CAP recommends using neoplastic cellularity as the number of neoplastic cells or nuclei compared to the overall or toal number of cells or nuclei.

Massive Parallel Squencing (Next Generation Sequencing) is ran on the Illumina iSeq 100, MiniSeq and NextSeq 500, using "sequencing by synthesis" chemistry, upon different targeted sequencing panels.

Genomic DNA QC : Quantified by Qubit and the integrity control by conventional PCR
Library QC : Check by automatic electrophoresis (Bioanalyser)
Sequencing QC : Coverage > 500x, Q30 > 80%

Bioinformatics :
Illumina Local Run Manager on a desktop PC DNA Amplicon (BaseSpace Workflow) 2.1.0.19 [DNA Amplicon Workflow (3.24.1.8+ master), BWA-MEM Whole-Genome(Aligner)0.7.9a-isis-1.0.2, Pisces Variant Caller 5.2.9.23, Illumina Annotation Engine 2.0.11-0- g7fb24a09, Bam Metrics v0.0.22, and SAMtools 0.1.19-isis-1.0.3, Annotation Dataset 91.26.44]


Curation :
Human GRCh37/hg19, ClinVar, Genome Nexus, Franklin, VarSome


Limitations : The workflow could detect SNV-indel type mutations with a variant allelic frequency greater or equal to 5%. However, variants with VAF<10% might fall short of Sanger sequencing confirmation. Due to the nature of FFPE specimen and limitations of the amplicon-based library preparation, genomic changes leading to false negativity, including, but not limited to, large deletion, large duplication and gross chromosomal abnormalities, cannot be completely excluded.

The Ion Torrent Genexus System automates sample and library preparation, sequencing, analysis, and reporting. The Genexus Purification System and Genexus Integrated Sequencer with Genexus Software work together seamlessly, tracking sample information and results automatically through the process. This automated system reduces manual steps and facilitates quality data and quick reports.

Genomic DNA/RNA QC : Quantified by Qubit and the integrity control by conventional PCR
Library QC : Integrated in the automated system
DNA Sequencing QC : >=Q20 bases, Uniformity
RNA Sequencing QC : >=Q20 bases, Mapped

Bioinformatics : Integrated
The factory bioinformatics reports a standardized table of known variants. An in-house bioinformatic pipeline (GATK-Mutect2 calling) is installed for screening uncalled variants for the 12-gene lung cancer panel. Sequencing data of selected cases should also be reviewed by Integrated Genome Viewer (IGV).

Curation :
Human GRCh37/hg19, ClinVar, Genome Nexus, Franklin, VarSome


Limitations : The workflow could detect SNV-indel type mutations with a variant allelic frequency greater or equal to 5%. However, variants with VAF less than 10% might fall short of Sanger sequencing confirmation. Due to the nature of FFPE specimen and limitations of the amplicon-based library preparation, false negative results cannot be completely excluded. These latter missed genomic changes might include large deletion, large duplication, gross chromosomal abnormalities, intronic variants, etc.

Somatic mutation testing is done on the FFPE block or cytology cell block.

Germline mutation testing is performed on FFPE block and peripheral white cells from the EDTA blood sample, and is also supplemented by MLPA.

Sequencing : The AmpliSeq for Illumina BRCA Panel is a targeted resequencing assay for somatic and germline variants across BRCA1 and BRCA2 genes, targeting all the coding exonic regions and the flanking intronic sequences of the BRCA1 and BRCA2 genes.

MLPA : SALSA MLPA Probemix kits (MRC, Holland): P002 BRCA1, P090 BRCA2 and P239 BRCA1 region;
confirmed by P087 BRCA1 Confirmation or P077 BRCA2 Confirmation whichever is applicable; Coffalyzer.Net software v.220513.1739 (database v.220401.0000) for quality control, visualization and analysis of data.

All findings are to be confirmed by orthogonal methods, including Sanger sequencing, RT-PCR and FISH.

