Detecting mutations in single cells from cancer specimens is now a major area of translational research. In a recent article in this journal, Khalique et al validated an immunohistochemistry assay for ARID1A that reliably identifies loss of function mutations in single cells in tissue sections. This work exemplifies best practice for developing and orthogonally validating immunohistochemical assays to provide clearly interpretable mutational results with spatial context

Allo

Anglesio

Bitler

Cerami

Chen

Chene

Coons

Gibson

Iggo

Ioannidis

Jelinic

Jones

Kadoch

Khalique

Kupryjanczyk

Köbel

Marrack

Matos

McBride

Miller

Ramos

Samartzis

Stamp

Wang

Wiegand

Williamson

Witkowski

English

Köbel, Martin

Anglesio, Michael S

Brenton, James D

Apollo (Cambridge)

The Journal of Pathology: Clinical ResearchJ Pathol Clin Res July 2018; 4: 149–153Published online in Wiley Online Library(wileyonlinelibrary.com). DOI: 10.1002/cjp2.108INVITED COMMENTARYYou won’t believe this old test … that does cheap single-cellmutation detection†Martin Köbel1* , Michael S. Anglesio2 and James D. Brenton31Department of Pathology and Laboratory Medicine, University of Calgary, Foothills Medical Centre, Calgary, Alberta, Canada2Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, British Columbia, Canada3Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK*Correspondence to: Dr Martin Köbel, Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services,1304 S29 St NW, Calgary, 2TN 2T9, Alberta, Canada. Tel +1 403 944 8504; Email: martin.koebel@cls.ab.ca†Invited commentary for Khalique et al. Optimised ARID1A immunohistochemistry is an accurate predictor of ARID1A mutational statusin gynaecological cancer. J Pathol Clin Res 2018; 4: 154–166.AbstractDetecting mutations in single cells from cancer specimens is now a major area of translational research. In arecent article in this journal, Khalique et al validated an immunohistochemistry assay for ARID1A that reliablyidentifies loss of function mutations in single cells in tissue sections. This work exemplifies best practice fordeveloping and orthogonally validating immunohistochemical assays to provide clearly interpretable mutationalresults with spatial context.Keywords: Immunohistochemistry; Female reproductive system; Ovary; Mutation; TestingReceived 22 June 2018; Accepted 10 July 2018The authors declare no conflict of interest.The reputation of immunohistochemistry assays(IHC) as biomarkers for precision medicine is notthe best. There is an impression in some quartersthat interpreting different shades of brown is likereading tea leaves. Clinical IHC has been around fordecades [1]—so why do we have such a jaundicedview of its value for clinical biomarkers? Incompre-hensibly, there is still wide use of low-specificityantibodies and poorly standardized assays, resultingin variable biological and clinical signals. Over20 000 publications using IHC are published eachyear and the majority should probably have neverseen the light of day [2]. By contrast, in a recentarticle in this journal Khalique and colleagues dem-onstrate what is needed to turn research-grade IHCinto a clinically useful assay for detecting mutationsin ARID1A [3].Their study rigorously validates three differentARID1A (AT-rich interactive domain-containing pro-tein 1A; also called BAF250a) antibodies using anautomated, standardized assay against the gold stan-dard of next-generation sequencing to detect ARID1Aloss of function mutations. Staining from all three anti-bodies shows (almost) perfect agreement with veryhigh concordance between loss of ARID1A expressionand detection of predicted loss of function ARID1Amutations.This finding is of immediate clinical relevance fordiagnostic questions such as distinguishing clear celland endometrioid histotypes of ovarian or