Expanding on WHO guideline compliant molecular testing of central nervous system tumors by low density whole genome sequencing. Karl Kashofer, Phd Institut für Pathologie Medizinische Universität Graz
Disclosures Member of Clinical Oncology Advisory Board of hermo Fisher alks for Bristol-Myers Squibb alks for AstraZeneca
owards an integrated diagnosis incorporation of genetic markers integration of genetic and morphologic findings genetic markers often more reliable than histology genetic markers sometimes trump the histological phenotype Louis et.al, Acta Neuropathol (2016) 131:803 820
High-Grade Malignant Glioma: ESMO Clinical Practice Guidelines Stupp et. al. Ann Oncol (2014) 25 (suppl 3): iii93-iii101. Clinical relevance
MGM Methylation Qiagen MGM Pyro Kit Bisulfit Conversion Conversion of all Cytosins to Uracils No conversion of methylated Cytosine Sequencing of 4 CpG sites in Exon 1 of MGM by Pyrosequencing A => unmethylated C => methylated Ratio A/C Methylation is expressed as average of 4 sites
Qiagen MGM Pyro Kit unmethylated A4: YGAYGYGAGGYG 2% 2% 2% 4% 1000 800 600 400 200 0 E S G C G A C A 5 G C G 10 C A G 15 C G C G 20 no CpG 48% methylated A6: YGAYGYGAGGYG 45% 48% 50% 49% 600 500 400 300 200 100 0 E S G C G 5 A C A G 10 C G C A 15 G 20 no CpG
Meth+ Meth- Bisulfite Sequencing with Ion orrent
Mutation analysis
Mutation analysis Ion orrent Ampliseq Panel highly multiplexed PCR (449 amplicons in 2 pools) reliable results from 10ng DNA 48 genes implicated in neurological tumors ACVR1, AK1, APC, ARX, BRAF, CDKN1A, CIC, CNNB1, DDX3X, EIF1AX, EGFR, FGFR1, FUBP1, GNA11, GNAQ, GNAS, H3F3A, HIS1H3B, HIS1H3C, IDH1, IDH2, KI, KLF4, KLLN, KRAS, LZR1, MAX, ME, MLH1, NOCH1, NRAS, PIK3CA, PCH1, PCH2, PEN, RB1, RE, SF3B1, SMARCA4, SMARCB1, SMO, SUFU, CF12, P53, RAF7, SC1, SC2 and VHL.
Copy Number Variation Gain and Loss of Chromosomes Amplification of defined regions 1p and 19q
Copy Number Variation Gain and Loss of Chromosomes Amplification of defined regions 1p and 19q and many more CN (fat = prognostic) 10p 10q umor glioblastoma IDHwt 50% glioblastoma IDHwt 70%, medulloblastoma Alteration 17q 19q13.42 medulloblastoma embryomal tumor with multilayered rosettes, C19MC alterated high level 1p, 6q, 10, 14q, 18q 1p19q 1q, 9q, 12q, 15q, 17q, 20q 2, 7p, 8q, 14, 17q, 16p 2, 7q, 11q meningeoma grade II oligodendroglioma meningeoma grade II malignant peripheral nerve sheath tumor embryomal tumor with multilayered rosettes, C19MC alterated codeletion gain gain gain 22q 22q11.2 6q meningeoma grade I atypical teratoid / rhabdoid tumor embryomal tumor with multilayered rosettes, C19MC alterated 9p 9p19q meningeoma grade III higher freuqency in anaplastic astrocytoma IDHmut 9q, 11q, 13q, 17p 18, 10q or Xq CCND2 CDK4 CDKN2A malignant peripheral nerve sheath tumor astrocytma IDHmut CDKN2B Chr. 10 Chr. 7 Chr.6 EGFR GLI2 MDM4 ME MYC MYCN MYCN NF1 glioblastoma IDHwt glioblastoma adult glioblastoma adult medulloblastoma glioblastoma medulloblastoma glioblastoma glioblastoma medulloblastoma, astrocytoma IDHmut medulloblastoma medulloblastoma schwannoma, malignant peripheral nerve sheath tumor gain monosomy (region 17q) NF2 PDGFRA PRC2 PRKAR1A schwannoma glioblastoma malignant peripheral nerve sheath tumor schwannoma, malignant peripheral nerve sheath tumor (22q) (region 17q) PCH1 PEN medulloblastoma glioblastoma deletion biallelic deletion malignant peripheral nerve sheath tumor, glioblastoma IDHwt
