Clinically Useful Next Generation Sequencing and Molecular Testing in Gliomas MacLean P. Nasrallah, MD PhD

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1 Clinically Useful Next Generation Sequencing and Molecular Testing in Gliomas MacLean P. Nasrallah, MD PhD Neuropathology Fellow Division of Neuropathology Center for Personalized Diagnosis (CPD)

2 Glial neoplasms infiltrating gliomas Astrocytic tumors Diffuse astrocytoma II Anaplastic astrocytoma III Glioblastoma Giant cell glioblastoma IV Gliosarcoma Oligodendroglial tumors Oligodendroglioma II Anaplastic oligodendroglioma III Oligoastrocytic tumors Oligoastrocytoma II Anaplastic oligoastrocytoma III Courtesy of Dr. Maria Martinez-Lage 2

3 2016 3

4 The 2016 WHO classification of tumours of the central nervous system Louis et al., Acta Neuropathologica

5 Talk Outline Genetic, epigenetic and metabolic changes in gliomas Mechanisms/tumor biology Incorporation into daily practice and WHO classification Penn s Center for Personalized Diagnostics Tests performed Results and observations to date Summary 5

6 The 2016 WHO classification of tumours of the central nervous system Louis et al., Acta Neuropathologica

7 Mechanism of concurrent 1p and 19q chromosome loss in oligodendroglioma lost FUBP1 CIC Whole-arm translocation Griffin et al., Journal of Neuropathology and Experimental Neurology

8 Oligodendroglioma: 1p19q co-deletion Since the 1990s Diagnostic Prognostic Predictive Li et al., Int J Clin Exp Pathol

9 Mutations of Selected Genes in Glioma Subtypes GBM Astrocytoma Oligodendroglioma Oligoastrocytoma Killela et al., PNAS

10 Escaping Senescence Telomerase reverse transcriptase gene Oligodendrogliomas Primary GBMs Alternative lengthening of telomeres (ALT)/ATRX Astrocytomas Secondary GBMs Cohesion between sister chromatids provides a template for recombination and repair during and after DNA replication in S and G 2 phases of the cell cycle Reitman et al., Acta Neuropathol 2013, Ramamoorthy & Smith, Cancer Cell

11 Mutations of Selected Genes in Glioma Subtypes GBM Astrocytoma Oligodendroglioma Oligoastrocytoma Killela et al., PNAS

12 Association of IDH1 status with PFS mutated IDH1 Hartmann et al., Acta Neuropathologica

13 Mutations of Selected Genes in Glioma Subtypes GBM Astrocytoma Oligodendroglioma Oligoastrocytoma Killela et al., PNAS

14 Diagnosis of GBM Pseudopalisading necrosis Endothelial proliferation 14

15 Glioblastoma subtypes genomic changes Cell 155, , October 10,

16 Sturm et al., Cancer Cell

17 IDH Labussiere et al.,

18 IDH mutations result in Glioma- CpG methylation phenotype (G-CIMP) and histone methylation in gliomas demethylases Block to differentiation DNA hydroxylases Venneti & Thompson, Brain Pathology

19 Simplified algorithm for classification of diffuse gliomas based on histology and genetics Louis et al., Acta Neuropathologica

20 EGFR p53 mutated IDH1 Ki-67 20

21 Center for Personalized Diagnostics David Roth, MD PhD Chair of Pathology Jennifer Morrissette, PhD Clinical Director of the CPD Kojo Elanitoba-Johnson, MD Director of the CPD 21

22 The CPD gene panel 47 genes targeted (Illumina TruSeq Cancer Panel) MiSeq instrument In-house algorithms to identify variants 22

23 Solid Tumor Panel Version 2: 153 genes ABL1 BAP1 CDK6 EPHA3 FGFR3 JAK1 MAP2K2 MRE11A NOTCH1 PTPN11 SETD2 TERT AKT1 BRAF CDKN2A ERBB2 FGFR4 JAK2 MAP2K4 MSH2 NOTCH2 RAB35 SF3B1 TET2 AKT2 BRCA1 CHEK2 ERBB3 FLT3 JAK3 MAPK1 MSH6 NOTCH3 RAC1 SLIT2 TGFBR2 AKT3 BRCA2 CIC ERBB4 FUBP1 KCNG1 MAPK3 MTOR NRAS RAD50 SMAD4 TP53 ALK BRIP1 CRKL ERCC2 GATA3 KDM5A MAX MYC PAK1 RAD51 SMARCA4 TRAF7 APC BTK CSF1R ERG GNA11 KDM5C MCL1 MYCN PALB2 RAD51B SMO TSC1 AR CREBBP CTNNB1 ESR1 GNAQ KDM6A MDM2 NBN PBRM1 RAD51C SPOP TSC2 ARAF CCND1 DAXX ESR2 GNAS KDR MDM4 NF1 PDGFRA RAD51D SRC ARID1A CCND2 DDR2 EZH2 HRAS KIT MED12 NF2 PIK3CA RAF1 STAG2 U2AF1 ARID2 CCND3 DNMT3A FBXW7 H3F3A KMT2C MEN1 NTRK1 PIK3CB RB1 STK11 VHL ATM CCNE1 EGFR FGF3 IDH1 KRAS MET NTRK2 PIK3R1 RET SUFU WT1 ATRX CDH1 FGFR1 IDH2 LRRK2 MITF NTRK3 PTCH1 RHOA SUZ12 XRCC2 AURKA CDK4 EP300 FGFR2 IGF1R MAP2K1 MLH1 NKX2-1 PTEN RNF43 SYK 23

