray Instrumentation System – Analytical Performance Precision/Reproducibility Intensity readings generated by Scanner 3000DX were not changed, manipulated or revised after scanning was completed Raw .CEL data were analyze for Uniformity Intra-, inter-chip, intra-, inter-scanner, intra-, inter-fluidics port reproducibility Random effect of FS, For port or scanner by ANOVA No scaling nor normalization to observe variability in all system components with raw data 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Affymetrix GeneChip Microarray Instrumentation System – Analytical Performance Array Design Array to assess scanner performance without hybridization step 32x32 cell array to have photobleaching stability and can be scanned over 100 times A single array was scanned 4 times for each of three scanners Global uniformity: CV < 10% Local uniformity: CV < 1% (each 400 mM2 gridded cell with surrounding 4 cells; averaged over alternating cells of the 32x32 array) 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Affymetrix GeneChip Microarray Instrumentation System – Analytical Performance Array to assess overall system performance Control probes Random generated synthetic sequence Located in a specific pattern across the array Lengths of probes: 16-25mer For grid alignment and To evaluate variability introduced by hybridization Discrimination controls 4 control probe sets 30 tiled PM/MM for each probe set Measurement of discrimination of PM from MM 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Affymetrix GeneChip Microarray Instrumentation System – Analytical Performance Sample Type Two target sets for control and discrimination at concentrations of 0.1, 0.5, 1 and 3 pM Study Design Goal to generate reproducibility data for consistent system performance independent of assay type 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Affymetrix GeneChip Microarray Instrumentation System – Analytical Performance Study Design (Continued) Metric: Discrimination score (PM vs. MM) Design: Replicated fully factorial fully nested One target aliquot was hybridized to 12 chips on 3 Fluidics Stations Each array was scanned in duplicated on each of three scanners 72 scans for one target aliquot 3 replicates/aliquot 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Affymetrix GeneChip Microarray Instrumentation System – Analytical Performance 643 identical features on the array The overall average CV was 8.0% Between-scan CV 4 scans per day Scanner 1: % Scanner 2: % Scanner 3: % 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Affymetrix GeneChip Microarray Instrumentation System – Analytical Performance Between-scanner CV Time Point CV% Range – 6.8 – 7.4 – 9.2 – 10.9 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Affymetrix GeneChip Microarray Instrumentation System – Analytical Performance Discrimination Score: 36 average discrimination scores from all arrays fell within 95% CI (average = average of 2 scanned images/array x 36 arrays) P-values for variability of FS, FS ports, and scanner > 0.05 Chip-to-chip variability One PM CV > 13% All MM Cvs <=15% 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Roche AmpliChip CYP450 Microarray – 2C19 Test

A microarray-based genotyping test Processes PCR amplification of purified genomic DNA (a 35 cycle program) from a whole blood sample Fragmentation and labeling of the amplified products ( nucleotides labeled with biotin) Hybridization of the amplified products (with a hybridization control) to a microarray and scanning the microarray Determination of the CYP450 genotype and predict the phenotype 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

To identify specific nucleic acid sequences Query for the presence of certain known sequence polymorphisms (240 probes for each polymorphism) by the analysis of the pattern of hybridization to a series of probes (15,000 different probes and copies of each probe) that are specifically complementary either to wild-type or mutant sequences The assay (20x20 m2) is designed to distinguish 3 alleles of the CYP2C19 gene 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Roche AmpliChip CYP450 2C19 Test Analytical Performance
Precision/Reproducibility 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

23 system errors 21 scanner failures 2 fluidics failures One miscall: a called 2*/*2 sample but later sequencing revealed a 2*/*10 sample 100 replicates for the failure rate of the AmpliChip CYP450 system One whole system failure: 1% failure rate with 95% CI to 99.97% - one chip failed to scan and subsequent attempts also failed 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Detection Limit: >= 95% positive rate 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Specificity 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Genotype Detection 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Predicted Enzyme Activity
Allele Nucleotide Predicted Enzyme Activity *1 None Normal *2ABD -1584G, 1039C>T, 1661G>C, 2850C>T, 4180G>C *3 2549A del *4ABDJK 100C>T, 1039C>T, 1661G>C, 1846G>A, 2850C>T, 4180G>C *5 Entire CYP2D6 Gene deleted *6ABC 1707Tdel, 1976G>A, 4180G>C *7 A2935C *8 1661G>C, 1758G>T, 2850C>T, 4180G>C 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

