Content Highlight Test Specification Test Content Performance Validation Test Report I II III IV V The NovoPM TM comprehensive cancer genomic profiling test Comprehensive genomic profiling for various solid tumors The Novogene Precision Medicine (NovoPM TM ) comprehensive cancer genomic profiling test is a next-generation sequencing (NGS)-based assay that analyzes more than 5 genes for clinically important alterations. These genes are known to be relevant for the diagnosis and/or treatment of various solid tumors according to National Comprehensive Cancer Network (NCCN) guidelines and medical literatures. Flexible Test This test is applicable to both tissue and ctdna samples. Paired tumor/normal samples or tumor sample alone are acceptable. Reliable Performance Comprehensive analytical validation has been completed and the test includes rigorous quality control measures. Competitive Price With the world' s largest sequencing capacity and superior process efficiency, Novogene provides highly competitive prices for all our services. Ultra-fast Sample Importation (when necessary) As a high-tech life science company in Beijing, Novogene can utilize a government-authorized green channel for ultra-fast importation of clinical samples through the Beijing Customs, when necessary. (Figure 1) Highlight Sample importation request Sample transportation Sample receipt Comprehensive Results NovoPM TM interrogates the complete coding regions of 548 genes and the introns of 21 genes, can detect all four types of genomic abnormalities (SNV, InDel, CNV and fusion) and also generates results (TMB, btmb and MSI) that can help guide cancer immunotherapies. Apply for CIQ permits Customs clearance Within 2 weeks Figure 1. Novogene's ultra-fast importation of samples to be analyzed in China
Test Specification Sample Requirement Sample Type FFPE tissue Extracted tissue DNA Plasma Extracted ctdna Whole blood Extracted white blood cell DNA Sample Requirement Approximately ten 4-μm sections, each with tissue area 25 mm 2, tumor content 2% Total DNA 5 ng; DNA concentration (quantified by Qubit) 2 ng/μl, total volume 1 μl; Purity: OD26/28 = 1.8-2. without degradation or RNA contamination 4 ml (for ctdna extraction) Total ctdna 6 ng; DNA concentration (quantified by Qubit) 3 ng/μl, total volume 1 μl; Purity: without genomic DNA contamination 1 ml (for ctdna and white blood cell DNA extraction) Total DNA 1 μg; DNA concentration (quantified by Qubit) 2 ng/μl, total volume 1 μl; Purity: OD26/28 = 1.8-2. without degradation or RNA contamination Note: Paired tumor/normal samples are recommended while tumor sample alone is also acceptable. Sequencing Strategy HiSeq X Ten or NovaSeq 6, PE 15. Data Quality Guarantee We guarantee that 8% of bases have a sequencing quality score Q3, which exceeds Illumina s official guarantee of 75%. Recommended Sequencing Depth To achieve the limit of detection (1.% and.5% for SNV in tissue DNA and ctdna, respectively), we recommend at least 1X and 2X effective average sequencing depth for tissue DNA and ctdna, respectively. Bioinformatics and Result Interpretation Variant calling in NovoPM TM is done using Novogene s state-of-the-art bioinformatics pipeline. Clinical significance of the variants is determined according to well-recognized databases including COSMIC, dbsnp, 1 Genomes, ExAC, OncoKB, ClinVar, PharmGKB, SIFT and Polyphen2. Turn-around Time Sample Types Deliverables Calendar Days Extracted ctdna samples Extracted tissue DNA samples FFPE tissue samples Raw data Raw data & Report Raw data Raw data & Report Raw data Raw data & Report 9 1 1 11 12 13
