idh1 r132h/atrx immunohistochemical validation...idh1 r132h/atrx immunohistochemical validation...
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IDH1 R132H/ATRX Immunohistochemical validation
Stephen Yip, M.D., Ph.D., FRCPC
CIQC/DSM 2016 12 June 2016
0835-0905
University of British Columbia
Disclosure Statement
• I have nothing to disclose
• I will not discuss off label use and/or investigational use in my presentation.
Objectives
• To appreciate the algorithm for molecular classification of low- grade glioma
• Understand the central role of identification of IDH mutation and ATRX loss in LGG classification
• To describe cIQc effort of ensuring consistent
implementation and interpretation of biomarker testing in neuro-oncology
2007
Clinical behaviour
Histology
Cytogenetics Molecular genetics
Identification, validation of
novel marker – IHC, SNaPshot,
MLPA testing
#1
IDH1 R132H mutation in 2° GBM
Sequencing, HRM, IHC
Better prognosis ?
IDH1/2 testing
Parsons DW, Jones S, Zhang X, et al: An Integrated Genomic Analysis of Human Glioblastoma Multiforme. Science, 2008 Yan H, Parsons DW, Jin G, et al: IDH1 and IDH2 mutations in gliomas. N Engl J Med 360:765-73, 2009
Yan H, Parsons DW, Jin G, et al: IDH1 and IDH2 mutations in gliomas. N Engl J Med 360:765-73, 2009
IDH1 R132H mutation in malignant glioma Argument for more unbiased genomic scanning of cancers
loosterhof NK, Bralten LB, Dubbink HJ, et al: Isocitrate dehydrogenase-1 mutations: a fundamentally new understanding of diffuse glioma? Lancet Oncol 12:83-91, 2010
Yan H, Parsons DW, Jin G, et al: IDH1 and IDH2 mutations in gliomas. N Engl J Med 360:765-73, 2009
IDH1/2 mutations in gliomas
IHC test available for the most common mutation R132H
IDH1 R132H
Tumour exome
Matched normal
IDH1 R132H ACG->ATG
H09 – Mutation- specific antibody Genotyping assay Sequencing- based assay
Anaplastic astrocytoma (WHO III) – IDH1 R132H POSITIVE
Allelic discrimination assays for IDH1/2 hotspot mutations
p.R132C p.R132S
Jessica Que & Julie Ho - VGH
Intrahepatic cholangioCa, AML, melanoma
Cancer Genome Atlas Research, et al. (2015). Comprehensive, Integrative Genomic Analysis of Diffuse Lower-Grade Gliomas. N Engl J Med, 372(26), 2481-2498. doi: 10.1056/NEJMoa1402121
Molecular segregation of low grade gliomas
1p19q LOH aka ODG
1p19q ROH aka “astrocytomas”
1p19q ROH, IDH wildtype Behaves like GBM
ATRX
IDH1 R132H IDH1 R132H
ATRX ATRX
IDH1 R132H
EGFR
OLIGODENDROGLIOMA
H&E ATRX
IDH1 R132H 1p19q FISH
1p19q Co-deleted
ATRX_20X
ATRX_40X
ATRX loss in tumour cells with preservation of expression in entrapped neurons
Matija Snuderl – NYU Craig Horbinski – U Kentucky
1p19q retained
2016
BRAF V600E
TMA
• VGH low grade and high grade gliomas as well as non-neoplastic lesions
• IDH1 R132H and ATRX IHC status identified
• IDH1/2 hotspot sequencing performed
• 1p19q FISH performed on LGG and GBM samples
IDH1 R132H cIQc challenge
Run 1
Lab ID Total n % Scorable Pairwise complete
observations
Concordance with reference
(%) Sensitivity Specificity
PPV (positive
predictive value)
NPV (negative predictive
value)
Cohen's kappa
101 22 100 22 19/22 (86%) 0.73 1 1 0.79 0.73 102 22 90.91 20 20/20 (100%) 1 1 1 1 1 103 22 100 22 22/22 (100%) 1 1 1 1 1 107 22 100 22 22/22 (100%) 1 1 1 1 1 110 22 95.45 21 14/21 (67%) 0.45 0.9 0.83 0.6 0.35 111 22 100 22 19/22 (86%) 0.73 1 1 0.79 0.73 112 22 100 22 21/22 (95%) 1 0.91 0.92 1 0.91 114 22 100 22 22/22 (100%) 1 1 1 1 1 123 22 100 22 22/22 (100%) 1 1 1 1 1 125 22 95.45 21 21/21 (100%) 1 1 1 1 1 126 22 100 22 22/22 (100%) 1 1 1 1 1 149 22 100 22 22/22 (100%) 1 1 1 1 1 162 22 100 22 22/22 (100%) 1 1 1 1 1 175 22 95.45 21 21/21 (100%) 1 1 1 1 1 191 22 100 22 22/22 (100%) 1 1 1 1 1 202 22 100 22 19/22 (86%) 1 0.73 0.79 1 0.73
