Canadian Journal of

Volume 10 | Issue/numéro 2

pathology pathologie Revue canadienne de

Survey of PhySician SatiSfaction

PM 43490512

www.cap-acp.org

Irvaym B. Barsoum MD, MSc, PhD; Michael Carter MD, PhD; Hala Faragalla MD FRCPC; Louis Gaboury MD, PhD, FRCPC, FCAP; John Gartner MD CM, FRCPC; Laurette Geldenhuys MBBCH, FFPATH, MMed, FRCPC, MAEd, FIAC; Zeina Ghorab MD, MSc; Nadia Ismiil MBChB, FRCPC; Jason Karamchandani MD; Adriana Krizova MD, MSc, FRCPC; David Munoz, MD, MSc, FRCPC; Christopher Naugler MD, FRCPC; Tony Ng MD, PhD FRCPC; Sharon Nofech-Mozes MD; Maria Pasic PhD, FCACB; Aaron Pollett MD, MSc, FRCPC; Rola Saleeb MD; Harman Sekhon MD, PhD, FCAP; Monalisa Sur MBBS, FCPath, MMed., MRCPath, FRCPC; Aducio Thiesen MD, PhD, MSc, FRCPC; Stephen Yip MD, PhD, FRCPC.

internationaL eDitoriaL BoarD Emma H. Allott PhD, University of North Carolina, USA; Fredrik Bosman MD, PhD, University of Lausanne, Switzerland; Daniel Chan PhD, DABCC, FACB, Johns Hopkins University School of Medicine, USA; Runjan Chetty MB BCh, FRCPA, FFPath, FRCPath, FRCPC, FCAP, Dphil, University of Toronto, Canada; Kumarasen Cooper MBChB, Dphil, FRCPath, University of Pennsylvania, USA; Brett Delahunt BSc MD, BSc Hons, BMedSC, MB ChB, FRCPA, FFSc, FRCPath, University of Otago, New Zealand; Sunil R Lakhani BSc (Hon), MBBS, MRCPath, MD, FRCPath, FRCPA, The University of Queensland, Australia; Virginia A. LiVolsi MD, MASCP, University of Pennsylvania, USA; Ricardo Lloyd MD, PhD, University of Wisconsin, USA; Jesse Mckenney MD, Cleveland Clinic, USA; Chris Meijer MD, PhD, VU University, The Netherlands; George Netto MD, PhD, Johns Hopkins University School of Medicine, USA; Isobel Scarisbrick PhD, Mayo Clinic, USA; Manfred Schmitt, Dr. rer. nat., Dr. med. habil. (Ph. D., M.D. sci.), Dipl.-Biologist, Technical University, Munich, Germany; Iris Schrijver MD, Stanford University, USA; Andreas Scorilas PhD, University of Athens, Greece; Ming Tsao, MD FRCPC, University of Toronto, Canada; Mark Wick MD, University of Virginia, USA.

coMitÉ De rÉDaction Irvaym B. Barsoum M. D., M. Sc, Ph.D; Michael Carter M. D., Ph. D; Hala Faragalla M. D., FRCPC; Louis Gaboury M. D., Ph. D., FRCPC, FCAP; John Gartner M. D. CM, FRCPC; Laurette Geldenhuys MBBCH, FFPATH, MMed, FRCPC, MAEd, FIAC; Zeina Ghorab M. D.; Nadia Ismiil MBChB, FRCPC; Jason Karamchandani M. D.; Adriana Krizova M. D., M. Sc, FRCPC; David Munoz M. D., M. Sc, FRCPC; Christopher Naugler M. D., FRCPC; Tony Ng, M. D., Ph. D., FRCPC; Sharon Nofech-Mozes M. D.; Maria Pasic Ph. D., FCACB; Aaron Pollett M. D., M. Sc, FRCPC; Rola Saleeb M. D.; Harman Sekhon M. D., Ph. D, FCAP; Monalisa Sur MBBS, FCPath, MMed., MRCPath, FRCPC; Aducio Thiesen M. D., Ph. D, M. Sc, FRCPC; Stephen Yip M. D., Ph. D, FRCPC.

coMitÉ De rÉDaction internationaL Emma H. Allott Ph. D, Université de North Carolina, É.-U.; Fredrik Bosman M. D., Ph. D., Université de Lausanne, Suisse; Daniel Chan Ph. D., DABCC, FACB, faculté de médecine de l’Université Johns Hopkins, É.-U.; Kumarasen Cooper MBChB, Dphil, FRCPath, Université de la Pennsylvania, É.-U.; Brett Delahunt B. Sc Hons BMedSc MB ChB MD FRCPA FFSc FRCPath, Université d’Otago, Nouvelle-Zélande; Sunil R Lakhani M. D. B. Sc (Hon), MBBS, MRCPath, FRCPath, FRCPA, Université de Queensland, Australie; Virginia A. LiVolsi M. D., MASCP, Université de la Pennsylvanie, É.-U.; Ricardo Lloyd M. D., Ph. D, Université du Wisconsin, É.-U.; Jesse Mckenney M. D., Clinique de Cleveland, É.-U.; Chris Meijer M. D., Ph. D., Université VU, Pays-Bas; George Netto M. D., Ph. D., faculté de médecine de l’Université Johns Hopkins, É.-U.; Isobel Scarisbrick, Ph. D., Clinique Mayo, É.-U.; Manfred Schmitt, Dr. rer. nat., Dr. med. habil. (Ph. D., M.D. sci.), Dipl.-Biologist, Université polytechnique, Munich, Allemagne; Iris Schrijver M. D., Université Stanford, É.-U.; Andreas Scorilas Ph. D., Université d’Athènes, Grèce; Ming Tsao M. D. FRCPC, Université de Toronto, Canada; Mark Wick M. D., Université de la Virginie, É.-U. 2

Canadian Journal of Pathology | Volume 10, Issue 2 | www.cap-acp.org

coMitÉ De rÉDaction

eDitoriaL BoarD

eDitoriaL BoarD

Canadian Journal of

Volume 10 | issue 2 eDitor-in-chief George M Yousef MD, PhD, FRCPC eDitor eMerituS J. Godfrey Heathcote MA, MB, BChir, PhD, FRCPC founDinG eDitor Jagdish Butany MBBS, MS, FRCPC

pathology

Official Publication of the Canadian Association of Pathologists

table of Contents

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art Director Sherri Keenan tranSLation Magali Cloutier Provencher Éliane Fréchette PuBLiShinG aGency clockwork communications inc. PO Box 33145, Halifax, NS, B3L 4T6 902.442.3882 / [email protected] Canadian Journal of Pathology is a peer-reviewed journal published four times per year, by Clockwork Communications Inc., on behalf of the Canadian Association of Pathologists. We cannot assume responsibility or commitment for unsolicited material. Any editorial material, including photographs, that are accepted from an unsolicited contributor, will become the property of the Canadian Association of Pathologists. Copyright Canadian Association of Pathologists (CAP-ACP). All rights reserved. Reprinting in part or in whole forbidden without express written consent from CAP-ACP. We welcome editorial submissions to www.CanadianJournalOf Pathology.ca Publications Mail Agreement No. 43490512 ISSN 1918-915X (print) ISSN 1918-9168 (online) Return undeliverable Canadian addresses to: clockwork communications inc PO Box 33145 Halifax, NS B3L 1K3

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Molecular Pathology Corner: The role of next generation sequencing in current laboratory diagnostics Authors: Henriett Butz MD, PhD, Attila Patócs MD, MSc, PhD.

aSSociate eDitor Martin J. Trotter MD, PhD, FRCPC PuBLiShinG eDitor Deborah McNamara

Letter from the Editor-in-Chief: Funding your research project: Time to think outside the box?

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pathologists' assistants CoRneR Case of the Month: Rhabdomysarcoma Author: Martin Grealish MLT, MTM, PA(CCCPA).

ReseaRCh aRtiCle Survey of physician satisfaction with anatomical pathology services in Alberta Authors: Denise La Perle MLT, Angela Thompson MD, FRCPC, Allie Moskalyk MLT, Reanne Cunningham MLT, and Máire A. Duggan MD, FRCPC.

HER2 overexpression and amplification in diffuse gastric cancer in Newfoundland and Labrador Authors: Robyn Ndikumana MD, BScN, Altaf Taher MD, FRCPC, FCAP, Polycarp Erivwo MBBS, FMCPath, FRCPC, Melanie Seal MD, FRCPC.

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Case RepoRt & ReViews Update on the molecular pathology of diffusely infiltrating gliomas Authors: Andrew F. Gao MD and David G. Munoz MD, MSc.

Primary cardiac angiosarcoma with C-KIT expression: A case report and review of literature Authors: Mubarak Al-Shraim MD, FRCPC, Howadah Elhakeem MD, Ahmed Rezk MD, Mahmoud Rezk Abdelwahed Hussein MD, PhD, Jagdish W. Butany MD, FRCPC.

Appendiceal vasculitis with subsequent macrophage activation syndrome Authors: Jolanta Jedrzkiewicz MD, Liane Heale MD, Robert H. Riddell MBBS, William Dubinski MD, Ronald M. Laxer MD, Iram Siddiqui MBBS.

Spermatic cord paraganglioma: A case report and literature review Authors: Ryan DeCoste MD, Jonathan Moore MD, Padraic O’Malley MD, Jennifer Merrimen MD. Revue canadienne de pathologie | volume 10, numéro 2 | www.cap-acp.org

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LETTER FROM THE EDITOR-IN-CHIEF

FUNDING YOUR RESEARCH PROJECT: TIME TO THINK OUTSIDE THE BOX?