*9 delAGA Reduced *10AB 100C>T, 1039C>T, 1661G>C, 4180G>C *11 883G>C, 1661G>C, 2850C>T, 4180G>C None *15 T138ins *17 1023C>T, 1661G>C, 2850C>T, 4180G>C *19 1661G>C, delAACT, 2850C>T, 4180G>C *20 1661G>C, 1973insG, 1978C>T, 1979T>C, 2850C>T, 4180G>C *29 1659G>A, 1661G>C, 2850C>T, 3183G>A, 4180G>C *35 -1584C, G31A, 1661G>C, 2850C>T, 4180G>C Normal *36 100C>T, 1039C>T, 1661G>C, 4180G>C, gene conversion to CYP23T in exon 9 *40 1023C>T, 1661G>C, 1863ins(TTT CGC CCC)2; 2850C>T, 4180G>C *41 –1548C, 1661G>C, 2850C>T, 4180G>C 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

*1XN duplicate active *1 genes (n is not determined-range 2 -13) Increased *2XN duplicate active *2 genes (n is not determined-range 2 -13) *4XN duplicate active *4 genes (n is not determined) None *10XN duplicate partially active *10 genes (n is not determined) Reduced *17XN duplicate partially active *17 genes (n is not determined) *35XN duplicate active *35 genes (n is not determined) *41XN duplicate partially active *41 genes (n is not determined) 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Allele Japan China Caucasian EU Caucasian US Black – US Black -Africa Amer-indian Saudi Arabia Turkey *1 42-43% 23% 33-37% 37-40% 29-34% 28-56% 66% * 37% *2 9-13% 20% 22-33% 26-34% 20-27% 11-45%[1] 19% 35% *3 1% 1-4% <2% <1% *4 0-1% 12-23% 18-23% 7-9% 1-7% 4% 11% *5 5-6% 6% 2-7% 2-4% 6-7% 1-6% 15% *6 7% *9 0-3% 2-3% *10 39-41% 50-70% 1-2% 4-8% 3-8% 3-9% 1-17% *17 15-26% 9-34% *41 *1xN 3% *2xN 10% *4xN 2% Note: Percentages represent ranges of allelic frequencies reported in various published studies. *No published data available 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Roche AmpliChip CYP450 2D6 Test
15,129 probes with ~107 of the specific oligonucleitide probe each Length of probe sequence: bases A single Probe Set consists of 4 Probes (or Features) which have a fixed target except for at the substitution position wherean A, C, G, and T are included to generate four unique probes: one Perfect Match (PM) and three Mismatch (MM) 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

A Probe Set Pair consists of a Wild-type Probe Set (for Wild-type allele) and a Mutant Probe Set (for a known polymorphism). To distinguish 29 polymorphisms in CYP2D6 including gene duplications and gene deletion To identify 27 distinct alleles including 7 CYP2D6 gene duplication genes 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

A microarray-based genotyping test Processes PCR amplification of purified genomic DNA (a 35 cycle program) from a whole blood sample Fragmentation and labeling of the amplified products ( nucleotides labeled with biotin) Hybridization of the amplified products (with a hybridization control) to a microarray and scanning the microarray Determination of the CYP450 genotype and predict the phenotype 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