endometrialcarcinomas from histotypes in which ARID1A alter-ations do not occur [4,5]. While the prognostic valueof ARID1A mutation is controversial, even contradic-tory, this well validated assay now enables large-scalestudies to definitively test prognostic utility [6]. Look-ing forward, there is increasing clinical interest in thedevelopment of synthetic-lethal treatments with loss offunction of ARID1A mutations across a broad spec-trum of tumors [7–9]. The results from Khalique andcolleagues now provide a robust biomarker for clinicaltrial inclusion and potentially a predictive biomarkerfor future therapies.The history of ARID1A as a cancer-relevant gene isrelatively short. In 2010, two groups simultaneously© 2018 The Authors. The Journal of Pathology: Clinical Research published by The PathologicalSociety of Great Britain and Ireland and John Wiley & Sons Ltd.J Pathol Clin Res July 2018; 4: 149–153This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use anddistribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.identified recurrent ARID1A mutations in slightly lessthan half of endometriosis-associated clear cell [10,11]and endometrioid ovarian carcinomas [10]. Since then,ARID1A mutations have been detected in many cancertypes including endometrial endometrioid and clearcell carcinomas, urinary bladder carcinomas, Hodgkinlymphoma, gastroesophageal, colorectal and hepato-biliary carcinomas among others [12]. ARID1Aencodes one component of the SWI/SNF chromatinremodeling complex that facilitates target-specificDNA binding and regulation of transcription by repo-sitioning nucleosomes [6]. Cancers with alterations inthe SWI/SNF chromatin remodeling complex are notsensitive to conventional chemotherapy and have dis-tinct clinical features [13,14]. Co-inactivation ofARID1A and its paralog ARID1B give rise to particu-larly aggressive dedifferentiated endometrial and ovar-ian endometrioid carcinomas [15] and mutations inanother SWI/SNF component SMARCA4 causes lethalsmall cell carcinoma of the ovary [16–19]. ARID1A-targeted therapies are now in clinical developmentwith trials focusing on cancers with loss of functionARID1A mutations (NCT02059265, NCT03297424;see https://clinicaltrials.gov/).Although DNA sequencing is the gold standard fordetecting ARID1A mutations, nonsequencing-basedassays still have important clinical utility. Firstly, next-generation sequencing of ARID1A has been difficultowing to its large size and high GC content in exon1. Secondly, the implementation of an IHC based test(once properly validated) has fewer barriers to imple-mentation as the infrastructure required is inexpensiveand has ISO accreditation in most pathology centers.The origins of IHC can be traced back to the 1930s[20,21] but wider clinical adoption had to wait untilthe 1980s, enabled by commercial-scale production ofmonoclonal antibodies and other refinements allowinguse of formalin-fixed paraffin embedded tissue [1].Publications from the 1990s then showed that IHCcould be used to identify mutant p53 [22]. However, itwas not until very recently that an IHC assay couldprovide near-perfect accuracy in predicting TP53mutation status [23]. Advancements have depended on3 main factors: first, significantly improved sensitivityand specificity by using higher affinity rabbit monoclo-nal antibodies and newer polymer-based detection sys-tems with increased signal amplification. Second,robust standardization of experimental conditions fromimproved automated staining platforms. Third,improvements in interpretation by mandating use ofnormal cells as intrinsic controls for judging loss ofprotein expression [23]. This has enabled sophisticatedinterpretation of abnormal p53 staining that maps thedifferent classes of TP53 mutations to their functionalconsequences (Table 1).There are limitations to the