B

efore a research project is launched, investigators must typically overcome a major obstacle - the availability of funding to support the research work. Obtaining funding from government agencies such as the Canadian Institutes of Health Research (CIHR) or U.S. National Institute of Health (NIH) is highly competitive and the likelihood of receiving grant funding is low. The CIHR Fall 2017 project grants competition had an overall success rate of only 15%.2 Despite some recent funding increases, obtaining funding support from these agencies will always remain difficult, especially for researchers who are not fulltime in the laboratory, but also hold clinical responsibilities. Laboratory physicians can increase their probability of grant funding success by collaboration with larger, especially multidisciplinary research groups. However, many clinical researchers must search for alternative or nonconventional sources of research funding.

Academic institutions may provide small amounts of research support as start-up funding for new investigators or seedmoney for small projects. Institutions may also be able to provide indirect funding, including salary for trainees and technicians and the in-kind contribution of research/office space or equipment. University- or hospital-associated foundations may also be a source of research support. Increasingly, investigators are looking to private, nongovernmental sources for research funding, primarily non-profit organizations and for-profit commercial industry. Non-profit organizations such as Heart and Stroke Foundation, Cancer Research Society, Kidney Foundation, and Alzheimer Society provide significantly less funding compared to government agencies, but allocation of funding remains a peer-review process and compared to larger agencies, the competition is usually more limited. This can be a great funding option in the initial phase of a research project when the principal investigator does not have enough preliminary evidence to support a large scale grant application.2

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Collaboration/partnership with industry is also a feasible funding route. Pharmaceutical and device manufacturers fund more than half of medical research funding in the United States. If stringent ethical guidelines are followed, private sector companies can be an important source of funding for biomedical research.3 There are many examples of successful collaborations in specialized projects that are of mutual benefit to both the research institute and the industry partners.4 Francis Collins recently highlighted the need to forge innovative partnerships between the traditional strengths of the private sector and academic laboratories.5 Another attractive alternative for clinician scientists such as pathologists may be to execute “low budget, high yield research”. This includes morphology-based research projects; analyzing morphological parameters and their value in providing prognostic or predictive information. An interesting and well-established example of this is the development of criteria for hereditary colon cancer which was based on morphological assessment. Other famous success stories include the Gleason grading of prostate cancer and the Fuhrman grading system for renal cell carcinoma. Immunohistochemistry-based analysis can also have high clinical value while maintaining a substantially lower research budget. Morphology research has been significantly boosted recently due to advancement in digital pathology enabling a highly sophisticated morphometric assessment.6,7 Research in quality assurance, data management, and medical education are other spheres that may require less funding support than traditional bench research. Finally, success in obtaining research funding, especially for junior investigators, requires mentorship and professional training in writing grant applications as well as drafting research manuscripts. Many resources for successful grant continued on page 6

LETTRE DU RÉDACTEUR EN CHEF

FINANCEMENT DES PROJETS DE RECHERCHE : LE MOMENT EST-IL VENU DE SORTIR DES SENTIERS BATTUS?

A

grande taille, la compétition est généralement moins forte. Il peut s’agir d’une option de financement intéressante durant la phase initiale d’un projet de recherche, où le chercheur principal ne détient pas encore suffisamment de données pour faire une demande de subvention de plus grande envergure2.

vant qu’un projet de recherche soit entrepris, les chercheurs doivent habituellement surmonter un obstacle majeur : trouver du financement pour soutenir leurs travaux. Le processus de demande de subventions auprès d’organismes gouvernementaux, comme les Instituts de recherche en santé du Canada (IRSC) ou les National Institutes of Health (NIH) des États-Unis, est hautement compétitif, et les chances de recevoir des fonds sont minces. À titre d’exemple, le concours de subventions Projet de l’automne 2017 a connu un taux de réussite global de seulement 15 %1. En effet, malgré l’augmentation récente du financement accordé à la recherche, obtenir des fonds de ces organismes demeurera toujours difficile, particulièrement pour les chercheurs qui ne travaillent pas en laboratoire à temps plein, mais qui assument aussi des responsabilités cliniques. Les médecins biologistes peuvent accroître la probabilité d’obtenir une subvention en collaborant avec des groupes de recherche multidisciplinaires de plus grande envergure. Toutefois, nombreux sont les cliniciens qui doivent sortir des sentiers battus pour financer leurs travaux de recherche.

Les collaborations et les partenariats avec l’industrie sont une autre source de financement envisageable. Aux États-Unis, l’industrie pharmaceutique et les fabricants d’instruments médicaux financent plus de la moitié des travaux de recherche médicale. Si des lignes directrices rigoureuses en matière d’éthique sont suivies, les sociétés privées peuvent être une importante source de financement pour la recherche biomédicale3. Il existe de nombreux exemples de collaborations réussies développées dans le cadre de projets spécialisés qui avantageaient à la fois l’institut de recherche et les partenaires de l’industrie4. Francis Collins a récemment souligné la nécessité d’établir des partenariats novateurs pour tirer profit des forces traditionnelles respectives du secteur privé et des laboratoires universitaires5.

Les établissements universitaires peuvent octroyer de petits montants aux nouveaux chercheurs pour démarrer leurs projets ou accorder des subventions de démarrage aux projets de faible ampleur. Certains peuvent également être en mesure d’offrir du financement indirect, en payant par exemple le salaire des internes et des techniciens, et en fournissant un espace de bureau ou de recherche, ou de l’équipement. Les fondations affiliées à des universités ou à des hôpitaux peuvent elles aussi servir à soutenir la recherche. De plus en plus de chercheurs se tournent vers des sources privées non gouvernementales, pour la plupart des organismes sans but lucratif et des entreprises à but lucratif, pour financer leurs travaux de recherche. Les organismes sans but lucratif, notamment la Fondation des maladies du cœur et de l’AVC du Canada, la Société de recherche sur le cancer, la Fondation canadienne du rein et la Société Alzheimer du Canada, offrent des subventions beaucoup moins importantes que celles allouées par les organismes gouvernementaux. Cela dit, l’octroi du financement passe par un processus d’évaluation par les pairs et, comparativement aux organisations de plus

Une autre solution intéressante pour les cliniciens-chercheurs comme les pathologistes consiste à réaliser des travaux de recherche qui, malgré un budget restreint, présentent un rendement élevé. On peut entre autres penser aux projets de recherche morphologique, qui permettent d’analyser différents paramètres morphologiques et leur valeur au moment de fournir de l’information pronostique ou prédictive. Un bon exemple de ce type de projets est l’établissement de critères en lien avec le cancer héréditaire du côlon, qui étaient basés sur une analyse morphologique. D’autres modèles de réussite bien connus comprennent le système de classification de Gleason pour le cancer de la prostate et celui de Fuhrman pour l’hypernéphrome. Les analyses immunohistochimiques peuvent aussi avoir une valeur clinique importante, tout en permettant le maintien d’un budget de recherche considérablement plus faible. Récemment, l’intérêt pour la recherche morphologique a fortement augmenté en raison de progrès accomplis en pathologie numérique qui ont permis la réalisation d’analyses morphométriques hautement sophistiquées6,7. Les domaines de l’assurance de la qualité, de

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5

Lettre Du rÉDacteur en chef (suite) la gestion des données et de la formation médicale sont d’autres sphères qui peuvent nécessiter un soutien financier moins élevé que les secteurs associés à la recherche fondamentale. En conclusion, pour réussir à aller chercher du financement pour leurs travaux de recherche, les chercheurs, particulièrement ceux qui en sont à leurs débuts, ont besoin de mentorat et de formation professionnelle pour apprendre à rédiger des demandes de subventions et des manuscrits de recherche. De nombreuses ressources sont déjà disponibles à ce propos8, mais la ténacité demeure la principale clé, et l’obtention de subventions pour la recherche restera toujours un défi.

of medical research: important opportunity or treacherous pitfall? J Gen Intern Med. 2016;31(2): 228-233. 4. Di Meo A, Saleeb R, Wala SJ, Khella HW, Ding Q, Zhai H et al. A miRNA-based classification of renal cell carcinoma subtypes by PCR and in situ hybridization. Oncotarget. 2018;9(2):2092-2104. 5. Collins F. Has the revolution arrived? Nature. 2010;464(7289):674-675. 6. Bellis M, Metias S, Naugler C, Pollett A, Jothy S, Yousef GM. Digital pathology: attitudes and practices in the Canadian pathology community. J Pathol Inform. 2013;4:3.

George M. Yousef, M. D., Ph. D., FRCPC Rédacteur en chef, Revue canadienne de pathologie

1. Instituts de recherche en santé du Canada [En ligne]. Résultats du concours de subventions Projet de l’automne 2017. [cité le 17 avr. 2018]. Disponible : http://www.cihr-irsc.gc.ca/f/50802.html 2. Private funding for science (éditorial). Nat Methods. 2016;13:537. 3. Tierney WM, Meslin EM, Kroenke K. Industry support

7. Mirham L, Naugler C, Hayes M, Ismiil N, Belisle A, Sade S et al. Performance of residents using digital images versus glass slides on certification examination in anatomical pathology: a mixed methods pilot study. CMAJ open. 2016;4(1):E88-E94 8. Instituts de recherche en santé du Canada [En ligne]. L’art d’écrire une demande aux IRSC. [cité le 18 avr. 2018]. Disponible : http://www.cihr-irsc.gc.ca/f/45281.html

Letter froM the eDitor-in-chief (cont.) writing are available8 but persistence is usually the key and obtaining research funding will always be a struggle.

carcinoma subtypes by PCR and in situ hybridization. Oncotarget. 2018;9(2):2092-2104.

