Platform Molecular Diagnostics UZ Ghent (MDG)

The MDG platform's core activity is the detection of specific somatic defects in the DNA and RNA of the malignant tissues, blood or bone marrow of patients with cancer.

The tests are requested through the Department of Pathological Anatomy UZ Ghent or through the Laboratory of Clinical Biology UZ Ghent (hemato-oncology). Laboratory tests are performed at the Center for Medical Genetics Ghent.

Please refer to the Guidelines for test requests and Sample Instructions.

Accredited MDG tests in routine (ISO 15189:2012):

SOLID TUMORS:

• DNA NGS SOLID TUMOR panel: detection of SNVs and indels in 112 genes: AKT1, ALK, APC, AR, ARID1A, ATM, BAP1, BARD1, BCOR, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CDH1, CDK4, CDK6, CDK12, CDKN2A, CDKN2B, CHEK1, CHEK2, CTNNB1, DDX3X, DGCR8, DICER1, DPYD, DROSHA, EGFR, EIF1AX, ELP1, ERBB2, ERBB3, ESR1, FBXW7, FGFR1, FGFR2, FGFR3, FGFR4, FOXL2, FRK, GATA3, GNA11, GNAQ, GNAS, GPR161, H3-3A, H3-3B, H3C2, H3C3, HNF1A, HRAS, IDH1, IDH2, IL6ST, JAK1, JAK2, KBTBD4, KEAP1, KIT, SCRATCH, MAP2K1, MDM2, MET, MRE11, MTAP, MYC, MYCN, MYOD1, NBN, NF1, NF2, NOTCH1, NRAS, NTRK1, NTRK2, NTRK3, PALB2, PDGFRA, PDGFRB, PIK3CA, PIK3R1, POLE, PRKCA, PTCH1, PTEN, PTPN11, RAD50, RAD51B, RAD51C, RAD51D, RB1, RET, RNF43, ROS1, SDHA, SDHB, SDHC, SDHD, SF3B1, SMAD4, SMARCA4, SMARCB1, SMO, SPOP, STAT3, STK11, SUFU, TERT, TP53, VHL using the KAPA HyperCap technology (Roche).The specifications of the regions examined and the reference sequences of the genes can be accessed here. The quality of the DNA sample extracted from tumor material is checked using a DNA quality test. A capture-based NGS technology is used for enrichment of the regions of interest of the 112 genes followed by sequencing with the Illumina technology. Data analysis is performed using an in-house bcbio workflow. The sequencing reads are aligned with a reference sequence (hg38). For release of NGS results there is aimed for a minimum coverage of 300x. All 112 genes are looked at in the tumor samples. Only pathogenic variants, presumably pathogenic variants and variants of unknown significance (VUS) are reported (Richards et al. Genet Med 2015).The limit for detecting a variant is 5%. As an exception, known hotspot variants are also reported when the variant's allele frequency (VAF) is between 2 - 5% and the coverage is > 300x and the variant is present in > 10 reads. With this test, somatic and a germline variants cannot be distinguished.
  Additionally, this panel allows MSI analysis based on 17 microsatellite regions: BAT25, BCL2L11, BTBD7, D18S55, EML4, GRIN2A, GTF2IP1, KDM6A, KIF5B, MRE11A, NR21, NR24, NR27, PIP5K1A, RYR3, SMARCB1, TGFBR2 using the mSINGS script (Salipante et al. Clinical Chemistry 2014). This analysis has a sensitivity of 74% and specificity of 100%. The limit for detecting microsatellite instability is 30% tumor cells.

• RNA NGS SOLID TUMOR panel: detection of fusions and splice variants in 26 genes: ALK, BRAF, EGFR, ERG, FGFR1, FGFR2, FGFR3, KRAS, MET, MYB, MYBL1, MYC, NRG1, NTRK1, NTRK2, NTRK3, PPARG, PRKCA, RAF1, RELA, RET, ROS1, RSPO2, RSPO3, TMPRSS2, YAP1 using Archer FusionPlex technology. The specifications of the investigated regions and the reference sequences of the genes can be accessed here. The quality of the RNA extracted from tumor tissue is checked using an RNA quality assay. An amplicon-based NGS technology is used for enrichment of the regions of interest from the 26 genes followed by sequencing with the Illumina technology. Data analysis is performed using Archer Analysis Software. The sequencing reads are aligned to a reference sequence (hg19) using the Archer Analysis Software module. For release of results, we aim for a minimum of 10 unique reads, > 1% percentage of reads and minimum 5 unique start sites for the fusions/splice variants and minimum 0.6M total reads per sample.
  