Sanger sequencing verified
Lab ID Clone Dilution Supplier Ag Retrieval
Ab Incubation Time Detection Enhancement Chromogen
101 H09 1:100 Dianova CC1 32 min Ventana Optiview Copper DAB
102 H09 1/250 Dianova DAKO 3in1 High
pH 30" DAKO FLEX+ Copper
Sulphate DAKO DAB+
103 IDH
R132H 1/50 DIANOVA CC1 64, 32 ULTRA VIEW DAB COPPER DAB
107 H09 1:20 Dianova Ventana CC1
32min 32min Ventana Optiview DAB none DAB 110 H09 1:50 Dianova High pH (pH 9) 30 min Dako Flex Mouse linker DAB
111 H09 1/50 Histobiotec CC1 - 36 minutes 32 minutes Ultraview ---- Dab
112 H09 1:100 Dianova CC1 30 min 40 min iView copper DAB 114 H09 1/200 Dianova CC1 32min 16min Optiview-Ventana Copper DAB 123 H09 1:50 Cedarlane CC1 standard 60 min UltraView none DAB
125 111219/18 1/2000 dianova ER1-30 (bond) 15 min Bond Polymer Refine
Detection Bond DAB enhancer DAB
126 H09 1:500 = 0.4ug/ml OPTISTAIN pH9.0 Tris-EDTA 30 minutes Quanto polymer None Dako DAB+
149 DIA HO9
M 1:25 Dianova PT Link, high pH 30 min Envision Flex Yes DAB
162 H09 1:80 Dianova Ventana CC1 48
min. 32 min, 36 C Ventana OptiView - Ventana
OptiView DAB 175 H09 1:200 Dianova CC1 32 min Optiview None DAB 191 H09 1/10 Dianova CC1 32' ultraview none DAB
202 H09 1/50 HISTOBIOT
ECH HIGH PH 16 MIN LEICA REFINE
DETECTION KIT NONE DAB
RUN1
Run 1 Run 2
Lab ID Concordance
with sequencing
Cohen's kappa Lab ID
Concordance with
sequencing
Cohen's kappa
101 86% 0.73 101 100% 1 102 100% 1 102 100% 1 103 100% 1 103 100% 1 107 100% 1 107 100% 1 110 67% 0.35 110 100% 1 111 86% 0.73 111 87% 0.74 112 95% 0.91 112 100% 1 114 100% 1 114 100% 1 123 100% 1 123 100% 1 125 100% 1 125 100% 1 126 100% 1 126 100% 1 149 100% 1 149 96% 0.93 162 100% 1 162 96% 0.92 175 100% 1 175 100% 1 191 100% 1 191 96% 0.92 202 86% 0.73 202 100% 1
- - - 215 100% 1 - - - 217 89% 0.78
IDH1 R132H cIQc challenge (runs 35 & 44)
Run 1 Run 2
Lab ID Concordance
with sequencing
Cohen's kappa Lab ID
Concordance with
sequencing
Cohen's kappa
101 86% 0.73 101 100% 1 102 100% 1 102 100% 1 103 100% 1 103 100% 1 107 100% 1 107 100% 1 110 67% 0.35 110 100% 1 111 86% 0.73 111 87% 0.74 112 95% 0.91 112 100% 1 114 100% 1 114 100% 1 123 100% 1 123 100% 1 125 100% 1 125 100% 1 126 100% 1 126 100% 1 149 100% 1 149 96% 0.93 162 100% 1 162 96% 0.92 175 100% 1 175 100% 1 191 100% 1 191 96% 0.92 202 86% 0.73 202 100% 1
- - - 215 100% 1 - - - 217 89% 0.78
IDH1 R132H cIQc challenge (runs 35 & 44)
Run 1 Run 2
Lab ID Concordance
with sequencing
Cohen's kappa Lab ID
Concordance with
sequencing
Cohen's kappa
101 86% 0.73 101 100% 1 102 100% 1 102 100% 1 103 100% 1 103 100% 1 107 100% 1 107 100% 1 110 67% 0.35 110 100% 1 111 86% 0.73 111 87% 0.74 112 95% 0.91 112 100% 1 114 100% 1 114 100% 1 123 100% 1 123 100% 1 125 100% 1 125 100% 1 126 100% 1 126 100% 1 149 100% 1 149 96% 0.93 162 100% 1 162 96% 0.92 175 100% 1 175 100% 1 191 100% 1 191 96% 0.92 202 86% 0.73 202 100% 1
- - - 215 100% 1 - - - 217 89% 0.78
- In Run 1, Lab 101 possessed weak staining that led to false negative results. They reduced their dilution from 1:100 to 1:50 after cIQc feedback, and achieved 100% concordance with sequencing for Run 2. - In Run 1, Lab 110 had significant background staining and decreased overall sensitivity that led to many discordant results. After complete protocol re-optimization based on another cIQc participant’s successful staining protocol, Lab 110 achieved 100% concordance with sequencing for Run 2. - In Run 1, Lab 202 had significant background staining that led to false positive results. They increased their dilution from 1:50 to 1:100 and reduced antibody incubation time after cIQc feedback, and achieved 100% concordance with sequencing for Run 2.