George M. Yousef MD, PhD, FRCPC Editor-in-Chief, Canadian Journal of Pathology

5. Collins F. Has the revolution arrived? Nature. 2010;464(7289):674-5.

1. Canadian Institutes of Health Research [Internet]. Fall 2017 Project Grant Results. [cited 2018 Apr 17]. Available from: http://www.cihr-irsc.gc.ca/e/50802.html

6. Bellis M, Metias S, Naugler C, Pollett A, Jothy S, Yousef GM. Digital pathology: Attitudes and practices in the Canadian pathology community. J Pathol Inform. 2013;4:3

2. Private funding for science (Editorial). Nat Methods. 2016;13:537. 3. Tierney WM, Meslin EM, Kroenke K. Industry support of medical research: important opportunity or treacherous pitfall? J Gen Intern Med. 2016;31(2):22833. 4. Di Meo A, Saleeb R, Wala SJ, Khella HW, Ding Q, Zhai H, et al. A miRNA-based classification of renal cell 6

Canadian Journal of Pathology | Volume 10, Issue 2 | www.cap-acp.org

7. Mirham L, Naugler C, Hayes M, Ismiil N, Belisle A, Sade S, et al. Performance of residents using digital images versus glass slides on certification examination in anatomical pathology: a mixed methods pilot study. CMAJ open. 2016;4(1):E88-94 8. Canadian Institutes of Health Research [Internet]. The art of writing a CIHR application. [cited 2018-Apr 18]. Available from: http://www.cihr-irsc.gc.ca/e/45281.html

WHY DOES

PD-L1 TESTING MATTER?

Immunotherapy decisions are informed by PD-L1 expression1-5 Current antibodies used in immuno-oncology target the PD-1/PD-L1 and CTLA-4 inhibitory checkpoint pathways. The treatment prevents tumour cells from taking advantage of the T-cell inhibitory pathways and escaping the immune response.

PD-L1 is not a binary marker6 Depending on the tumour expression levels of PD-L1, patients are likely to respond differently to different therapies.7 PD-L1 High PD-L1 Low/Neg

More likely to respond to PD-1/ PD-L1 inhibition with monotherapy2 May be best suited for combination therapy2

PD-L1 test to inform treatment decision

AstraZeneca is committed to enabling access to high-quality, validated assays for the purposes of treatment decision making Striving for the utmost confidence in patient treatment decisions led AstraZeneca to commission large-scale PD-L1 assay concordance studies in NSCLC8,9, UC10 and HNSCC11, and to be actively involved in the cross-industry Blueprint initiative to establish assay concordance in NSCLC.12 Data from Blueprint12, AZ9 and other8,13,14 studies in NSCLC build optimism that most PD-L1 assays (22C3, SP263, 28-8) are compatible and can potentially be interchanged using different algorithms. PD-L1 assays

Visit www.iDbladder.ca and www.iDlung.ca for more information References: 1. He J. et al. Development of PD-1/PD-L1 pathway in tumor immune microenvironment and treatment for non-small cell lung cancer. Sci Rep. 2015; 5:13110. doi:10.1038/srep13110. 2. Postow M.A. et al. Immune checkpoint in blockade in cancer therapy. J Clin Oncol. 2015;33(17):1974-82. 3. Gettinger SN et al. First-line monotherapy with nivolumab (NIVO; anti-programmed death-1 [PD-1]) in advanced non-small cell lung cancer (NSCLC): Safety, efficacy and correlation of outcomes with PD-1 ligand (PD-L1) expression. J Clin Oncol. 2015;33(Suppl):abstract 8025. 4. Garon EB et al. Pembrolizumab for the Treatment of Non–Small-Cell Lung Cancer. N Engl J Med. 2015;372:2018-2028. 5. Hellman MD et al. CheckMate 012: Safety and efficacy of first-line (1L) nivolumab (nivo; N) and ipilimumab (ipi; I) in advanced (adv) NSCLC. J Clin Oncol. 2016;34(Suppl):abstract 3001. 6. Patel SP and Kurzrock R. et al. PD-L1 expression as a predictive biomarker in cancer immunotherapy. Mol Cancer Ther. 2015;14(4):847-56. 7. Antonia S. et al. Safety and antitumour activity of durvalumab plus tremelimumab in non-small-cell lung cancer: a multicenter, phase 1b study. Lancet Oncol. 2016;17(3):299-308. 8. Rimm DL. et al. A prospective, multi-institutional, pathologist-based assessment of 4 immunohistochemistry assays for PD-L1 expression in non-small cell lung cancer. JAMA Oncol. 2017; doi: 10.1001/jamaoncol.2017.0013. 9. Ratcliffe MJ et al. Agreement between programmed cell death ligand-1 diagnostic assays across multiple protein expression cutoffs in non-small cell lung cancer. Clin Cancer Res 2017; 23(14); 3585–91. 10. Scott M.L. et al. Assessment of heterogeneity of PD-L1 expression in NSCLC, HNSCC, and UC with Ventana SP263 assay. Journal of Clinical Oncology 2016; 35(15suppl) published ahead of print. 11. Ratcliffe MJ. et al. A comparative study of PD-L1 diagnostic assays in squamous cell carcinoma of the head and neck. Annals of Oncology 2016; 27 (Suppl. 6): VI328-350. 12. Hirsch F.R. et al. PD-L1 IHC assays for lung cancer: Results from Phase I of the Blueprint PD-L1 IHC assay comparison project. J Thorac Oncol. 2017;12(2):208-22. 13. Brunnström H et al. PD-L1 immmunohistochemistry in clinical diagnostics of lung cancer: inter-pathologist variability is higher than assay variability. Modern Pathology 2017; 1-11. 14. Adam J et al. Multicentric French harmonization study for PD-L1 IHC testing in NSCLC. J Thorac Oncol. 2017;12(15): PL04a.04.

ONC1702E

The AstraZeneca logo is a registered trademark of AstraZeneca AB, used under license by AstraZeneca Canada Inc. © AstraZeneca Canada Inc. 2017.

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.DPORRSV&UDQEURRN7UDLO9HUQRQ 3HQWLFWRQ Rural Incentives* ~ Relocation Support ~ Diverse Practice Options

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MoleCulaR pathology CoRneR This article was peer-reviewed.

KeywoRds: next generation sequencing; laboratory diagnostics, genetic testing

the roLe of next Generation SequencinG in current LaBoratory DiaGnoSticS authors:

Henriett Butz1,2 MD, PhD, Attila Patócs1,2 MD, MSc, PhD

affiliations:

1

MTA-SE, Lendület Hereditary Endocrine Tumours Research Group, Hungarian Academy of Sciences and Semmelweis University, Hungary. 2 Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary

acknowledgements: This work has been funded by the National Research, Development and Innovation Office – NKFIH PD116093 and by Semmelweis Research-Innovation Fund (STIA-KF-17) to Henriett Butz, and by National Bionics Program to Attila Patócs. Attila Patócs is the recipient of “Lendület” grant from Hungarian Academy of Sciences. Henriett Butz is the recipient of a Bolyai Research Fellowship of Hungarian Academy of Sciences

The authors declare that there are no conflicts of interest regarding the publication of this paper. All authors have provided CAP-ACP with non-exclusive rights to publish and otherwise deal with or make use of this article, and any photographs/images contained in it, in Canada and all other countries of the world.

ABSTRACT In this article, we briefly review the current applications of next generation sequencing (NGS) with an overview of the technical aspects, the potential pitfalls of the methodology and bioinformatics, quality assurance and available guidelines. We summarize the role of NGS in current practice, introducing its advantages and potential drawbacks.

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MoleCulaR pathology CoRneR next Generation SequencinG (cont.)

Next generation sequencing (NGS) allows assessment of multiple genomic alterations at the same time.

Next generation sequencing (NGS) allows assessment of multiple genomic alterations at the same time. Due to its time and cost effectiveness it is being rapidly integrated into laboratory diagnostics. For both identification of germline mutations in inheritable diseases and somatic alterations in sporadic tumours, NGS provides a platform to improve personalized patient care through more precise diagnosis, prognosis and therapy.1,2 Although whole genome sequencing (WGS - sequencing of the entire genome) and whole exome sequencing (WES - sequencing all of the proteincoding genes) are available, the most prevalent applications of NGS are evaluation of specific genes using targeted panels.3 At present, the number of clinically significant genes with diagnostic or prognostic implications or necessary for indication of targeted therapies is low, therefore the majority of variants identified by WGS and WES cannot be easily interpreted from a clinical point of view.3 In targeted panels the interpretation of results is more direct and straightforward. The methodology is based on selection of a set of genes (from a few hundred to thousands depending on platform) by multiple polymerase chain reaction (PCR) or hybridization probes. During library preparation these fragments are tagged and bar-coded with platform specific adapters and sample barcodes. The sequencing libraries then are sequenced by synthesis. The bioinformatics analysis consists of three main steps.4 During primary analysis the instrument processes raw signals into nucleotide bases with various length (reads, see glossary) depending on the chemistry used. During the secondary analysis reads are aligned to a reference sequence and variants present in samples are identified (this process is named variant calling). During tertiary bioinformatics analyses the detected variants are annotated using several variant or mutation databases (variant analysis and

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interpretation).4 The entire process needs verification and validation. As approved NGS-based in vitro diagnostic (IVD) assays are not widely available, each laboratory is required to develop and validate its own protocols including sample and library preparation, bioinformatics analysis and quality assurance. This process of monitoring quality and performance, such as analytical sensitivity, specificity, repeatability, reproducibility, accuracy, robustness, limit of detection, limitations and uncertainty, is extremely Therefore, many challenging).5 laboratories choose commercial kits developed for specific-purposes, today most panels are used in oncology. However, even in this case verification is still required to ensure that the sequencing reaches the pre-defined performance specifications.5 Pre-analytical factors (e.g. sample handling, storage and nucleic acid extraction) are also important and highly influence NGS performance. For example, in surgical pathology specimens sample sizes can be limited, formalin fixation can impair DNA quality, and there is risk of crosscontamination while cutting. Additionally, morphological assessment is also needed to rule out contamination with normal tissue and the proportion of neoplastic cells in the analysed sample is also needed for correct interpretation.5 Currently there is no gold standard freely-available tool or filtering settings for bioinformatics analysis related to clinical NGS applications. Each laboratory, therefore, has to develop its own pipeline, but commercially available software packages designed for NGS data analysis are preferred over homemade bioinformatics solutions.5 Quality filtering of read alignment defines sensitivity and specificity of the test. Using very strong filtering could lead to loss of variants present at low

MoleCulaR pathology CoRneR next Generation SequencinG (cont.)

