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Useful For
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The next generation sequencing (NGS) is a high throughput cost-effective sequencing technology which is useful for comprehensive molecular analysis of tumor’s genomic components of the tumor site for each cancer patient. It is used to identify molecular biomarkers within the patient’s genetic profile which will help to select targeted therapy against the identified actionable mutations or genes or their related pathways to allow accurate management for cancer patients. It helps to deliver high quality clinical services for different groups of cancer patients to improve their quality of life.
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Method name and description
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Next Generation Sequencing for Solid Tumors. Different panels are used to serve different types of cancers.
The tumor area is collected from slides of a formalin-fixed paraffin-embedded (FFPE) specimen; this area was identified by the consultant pathologist from which genomic DNA/RNA was extracted and analyzed by using Next Generation Sequencing NGS. Data generated were analyzed for alterations in the following Hotspots genes, copy number variations (CNV) and fusion drivers.
The lower limit of mutation detection using NSG assay is 5 – 10 % mutant allele frequency. This test did not distinguish between somatic and germline mutation. Any interpretation has been provided with the assumption that the variants detected are somatic mutations. Results are reported based on this technique only, no additional confirmation was performed.
The method is designed to detect point mutations, deletion, insertion, hotspots, fusions and copy number variant. For point mutations, deletion, insertion; DNA is required from the sample. For gene fusions; RNA is required from the sample.
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Reporting name
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The NGS report includes information about the following:
Hotspots genes (DNA sequencing).
Copy number variations (CNV).
Fusion drivers (RNA sequencing).
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Clinical information
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Next-generation sequencing technologies have developed in clinical laboratories, enabling rapid transformations in genomic medicine. The NGS reduced the cost of large-scale sequencing by several orders. It allows to analyze each patients’ genomic components to assist in the diagnosis and management of different cancer types. Next-generation sequencing technologies are also facilitating further advances in therapeutic decision making and disease prediction for at-risk patients.
Worldwide, three main levels of analysis, with increasing degrees of complexity, are possible to be performed using NGS: disease targeted gene panels, exome sequencing (ES), and genome sequencing (GS).
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Specimen type / Specimen volume / Specimen container
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FFPE tissue:
Option 1: Tissue sections of 10X (5-7 µm) fixed on non-charged slides with an H&E stained slide with marked tumor area.
Option 2: Tissue curls of 6 sections (10 µm) collected in 2 mL tube (e.g. Eppendorf).
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Collection instructions / Special Precautions / Timing of collection
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Specimens are arranged from Sunday to Wednesday from the Anatomical Pathology Department to the Molecular Genetics Laboratory at room temperature. Please avoid direct sun light exposure.
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Relevant clinical information to be provided
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The following points must be provided:
- The tumor rich area on the must be marked by a consultant pathologist.
- Tumor percentage.
- Cancer type.
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Storage and transport instructions
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The specimens are stored and transported at Room Temperature (16 - 25° C).
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Specimen Rejection Criteria
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Biological reference intervals and clinical decision values
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The lower limit of detection is 5%.
Differents NGS panels are used for Solid Tumors. The used panel is selected based on the cancer type of each patient.
Panel-1:
This assay is used to unique variants (hotspot mutations, CNV genes, and fusion isoforms) in different cancer types mainly lung, colorectal, melanoma and GIST.
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Hotspot genes and screened genes
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Copy number variants (CNV)
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Fusion drivers
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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.
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ALK, AR, BRAF, CCND1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, FGFR4, KIT, KRAS, MET, MYC, MYCN, PDGFRA, PIK3CA
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ABL1, AKT3, ALK, AXL, BRAF, ERG, ETV1, ETV4, ETV5, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, MET, NTRK1, NTRK2, NTRK3, PDGFRA, PPARG, RAF1, RET, ROS1.
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Panel-2:
This assay is used to detect 2,769 total unique variants (hotspot mutations, CNV genes, and fusion isoforms), with some key statistics: 269 TP53 mutations which accounts for approximately 90% of known, recurrent hotspots, 151 NTRK1/2/3 fusion isoforms across varying partners and breakpoints, 156 FGFR1/2/3 fusion isoforms across varying partners and breakpoints, 89 RET fusion isoforms across varying partners and breakpoints, 3 intra-genetic variants (MET Exon 14 Skip, EGFRviii, and ARv7) and 218 potential resistance mutations across 22 genes.
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Hotspot genes and screened genes
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Copy number variants (CNV)
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Fusion drivers
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AKT1, AKT2, AKT3, ALK, AR, ARAF, BRAF, CDK4, CDKN2A, Check2, CTNNB1, EGFR, ERBB2, ERBB3, ERBB4, ESR1, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, GNA11, GNAQ, HRAS, IDH1, IDH2, KIT, KRAS, MAP2K1, MAP2K2, MET, MTOR, NRAS, NTRK1, NTRK2, NTRK3, PDGFRA, PIK3CA, PTEN, RAF1, RET, ROS1, SMO, TP53.
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ALK, AR, BRAF, CD274, CDKN2A, EGFR, ERBB2, ERBB3, FGFR1, FGFR2, FGFR3, FGFR4, KRAS, MET, PIK3CA, PTEN.
