Assessment performed on Friday, September 18, 2015, at Vancouver General Hospital

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1 Assessors report for ciqc Run 49: ATRX (June 2015) Assessors: S Yip and J Won (recorder) Assessment performed on Friday, September 18, 2015, at Vancouver General Hospital Background The combined application of IDH1 R132H and ATRX immunohistochemistry and 1p/19q co-deletion analysis can significantly increase the diagnostic and prognostic accuracy of low grade gliomas. Constituting a key parameter in this integrated diagnosis, abrogated ATRX protein expression based on immunohistochemistry is used as a surrogate for ATRX mutation, which is strongly associated with IDH1/2 mutated astrocytomas and not oligodendrogliomas. ATRX immunohistochemistry can refine the diagnostic accuracy of low grade glioma; however, it is heavily influenced by the quality of tissue material, and interpretation is particularly challenging as nuclear positivity is seen in endothelial cells, entrapped neurons, microglia and reactive astrocytes. Overview Participating laboratories were asked to stain a tissue microarray consisting of 28 single-core gliomas and nonneoplastic lesions that have been previously subjected to molecular analyses for IDH1/2 mutations and 1p/19q co-deletion analysis for select cases. The same TMA was also previously sent out to many of the same laboratories for proficiency testing of IDH1 R132H immunohistochemistry. Overall, self-assessments from participating labs were excellent. All participating labs returned slides to the ciqc office in time for the assessment meeting, and slides were blindly reviewed by ciqc assessors. Independent review led to infrequent alteration of original self-reported results due to a score being deemed as discordant between self-assessment and final ciqc review then re-classified based on ciqc assessor consensus. Specific comments from ciqc assessors are listed in the following table: Lab IHC Status* ciqc Comments 101 Adequate 102 Optimal Little to no background staining and nice staining intensity in positive cores. 110 Adequate 112 Adequate 113 Adequate Slightly weak intensity resulting in a non-homogenous staining pattern observed in some cores (e.g. Core 11). Counter stain too intense, leading to difficulties in interpretation of staining (e.g. results for 5 cores were changed from positive to negative after ciqc review). 123 Optimal 125 Adequate

2 149 Adequate 175 Optimal 193 Optimal 215 Sub-optimal While interpretation of staining (i.e. self-assessment) was accurate, considerable background staining overall. 217 Adequate Slight background staining. *Based on ciqc assessment Conclusion This inaugural ATRX immunohistochemistry proficiency testing challenge has demonstrated that ATRX is a challenging immunohistochemical marker. The staining variability is illustrated below in Figures 1. Interpretation must be done by an experienced neuropathologist and continued participation in external quality assurance is necessary. When correctly interpreted, ATRX immunohistochemistry is valuable as a subsequent test for distinguishing between astrocytomas and oligodendrogliomas after IDH1 R132H immunohistochemistry has been performed for differential diagnosis of glioma.

3 Figure 1. Variable IHC staining of ATRX by ciqc participants. a) Consensus negative core in all labs. b) Consensus positive core in all labs. The Garrattogram from self-assessment and ciqc-assessment for ATRX IHC results is provided in Supplementary Figure 1. Supplementary Table 1 summarizing staining protocols can also be found at the end of this document. Your regular participation in ciqc is greatly appreciated and we look forward to continually working with you and the Canadian Association of Pathologists Association Canadienne des Pathologistes.

4 Figure S1. Garrattograms after self-assessment and ciqc-assessment of ATRX IHC. *Self-assessment not submitted.

5 Table S1. Reported ATRX IHC staining protocols. Lab ID Ag Retrieval Time for Ag Ab Ab Time for Ab Detection Amplification Enhancement Ab Clone Ab Lot# Chromogen Method Retrieval (min) Dilution Supplier/Vendor Incubation (min) System (Y/N) (Y/N) 101 CC1 40 minutes POLYCLONAL 1:200 Sigma B minutes OptiView N COPPER DAB PT low ph C polyclonal 1:200 Sigma B min Dako Flex HRP N N DAB 112 BOND Epitope Sigma Life BOND Polymer 60 minutes polyclonal 1:250 G minutes Retrieval 1 Science Refine none none DAB 113 High ph Buffer 30' Polyclonal 1/1000 Sigma-Aldrich G ' Flex +30 Y N DAB 125 ER min polyclonal 1/300 Sigma A min Bond Polymer Refine Detection N Y DAB 149 PT Link high ph 20 Sigma HPA :500 Sigma 00D EnVision Flex yes no DAB 164 ultracc1 64 min polyclonal 1:200 HPA E hres Optiview n n DAB 175 HIER 48 ATRX 1:100 Sigma F Opti-kit N Y DAB 193 CC2 32 minutes rabbit poly 1/400 Sigma C minutes 37 deg polymer Optiview no no DAB 215 CC1 48 Prod # HPA :100 Sigma E min Optiview No No DAB 217 cc1 92 polyclonal 1:200 Sigma abcd 120 ultraview y y dab