Quality filtering of read alignment defines sensitivity and specificity of the test. level which can be problematic for identification of mutations present at low level in somatic tumours due to the heterogeneity and/or low tumour cell ratio. Alternatively, inclusive filters can minimize false negative results but will increase the burden of confirmatory analysis.3 As read error ratio increases with the increase of coverage (read number aligned to the reference sequence) practically 300-500 reads/target has been suggested to be enough for diagnostics in molecular pathology, and variants under 5 reads are usually considered as likely false calls.3,5 During variant calling and analysis it is important to identify false sequence variants and to determine variant allele frequency (VAF, see glossary). In germline testing (diploid zygosity) VAF represents near 0 and 100% for homozygosity and near 50% for heterozygosity. In somatic testing, due to the above-mentioned factors (contamination with normal tissue, neoplastic cell ratio and tumour heterogeneity) VAF can be unpredictable. Also, in case of indels (insertion and/or deletion) it is recommended to confirm the variants by manual visualization of sequencing data using software e.g. Integrative Genomics viewer (IGV)6 or Tablet7 to reduce the risk of false positive or incorrect calls.5 After bioinformatics, in order to maintain technical validity, confirmatory tests are recommended and needed. For validation of germline variants, Sanger sequencing is generally accepted but due to its 10-20% VAF sensitivity in case of somatic tumour mutations it is not the ideal method for confirmation of all variants revealed.3 Some recommend duplicate sequencing of the entire panels using other methodologies or

performing single-locus test for every targeted gene.3 This significantly increases costs. One needs to keep in mind that some alterations are not detectable by NGS methods due to its methodology like repetitive sequences; copy-number variations; long insertiondeletions; structural variants; aneuploidity; or epigenetic alterations.8 In summary, handling large data sets generated by high-throughput technologies can result in false-positive and incidental findings.9 As a consequence some authors highlight overtesting, overdiagnosis, and overtreatment as major side effects of translational omics.10,11 To achieve clinically relevant results of molecular profiling, validation with large independent data sets is key.9 It is important to pay attention to other factors such as tumour type (different mutations can have different impact in different tumours) and clinical questions (diagnosis, prognosis and recommendation for targeted therapy). Both somatic and germline tissues can be assessed at the same time and the tumour tissue-specific variant subtraction can be helpful to find the clinically relevant mutations or variants3. Otherwise VAF can be taken into consideration. Generally, any germline variant presented >1% in the population is usually not relevant for hereditary cancer and various databases containing allele frequency data can help in filtering of relevant variants.3,5,12 Tumour specific mutation resources (COSMIC - Catalogue of Somatic Mutations in Cancer, TCGA – The Cancer Genome Atlas, MCG – My Cancer Genome) and literature mining could add relevant information for variant interpretation. At germline level ClinVar, dbSNP, Exome Variant Server and HGMD (The Human Gene Mutation Database) can help the interpretation. Hence, the continual development of high-quality and freely accessible databases will have a profoundly positive effect on the progress of genomic medicine.9

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MoleCulaR pathology CoRneR next Generation SequencinG (cont.) To estimate the significance of variants of uncertain significance (VUS) is challenging. Multiple source of information (variant frequency, predictions, and subsidiary functional studies) need to be taken into account in order to achieve recommendations of guidelines (e.g. ACMG recommendations) in categorization of a particular variant.12 Laboratories should also have a clear protocol about reporting secondary findings.13 Laboratory reports of NGS results also have special obligatory requirements. The report should focus on the clinically relevant information but a brief description of technical characteristics, bioinformatics pipelines, validation reports, variant annotations and classification should be included while additional, more detailed, data have to be available on request.5 RNA sequencing, also called whole transcriptome sequencing (WTS) is available although with less importance in clinical diagnostic testing. Nevertheless, in some rare cases like different splicing defects it can be particularly useful.14 Splicing is the process of intron removal from the primary transcripts that is an essential step in most mammalian gene expression. Mutations affecting splice sites or in genes involved in the protein complex of spliceosome can perturb this cellular function and lead to a perplexed balance of critical alternative mRNA isoforms.15 Variants leading to splicing defects have been described in hereditary, rare Mendelian disorders (e.g. Microcephalic Osteodysplastic Primordial Dwarfism type I (MOPD I, or also known as Taybi-Linder Syndrome) with characteristic neurological and skeletal abnormalities and with a life span from a few months to close to 13 years).15 More recently acquired somatic mutations in splicing factors (SF3B1, ZRSR2, SF1,

Next generation sequencing is usually not used in the classical meaning of screening (testing people without symptoms to diagnose a frequent illness). 12

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PRPF40B, U2AF1, SF3A1) has been also described in hematological malignancies including myelodysplastic syndrome, acute myeloid leukemia, chronic lymphocytic leukemia and chronic myelomonocytic leukemia.15 In research settings DNA and RNA sequencing are more widely used. Both methodologies have been successfully used in identification of novel pathogenic factors like new disease-causing mutations or on determination of differentially functioning pathways and mechanisms through altered gene expression patterns.16,17 The purpose of a laboratory test can be screening, diagnosis, prediction of prognosis or indication of targeted therapy (so called companion-test). Next generation sequencing is usually not used in the classical meaning of screening (testing people without symptoms to diagnose a frequent illness). Although the use of cancer susceptibility gene panels is expanding in cancer risk assessment it is hard to estimate the appropriate risk (and management) for people harbouring a particular mutation when there is no evidence of illness especially when disease penetrance is low.18 The diagnosis of a genetic disease is usually based on clinical manifestation and genetic tests are used for confirmation of the clinical diagnosis. Therefore, genetic testing applied for testing of a special gene or a specific gene set in Mendelian disorders.19 However in clinical genetics practice of e.g. mental retardation or development disorders exome (or clinical exome: genes having known disease-causing mutations) sequencing can help to reveal the pathogenic variations. Selected gene panel testing may be particularly useful in situations where there are multiple genes and mutations associated with a specific hereditary syndrome (e.g. dementia panels, epilepsy panels etc.) or hereditary cancer syndromes (breast and ovarian cancer, pheochromocytoma) and it is

MoleCulaR pathology CoRneR next Generation SequencinG (cont.)

As targeted therapy and molecular diagnostics are growing, a new term “theranostics” has been introduced.

difficult to predict which gene would be mutated due to the lack of phenotypegenotype correlation.18 In the genetic susceptibility of pheochromocytomaparaganglioma (PPGL) syndrome there are 16 different genes which together are responsible at least for one-third of all PPGL patients. Therefore germline mutations testing is recommended for all patients having PPGL to establish diagnosis of the hereditary disease. Targeted NGS library preparation kits are also available for sequencing large regions or genes such as mitochondrial genome, BRCA or CF genes that are particularly time and labour intensive and costly if performed by traditional Sanger sequencing.

inhibitor (crizotinib) is recommended.21 However, following first-line EGFR targeted therapy if the tumour has T790M mutation that is responsible for resistance first generation EGFR inhibitors has to be changed to newer agents (such as afatinib or osimertinib).21 As targeted therapy and molecular diagnostics are growing, a new term “theranostics” has been introduced. To date in clinical molecular diagnostics, applications of IVD kits are only available for targeted panels mostly related to therapy. In the near future, it is expected, that companies will develop more IVD kits to meet the clinical needs. Conclusion:

Prognosis prediction is important in cancer with somatic mutations. For different cancer panels covering oncogenes (e.g. EGFR, MET, RET, PDGFRA etc.) even IVD targeted panels are available. Some mutations are predictors for prognosis (e.g. KRAS mutations in lung cancer) and also estimate response to targeted therapy.21,22 This leads to “companion diagnostics” giving information that is essential for the effective use of a corresponding drug. The methods and kits used for companion diagnostics are mostly predetermined and those are criteria for treatment of the particular drug. Therapy related genetic tests are indispensable because most of the targeted therapy drugs are effective only when a certain mutation is present. Also mutations of resistance can serve as contraindications to therapy. For example, for lung cancer patients with stage IV non-small cell lung cancer (NSCLC) having a sensitizing EGFR mutations first generation EGFR tyrosine kinase inhibitors are recommended (e.g. erlotinib) while for tumours having ALK or ROS1 gene rearrangements ALK

NGS is extremely useful given its very high-throughput and cost-effectiveness. Due, however, to the complexity of the assays, high number of potential pitfalls, lack of standardization of laboratory processes, lack of IVD kits and bioinformatics pipelines its application in clinical diagnostics requires expert team work from both clinical and laboratory fields.5 The development of laboratory processes, bioinformatics pipelines and available data for clinical interpretation will pave the road for more guidelines and recommendations that have to be followed in order to give clinically reliable results. Glossary Adapter: short oligonucleotide sequence linked to the ends of DNA to be sequenced for allowing the sequencing primers to hybridize Barcode: individual short oligonucleotide sequences linked to the library fragments of each sample so they can be

distinguished during data analysis. Therefore multiple samples can be sequenced at the same time in the same reaction Alignment: a process used for aligning short sequence reads to a reference sequence Companion diagnostics: a diagnostic test used as a companion to a therapeutic drug to determine its applicability Coverage: coverage is the number of reads that include a given nucleotide in the reconstructed sequence. It can be given as read/base or read/target Filtering, quality filtering: a process used for filtering sequence reads based on various criteria including read quality scores, library preparation related quality (i.e. primer/adaptor specific sequences), homopolymers (DNA sequences containing several base pairs of the same nucleotide). Not uniquely aligned reads are also eliminated from further processing Pipeline: a complex workflow with several steps of bioinformatics analysis of NGS data Raw read: short sequence generated (“read”) by sequencing instrument. Theranostics: combines specific targeted diagnostic tests with specific targeted therapy based on the result of the former Variant allele frequency (VAF): the fraction of sequencing reads overlapping a genomic coordinate that support the non-reference (mutant/alternate) allele. (For instance in case of a germline mutation we can detect e.g. 321 wild type and 340 mutant base containing reads then VAF=340/(321+340). This means 51% of the reads are mutant. In germline variations we expect values close to 0, 100 or 50% for homozygote wild type, homozygote mutant or heterozygote variant. In case of somatic mutations these numbers can be more