Test Content SNV InDel CNV Fusion EGFR,BRAF KRAS, EGFR, HER2,MET, ALK,ROS1 RET,NTRK, TMB btmb MSI CNV: Copy Number Variation TMB: Tumor Mutation Burden btmb: blood Tumor Mutation Burden MSI: Microsatellite Instability Figure 2. Genomic profiling results generated by NovoPM TM Gene Targets NovoPM TM interrogates the complete coding regions of 548 genes and the introns of 21 genes for all four types of genomic abnormalities: SNV, InDel, CNV and fusion. Approximately 1 mutations can guide the potential application of multiple FDA-approved targeted and/or immunotherapies as shown in Table 1. The other genes are also analyzed for their relevance to the diagnosis and/or treatment of various solid tumors according to medical literatures. The interpretation of detected mutations in those genes is done according to Novogene s in-house oncology knowledgebase constructed on the basis of public resources such as OncoKB. Table 1. Gene targets in NovoPM TM that are associated with FDA-approved therapies Indication Biomarker FDA-approved Therapy Non-Small Cell Lung Cancer (NSCLC) Melanoma Breast Cancer Colorectal Cancer MSI-high Solid Tumors MSI-high Colorectal Cancer EGFR 19Del and L858R EGFR T79M ALK rearrangements BRAF V6E ROS1 fusion BRAF V6E ERBB2 (HER2) amplification KRAS wild-type MSI MSI Gilotrif (afatinib), Iressa (gefitinib) or Tarceva (erlotinib) Tagrisso (osimertinib) Alecensa (alectinib), Xalkori (crizotinib) or Zykadia (ceritinib) Tafinlar (dabrafenib) in combination with Mekinist (trametinib) Xalkori (crizotinib) Tafinlar (dabrafenib) or Zelboraf (vemurafenib) Herceptin (trastuzumab) etc Erbitux (cetuximab) KEYTRUDA (pembrolizumab) OPDIVO (nivolumab)...
Performance Validation Validation with reference standards Table 2 shows the sequencing performance metrics of NovoPM TM with 1 ng DNA input and 1X average sequencing depth on HiSeq X Ten or NovaSeq 6, PE 15. Further validation was done with various mixtures of DNA from 5 cell lines with known germline mutations and 3 cancer cell lines with known somatic mutations. Accuracy The sensitivity of SNV and InDel detection was 96.58% and 94.91%, respectively, for variants with MAF 1%. The concordance of HER2 CNV detection between digital PCR and NovoPM TM : R 2 >.99. ROS1 and ALK fusion detection rate was 1% when fusion-positive tumor cell content 2%. Reproducibility NovoPM TM results were highly reproducible in across-operator and across-time point tests for SNV (Table 3), InDel, HER2 CNV and ROS1/ALK fusions. Table 2. Sequencing performance metrics of NovoPM TM with reference standards Catalog Mean CV (%) Max Min Raw bases (G) 9.16 15.7 11.64 5.73 Clean bases (G) 8.54 16.91 11.7 5.14 Average sequencing depth on target 118 15.88 148 71 Probe capture ratio after Rmdup (%) 5.1 7.18 54.4 41.96 Mapping rate (%) 99.3.9 99.46 99.16 Table 3. Reproducibility of SNV detection by NovoPM TM with reference standards Operator Time Point Replicate SNVs Detected Sensitivity (Out of 1,25 SNVs) (%) #1 1162 96.43 A I #2 1155 95.85 #3 1159 96.18 #1 1144 94.94 B I #2 #3 1146 1128 95.1 93.61 #1 118 97.93 A II #2 1166 96.76 #3 1164 96.6 Validation with clinical FFPE samples High concordance between NovoPM TM results and those of digital PCR or FISH was observed on 17 clinical FFPE samples including 6 SNV/InDel-positive and 1 SNV/InDel-negative, 5 fusion-positive, 5 HER2 CNV-positive and 2 HER2 CNV-negative (Table 4). Table 4. Concordance of NovoPM TM results with clinical FFPE samples Number of Concordant Alterations Number of Discordant Alterations Concordance (%) Reference Test Platform SNV & InDel 18 2 9 digital PCR CNV 7 1 FISH Fusion 5 1 FISH Total 3 2 94