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ALK, BRAF, ESR1, FGFR1, FGFR2, FGFR3, MET, NRG1, NTRK1, NTRK2, NTRK3, NUTM1, RET, ROS1, RSPO2, RSPO3.
AR, EGFR, MET
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Panel-3:
This assay to detect somatic mutations in BRCA1 and BRCA 2 genes, that are associated with several cancer types such as breast, ovarian, pancreatic, prostate and other cancer types; and their importance as biomarkers for approved cancer therapies and patient management.
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Comprehensive mutation coverage
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BRCA1/BRCA2
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Panel-4:
This molecular diagnostic test for solid tumor is a comprehensive genomic profiling of cancers affecting children and young adults such as Sarcomas and Gliomas. This assay includes 203 unique genes including thousands of fusion drivers (and comprehensive mutation coverage).
It includes a large 97-gene translocation/fusion panel (>1700 fusion isoforms variants), as fusions are relatively more common in childhood sarcomas, but also an 82-DNA target panel with comprehensive coverage of all relevant mutations, 44 targets with full exon coverage, specifically tumor suppressor genes, and 24 CNV targets from DNA and RNA in a single workflow.
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Comprehensive mutation coverage (86): ABL1, ABL2, ALK, ACVR1, AKT1, ASXL1, ASXL2, BRAF, CALR, CBL, CCND1, CCND3, CCR5, CDK4, CIC, CREBBP, CRLF2, CSF1R, CSF3R, CTNNB1, DAXX, DNMT3A, EGFR, EP300, ERBB2, ERBB3, ERBB4, ESR1, EZH2FASLG, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GATA2, GNA11, GNAQ, H3F3A, HDAC9, HIST1H3B, HRAS, IDH1, IDH2, IL7R, JAK1, JAK2, JAK3, KDM4C, KDR, KIT, KRAS, MAP2K1, MAP2K2, MET, MPL, MSH6, MTOR, MYC, MYCN, NCOR2, NOTCH1, NPM1, NRAS, NT5C2, PAX5, PDGFRA, PDGFRB, PIK3CA, PIK3R1, PPM1D, PTPN11, RAF1, RET, RHOA, SETBP1, SETD2, SH2B3, SH2D1A, SMO, STAT3, STAT5B, TERT, TPMT, USP7, ZMYM3.
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Full exon coverage (44): APC, ARID1A, ARID1B, ATRX, CDKN2A, CDKN2B, CEBPA, CHD7, CRLF1, DDX3X, DICER1, EBF1, EED, FAS, GATA1, GATA3, GNA13, ID3, IKZF1, KDM6A, KMT2D, MYOD1, NF1, NF2, PHF6, PRPS1, PSMB5, PTCH1, PTEN, RB1, RUNX1, SMARCA4, SMARCB1, SOCS2, SUFU, SUZ12, TCF3, TET2, TP53, TSC1, TSC2, WHSC1, WT1, XIAP.
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Copy number variation (CNV) (28): ABL2, ALK, BRAF, CCND1, CDK4, CDK6, EGFR, ERBB2, ERBB3, FGFR1, FGFR2, FGFR3, FGFR4, GLI1, GLI2, IGF1R, JAK1, JAK2, JAK3, KIT, KRAS, MDM2, MDM4, MET, MYC, MYCN, PDGFRA, PIK3CA.
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Fusion and expression (98): ABL1, ABL2, AFF3, ALK, BCL11B, BCOR, BCR, BRAF, CAMTA1, CCND1, CIC, CREBBP, CRLF2, CSF1R, DUSP22, EGFR, ETV6, EWSR1, FGFR1, FGFR2, FGFR3, FLT3, FOSB, FUS, GLI1, GLIS2, HMGA2, JAK2, KAT6A, KMT2A, KMT2B, KMT2C, KMT2D, LMO2, MAML2, MAN2B1, MECOM, MEF2D, MET, MKL1, MLLT10, MN1, MYB, MYBL1, MYH11, MYH9, NCOA2, NCOR1, NOTCH1, NOTCH2, NOTCH4, NPM1, NR4A3, NTRK1, NTRK2, NTRK3, NUP214, NUP98, NUTM1, NUTM2B, PAX3, PAX5, PAX7, PDGFB, PDGFRA, PDGFRB, PLAG1, RAF1, RANBP17, RARA, RECK, RELA, RET, ROS1, RUNX1, SS18, SSBP2, STAG2, STAT6, TAL1, TCF3, TFE3, TP63, TSLP, TSPAN4, UBTF, USP6, WHSC1, YAP1, ZMYND11, ZNF384. Gene Expression: BCL2, BCL6, FGFR1, FGFR4, IGF1R, MET, MYCN, MYC, TOP2A.
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Factors affecting test performance and result interpretation
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This test was validated for DNA extraction from FFPE, the FFPE process has effect on the DNA quality and might result on DNA degradation. In addition, PCR inhibitors due to FFPE fixation might affect the PCR amplification and library preparation.
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Turnaround time / Days and times test performed / Specimen retention time
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Days Test is Performed: Arranged by laboratory Rota.
Single gene testing: 10 working days.
Reflex testing: 10 working days for the first test and an additional week (5 working days) for every following test.
For NGS depending on the used panel the TAT is 4 weeks for urgent samples and up to 8 weeks for routine batch.
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