  
• RNA NGS SARCOMA panel: detection of fusions and splice variants in 55 genes: ALK, BCOR, BRAF, CAMTA1, CIC, CSF1, CTNNB1, EGFR, EPC1, ERG, ESR1, EWSR1, FGFR1, FGFR2, FGFR3, FOS, FOSB, FOXO1, FUS, GLI1, HMGA2, JAZF1, MDM2, MEAF6, MET, MGEA5, MKL2, MYOD1, NCOA1, NCOA2, NR4A3, NTRK1, NTRK2, NTRK3, NUTM1, PAX3, PDGFB, PHF1, PLAG1, PRKCA, PRKCB, PRKCD, RAF1, RET, ROS1, SS18, STAT6, TAF15, TCF12, TFE3, TFG, USP6, VGLL2, YAP1, YWHAE using the Archer FusionPlex technology. The specifications of the investigated regions and the reference sequences of the genes can be accessed here. The quality of the RNA sample extracted from tumor tissue is checked using an RNA quality assay. An amplicon-based NGS technology is used for enrichment of the regions of interest from the 55 genes followed by sequencing with the Illumina technology. Data analysis is performed using Archer Analysis Software. The sequencing reads are aligned with a reference sequence (hg19) using the Archer Analysis Software module. For release of results, we aim for a minimum of 10 unique reads, > 1% percentage of reads and minimum 5 unique start sites for the fusion/splice variants and minimum 1.8M total reads per sample.
• MDG-MGMT assay: detection of MGMT promoter methylation in glioblastoma.

For the examination of somatic mutations in solid tumors and sarcomas, the tumor tissue in FFPE should be sent to the Department of Pathological Anatomy UZ Ghent, using the request form of that department. See the laboratory guide of the Department of Pathological Anatomy UZ Ghent. In exceptional cases, fresh or frozen tumor tissue can also be sent to Center Medical Genetics. Sample instructions and request form via acquired genetic disorders (https://www.cmgg.be/en/professionals/labguide/acquired-genetic-disorders)

HEMATOLOGIC DISORDERS:

• DNA NGS HEMATO panel: detection of SNVs and indels in 116 genes: ANKRD26, ARID1A, ASXL1, ATM, B2M, BCL2, BCOR, BCORL1, BIRC3, BRAF, BTG1, BTK, CALR, CARD11, CBL, CBLB, CCL22, CCND1, CCND3, CD28, CD33, CD58, CD79A, CD79B, CDKN2A, CEBPA, CREBBP, CSF3R, CUX1, CXCR4, DDX3X, DDX41, DIS3, DNMT3A, EGR2, EP300, ETHNK1, ETV6, EZH2, FAS, FBXW7, FLT3, FOXO1, FYN, GATA1, GATA2, GNA13, GNB1, HAVCR2, HRAS, ID3, IDH1, IDH2, IL7R, IRF4, JAK1, JAK2, JAK3, KIT, KLF2, KMT2A, KMT2D, KRAS, MAP2K1, MEF2B, MPL, MYC, MYD88, NF1, NFE2, NFKBIE, NOTCH1, NOTCH2, NPM1, NRAS, NSD2, NUDT15, PHF6, PIK3CA, PIM1, PLCG1, PLCG2, POT1, PPM1D, PRKCB, PRPF8, PTEN, PTPN11, RAD21, RHOA, RUNX1, SETBP1, SF3B1, SGK1, SH2B3, SMC1A, SMC3, SOCS1, SRSF2, STAG2, STAT3, STAT5B, STAT6, TCF3, TET2, TNFAIP3, TNFRSF14, TP53, TPMT, TRAF2, U2AF1, UBA1, UBTF, WT1, XPO1, ZRSR2 using the KAPA HyperCap technology (Roche). The specifications of the regions examined and the reference sequences of the genes can be consulted in the Clinical Biology laboratory guide https://labgids.uzgent.be/. A capture-based NGS technology is used for enrichment of the regions of interest from the 116 genes followed by sequencing with the Illumina technology. Data analysis is performed using an in-house bcbio workflow. The sequencing reads are aligned with a reference sequence (hg38). A minimum coverage of 300x for regions of interest is targeted for release of NGS results. All 116 genes are looked at in the hemato-onco samples. Only pathogenic variants, suspected pathogenic variants and variants of unknown significance (VUS) are reported (Richards et al. Genet Med 2015). The limit for detecting a variant is 5%. As an exception, known hotspot variants that are also reported between when the variant's allele frequency (VAF) is between 2 - 5% and the coverage is > 300x and the variant is present in > 10 reads. With this test, somatic and a germline variants cannot be distinguished.
• MDG-MPN1-mini assay: detection of SNVs and indels in CALR, JAK2 and MPL in MPN. An in-house PCR-based NGS enrichment is used followed by sequencing with Illumina technology. Data analysis is performed using an in-house bcbio workflow. The sequencing reads are aligned with a reference sequence (hg38). A minimum coverage of 300x for the regions of interest is targeted for release of the NGS results. Only pathogenic variants, presumably pathogenic variants and variants of unknown significance (VUS) are reported (Richards et al. Genet Med 2015). The limit for detecting a variant is 5%. As an exception, known hotspot variants that are also reported when the variant's allele frequency (VAF) is between 2 - 5% and the coverage is > 300x and the variant is present in > 10 reads. With this test, somatic and a germline variants cannot be distinguished.
• RNA NGS ALL panel: detection of fusions and splice variants in 64 genes: ABL1, ABL2, BCL11B, BCL2, BCL6, BCR, BRAF, CHD1, CREBBP, CRLF2, CSF1R, DNM2, EBF1, EPOR, ETV6, EZH2, FBXW7, FGFR1, FLT3, IDH1, IDH2, IKZF1, IKZF2, IKZF3, IL7R, JAK1, JAK2, JAK3, KDM6A, KLF2, KMT2A (MLL), KRAS, MLLT4, MPL, MYC, NF1, NOTCH1, NRAS, NT5C2, NTRK3, NUP214, NUP98, P2RY8, PAG1, PAX5, PBX1, PDCD1LG2, PDGFRA, PDGFRB, PICALM, PTK2B, PTPN11, RUNX1, SEMA6A, SETD2, SH2B3, STAT3, STAT5B, STIL, TAL1, TCF3, TYK2, WT1, ZCCHC7 using Archer FusionPlex technology. The specifications of the regions examined and the reference sequences of the genes can be consulted in the Laboratory Guide of the Clinical Biology https://labgids.uzgent.be/. An amplicon-based NGS technology is used for enrichment of the regions of interest of the 64 genes followed by sequencing with the Illumina technology. Data analysis is performed using Archer Analysis Software. The sequencing reads are aligned with a reference sequence (hg19) using the Archer Analysis Software module.
• RNA NGS HEME panel: detection of fusions and splice variants in 87 genes: ABL1, ABL2, ALK, BCL11B, BCL2, BCL3, BCL6, BCR, BIRC3, CBFB, CCND1, CCND2, CCND3, CD274, CDK6, CDKN2A, CEBPA, CEBPD, CEBPE, CEBPG, CHD1, CHIC2, CIITA, CREBBP, CRLF2, CSF1R, CTLA4, DEK, DUSP22, EBF1, EIF4A1, EPOR, ERG, ETV6, FGFR1, FOXP1, GLIS2, ID4, IKZF1, IKZF2, IKZF3, IRF4, IRF8, JAK2, KAT6A, KLF2, KMT2A, MALT1, MECOM, MKL1, MLF1, MLLT10, MLLT4, MUC1, MYC, MYH11, NF1, NFKB2, NOTCH1, NTRK3, NUP214, NUP98, P2RY8, PAG1, PAX5, PDCD1, PDCD1LG2, PDGFRA, PDGFRB, PICALM, PML, PRDM16, PTK2B, RARA, RBM15, ROS1, RUNX1, RUNX1T1, SEMA6A, SETD2, STIL, TAL1, TCF3, TFG, TP63, TYK2, ZCCHC7 using Archer FusionPlex technology. The specifications of the regions examined and the reference sequences of the genes can be consulted in the Laboratory Guide of the Clinical Biology https://labgids.uzgent.be/. An amplicon-based NGS technology is used for enrichment of the regions of interest of the 87 genes followed by sequencing with the Illumina technology. Data analysis is performed using Archer Analysis Software. The sequencing reads are aligned with a reference sequence (hg19) using the Archer Analysis Software module.

To request an MDG test for a patient with a hemato-oncology condition, send the blood or bone marrow to the Clinical Biology UZ Gent Laboratory, using that department's request form. See the Laboratory Guide of the Clinical Biology Laboratory UZ Gent: https://labgids.uzgent.be/ (choose Request forms - Request form special hematology - cytomorphology | immunophenotyping | molecular analyses).

MDG tests under development:

- ctDNA NGS SOLIDE TUMOR panel on liquid biopsies from solid tumor patients

- methylation profiling for solid tumor samples

- comprehensive genomic profiling (CGP) of solid tumor samples on liquid biopsies and on FFPE samples

- homologous recombination deficiency (HRD) testing of solid tumor samples