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