ARID1A IHC assayfrom Khalique and colleagues that should be kept inmind. First, while most ARID1A mutations are loss offunction mutations, there are a number of recurrentmissense mutations that may impair ARID1A func-tion, but do not appear to change protein expression.For a clinical trial, using only IHC could result in con-tamination of ARID1A-competent trial populations.The extent to which IHC could miss deleteriousARID1A missense mutations is unknown and requiresfurther study.Second, ARID1A loss can be subclonal, indicating alater mutational event during tumor evolution. Tempo-ral modeling of endometrial endometrioid carcinomasshowed that only a portion of ARID1A mutations weretruncal [24]. Subclonal ARID1A mutations are com-mon in mismatch repair deficient (MMR-D) cancersbecause ARID1A has numerous short mononucleotiderepeats that have a high chance of slippage errors(Figure 1) [25,26]. In such cases, convergent evolutionof different ARID1A alterations resulting in phenotypi-cally indistinguishable but mechanistically distinct lossof ARID1A protein can occur [24].Subclonal loss may also be critical in identifyingpremalignant lesions. In the example of endometriosis,malignant transformation is rare and no biomarkersexist to distinguish “premalignant” endometriosis fromthe much more common chronic disease. A handful ofstudies have implicated subclonal loss of ARID1Aimmunoreactivity in endometriosis without co-existingcancer (i.e. loss in only a subset of endometriosisglands and/or loss limited to contiguous epitheliumTable 1. Correlation of p53 immunohistochemical phenotype withTP53 genotypep53 staining pattern Interpretation TP53 mutation statusNuclear staining in variabledistribution andintensityWild typepatternWild type*Diffuse strongoverexpression invirtually all tumor cellsMutant Nonsynonymous/missensemutationComplete absence withretained internal controlMutant Loss of function mutationsincluding indels,stopgains and splicingmutationsCytoplasmic (uncommon) Mutant Loss of function mutationdisrupting nuclearlocalization domain*with the exception of truncating or splicing TP53 mutations that may resultin wild type IHC staining observed in 2–4% of tubo-ovarian high-grade serouscarcinomas.150 Köbel et al© 2018 The Authors. The Journal of Pathology: Clinical Research published by The PathologicalSociety of Great Britain and Ireland and John Wiley & Sons Ltd.J Pathol Clin Res July 2018; 4: 149–153within a gland) [27–29]—although validation of thesefindings with orthogonal sequencing data is very lim-ited [30]. Now that we have high quality IHC tools,there is certainly sufficient justification for larger scaleinvestigation of ARID1A loss (and mutation) in endo-metriosis, particularly if cohorts can be identified whereprogression to cancer has occurred. A clear understand-ing of how to interpret heterogeneous/subclonalARID1A staining will undoubtedly accelerate suchstudies.Finally, the mechanisms explaining total loss ofARID1A protein are still elusive. Unlike TP53, loss ofheterozygosity at the ARID1A locus has not beenwidely reported, and at least for clear cell andendometrioid ovarian carcinomas, does not appear tobe common [31,32]. In the cohort by Khalique andcolleagues 8/45 (18%) cases harbored more than oneARID1A mutation and similar rates (6/27; 22%) havepreviously been reported [32]. Systematic analyses arerequired to validate whether (or when) both alleles areaffected and to rule out alternative possibilities, suchas the presence of two co-existing subclones carryingunique alterations.Despite these caveats, the report from Khalique andcolleagues suggests that ARID1A IHC can reliablydetect loss of function mutations and is ready to beaccepted as a clinical-grade test. A categorical scoringsystem should now be agreed upon to distinguishFigure 1. (A) H&E photomicrograph