NovoPM-TMB (Tumor Mutation Burden Analysis) TMB represents the total number of mutations per coding area of a tumor genome. It s usually calculated through genomic sequencing of tumor DNA. The value of TMB has been found to correlate with the efficacy of some anti-pd1/pd-l1 immunotherapies in certain tumor types. NovoPM-TMB is an alternative to TMB calculated from whole exome sequencing (WES) as there is strong linear correlation between the two (Figure 3). We also found that our NovoPM-TMB results showed a statically significant correlation with mismatch repair (MMR) status or POLE mutation status in 262 Chinese lung cancer samples (Table 5), which further proves the validity of this assay. NovoPM Cancer Panel-Mutation Count A. TCGA dataset LUAD, N=514 8 7 6 5 4 R 2 =.91 3 2 1 2 4 6 8 1 12 14 WES-Mutation Count NovoPM Cancer Panel-Mutation Count 6 B. TCGA dataset LUSC, N=492 5 4 3 R 2 =.83 2 1 2 4 6 8 1 12 WES-Mutation Count 25 C. FFPE LC and CRC samples N=15 8 D. Reproducibility test, 3 aliquots/sample NovoPM-TMB (mut/mb) 2 15 1 5 R 2 =.88 NovoPM-TMB (mut/mb) 6 4 2 4.35 ±.1 7.82 ±.66 72.5 ±.52 1.73 ±.5 44.78 ±.25 5 1 15 2 WES-TMB (mut/mb) P1 P2 P3 P4 P5 Sample ID Figure 3. Validation of NovoPM-TMB NovoPM-TMB shows strong linear correlation with WES-TMB for two TCGA lung cancer datasets (A and B) and a Novogene in-house dataset from FFPE samples (C). NovoPM-TMB results from the same set of FFPE samples are also highly reproducible across different time points of analysis (D). (Shan et al., WCLC 217-poster presentation by Novogene) LUAD: Lung Adenocarcinoma; LUSC: Lung Squamous Cell Carcinoma; LC: Lung Cancer; CRC: Colorectal Cancer. Table 5. Correlation between NovoPM-TMB and the MMR status or POLE mutation status of 262 Chinese lung cancer samples TMB MMR status (p = 5.71 E-1) POLE status (p = 2.17 E-7) Normal MMR (N = 234) dmmr (N = 28) POLE wt (N = 255) POLE mut (N = 7) High 16 (6.8%) 16 (57.1%) 25 (9.8%) 7 (1%) Low 218 (93.2%) 12 (42.9%) 23 (9.2%) (%) Cut-offs calculated according to Zehir et al., Nat Med, 217
NovoPM-MSI (Microsatellite Instability Analysis) MSI is the condition of genetic hypermutability (predisposition to mutation) that results from impaired DNA MMR. FDA approval of KEYTRUDA for MSI-high (MSI-H) solid tumors in May 217 and OPDIVO for MSI-H colorectal cancer in August 217 showed that MSI is a predictive biomarker for these anti-pd1 immunotherapies. Using a training set of paired tumor/normal samples with known MSI status (measured by PCR, the current gold standard in clinical testing for MSI status), we identified 19 out of 43 mononucleotide microsatellite loci covered by NovoP- M TM whose repeat lengths can reliably reflect the MSI status of the tumor samples. These 19 loci are defined as the ''MSI marker set''. For each tumor sample to be analyzed, the histogram of read counts for different repeat lengths of each locus in the MSI marker set are compared to that of the paired normal sample using the Mann-Whitney U test. A locus is considered unstable if the P-value is less than.5. The percentage of unstable loci in the MSI marker set is calculated as the NovoPM-MSI score. To analytically validate the NovoPM-MSI algorithm, we analyzed 113 colorectal cancer FFPE samples with both PCR and NovoPM TM. The results are shown in Figure 4 and Table 6. The NovoPM-MSI algorithm achieved 88.9% of sensitivity and 96.2% of specificity..6 NovoPM-MSI results PCR-MSI positive MSI Score.4.2 P1 P3 P5 P7 P9 P11 P13 P15 P17 P19 P21 P23 P25 P27 P29 P31 P33 P35 P37 P39 P41 P43 P45 P47 P49 P51 P53 P55 P57 P59 P61 P63 P65 P67 