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MoleCulaR pathology CoRneR next Generation SequencinG (cont.) variable due to tumour heterogeneity. This can have therapeutic consequence as mutation positivity can usually be established at a low VAF for targeted therapy (depending on the diagnostic kit used) Variant calling: a process used for identifying nucleotide variants from results of next generation sequencing data. References 1. Yousef GM. Personalized cancer genomics: the road map to clinical implementation. Clin Chem. 2012;58(4):661-3. 2. Pasic MD, Samaan S, Yousef GM. Genomic medicine: new frontiers and new challenges. Clin Chem. 2013;59(1):158-67. 3. Strom SP. Current practices and guidelines for clinical nextgeneration sequencing oncology testing. Cancer Biol Med. 2016;13:3-11. 4. Oliver GR, Hart SN, Klee EW. Bioinformatics for clinical next generation sequencing. Clin Chem. 2015;61(1):124-35. 5. Deans ZC, Costa JL, Cree I, Dequeker E, Edsjö A, Henderson S, et al. Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL. Virchows Arch. 2017;470(1):5-20.

and mappings. Methods Mol Biol. 2016;374:253-68. 8. Yousef GM. Personalized medicine in kidney cancer: learning how to walk before we run. Eur Urol. 2015;68(6):1021-2. 9. Diamandis EP, Li M. The side effects of translational omics: overtesting, overdiagnosis, overtreatment. Clin Chem Lab Med. 2016;54(3):389– 396. 10. Ibrahim R, Pasic M, Yousef GM. Omics for personalized medicine: defining the current we swim in. Expert Rev Mol Diagn. 2016;16(7):719-22. 11. Biesecker LG, Green RC. Diagnostic clinical genome and exome sequencing. N Engl J Med. 2014;370(25):2418-25. 12. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405-24. 13. Kalia SS, Adelman K, Bale SJ, Chung WK, Eng C, Evans JP, et al. Recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2016 update (ACMG SF v2.0): a policy statement of the American College of Medical Genetics and Genomics. Genet Med. 2017;19(2):249-255.

6. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29(1):24-6.

14. Wrighton KH. Genetic testing: The diagnostic power of RNA-seq. Nat Rev Genet. 2017;18(7): 392-393.

7. Milne I, Bayer M, Stephen G, Cardle L, Marshall D. Tablet: visualizing next-generation sequence assemblies

15. Padgett RA. New connections between splicing and human disease. Trends Genet. 2012;28(4):147-54.

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16. Comino-Méndez I, Gracia-Aznárez FJ, Schiavi F, Landa I, LeandroGarcía LJ, Letón R, et al. Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. Nat Genet. 2011;43(7):663-7. 17. Butz H, Ding Q, Nofech-Mozes R, Lichner Z, Ni H, Yousef GM. Elucidating mechanisms of sunitinib resistance in renal cancer: an integrated pathologicalmolecular analysis. Oncotarget. 2018;9:4661-4674. 18. Robson ME, Bradbury AR, Arun B, Domchek SM, Ford JM, Hampel HL, et al. American Society of Clinical Oncology policy statement update: genetic and genomic testing for cancer susceptibility. J Clin Oncol. 2015;33(31):3660-7. 19. Lee H, Deignan JL, Dorrani N, Strom SP, Kantarci S, QuinteroRivera F et al. Clinical exome sequencing for genetic identification of rare Mendelian disorders. JAMA. 2014;312(18):1880-7. 20. Lenders JW, Duh QY, Eisenhofer G, Gimenez-Roqueplo AP, Grebe SK, Murad MH, et al.; Endocrine Society. Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2014;99(6):1915-42. 21. Mayekar MK, Bivona TG. Current landscape of targeted therapy in lung cancer. Clin Pharmacol Ther. 2017;102(5):757-64. 22. Marabese M, Ganzinelli M, Garassino MC, Shepherd FA, Piva S, Caiola E, et al. KRAS mutations affect prognosis of non-small-cell lung cancer patients treated with first-line platinum containing chemotherapy. Oncotarget. 2015;6(32):34014-22.

Ap pttima® HP PV assay Do on n’tt just se ense a presence. Se en nse a thre eat.

New expanded Health Canad appro approval oval for Ap ptima® HPV Va now specifies in ntended uses `As a first-line primary screening g test. `For reflex testing of abnormal Pap P test results. ` For adjunctive testing together with w cervical cytology (co-tes The Aptima HPV assay offers a unique appro oach to HPV testing by targeting the E6/E7 mRNA of 14 high-risk HPV types associated with cervical cancer and precancerous lesions. The assay offers the t same excellent sensitivity and improved specificity compared to HPV DNA tests.2

Diagnostic Solutions | hologic.ca | info-canad [email protected] ADS-01657-CAN-EN Revv..001 Hologic, Inc. All rights reserved. Hologic, T The Science of Sure, Aptima, Panther and associated logos are trademarks and/or registered trademarks emarks of Hologic, Inc., and/or its subsidiaries in the United States and/or other countries. This information is intended for medical professionals and is not intended as a product solicitation or promotion where such activities are prohibited. Because Hologic materials are distributed through websites, eBroadcassts and tradeshows, it is not always possible to control where such materials appearr.. For specific information nformation on what products are available for sale in a particular country, please contact your local Holog gic representative or write to [email protected]. References: 1. Aptima HPV Assay [package insert, AW AW-14518-001 Revv..00 01.] 2. Haedicke & Iftnerr,, A review of the clinical performance of the Aptima HPV assayy,, Journal of Clinical Virologyy, 2016; 76: S40-S48.

RubRiQue de pathologie MolÉCulaiRe Cet article a été révisé par des pairs.

Mots-ClÉs : séquençage de nouvelle génération; diagnostics de laboratoire, dépistage génétique

Le rôLe Du SÉquençaGe De nouveLLe GÉnÉration DanS LeS DiaGnoSticS De LaBoratoire actueLS auteurs :

Henriett Butz M.D., Ph. D.1,2; Attila Patócs M.D., M. Sc., Ph. D.1,2

affiliations : 1MTA-SE, Groupe de recherche Lendület sur les tumeurs endocrines héréditaires, Académie hongroise des sciences et Université Semmelweis, Hongrie. 2 Département de médecine de laboratoire, Université Semmelweis, Budapest, Hongrie. remerciements : Ce travail a été financé par le Bureau national de la recherche, du développement et de l’innovation – NKFIH PD116093 et le Fonds recherche-innovation de Semmelweis (STIA-KF-17) pour Henriett Butz, par le programme national de biomimétisme pour Attila Patócs. Attila Patócs est récipiendaire de la bourse « Lendület » de l’Académie hongroise des sciences. Henriett Butz est récipiendaire de la bourse de recherche Bolyai de l’Académie hongroise des sciences.

Les auteurs ne déclarent aucun conflit d’intérêts relativement à la publication de cet article. Tous les auteurs ont accordé à la CAP-ACP le droit non exclusif de publier et d’utiliser cet article et toute photographie ou image qu’il renferme, ou d’en disposer autrement, au Canada et partout ailleurs dans le monde.

RÉSUMÉ Dans cet article, nous passons brièvement en revue les applications actuelles du séquençage de nouvelle génération (SNG) et donnons un aperçu de ses aspects techniques, des pièges potentiels de la méthodologie et de la bio-informatique, de l’assurance de la qualité et des lignes directrices disponibles. Nous résumons le rôle du SNG dans la pratique actuelle, et présentons ses avantages et ses inconvénients potentiels. 16

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RubRiQue de pathologie MolÉCulaiRe SÉquençaGe De nouveLLe GÉnÉration (suite)

Le séquençage de nouvelle génération (SNG) permet d’évaluer de nombreuses altérations génomiques en même temps.