Consent is necessary for ordering this test (either somatic test alone, or somatic and germline tests). (Engish) (Chinese)

Pamphlets for BRCA testing in high staged, high grade ovarian cancer (English)(Chinese)

The Oncomine Precision Assay, Ion Torrent Genexus System, is an automatic targeted assay for genetic analysis of 50 genes with known relevance to solid tumors. Variants can be confirmed by orthogonal methods (PCR-Sanger sequencing, RT-PCR + Sanger, FISH, etc.). SNV/indel, CNV and fusion variants are included.

Actionable variants in a 12-gene lung cancer somatic mutation panel are reported:
ALK, BRAF, EGFR, ERBB2, KRAS, MET, NTRK1, NTRK2, NTRK3, PIK3CA, RET and ROS1 genes

Hot spot mutations (45):
AKT1 AKT2 AKT3 ALK AR ARAF BRAF CDK4 CDKN2A CHEK2 CTNNB1 EGFR ERBB2 ERBB3 ERBB4 ESR1 FGFR1 FGFR2 FGFR3 FGFR4 FLT3 GNA11 GNAQ GNAS HRAS IDH1 IDH2 KIT KRAS MAP2K1 MAP2K2 MET MTOR NRAS NTRK1 NTRK2 NTRK3 PDGFRA PIK3CA PTEN RAF1 RET ROS1 SMO TP53

Copy Number Variants (14):
ALK AR CD274 CDKN2A EGFR ERBB2 ERBB3 FGFR1 FGFR2 FGFR3 KRAS MET PIK3CA PTEN

Fusion Drivers (18):
ALK AR BRAF EGFR ESR1 FGFR1 FGFR2 FGFR3 MET NRG1 NTRK1 NTRK2 NTRK3 NUTM1 RET ROS1 RSPO2 RSPO3

As backup: Illumina Focus Panel on iSeq 100: The AmpliSeq for Illumina Focus Panel is a targeted resequencing assay for biomarker analysis of 52 genes with known relevance to solid tumors. Variants can be confirmed by orthogonal methods (PCR-Sanger sequencing, RT-PCR + Sanger, FISH, etc.). SNV/indel and fusion variants are reported. Detection of CNV variant required cloud computation and is not routinely performed.

Actionable variants in a 12-gene lung cancer somatic mutation panel are reported:
ALK, BRAF, EGFR, ERBB2, KRAS, MET, NTRK1, NTRK2, NTRK3, PIK3CA, RET and ROS1 genes

Hot spot mutations (35):
AKT1, ALK, AR, BRAF, CDK4, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ESR1, FGFR1, FGFR2, FGFR3, GNA11, GNAQ, HRAS, IDH1, IDH2, JAK1, JAK2, JAK3, KIT, KRAS, MAP2K1, MAP2K2, MET, MTOR, NRAS, PDGFRA, PIK3CA, RAF1, RET, ROS1, SMO

Copy Number Variants (19):
ALK, AR, BRAF, CCND1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, FGFR4, KIT, KRAS, MET, MYC, MYCN, PDGFRA, PIK3CA

Fusion Drivers (23):
ABL1, ALK, AKT3, AXL, BRAF, EGFR, ERBB2, ERG, ETV1, ETV4, ETV5, FGFR1, FGFR2, FGFR3, MET, NTRK1, NTRK2, NTRK3, PDGFRA, PPARG, RAF1, RET, ROS1

The AmpliSeq for Illumina Focus Panel is a targeted resequencing assay for biomarker analysis of 52 genes with known relevance to solid tumors. Variants can be confirmed by orthogonal methods (PCR-Sanger sequencing, RT-PCR + Sanger, FISH, etc.). SNV/indel and fusion variants are reported. Detection of CNV variant required cloud computation and is performed infrequently.