P69 P71 P73 P75 P77 P79 P81 P83 P85 P87 P89 P91 P93 P95 P97 P99 P11 P13 P15 P17 P19 P111 P113 Sample ID Figure 4. Validation of NovoPM-MSI NovoPM-MSI analytical validation with 113 colorectal cancer FFPE samples. X-axis shows the sample ID and Y-axis shows the MSI score. The green line shows results from the NovoPM-MSI algorithm. The horizontal dotted green line shows the threshold for MSI status (MSI score =.2). The red dots indicate samples that were tested MSI-high with a PCR method. Table 6. Concordance between PCR and NovoPM-MSI results on 113 colorectal cancer FFPE samples NovoPM-MSI NovoPM-MSS Sensitivity Specificity PCR-MSI(N=9) 8 1* 88.9% PCR-MSS(N=14) 4* 1 96.2% Note: * Ambiguous samples that usually should be further tested with a PCR method; MSS: Microsatellite Stable.
Test Report The report for the NovoPM TM comprehensive cancer genomic profiling test is automatically generated as a PDF which includes the relevant patient, sample, and test information extracted from Novogene s laboratory information management system (LIMS). In addition to the standard test report format, Novogene also provides tailored test reports and full test results upon request. An example of the standard test report is shown below. Tower A, C1 Building, C District, IT Industrial Park, No.1 Jiuxianqiao North Road, Chaoyang District, Beijing, China biopharma-cs@novogene.com https://en.novogene.com/ Patient ID: xxxx Date of Report: 22/1/218 Sample Details Patient ID : xxxx Cancer Type : Colon Cancer Gender : Male Sample Type: FFPE, Slides Date of Birth : 15/3/1978 Sample ID : 218112XX Test Results 1 Section I: Includes the patient ID, gender, date of birth, date of the report and specifics such as the cancer type and sample type. Gene Variants Gene Variant Variant Allele Frequency Therapy Class I Class II Class III Potential Drug Resistance Information TP53 Exon9 c.991c>t p.q331x 87.5% / / Alisertib (MLN8237), AZD1775 / 2 Section II: Includes the variant detection results that have therapies. AMER1 Exon2 c.1591c>t p.r531x 66.9% / / / / Note: Class I: FDA/CFDA approved drugs in this tumor type; Class II: FDA/CFDA approved drugs in other tumor types; Class III: Drugs in clinical trials. Tumor Mutation Burden (TMB) Test Item Test Result TMB Microsatellite Instability (MSI) 2.22 mut/mb 3 Section III: Contains results of TMB and MSI which indicate potential benefit from Test Item MSI Score Test Result certain immunotherapies. MSI.4 High Note: if the value of the MSI is.17~.23, a PCR method is recommended to confirm the result. Disclaimer A finding of driver gene alteration does not necessarily indicate pharmacologic effectiveness of any drug or treatment; a finding of no driver gene alteration does not necessarily indicate lack of pharmacologic effectiveness of any drug or treatment. This report makes no promises or guarantees that a particular drug will be effective in the treatment of disease in any patient. This report also makes no promises or guarantees that a drug with potential lack of clinical benefit will in fact provide no clinical benefit. The information in this report should be considered in conjunction with all other relevant information regarding a particular patient, before the patient s treating physician recommends a course of treatment. Decision on patient care and treatment should be based on the independent medical judgement and information from other diagnostic tests, and patient preferences, in accordance with the standard of care in a given community. Tested by: Reviewed by : Report Date: 22/1/218
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