Copy Number Variation Gain and Loss of Chromosomes Amplification of defined regions 1p and 19q and many more echnique: low density WGS CN (fat = prognostic) 10p 10q umor glioblastoma IDHwt 50% glioblastoma IDHwt 70%, medulloblastoma Alteration 17q 19q13.42 medulloblastoma embryomal tumor with multilayered rosettes, C19MC alterated high level 1p, 6q, 10, 14q, 18q 1p19q 1q, 9q, 12q, 15q, 17q, 20q 2, 7p, 8q, 14, 17q, 16p 2, 7q, 11q meningeoma grade II oligodendroglioma meningeoma grade II malignant peripheral nerve sheath tumor embryomal tumor with multilayered rosettes, C19MC alterated codeletion gain gain gain 22q 22q11.2 6q meningeoma grade I atypical teratoid / rhabdoid tumor embryomal tumor with multilayered rosettes, C19MC alterated 9p 9p19q meningeoma grade III higher freuqency in anaplastic astrocytoma IDHmut 9q, 11q, 13q, 17p 18, 10q or Xq CCND2 CDK4 CDKN2A malignant peripheral nerve sheath tumor astrocytma IDHmut CDKN2B Chr. 10 Chr. 7 Chr.6 EGFR GLI2 MDM4 ME MYC MYCN MYCN NF1 glioblastoma IDHwt glioblastoma adult glioblastoma adult medulloblastoma glioblastoma medulloblastoma glioblastoma glioblastoma medulloblastoma, astrocytoma IDHmut medulloblastoma medulloblastoma schwannoma, malignant peripheral nerve sheath tumor gain monosomy (region 17q) NF2 PDGFRA PRC2 PRKAR1A schwannoma glioblastoma malignant peripheral nerve sheath tumor schwannoma, malignant peripheral nerve sheath tumor (22q) (region 17q) PCH1 PEN medulloblastoma glioblastoma deletion biallelic deletion malignant peripheral nerve sheath tumor, glioblastoma IDHwt
CNV Analysis by ldwgs Extract DNA Fragment DNA, ligate Adapters Sequence ~2-3 Mio fragments Map to human genome Analyze distribution Normal umor 2n
CNV Analysis by ldwgs no CNV 1p19q co-deletion
More complex genotypes
More complex genotypes
More complex genotypes
More complex genotypes
Amplification of 4q12
Amplification of 4q12 he 4q12 amplicon in malignant peripheral nerve sheath tumors: consequences and implications for sunitinib treatment. Zietsch et al, PLoS One. 2010 Jul 29;5(7):e11858. Amplification of the PDGFRA, KI and KDR genes in glioblastoma: a population based study Nobusawa et al, Neuropathology, 7 March 2011
Integrated Diagnosis for all CNS tumors
Scientific Analysis
Scientific Analysis Summary of 79 cases
Scientific Analysis Random forest analysis of 79 cases
Summary Integrated diagnosis is requested by WHO and clinicians Integrated diagnosis is feasible and should be implemented Established NGS technology (currently) sufficient Broader designs should be preferred larger gene panels ldwgs
Acknowledgements Martin Asslaber Gerald Höfler DGA lab members Molecular Diagnostics Lab karl.kashofer@medunigraz.at tel: +43 316 385 71752