Le séquençage de nouvelle génération (SNG) permet d’évaluer de nombreuses altérations génomiques en même temps. En raison de sa grande vitesse d’exécution et de son bon rapport coûtefficacité, il est rapidement intégré dans les diagnostics de laboratoire. Que ce soit pour repérer des mutations germinales dans les maladies héréditaires ou des mutations somatiques dans les tumeurs sporadiques, le SNG fournit une plateforme pour l’amélioration des soins personnalisés aux patients grâce à un diagnostic, à un pronostic et à un traitement plus précis1,2. Même si le séquençage du génome entier (SGE – séquençage du génome complet) et le séquençage de l’exome entier (SEE – séquençage de tous les gènes codant pour des protéines) sont accessibles, le SNG est plus souvent utilisé pour l’analyse de gènes précis au moyen de panels ciblés3. À l’heure actuelle, le nombre de gènes importants sur le plan clinique et ayant des répercussions sur le diagnostic ou le pronostic ou nécessaires à l’indication de traitements ciblés est faible, donc la majorité des variants identifiés par le SGE ou le SEE ne peuvent pas être facilement interprétés d’un point de vue clinique3. Par contre, en ce qui concerne les panels ciblés, l’interprétation des résultats est plus directe et plus simple. La méthodologie se fonde sur la sélection d’un ensemble de gènes (de quelques centaines jusqu’à plusieurs milliers, selon la plateforme) par plusieurs amplifications en chaîne par polymérase (PCR) ou sondes d’ADN. Durant la préparation des bibliothèques, ces fragments d’ADN sont étiquetés et on leur affecte un code à barres aux adaptateurs propres à la plateforme utilisée ainsi qu’un code à barres pour chaque échantillon. On effectue ensuite leur séquençage par synthèse. L’analyse bio-informatique comprend trois étapes principales4. Durant l’analyse primaire, l’instrument transforme les signaux bruts en bases nucléotidiques de différentes longueurs (lectures, voir le glossaire) selon le procédé chimique utilisé. Durant l’analyse secondaire, les lectures sont alignées à une séquence de référence et les

variants présents dans les échantillons sont repérés (ce processus se nomme la détection des variants). Durant l’analyse bio-informatique tertiaire, les variants détectés sont identifiés à partir de plusieurs bases de données de variants ou de mutations (analyse et interprétation des variants)4. L’ensemble du processus doit être vérifié et validé. Puisque les méthodes diagnostiques in vitro (DIV) fondées sur le SNG ne sont pas largement accessibles, chaque laboratoire doit élaborer et valider ses propres protocoles, incluant la préparation des échantillons et de la bibliothèque, son analyse bioinformatique et son assurance de la qualité. Ce processus de surveillance de la qualité et du rendement, qui comprend la sensibilité analytique, la spécificité, la répétabilité, la reproductibilité, l’exactitude, la robustesse, la limite de détection, les limites et l’incertitude, est extrêmement ardu5. Par conséquent, de nombreux laboratoires choisissent des trousses commerciales conçues à des fins précises et, de nos jours, la plupart des panels sont utilisés en oncologie. Cependant, même dans ce cas, la vérification est requise pour s’assurer que le séquençage atteint les caractéristiques de rendement prédéfinies5. Les facteurs préanalytiques (p. ex. la manipulation des échantillons, l’entreposage et l’extraction de l’acide nucléique) sont également importants et influencent grandement le rendement du SNG. Par exemple, en pathologie chirurgicale, la taille des échantillons prélevés peut être limitée, la fixation au formol peut nuire à la qualité de l’ADN, et il y a un risque de contamination croisée pendant la coupe. De plus, une évaluation morphologique est également nécessaire pour écarter la contamination par le tissu sain et la proportion de cellules néoplasiques dans l’échantillon analysé est aussi nécessaire pour bien interpréter les résultats5. Actuellement, il n’y a ni outil de référence accessible gratuitement, ni paramètres de filtration pour l’analyse bio-informatique relativement aux

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RubRiQue de pathologie MolÉCulaiRe SÉquençaGe De nouveLLe GÉnÉration (suite)

La filtration selon la qualité de l’alignement des lectures définit la sensibilité et la spécificité de l’analyse.

applications cliniques du SNG. Par conséquent, chaque laboratoire doit élaborer sa propre chaîne de traitement (pipeline), mais les progiciels disponibles sur le marché et conçus pour l’analyse des données du SNG sont préférés aux solutions bio-informatiques maison5. La filtration selon la qualité de l’alignement des lectures définit la sensibilité et la spécificité de l’analyse. L’utilisation de filtres très puissants pourrait entraîner la perte de variants faiblement exprimés, ce qui peut être problématique pour l’identification de mutations présentes en faible quantité dans les tumeurs somatiques en raison de l’hétérogénéité ou de la faible concentration des cellules tumorales. L’utilisation de filtres moins restrictifs peut quant à elle permettre de minimiser les faux négatifs, mais risque d’augmenter le fardeau de l’analyse de confirmation3. Puisque le taux d’erreur de lecture augmente avec l’augmentation de la couverture (nombre de lectures alignées à la séquence de référence), dans la pratique, de 300 à 500 lectures par cible seraient suffisantes pour le diagnostic en pathologie moléculaire, et les variants détectés dans moins de cinq lectures sont habituellement considérés comme étant vraisemblablement de fausses détections3,5. Durant la détection et l’analyse des variants, il est important d’identifier les faux positifs et de déterminer la fréquence allélique d’un variant (FAV, voir glossaire). Dans les tests germinaux (zygosité diploïde), la FAV est près de 0, et de 100 % pour l’homozygosité et près de 50 % pour l’hétérozygosité. Dans les tests somatiques, en raison des facteurs susmentionnés (contamination par le tissu sain, taux de cellules néoplasiques et hétérogénéité de la tumeur), il peut être difficile de prévoir la FAV. De plus, dans les cas d’indels (insertion ou délétion), il est recommandé de confirmer les variants par analyse manuelle des données de séquençage au moyen d’un logiciel comme Integrative Genomics Viewer (IGV)6 ou Tablet7 pour réduire le risque de faux positif ou de détection inexacte5.

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Afin de maintenir la validité technique après l’analyse bio-informatique, des tests de confirmation sont recommandés et nécessaires. Pour la validation des variants germinaux, le séquençage Sanger est généralement accepté, mais en raison de l’incertitude de 10 à 20 % de la FAV en cas de mutations d’une tumeur somatique, ce n’est pas la méthode idéale pour la confirmation de tous les variants détectés3. Certains recommandent de dupliquer le séquençage de l’ensemble des panels au moyen d’autres méthodologies ou de vérifier le locus pour chaque gène ciblé3. Or, cela fait grimper considérablement les coûts. Il faut garder à l’esprit que certaines altérations, comme les séquences répétitives, la variation du nombre de copies, les insertions-délétions longues, les variants structuraux, l’aneuploïdie ou les altérations épigénétiques8 ne sont pas détectables par les méthodes de SNG. En résumé, la manipulation de gros ensembles de données générés par des technologies à haut débit peut entraîner des faux positifs et des découvertes fortuites9. Par conséquent, certains auteurs soulignent que les tests excessifs, le surdiagnostic et le surtraitement sont des effets secondaires importants des approches « omiques »10,11. Pour parvenir à des résultats de profilage moléculaire pertinents sur le plan clinique, la validation au moyen de gros ensembles de données indépendants est essentielle9. Il est important de porter attention aux autres facteurs, comme le type de tumeur (des mutations différentes peuvent avoir des répercussions différentes sur des tumeurs différentes) et les sujets d’ordre clinique (diagnostic, pronostic et recommandation de traitement ciblé). Les tissus somatiques et germinaux peuvent être analysés en même temps et la soustraction du variant propre au tissu tumoral peut être utile pour trouver les mutations ou les variants pertinents sur le plan clinique3. Autrement, la FAV peut être prise en considération. Généralement, tout variant germinal présent chez moins de 1 % de la population n’est habituellement pas pertinent pour les

RubRiQue de pathologie MolÉCulaiRe SÉquençaGe De nouveLLe GÉnÉration (suite) cancers héréditaires, et les diverses bases de données sur la fréquence des allèles peuvent aider à filtrer les variants pertinents3,5,12. Les ressources sur les mutations propres aux tumeurs (COSMIC – Catalogue of Somatic Mutations in Cancer, TCGA – The Cancer Genome Atlas, MCG – My Cancer Genome) et une exploration de la littérature pourraient ajouter de l’information utile à l’interprétation des variants. Sur le plan germinal, ClinVar, dbSNP, Exome Variant Server et HGMD (The Human Gene Mutation Database) peuvent aider à l’interprétation. Ainsi, le développement continu de bases de données de grande qualité et accessibles gratuitement aura un effet positif important sur les avancées de la médecine génomique9. Il est difficile de juger de la pertinence des variants de signification incertaine (VSI). De multiples sources d’information (fréquence des variants, prévisions et études fonctionnelles secondaires) doivent être prises en compte afin de parvenir à respecter les recommandations des lignes directrices (p. ex. les recommandations de l’ACMG) pour la catégorisation d’un variant particulier12. Les laboratoires devraient également détenir un protocole bien défini sur la déclaration des observations secondaires13. Les rapports de laboratoire sur les résultats du SNG doivent aussi se conformer à des exigences particulières. Le rapport devrait présenter principalement l’information pertinente sur le plan clinique, avec une brève description des caractéristiques techniques, des chaînes de traitement bioinformatiques, des rapports de validation, des annotations et de la classification des variants, les données additionnelles, plus détaillées devant être disponibles sur demande5. Le séquençage de l’ARN, aussi appelé séquençage de transcriptome entier (STE), est possible, mais prend une moins grande place dans les tests diagnostiques cliniques. Néanmoins, dans certains cas rares, comme différents défauts d’épissage, il peut être particulièrement utile14. L’épissage est le processus au cours

duquel les introns sont éliminés des transcrits primaires, une étape essentielle à l’expression de la plupart des gènes chez les mammifères. Les mutations affectant les sites d’épissage ou se retrouvant dans les gènes impliqués dans le complexe protéique du splicéosome peuvent perturber cette fonction cellulaire et mener à un équilibre instable des isoformes d’ARNm alternatifs fondamentaux15. Les variants menant aux erreurs d’épissage ont été décrits dans des troubles mendéliens rares et héréditaires (p. ex. le nanisme microcéphalique ostéodysplasique primordial type 1 (MOPD type I, aussi connu sous le nom de syndrome de Taybi-Linder) présentant des anomalies squelettiques et neurologiques caractéristiques, l’espérance de vie des personnes atteintes variant de quelques mois à près de 13 ans)15. Des mutations somatiques plus récemment acquises dans les facteurs d’épissage (SF3B1, ZRSR2, SF1, PRPF40B, U2AF1, SF3A1) ont également été décrites dans des hémopathies malignes, dont le syndrome myélodysplasique, la leucémie myéloïde aiguë, la leucémie lymphoïde chronique et la leucémie myélomonocytaire chronique15. Dans les milieux de recherche, le séquençage de l’ADN et de l’ARN est plus largement utilisé. Les deux méthodologies ont permis d’identifier de nouveaux facteurs pathogènes, comme de nouvelles mutations à l’origine de maladies, et de déterminer des voies et des mécanismes différents par l’entremise de profils d’expression génique modifiés16,17. L’objectif d’un examen de laboratoire peut être le dépistage, le diagnostic, la prévision d’un pronostic ou l’indication d’un traitement ciblé (appelé test compagnon). Le séquençage de nouvelle génération n’est habituellement pas utilisé pour le dépistage dans son sens habituel (effectuer des examens chez des gens sans symptômes pour diagnostiquer une maladie fréquente). Même si l’utilisation de panels de gènes associés à une probabilité de cancer est de plus en plus répandue pour l’évaluation de ce risque, il