To identify specific nucleic acid sequences Query for the presence of certain known sequence polymorphisms (240 probes for each polymorphism) by the analysis of the pattern of hybridization to a series of probes (15,000 different probes and copies of each probe) that are specifically complementary either to wild-type or mutant sequences The assay (20x20 m2) is designed to distinguish 27 distinct alleles of the CYP2D6 gene 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Some rare CYP450 alleles are not reported by the AmpliChip CYP450 Test. These alleles are listed below: Alleles Not Reported by AmpliChip CYP450 Test CYP2D6 12, 13, 14, 16, 18, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 37, 38, 39, 42, 43, 44, 45, 46 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Precision/Reproducibility - The genotypes of reproducibility panel
Roche AmpliChip CYP450 2D6 Test Analytical Performance Precision/Reproducibility - The genotypes of reproducibility panel CYP2D6 genotype *4 X n / *41 *4 / *5 *1 / *1 *10B / *10B *17 / *29 *2 X n / *17 *9 / *35 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Correct Genotype Calls Correct Call Rate Estimate (95% CI)
Roche AmpliChip CYP450 2D6 Test CYP2D6 genotype Predicted Phenotype No. Tested Genotype Calls N (%) Correct Genotype Calls Correct Call Rate Estimate (95% CI) *4 X n / *41 Intermediate 135 134 (99.3) 134 100.0 (0.98) *4 / *5 Poor 134 (100.0) 133 0.99 (0.97) *1 / *1 Extensive 135 (100.0) 1.00 (0.98) *10B / *10B *17 / *29 *2 X n / *17 Extensive* *9 / *35 Total 944 941 (99.7) 940 1.00 (0.99) 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Roche AmpliChip CYP450 2D6 Test Analytical Performance
23 system errors 21 scanner failures 2 fluidics failures Three result yield “no call” One incorrect call from Panel member CYP2D6 *4/*5 100 replicates for the failure rate of the AmpliChip CYP450 system (50ng DNA/PCR of 10*/10* genotype) One whole system failure: 1% failure rate with 95% CI to 99.97% - one chip failed to scan and subsequent attempts also failed 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

The lowest level of genomic DNA at which a >= 95% positivity rate was obtained for correct detection of CYP2D6 (*4DxN/*41 and *4/*5 samples) was 25ng. DNA Amount (ng) Number of Arrays # Correct Calls Positivity Rate 95% CI 50 144 100% 97.5 – 100% 25 2.5 134 93.1% 87.6 – 96.6% 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Analytical Specificity Interference test With and without spiking of Albumin – 6000 mg/dL Bilirubin – 60 mg/dL Triglycerides – 3000 mg/dL 10-fold greater than normal Carryover contamination No interference and carryover contamination 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Method Comparison Bi-directional DNA sequencing 246 clinical samples (492 alleles sequenced) 2 No Calls (0.8%) Genotype Detection Allele-specific PCG and PCR-RFLP 50 ng/PCR 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

CYP2D6 Allele Genotype Number of Alleles Sequenced AmpliChip Results Correct Calls Miscalls No Calls Percent Agreement *1 103 102 1 99.0% *2 64 63 98.4% *3 14 100.0% *4 73 *5 26 *6 8 *9 9 *10 40 *15 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

*17 28 100.0% *29 12 *35 32 *36 2 *40 *41 71 *1XN 1 0.0% *2XN *4XN *10XN *17XN *35XN *41XN Total 492 488 4 99.2% 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Genotype Detection CYP2D6 Genotype Number of Unique Alleles Tested Correct Calls Number Of Miscalls No Calls % Agreement Replicates *1 218 217 1 99.5% 246 *2 110 109 99.1% 148 *3 14 100.0% 22 *4 131 157 *5 48 73 *6 8 *9 10 11 *10 63 61 2 96.8% 90 *15 4 *17 36 71 *29 12 35 *35 46 *36 16 *40 *41 79 97 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

*1XN 16 15 1 93.8% *2XN 7 6 85.7% *4XN 4 100.0% *10XN *17XN 5 *35XN *41XN Total 806 800 99.3% 1088 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

By Sample CYP2D6 Genotype Total Unique Samples Number of Correct Calls Miscalls Numbe Of No Calls Percent Agreement Call Rate *1/*1 31 100.0% *1/*1XN 5 *1/*2A 30 *1/*2AXN 2 1 50.0% *1/*2D *1/*2DXN *1/*3 *1/*4A *1/*4AXN *1/*4D 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

