Madrid, Spain IMPLEMENTING NEXT GENERATION SEQUENCING IN A PATHOLOGY LABORATORY Dr. JL Rodríguez Peralto
NGS
Ion Torrent Oncomine Focus Assay - Implementation experience for EGFR mutation detection at HOSPITAL 12 DE OCTUBRE For Research Use Only. Not for use in diagnostic procedures.
Oncomine Focus Assay (Thermo Fisher) Hotspot genes, n=35 Copy Number Variants, n=19 Fusion drivers, n=23 AKT1 ALK AR BRAF CDK4 CTNNB1 DDR2 EGFR ERBB2 ERBB3 ERBB4 ESR1 FGFR2 FGFR3 GNA11 GNAQ HRAS IDH1 IDH2 JAK1 JAK2 JAK3 KIT KRAS MAP2K1 MAP2K2 MET MTOR NRAS PDGFRA PIK3CA RAF1 RET ROS1 SMO ALK AR BRAF CCND1 CDK4 CDK6 EGFR ERBB2 FGFR1 FGFR2 FGFR3 FGFR4 KIT KRAS MET MYC MYCN PDGFRA PIK3CA ALK RET ROS1 NTRK1 NTRK2 NTRK3 FGFR1 FGFR2 FGFR3 MET BRAF RAF1 ERG ETV1 ETV4 ETV5 ABL1 AKT3 AXL EGFR ERBB2 PDGFRA PPARG DNA Panel 52 genes RNA Panel For Research Use Only. Not for use in diagnostic procedures.
Oncomine Focus Assay Workflow Tumor Sample Oncomine Assay* DNA/RNA Kit Ion PGM System* Next-generation Sequencing Ion Reporter Workflow* Automated analysis for SNVs, indels, CNVs and gene fusions Positive Variants Mutations Copy Number Variants Gene Fusions Ion 318 Select Chip* For Research Use Only. Not for use in diagnostic procedures.
NGS-DNA (41) - 31 adenocarcinomas (A) - 1 Large cell carcinoma (C) - 9 metastatic adenocarcinomas (M) Mutación CNV EGFR KRAS ALK BRAF AKT1 MTOR FGFR4 MET PIK3CA JAK ERBB3 BRACA1 APC GNAQ GNA11 KIT MYC A A A A A A A A A A A A A C A A A A M A A A A A M A A M A A A M A M A A M M M A M C5 C9 C12 C14 C15 C16 C22 C24 C25 C27 C11 C10 C21 C13 C1 C2 C18 C8 C33 C34 C36 C37 C26 C4 C39 C7 C32 C38 C19 C23 C28 C17 C3 C20 C29 C6 C30 C31 C35 C40 C41 Mutation HotSpot Mutación HotSpot for validation Amplification
NGS (41) EGFR Nº Cases % Cases WT 27 65,9 DEL19 5 12,2 L858R 6 14,6 L858R/V834L 1 2,4 INS20 1 2,4 DEL19/T790M 1 2,4 COBAS (40) EGFR Nº Cases % Cases WT 28 70,0 DEL19 5 12,5 L858R 6 15,0 INS20 1 2,5
DISCORDANT CASES 7,5%, 3/40 COBAS DEL19 WT L858R NGS DEL19-T790M L858R L858R-V834L
For Research Use Only. Not for use in diagnostic procedures. Oncomine Knowledgebase Reporter Software
NGS-RNA (37) - 27 adenocarcinomas (A) - 1 Large cell carcinoma (C) - 9 metastatic adenocarcinoma (M) Mutación CNV Fusion EGFR KRAS ALK BRAF AKT1 MTOR FGFR4 MET PIK3CA JAK ERBB3 BRACA1 APC GNAQ GNA11 KIT MYC ALK MET A A A A A A A A A A A A A C A A A A M A A A A A M A A M A A A M A M A A M M M A M C A A M A A A M C5 C9 C12 C14 C15 C16 C22 C24 C25 C27 C11 C10 C21 C13 C1 C2 C18 C8 C33 C34 C36 C37 C26 C4 C39 C7 C32 C38 C19 C23 C28 C17 C3 C20 C29 C6 C30 C31 C35 C40 C41 C42 C43 C44 C45 C46 C47 C48 C49 Mutation HotSpot Mutation HotSpot for validation Amplificaction Unvaluable Fusion - 1 ALK positive confirmed by FISH - 16 ROS1 negative IQ
For Research Use Only. Not for use in diagnostic procedures. Oncomine Knowledgebase Reporter Software
BEAR Project
BEAR Project - Participating Sites