24 Penn Precision Panel AKT1 ERBB2 KRAS PDGFRA ALK HRAS MAP2K1 PTEN BRAF IDH1 MET PIK3CA CSF1R IDH2 NOTCH1 RET EGFR KIT NRAS TP53 24

25 Amplification of EGFR detected by NGS Validation Often amplified in GBM and associated with EGFRvIII 25

26 NGS assay to detect EGFRvIII RNA Wild-type EGFR 1 8 EGFRvIII RNA-based assay RNA integrity assay built in ( amplification of 90, 120 and 250bp respectively) Detects RNA degradation in each submitted sample 26

27 Results February 2013 April 2016 ~600 gliomas have been sequenced O RRG E GBM Other AA AO DA AOA LGG PA PXA 27

28 Results February 2013 April Normal Variant Number of tumors QNS Abnormal 28

29 Genetic changes in glioblastomas MGMT meth EGFR amp TP53 EGFR mut PTEN EGFRvIII PIK3CA PTPN11 IDH1 KIT amp PDGFRA amp RB1 29

30 EGFR distribution of mutations Extracellular domain T M Protein kinase domain DB domain R108K (9x) A289V/D/T (14x) V774M (4x) 30

31 TP53 distribution of mutations TD PR DB domain OD NLS R175H (8x) R273H/C/P/L (17x) 31

32 Copy number variations 140 isolated EGFR 10 isolated PDGFRA 2 isolated KIT 1 isolated MET 19 PDGFRA + KIT amplifications 1 EGFR + PDGFRA + KIT amplifications 32

33 Summary of EGFR mutations detected 313 specimens have been received for EGFRvIII detection 54 positive specimens (17%) Positive 54 Low Positive Negative EGFRvIII can be present at different allele frequencies, suggestive of amplification of the wild type or mutant allele 33

34 Correlation of genetic changes in 157 GBMs Yule Correlation Coefficient ( normalized for mutation frequency mismatch) Analysis by Derek Oldridge 34

35 Co-occurrence of EGFR Mutations in GBM Analysis by Derek Oldridge 35

36 Genomic characterization of GBM A variety of genomic aberrations are associated with GBMs Heterogeneity appears to be common The clonal & subclonal composition of tumors are unique to individuals Subclonal structure may predict trajectory of disease progression Courtesy of Dr. Jennifer Morrissette, CPD, Penn 36

37 Determining whether a tumor is (mostly) clonal can be clinically useful One way this can be inferred is by analysis of allele frequencies of the different mutations detected If the allele frequencies differ, that could be interpreted as clonal evolution or different malignant clones If they are all approximately the same allele frequency then it could suggest a single clone Courtesy of Dr. Jennifer Morrissette 37

38 WHO 2016: Layered Diagnosis Integrated Diagnosis incorporating all aspects of tissue diagnosis Histological Classification WHO Grade reflects natural history, based on histology Molecular information Louis et al., Brain Pathology

39 Example: initial diagnosis 1. Integrated Diagnosis: pending 2. Histologic diagnosis: diffuse glioma with atypia, mitoses, microvascular proliferation and necrosis 3. WHO grade: at least III 4. Molecular studies: pending 39

40 1. Integrated Diagnosis: pending 2. Histologic diagnosis: diffuse glioma with atypia, mitoses, microvascular proliferation and necrosis 3. WHO grade: at least III 4. Molecular studies: pending Final Diagnosis Integrated Diagnosis: GBM (primary type), WHO IV, IDH intact, 1p19q intact, ATRX intact, +/- EGFR-AMP 40

41 1. Integrated Diagnosis: pending 2. Histologic diagnosis: diffuse glioma with atypia, mitoses, microvascular proliferation and necrosis 3. WHO grade: at least III 4. Molecular studies: pending Final Diagnosis Integrated Diagnosis: GBM (secondary type), WHO IV, IDH mutated, 1p19q intact, ATRX loss 41

42 1. Integrated Diagnosis: pending 2. Histologic diagnosis: diffuse glioma with atypia, mitoses, microvascular proliferation and necrosis 3. WHO grade: at least III 4. Molecular studies: pending Final Diagnosis Integrated Diagnosis: Anaplastic Oligodendroglioma, WHO III, IDH mutated, 1p19q codeleted, ATRX intact 42

43 Three different possible final diagnoses GBM (primary type), WHO IV, IDH intact, 1p19q intact, ATRX intact, +/- EGFR-AMP GBM (secondary type), WHO IV, IDH mutated, 1p19q intact, ATRX loss Anaplastic Oligodendroglioma, WHO III, IDH mutated, 1p19q codeleted, ATRX intact 43

44 Do we need a microscope? Yes! Must make histological diagnosis of glioma first WHO grade is based mainly on histology Some gliomas do not have these diagnostic genetic changes 44

45 Simplified algorithm for classification of diffuse gliomas based on histology and genetics More genetic changes will be found. Louis et al., Acta Neuropathologica

46 Molecular changes define tumors chr19q chr1p Slide courtesy of Dr. Arie Perry, USCAP 2016 presentation 46