*1/*4DXN 1 100.0% *1/*5 15 *1/*6B 3 *1/*9 2 *1/*10B 16 *1/*10BXN *1/*17 13 *1/*17XN *1/*29 *1/*35 *1/*35XN *1/*40 *1/*41 14 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

*1XN/*2A 3 2 1 66.7% *1XN/*4A 4 100.0% *1XN/*10A *1XN/*35 *1XN/*41 *2A/*2A 16 *2A/*3 *2A/*4A 20 *2A/*5 *2A/*6B *2A/*9 *2A/*10B *2A/*35 8 *2A/*41 5 *2AXN/*17 *2AXN/*41 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

*3/*3 2 100.0% *3/*4A 3 *3/*5 *3/*35 1 *3/*41 *4A/*4A 23 *4A/*4D *4A/*5 *4A/*6B *4A/*9 *4A/*15 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

4 100.0% *4A/*41 11 *4D/*5 1 *4D/*41 2 *4DXN/*5 *4DXN/*17 *5/*5 *5/*9 *5/*10B *5/*10BXN *5/*17 *5/*29 *5/*35 *5/*41 7 *6B/*41 *9/*17 *9/*41 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

*10B/*10B 17 16 1 94.1% *10B/*10BXN 2 100.0% *10B/*17 *10B/*35 *10B/*36 *10B/*40 *10B/*41 *10BXN/*41 *17/*17 4 *17/*29 *17/*41 3 *29/*29 *29/*36 *29/*41 *35/*35 *35/*41 *41/*41 9 *41/*41xN Total 403 400 99.3% 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Blended sample Expected genotype Number of Correct Calls
Plasmid Genotype Genomic DNA Genotype Blended sample Expected genotype Blended samples (n) Number of Correct Calls Number of Miscalls Number of No calls Genotype Call Rate *11 *1/*1 *1/*11 4 100% *2/*2 *2/*11 *4/*4 *4/*11 *5/*5 *11/*11 *41/*41 *41/*11 *7 *1/*7 *2/*7 *4/*7 *7/*7 *41/*7 *8 *1/*8 *2/*8 *4/*8 *8/*8 *41/*8 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

*19 *1/*1 *1/*19 4 100% *2/*2 *2/*19 *4/*4 *4/*19 *5/*5 *19/*19 *41/*41 *41/*19 *20 *1/*20 *2/*20 *4/*20 *20/*20 *41/*20 TOTAL 100 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Roche AmpliChip CYP450 2D6 Test Analytical Performance – Method Comparison Number of Number of Method(s) Samples Tested Correct calls Miscalls No Calls Allele-specific PCR 138 Allele-specific PCR and PCR-RFLP 1 PCR-RFLP 16 15 DNA Sequencing 14 DNA Sequencing and allele-specific PCR 191 189 2 DNA Sequencing and PCR-RFLP 28 DNA Sequencing, allele-specific PCR, and PCR-RFLP 13 PCR Size (for detection of *5/*5 genotype) 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Instrument AmpliChip CYP450 IVD software v2.0.0 will process data acquired from AmpliChip CE-IVD/US-IVD system and generate genotype report AmpliChip CYP450 IVD software on Affymetrix GeneChip workstation Affymetrix GCOS software 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

The AmpliChip CYP450 IVD software v2.0.0 shall analyze input CEL file(s) using the genotyping algorithm. The AmpliChip CYP450 IVD software v2.0.0 shall read information from GCOS database related to sample, experiment, chip identification, hybridization and scanning history. The AmpliChip CYP450 IVD software v2.0.0 shall write result to a report file for each input CEL file. Quality control – reagents for one replicate of the AmpliChip negative control and one replicate of the AmpliChip CYP450 positive control 2018/11/14 Copyright by Jen-pei Liu, PhD; Products are only for illustration

Statistical Methods for Biotechnology Products

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