BEAR Project - Samples 48 FFPE samples : each of the 8 sites provided 6 Samples : 4 samples for gene mutation analysis (DNA extraction) 2 samples for fusion transcript analysis (RNA extraction) Previously characterized samples: Colorectal adenocarcinoma Lung adenocarcinoma Melanoma Papillary thyroid carcinoma Pancreatic adenocarcinoma
BEAR Project - Technical Approach - Total number of FFPE samples processed - DNA samples: 32 in triplicate - RNA samples: 16 in triplicate - Material sent from samples: - 3 sections from each sample to each processing laboratory - Laboratories that processed samples: - Hospital Ramon y Cajal Madrid - Hospital 12 de Octubre Madrid - Laboratorio de Dianas Terapeuticas Madrid - Technical support: - FAS: Andrea Verardi, Marco Morey - CAC: Francesco Acquadro
Oncomine Solid Tumour DNA and Fusion Transcript Kits Hotspot Genes n=22 Fusion Drivers n=4 EGFR ALK ERBB2 ERBB4 FGFR1 FGFR2 FGFR3 MET DDR2 KRAS PIK3CA BRAF AKT1 PTEN NRAS MAP2K1 STK11 NOTCH1 CTNNB1 SMAD4 FBXW7 TP53 ALK ROS1 RET NTRK1
BEAR Project - Results DNA 23 New Variants 30 Samples Oncomine Solid Tumor DNA CE-IVD General Filter BEAR 32 Previously characterized Variants Sensitivity: 100% (32/32) *In addition 23 potentially pathogenic variants not previously tested were found
BEAR Project - Results DNA WT BRAF mut EGFR mut KRAS mut NRAS TP53 P L1 LQ LQ L2 L3 LQ Wild type cases BRAF mut cases EGFR mut cases KRAS mut cases NRAS mut cases TP53 mut cases KRAS +TP53 mut cases BRAF + EGFR mut cases Low quality results P: previous characterization L1, L2, L3: results from the 3 labs performing Ion Torrent analysis LQ: low quality
BEAR Project Results DNA- Concordance L1 30 Samples 100% Concordance L2 30 Samples L1 26 Samples * 96% Concordance L2 26 Samples L3 26 Samples *1/26 bad quality samples didn't give results
BEAR Project Results DNA- Conclusions - NGS method used shows a very strong inter-laboratory concordance of results obtained - A total of 55 variants were identified in the 30 processed samples - All the expected variants (n=32) were identified (Sensitivity 100%) - 23 potentially pathogenic extra variants (not previously tested) were identified in the samples
Laboratorio de Dianas Terapeuticas Bárbara Angulo Carolina Dominguez Susana Hernández Fernando López Rios Hospital 12 de Octubre Jose Luis Rodríguez Peralto Yolanda Ruano Diana Cantero Hospital Ramon y Cajal José Palacios Tamara Caniego Almudena Santón Hospital Virgen del Rocio Michele Biscuola Enrique de Álava Fundación Jiménez Diaz Federico Rojo Cristina Chamizo Nerea Carvajal Hospital del Mar Beatriz Bellosillo Raquel Longaron Erica Torres Sergi Serrano Hospital Universitario Vall d Hebrón Javier Hernández Losa Roso Mares Rosa Somoza Hospital Universitario Marques de Valdecilla Javier Gómez Miguel Angel Piris Thermo Fisher Scientific and Technical support: Francesco Acquadro, CAC Andrea Verardi, FAS ; Marcos Morey, FAS