of endometrial endometrioid carcinoma, grade 3; (B) absence of MSH6 suggesting mismatch repairdeficiency (additional MSH2 absence not shown). The arrow shows positive internal control stromal staining; (C) small focus of subclonalARID1A loss (asterisk); (D) focus of p53 overexpression overlying but extending beyond the ARID1A focus (asterisk). Taken together thesepanels suggest a prototypical somatic mismatch repair deficiency resulting in subclonal TP53 missense alteration, and subsequentARID1A loss of function mutation.151Single-cell mutation detection by immunohistochemistry© 2018 The Authors. The Journal of Pathology: Clinical Research published by The PathologicalSociety of Great Britain and Ireland and John Wiley & Sons Ltd.J Pathol Clin Res July 2018; 4: 149–153between normal retained/present ARID1A expressionand abnormal/absent expression, and whether this iscomplete or subclonal. As for p53 IHC, ARID1A IHChas the potential to achieve near-single-cell resolutionfor detecting mutations whilst also providing detailedinformation about the spatial context of mutated cells.IHC based prediction of ARID1A mutation now joins asmall and exclusive group of well-validated and clini-cally useful predictive IHC biomarkers.Author contributions statementMK, MSA and JDB conceptualized the commentary.MK wrote the initial draft, MSA and JDB contributedto subsequent versions and all authors approved thefinal version.Reference1. Matos LL, Trufelli DC, de Matos MG, et al. Immunohistochemis-try as an important tool in biomarkers detection and clinical prac-tice. Biomark Insights 2010; 5: 9–20.2. Ioannidis JP. Why most published research findings are false. PLoSMed 2005; 2: e124.3. Khalique S, Naidoo K, Attygalle AD, et al. Optimised ARID1Aimmunohistochemistry is an accurate predictor of ARID1A muta-tional status in gynaecological cancers. J Pathol Clin Res 2018; 4:154–166.4. Köbel M, Rahimi K, Rambau PF, et al. An immunohistochemicalalgorithm for ovarian carcinoma typing. Int J Gynecol Pathol2016; 35: 430–441.5. Chen W, Husain A, Nelson GS, et al. Immunohistochemical profil-ing of endometrial serous carcinoma. Int J Gynecol Pathol 2017;36: 128–139.6. Wu RC, Wang TL, Shih IM. The emerging roles of ARID1A intumor suppression. Cancer Biol Ther 2014; 15: 655–664.7. Williamson CT, Miller R, Pemberton HN, et al. ATR inhibitors asa synthetic lethal therapy for tumors deficient in ARID1A. NatCommun 2016; 7: 13837.8. Miller RE, Brough R, Bajrami I, et al. Synthetic lethal targeting ofARID1A mutant ovarian clear cell tumors with dasatinib. MolCancer Ther 2016; 15: 1472–1484.9. Bitler BG, Aird KM, Garipov A, et al. Synthetic lethality by target-ing EZH2 methyltransferase activity in ARID1A-mutated cancers.Nat Med 2015; 21: 231–238.10. Wiegand KC, Shah SP, Al-Agha OM, et al. ARID1A mutations inendometriosis-associated ovarian carcinomas. N Engl J Med 2010;363: 1532–1543.11. Jones S, Wang TL, Shih IM, et al. Frequent mutations of chroma-tin remodeling gene ARID1A in ovarian clear cell carcinoma. Sci-ence 2010; 330: 228–231.12. Cerami E, Gao J, Dogrusoz U, et al. The cBio cancer genomicsportal: an open platform for exploring multidimensional cancergenomics data. Cancer Discov 2012; 2: 401–404.13. Kadoch C, Hargreaves DC, Hodges C, et al. Proteomic and bioin-formatic analysis of mammalian SWI/SNF complexes identifiesextensive roles in human malignancy. Nat Genet 2013; 45:592–601.14. McBride MJ, Kadoch C. Disruption of mammalian SWI/SNF andpolycomb complexes in human sarcomas: mechanisms and thera-peutic opportunities. J Pathol 2018; 244: 638–649.15. Köbel M, Hoang LN, Tessier-Cloutier B, et al. Undifferentiatedendometrial carcinomas show frequent loss of core switch/sucrosenonfermentable complex proteins. Am J Surg Pathol 2018; 42:76–83.16. Kupryjanczyk J, Dansonka-Mieszkowska A, Moes-Sosnowska J,et