IDH1 R132H cIQc challenge (runs 35 & 44)
Allelic discrimination assays for IDH1/2 hotspot mutations
p.R132C p.R132S
Jessica Que & Julie Ho - VGH
Intrahepatic cholangioCa, AML, melanoma
IDH1/2 Testing
• Indication for IDH1 R132H IHC testing – ANY “glioma” – Differentiate between reactive gliosis, non- infiltrative vs
diffuse glioma– i.e. confirm diagnosis – IDH1 R132H IHC – robust reactivity in cytoplasm
• Indication for IDH1/2 genotyping • If Immunonegative • Any WHO II/III diffuse glioma • Not GBM >55 year old • IDH1/2 Sanger sequencing, genotyping, NGS
ATRX
CORE14- Anaplastic ODG (WHO III), IDH1 R132H, 1p19q co-deleted
102 110 113 123
125 149 175 215
217 112
All centres agreed on POSITIVE reactivity
CORE13- Oligoastrocytoma (WHO II), IDH1 R132H
102 110 113 123
125 149 175 215
217 112
All centres agreed on NEGATIVE reactivity
Practice pattern of 1p19q FISH ordering - VGH
Molecular prognostic marker
IDH mutated ATRX intact
IDH mutated ATRX loss Genotype -
Molecular Dx- Anaplastic ODG
IDH wildtype ATRX intact
Anaplastic Astrocytoma
GBM- like
IDH mutated ATRX loss
Anaplastic Astrocytoma
Conclusion
• New molecular insights into LGG have altered the practice of clinical neuropathology
• Molecular segregation based on 1p19q/IDH/ATRX status is superior than histology
• Multi- institution QA/QC initiatives such as cIQc will ensure consistent and accurate implementation and interpretation of above assays
Acknowledgements
• VGH Neuropathology • Ian MacKenzie, John Maguire, Wayne Moore • Max Signaevski
• cIQc • Jennifer Won, John Garratt, Blake Gilks • Staff of VGH Immunohistochemistry core
• Participants of IDH and ATRX challenges
• Six participating laboratories • 100% concordance of IDH1 R132H IHC calls • 2/6 labs had initial discordance in HRM/sequencing calls
• 44/50 cases show concordance between IHC and Sanger sequencing (DNA from frozen tumour) • All 6 cases were immunopositive for IDH1 R132H
IHC vs Sequencing/Genotyping Sequencing backup
Correlation of IDH1 R132H/ATRX IHC with 1p19q status
FINAL DIAGNOSIS WHO Grading
IDH1 R132H STATUS (Present/Not present)
ATRX IHC status (Present/Not Present)
1P/1Q 19Q/19P
ODG II P N/A 0.71 0.65 ODG II N N/A 0.67 0.7 ODG II P N 0.72 0.64 ODG II P N 0.61 0.63 ODG II P pending 0.67 0.68 ODG II P N/A 0.6 0.54 ANAPLASTIC ODG III P N/A 0.57 0.6 ODG II P P 0.6 0.59 Anaplastic oligoastrocytoma III N P 0.76 0.71 Oligoastrocytoma II P P 0.73 0.67 ODG II P P 0.66 0.61 ODG II P N 0.6 0.58 Anaplastic ODG III P P 0.57 0.6 Anaplastic ODG III P P 0.55 0.52 ANAPLASTIC ODG III P P 0.66 0.55 ODG II P N 0.72 0.68 ODG II P P 0.62 0.59 ODG II N P 0.74 0.73 ODG II P N/A 0.55 0.57 ODG II P P 0.71 0.65 ODG II P N/A 0.62 0.59 ODG II P P 0.69 0.64 ODG II P P 0.79 0.8