NGS strategies that we have tried for TMB: pros and cons Oncomine Tumor Mutation Load Assay TruSight Tumor 170 Qiaseq Comprehensive Cancer Panel Genes, Input sample and Technology 409 genes DNA RNA Amplicon-based 170 genes DNA RNA Hybridization capturebased 275 genes DNA RNA Hybridizationextension-based Detected alterations SNVs, CNVs, small InDels DNA: SNVs, CNVs, Indels RNA: Fusions, Splice Variants SNVs, CNVs, small Indels Horizontal coverage 1,75 Mb Mutational burden 0,52 Mb Mutational burden? 0,83 Mb Mutational burden? Data analysis Ion Reporter Software More artefacts Base Space Sequence Hub Software Data Less artefacts Insight Software Less artefacts (UMIs) Thermo Fisher Illumina Qiagen
TMB Mutations/Mb 32 NSCLCs from Hospital 12 de Octubre (Madrid) Oncomine Tumor Mutation Load Assay 9.3% High 44% Intermediate TMB Groups Mutations/Mb Low 1-5 Intermediate 6-19 High 20 Samples 46% Low For Research Use Only. Not for use in diagnostic procedures.
Comparison study of technologies using Foundation One as gold standard Patient # TMB MS Mutations Found VUS Total Tissue origin Type of tumour Illumina TST170 AmpliSeq CCP QiaSeq CCP Thermo TML D00/MCBM Med 7/Mb Stable STK11, MYC, KEAP1, TP53 ATR, BRIP1, CEBPA, EP300, FAM123B, IRS2, SDHA, TGFBR2, WT1 13 Brain Unknown primary adenocarcinoma 8,5 D01/MEER High 62/Mb Stable ERBB2, NF1, STK11, ERRFI1, MYC, ASXL1, CDKN2A, SMARCA4, TET2, TP53 44 54 Soft tissue Unknown primary adenocarcinoma 54 D02/ERR D03/MPCR D04/ARA Low 2/Mb Low 1/Mb Low 4/Mb Stable MAP2K1, ARID1A, CDKN2A/B, KDM5C, KDM6A BRCA2, STAT2, MAP3K1, SYK, TNFAIP3, MLL2, NSD1 12 Soft tissue Stable BCOR, MYB AKT1, AR, CTNNA1, FAT1 6 Lung Stable MLL3 FANCA, WISP 3 Lung Unknown primary squamous cell carcinoma Lung adenoid cystic carcinoma Lung adenoid cystic carcinoma 4 D08/MTLC Med 15/Mb Stable STK11, CDKN2A/B, KEAP1, TP53 20 24 Lung Lung adenocarcinoma 7,5 D10/MARP Low 4/Mb Stable KRAS, PIK3CA, APC, ARID1A, FAM1234B, TP53 BRIP1, SMAD2, MAP3K1, SMAD3, NOTCH1, ZNF703, POLE, PTCH1, ROS1 15 Peritoneum Unknown primary adenocarcinoma 10 70 60 y = 1.077x - 2.9544 R² = 0.9648 50 TML Thermo 40 30 20 10 0 0 10 20 30 40 50 60 70 FoM
Conclusions NGS platform must be previously tested before to implant in a pathology laboratory NGS with a specific platforms is an effective and efficient technology NGS is accurate, easily to implant, cheaper and saver tissue than sequential PCR NGS is an excellent and precise technology to detect actionable mutations Some platform reports ie. Oncomine Knowledgebase Reporter Software facilitate the selection of target drugs and the clinical trials available for these patients. Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo Fisher Scientific products presented by third parties during this symposium. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.