est difficile d’estimer le risque et la prise en charge appropriés chez les gens porteurs d’une mutation particulière lorsqu’il n’y a pas de signes de maladie, particulièrement lorsque la pénétrance du gène est faible18. Le diagnostic d’une maladie génétique est habituellement fondé sur une manifestation clinique et les analyses génétiques sont utilisées pour confirmer ce diagnostic clinique. Par conséquent, des analyses génétiques sont utilisées pour analyser un gène particulier ou un ensemble de gènes propres à des maladies mendéliennes19. Cependant, dans la pratique clinique de la génétique, par exemple pour un exome de retard mental ou de trouble du développement (ou un exome clinique : gènes ayant des mutations pathogènes connues), le séquençage peut aider à révéler des mutations pathogènes. Des analyses de panels de gènes sélectionnés peuvent être particulièrement utiles dans des situations où il y a plusieurs gènes et mutations associés à un syndrome héréditaire précis (p. ex. les panels de démence, de l’épilepsie, etc.) ou à des syndromes de cancers héréditaires (cancer du sein et de l’ovaire, phéochromocytome) et il est difficile de prévoir quel sera le gène muté en raison de l’absence de corrélation phénotype/génotype18. Dans la prédisposition génétique du syndrome paragangliome / phéochromocytome (PPGL), il existe 16 gènes différents qui, ensemble, sont responsables d’au moins un tiers des cas de PPGL. Par conséquent, une analyse des mutations germinales est recommandée pour tous les patients atteints du PPGL afin d’établir le diagnostic de maladie héréditaire. Les trousses de préparation de la bibliothèque de SNG ciblé sont aussi disponibles pour le séquençage de grandes régions ou de gènes comme le génome mitochondrial, le gène BRCA ou le gène responsable de la fibrose kystique, qui sont particulièrement exigeants sur le plan du temps et de la main-d’œuvre et

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RubRiQue de pathologie MolÉCulaiRe SÉquençaGe De nouveLLe GÉnÉration (suite) coûteux s’ils sont effectués séquençage Sanger traditionnel.

par

La prévision du pronostic est importante dans les cas de cancer avec mutations somatiques. Pour différents panels de cancers couvrant les oncogènes (p. ex. EGFR, MET, RET, PDGFRA, etc.) même des panels ciblant les DIV sont disponibles. Certaines mutations constituent des indications de pronostic (p. ex. les mutations KRAS dans le cancer du poumon) et permettent de prévoir la réponse au traitement ciblé21,22. Cela mène au « diagnostic compagnon » qui donne de l’information essentielle à l’utilisation efficace du médicament correspondant. Les méthodes et les trousses utilisées pour les diagnostics compagnons sont principalement prédéterminées et il s’agit de critères pour le traitement par un médicament particulier. Les analyses génétiques associées au traitement sont indispensables, car la plupart des médicaments ciblés sont efficaces seulement en présence d’une certaine mutation. De plus, les mutations de résistance peuvent être une contreindication au traitement. Par exemple, en ce qui concerne les patients atteints d’un cancer du poumon non à petites cellules de stade IV (CPNPC) présentant des mutations activatrices de l’EGFR, les inhibiteurs de la tyrosine kinase de l’EGFR de première génération sont recommandés (p. ex. l’erlotinib), alors que pour les tumeurs présentant des réarrangements d’ALK ou de ROS1, un inhibiteur d’ALK (crizotinib) est recommandé21. Cependant, après le traitement de première intention ciblant l’EGFR, si la tumeur présente une mutation T790M qui est responsable d’une résistance, les inhibiteurs de l’EGFR de première génération doivent être remplacés par de nouveaux agents (comme l’afatinib ou l’osimertinib)21. Comme les traitements ciblés et les diagnostics moléculaires sont de plus en plus présents, le terme « théranostique » a été inventé. À ce jour, dans les diagnostics moléculaires cliniques, les applications 20

des trousses de DIV sont seulement accessibles pour les panels ciblés principalement associés à un traitement. Dans un avenir rapproché, on s’attend à ce que les entreprises conçoivent davantage de trousses de DIV pour répondre aux besoins cliniques. Conclusion : Le SNG est extrêmement utile étant donné son haut débit et son bon rapport coût-efficacité. Cependant, en raison de la complexité des méthodes d’analyse, du grand nombre de pièges potentiels, de l’absence de normalisation des processus de laboratoire, du manque de trousses de DIV et de chaînes de traitement bioinformatiques, son application dans les diagnostics cliniques exige le travail d’une équipe d’experts des domaines cliniques et de laboratoire5. L’élaboration de procédures de laboratoire et de chaînes de traitement bio-informatiques et la mise à disposition de données pour l’interprétation clinique permettront la création de davantage de lignes directrices et de recommandations qui devront être suivies afin d’obtenir des résultats fiables sur le plan clinique. Glossaire Adaptateur : courte séquence d’oligonucléotides liée aux extrémités de l’ADN à séquencer pour permettre à l’amorce de séquençage de s’hybrider. Code à barres : courtes séquences individuelles d’oligonucléotides liées à la bibliothèque de fragments de chaque échantillon afin qu’ils puissent être différenciés durant l’analyse des données. Par conséquent, de nombreux échantillons peuvent être séquencés en même temps, dans la même réaction. Alignement : processus utilisé pour aligner des lectures de séquences courtes à une séquence de référence. Diagnostics compagnons : test diagnostique utilisé de pair avec un

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médicament thérapeutique déterminer son applicabilité.

pour

Couverture : désigne le nombre de lectures dans lesquelles un nucléotide donné est présent dans la séquence reconstruite. Elle peut être donnée en nombre de lectures par base ou en nombre de lectures par cible. Filtration, filtration selon la qualité : processus utilisé pour filtrer les lectures de séquence en fonction de divers critères, dont les scores de qualité de la lecture, la qualité de la préparation de la bibliothèque (c.-à-d. les séquences précises amorce/adaptateur), les homopolymères (séquences d’ADN contenant plusieurs paires de bases du même nucléotide). Les lectures qui n’ont pas d’alignement unique sont aussi éliminées du processus. Chaînes de traitement : flux des travaux complexe comprenant plusieurs étapes d’analyse bio-informatique des données de SNG. Lecture brute : séquence courte générée (« lecture ») par un instrument de séquençage. Théranostique : association des tests diagnostiques ciblés particuliers à un traitement ciblé précis en fonction du résultat de ces derniers. Fréquence allélique du variant (FAV) : fraction des lectures de séquençage chevauchant une coordonnée génomique qui soutient l’allèle mutant/alternatif. (Par exemple, en cas de mutation germinale, nous pouvons détecter 321 lectures comprenant le gène de type sauvage et 340 lectures contenant le gène muté, donc la FAV = 340/(321+340). Cela signifie que 51 % des lectures concernent une mutation. En ce qui concerne les variations germinales, nous nous attendons à des valeurs près de 0, 100 % ou 50 % pour le type sauvage homozygote, mutant homozygote ou variant hétérozygote. Dans le cas des mutations somatiques, ces chiffres peuvent être plus variables

RubRiQue de pathologie MolÉCulaiRe SÉquençaGe De nouveLLe GÉnÉration (suite) en raison de l’hétérogénéité de la tumeur. Cela peut avoir une conséquence sur le plan thérapeutique, puisque la présence de la mutation peut habituellement être détectée à une FAV faible pour le traitement ciblé (selon la trousse de diagnostic utilisée). Détection des variants : processus utilisé pour détecter les variants de nucléotides des résultats des données de séquençage de nouvelle génération. Références 1. Yousef GM. Personalized cancer genomics: the road map to clinical implementation. Clin Chem. 2012;58(4):661-3. 2. Pasic MD, Samaan S, Yousef GM. Genomic medicine: new frontiers and new challenges. Clin Chem. 2013;59(1):158-67. 3. Strom SP. Current practices and guidelines for clinical nextgeneration sequencing oncology testing. Cancer Biol Med. 2016;13:3-11. 4. Oliver GR, Hart SN, Klee EW. Bioinformatics for clinical next generation sequencing. Clin Chem. 2015;61(1):124-35. 5. Deans ZC, Costa JL, Cree I, Dequeker E, Edsjö A, Henderson S, et al. Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL. Virchows Arch. 2017;470(1):5-20. 6. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29(1):24-6. 7. Milne I, Bayer M, Stephen G, Cardle L, Marshall D. Tablet:

visualizing next-generation sequence assemblies and mappings. Methods Mol Biol. 2016;374:253-68. 8. Yousef GM. Personalized medicine in kidney cancer: learning how to walk before we run. Eur Urol. 2015;68(6):1021-2. 9. Diamandis EP, Li M. The side effects of translational omics: overtesting, overdiagnosis, overtreatment. Clin Chem Lab Med. 2016;54(3):389–396. 10. Ibrahim R, Pasic M, Yousef GM. Omics for personalized medicine: defining the current we swim in. Expert Rev Mol Diagn. 2016;16(7):719-22. 11. Biesecker LG, Green RC. Diagnostic clinical genome and exome sequencing. N Engl J Med. 2014;370(25):2418-25. 12. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405-24. 13. Adelman K, Bale SJ, Chung WK, Eng C, Evans JP, et al. Recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2016 update (ACMG SF v2.0): a policy statement of the American College of Medical Genetics and Genomics. Genet Med. 2017;19(2): 249-255. 14. Wrighton KH. Genetic testing: The diagnostic power of RNA-seq. Nat Rev Genet. 2017;18(7):392-393. 15. Padgett RA. New connections between splicing and human disease. Trends Genet. 2012;28(4):147-54.