Actionable variants in a 12-gene lung cancer somatic mutation panel are reported:
ALK, BRAF, EGFR, ERBB2, KRAS, MET, NTRK1, NTRK2, NTRK3, PIK3CA, RET and ROS1 genes

Hot spot mutations (35):
AKT1, ALK, AR, BRAF, CDK4, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ESR1, FGFR1, FGFR2, FGFR3, GNA11, GNAQ, HRAS, IDH1, IDH2, JAK1, JAK2, JAK3, KIT, KRAS, MAP2K1, MAP2K2, MET, MTOR, NRAS, PDGFRA, PIK3CA, RAF1, RET, ROS1, SMO

Copy Number Variants (19):
ALK, AR, BRAF, CCND1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, FGFR4, KIT, KRAS, MET, MYC, MYCN, PDGFRA, PIK3CA

Fusion Drivers (23):
ABL1, ALK, AKT3, AXL, BRAF, EGFR, ERBB2, ERG, ETV1, ETV4, ETV5, FGFR1, FGFR2, FGFR3, MET, NTRK1, NTRK2, NTRK3, PDGFRA, PPARG, RAF1, RET, ROS1

The AmpliSeq for Illumina Comprehensive Panel v3 offers coverage of key cancer genes, including kinase domains and genes involved in DNA repair, targetting 161 unique cancer-associated genes

Hot spot mutations:
AKT1, AKT2, AKT3, ALK, AR, ARAF, AXL, BRAF, BTK, CBL, CCND1, CDK4, CDK6, CHEK2, CSF1R, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ERCC2, ESR1, EZH2, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, FOXL2, GATA2, GNA11, GNAQ, GNAS, H3F3A, HIST1H3B, HNF1A, HRAS, IDH1, IDH2, JAK1, JAK2, JAK3, KDR, KIT, KNSTRN, KRAS, MAGOH, MAP2K1, MAP2K2, MAP2K4, MAPK1, MAX, MDM4, MED12, MET, MTOR, MYC, MYCN, MYD88, NFE2L2, NRAS, NTRK1, NTRK2, PDGFRA, PDGFRB, PIK3CA, PIK3CB, PPP2R1A, PTPN11, RAC1, RAF1, RET, RHEB, RHOA, ROS1, SF3B1, SMAD4, SMO, SPOP, SRC, STAT3, TERT, TOP1, U2AF1, XPO1

Full-length genes:
ARID1A, ATM, ATR, ATRX, BAP1, BRCA1, BRCA2, CDK12, CDKN1B, CDKN2A, CDKN2B, CHEK1, CREBBP, FANCA, FANCD2, FANCI, FBXW7, MLH1, MRE11A, MSH2, MSH6, NBN, NF1, NF2, NOTCH1, NOTCH2, NOTCH3, PALB2, PIK3R1, PMS2, POLE, PTCH1, PTEN, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RB1, RNF43, SETD2, SLX4, SMARCA4, SMARCB1, STK11, TP53, TSC1, TSC2

Copy number variants:
AKT1, AKT2, AKT3, ALK, AR, AXL, BRAF, CCND1, CCND2, CCND3, CCNE1, CDK2, CDK4, CDK6, CDKN2A, CDKN2B, EGFR, ERBB2, ESR1, FGF19, FGF3, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, IGF1R, KIT, KRAS, MDM2, MDM4, MET, MYC, MYCL, MYCN, NTRK1, NTRK2, NTRK3, PDGFRA, PDGFRB, PIK3CA, PIK3CB, PPARG, RICTOR, TERT, TSC1, TSC2

Fusion drivers:
AKT2, ALK, AR, AXL, BRAF, BRCA1, BRCA2, CDKN2A, EGFR, ERBB2, ERBB4, ERG, ESR1, ETV1, ETV4, ETV5, FGFR1, FGFR2, FGFR3, FGR, FLT3, JAK2, KRAS, MDM4, MET, MYB, MYBL1, NF1, NOTCH1, NOTCH4, NRG1, NTRK1, NTRK2, NTRK3, NUTM1, PDGFRA, PDGFRB, PIK3CA, PPARG, PRKACA, PRKACB, PTEN, RAD51B, RAF1, RB1, RELA, RET, ROS1, RSPO2, RSPO3, TERT