al. Ovarian small cell carcinoma of hypercalcemic type - evi-dence of germline origin and SMARCA4 gene inactivation. a pilotstudy. Pol J Pathol 2013; 64: 238–246.17. Ramos P, Karnezis AN, Craig DW, et al. Small cell carcinoma ofthe ovary, hypercalcemic type, displays frequent inactivating germ-line and somatic mutations in SMARCA4. Nat Genet 2014; 46:427–429.18. Jelinic P, Mueller JJ, Olvera N, et al. Recurrent SMARCA4 muta-tions in small cell carcinoma of the ovary. Nat Genet 2014; 46:424–426.19. Witkowski L, Carrot-Zhang J, Albrecht S, et al. Germline andsomatic SMARCA4 mutations characterize small cell carcinoma ofthe ovary, hypercalcemic type. Nat Genet 2014; 46: 438–443.20. Marrack J. Nature of antibodies. Nature 1934; 133: 292–293.21. Coons AH, Creech HJ, Jones RN, et al. The demonstration ofpneumococcal antigen in tissues by the use of fluorescent antibody.J Immunol 1942; 45: 159–170.22. Iggo R, Gatter K, Bartek J, et al. Increased expression of mutantforms of p53 oncogene in primary lung cancer. Lancet 1990; 335:675–679.23. Köbel M, Piskorz AM, Lee S, et al. Optimized p53 immunohisto-chemistry is an accurate predictor of TP53 mutation in ovarian car-cinoma. J Pathol Clin Res 2016; 2: 247–258.24. Gibson WJ, Hoivik EA, Halle MK, et al. The genomic landscapeand evolution of endometrial carcinoma progression and abdomi-nopelvic metastasis. Nat Genet 2016; 48: 848–855.25. Ye J, Zhou Y, Weiser MR, et al. Immunohistochemical detectionof ARID1A in colorectal carcinoma: loss of staining is associatedwith sporadic microsatellite unstable tumors with medullaryhistology and high TNM stage. Hum Pathol 2014; 45:2430–2436.26. Allo G, Bernardini MQ, Wu RC, et al. ARID1A loss correlateswith mismatch repair deficiency and intact p53 expression inhigh-grade endometrial carcinomas. Mod Pathol 2014; 27:255–261.27. Samartzis EP, Samartzis N, Noske A, et al. Loss ofARID1A/BAF250a-expression in endometriosis: a biomarker forrisk of carcinogenic transformation? Mod Pathol 2012; 25:885–892.28. Stamp JP, Gilks CB, Wesseling M, et al. BAF250a expression inatypical endometriosis and endometriosis-associated ovarian can-cer. Int J Gynecol Cancer 2016; 26: 825–832.152 Köbel et al© 2018 The Authors. The Journal of Pathology: Clinical Research published by The PathologicalSociety of Great Britain and Ireland and John Wiley & Sons Ltd.J Pathol Clin Res July 2018; 4: 149–15329. Chene G, Ouellet V, Rahimi K, et al. The ARID1A pathway inovarian clear cell and endometrioid carcinoma, contiguous endo-metriosis, and benign endometriosis. Int J Gynaecol Obstet 2015;130: 27–30.30. Anglesio MS, Papadopoulos N, Ayhan A, et al. Cancer-associatedmutations in endometriosis without cancer. N Engl J Med 2017;376: 1835–1848.31. Anglesio MS, George J, Kulbe H, et al. IL6-STAT3-HIF signalingand therapeutic response to the angiogenesis inhibitor sunitinib inovarian clear cell cancer. Clin Cancer Res 2011; 17: 2538–2548.32. Wang YK, Bashashati A, Anglesio MS, et al. Genomic conse-quences of aberrant DNA repair mechanisms stratify ovarian can-cer histotypes. Nat Genet 2017; 49: 856–865.153Single-cell mutation detection by immunohistochemistry© 2018 The Authors. The Journal of Pathology: Clinical Research published by The PathologicalSociety of Great Britain and Ireland and John Wiley & Sons Ltd.J Pathol Clin Res July 2018; 4: 149–153

You won't believe this old test … that does cheap single-cell mutation detection.

Crossref

You won't believe this old test … that does cheap single-cell mutation detection

https://www.repository.cam.ac.uk/bitstream/handle/1810/285056/K-bel_et_al-2018-The_Journal_of_Pathology%253A_Clinical_Research.pdf?sequence=3&isAllowed=y

You won't believe this old test … that does cheap single-cell mutation detection.

Abstract

Similar works

Full text

Available Versions

Apollo (Cambridge)

Crossref