16. Comino-Méndez I, Gracia-Aznárez FJ, Schiavi F, Landa I, LeandroGarcía LJ, Letón R, et al. Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. Nat Genet. 2011;43(7):663-7. 17. Butz H, Ding Q, Nofech-Mozes R, Lichner Z, Ni H, Yousef GM. Elucidating mechanisms of sunitinib resistance in renal cancer: an integrated pathologicalmolecular analysis. Oncotarget. 2018;9:4661-4674. 18. Robson ME, Bradbury AR, Arun B, Domchek SM, Ford JM, Hampel HL, et al. American Society of Clinical Oncology policy statement update: genetic and genomic testing for cancer susceptibility. J Clin Oncol. 2015;33(31):3660-7. 19. Lee H, Deignan JL, Dorrani N, Strom SP, Kantarci S, QuinteroRivera F et al. Clinical exome sequencing for genetic identification of rare Mendelian disorders. JAMA. 2014;312(18):1880-7. 20. Lenders JW, Duh QY, Eisenhofer G, Gimenez-Roqueplo AP, Grebe SK, Murad MH, et al.; Endocrine Society. Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2014;99(6):1915-42. 21. Mayekar MK, Bivona TG. Current landscape of targeted therapy in lung cancer. Clin Pharmacol Ther. 2017;102(5):757-64. 22. Marabese M, Ganzinelli M, Garassino MC, Shepherd FA, Piva S, Caiola E, et al. KRAS mutations affect prognosis of nonsmall-cell lung cancer patients treated with first-line platinum containing chemotherapy. Oncotarget. 2015;6(32): 34014-22.

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pathologists’ assistants CoRneR

Key woRds: rhabdomyosarcoma, robotic assisted, radical trachelectomy

caSe of the Month rhaBDoMyoSarcoMa authors:

Martin Grealish1, MLT, MTM, PA(CCCAP).

affiliations: 1Senior Pathologists’ Assistant, University Health Network

The author would like to thank Dr. Tatjana Terzic for contributions to this paper. The author declares that there are no conflicts of interest regarding the publication of this paper. The author has provided CAP-ACP with non-exclusive rights to publish and otherwise deal with or make use of this article, and any photographs/images contained in it, in Canada and all other countries of the world. Case Presentation The patient is a young teenage female presenting with weekly intermenstrual bleeding beginning soon after her first menstrual cycle. A year later the patient noticed a mass intermittently protruding through the vagina. Imaging studies at the time reported normal anatomy in the area. Over the next 12 months, the patient and her mother noticed the bulge was becoming more prominent. Repeat imaging showed an exophytic large vascular vaginal mass involving the lower aspect of the cervix and filling the midvagina with no obvious vaginal wall invasion. Surgical History: No past surgical history Medical History: No history of disease. A grandfather was diagnosed with prostate cancer and an aunt (maternal) was diagnosed with breast cancer. Imaging Studies: Magnetic resonance imaging (MRI) showed a 5.6 cm exophytic pedunculated mass originating 22

at the lower cervix and extending into the mid and lower vagina with no vaginal invasion. Blood Work/Diagnostic Tests: Pelvic exam showed a 4 cm mass filling the vagina, originating from the cervix and not attached to the vaginal. Vaginal wall was smooth. Biopsy Findings: Rhabdomyosarcoma embryonal/botryoid type. Surgical Procedure Robotic assisted trachelectomy including cervix, upper vagina and bilateral parametrial tissue. The planned surgical procedure of radical trachelectomy and abdominal cerclage was the initial approach. If, intra-operatively, the mass was unresectable then a decision would be made to perform a radical hysterectomy or provide neoadjuvant chemotherapy. Gross Pathological Findings See Figures 1 and 2.

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Gross Description The specimen labeled with the patient's name and as "radical trachelectomy with cervix, upper vagina and bilateral parametrial tissue" consists of an unoriented trachelectomy with attached vaginal cuff and parametrium received fresh. The entire specimen weighs 62 grams. The superior cervical resection margin is painted with green ink. The remaining soft tissue and parametrial tissue margins are painted with blue ink. The cervix has a maximum diameter of 3.5 cm and measures 4.5 cm in length. The external os is not identified at intraoperative consultation. At specimen preparation the area where the specimen is opened is identified as the 12 o'clock position. The vaginal cuff measures 9.0 cm in circumference and 2.5 cm in length. The right parametrium measures 2.0 x 1.0 cm. The left parametrium measures 2.0 x 1.0 cm. There is an almost circumferential exophytic mass located in the cervix occupying the entire clock face except the 4 to 5 position that measures 5.5 x 5.0 cm.

pathologists’ assistants CoRneR caSe of the Month rhaBDoMyoSarcoMa (cont.) The mass arises on the posterior cervix at the os extending into the vaginal space. The mass has a tan fleshy appearance and sectioning reveals a variegated pale white, shiny and hemorrhagic surface. The mass appears to invade to a depth of 3.1 cm, is 1.8 cm from the superior cervical excision margin, 0.8 cm from the inked soft tissue margin and 1.4 cm from the vaginal cuff margin. The superior cervical excision margin and a section of posterior cervix are submitted

for intraoperative consultation. Photographs are taken. Tissue is stored frozen. Block Submission Representative sections: A - superior cervical margin submitted en face B - representative section of posterior cervix C - 12 to 3 o'clock en face vaginal cuff margin

cáÖìêÉ=NK=

Tracelectomy Specimen cáÖìêÉ=OK=

Tumour with closest cervical margin.

D - 3 to 6 o'clock en face vaginal cuff margin E - 6 to 9 o'clock en face vaginal cuff margin F - 9 to 12 o'clock en face vaginal cuff margin G - right parametrium H - left parametrium I-K - sagittal section of specimen including cervix, vaginal cuff, inked soft tissue margin and tumour L-N - sagittal section of specimen including cervix, vaginal cuff, inked soft tissue margin and tumour O-Q - sagittal section of specimen including cervix, vaginal cuff, inked soft tissue margin and tumour Additional blocks: (Please note that the specimen is not oriented) R-AA - largest piece of cervix including vagina cuff and tumour circumferentially sectioned in 5 sections as follows (R, T, U, W, Y sections with inked margin) : R-S slice one bisected T - slice 2 bisected U-V - slice 3 bisected W-X - slice 4 bisected Y-AA - slice 5 bisected (Y-Z - ½ of tissue bisected) AB-AK second largest piece of cervix including vaginal cuff and tumour circumferentially sectioned into 7 sections as follows: (Please note all sections have inked margin) AB - slice one AC - slice 2 AD-AE - slice 3 bisected AF-AG - slice 4 bisected AH-AI - slice 5 bisected AJ - slice 6 AK - slice 7 AL-AN - third largest piece of cervix including vaginal cuff and tumour trisected (AL section with inked margin) AO-AP - smallest piece of cervix including vaginal cuff and tumour bisected (3AO section with inked margin) Microscopic findings See Figures 3, 4, and 5.

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pathologists’ assistants CoRneR caSe of the Month rhaBDoMyoSarcoMa (cont.) cáÖìêÉ=PK=

Diagnosis • Embryonal rhabdomyosarcoma with diffuse anaplastic features; • Surgical margins negative for tumour; • Heterologous (cartilage) differentiation present; • Negative for lymphovascular space invasion; and • Bilateral parametrial soft tissue negative for malignancy. Discussion

Tumour with cervix. cáÖìêÉ=QK=

Tumour with vaginal cuff. cáÖìêÉ=RK=

During embryonic development, rhabdomyoblast cells develop that will eventually form skeletal muscle. Rhabdomyosarcoma is a cancer made of cells that would normally develop into skeletal muscle. The embryonal origin of this type of cancer makes it much more common in children. Embryonal rhabdomyosarcoma (ERMS) is the most common type, arising in head/neck area, bladder, vagina and around prostate/testicles. ERMS tends to have a better prognosis. It is subtyped as botryoid and spindle cell rhabdomyosarcoma. Alveolar and anaplastic rhabdomyosarcomas are the more common types in young adults and adults. These are faster growing malignancies that require progressively intense treatment. ERMS has a good response to chemotherapy and most children are treated in this way. A combination of radiation therapy and surgery will be considered based on the size and location of the tumour. Resources Houghton JP, McCluggage WG. Embryonal rhabdomyosarcoma of the cervix with focal pleomorphic areas. J Clin Pathol. 2007;60(1):88-9 (http://www.ncbi.nlm.nih.gov/pmc/articl es/PMC1860605/)

Tumour with closest cervical margin. 24

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http://sarcomahelp.org/rhabdomyosarco ma.html

RubRiQue des assistants en pathologie

Mots-ClÉs : rhabdomyosarcome, assisté par robot, trachélectomie radicale

caS Du MoiS rhaBDoMyoSarcoMe auteur :

Martin Grealish1, TLM, MTM, AP(CCCAP).

affiliations : 1Assistant en pathologie principal, Réseau universitaire de santé

L’auteur aimerait remercier Dre Tatjana Terzic pour sa contribution à cet article. L’auteur ne déclare aucun conflit d’intérêts relativement à la publication de cet article. L’auteur a accordé à la CAP-ACP le droit non exclusif de publier et d’utiliser cet article, et toute photographie ou image qu’il renferme, ou d’en disposer autrement, au Canada et partout ailleurs dans le monde.

Présentation du cas La patiente est une jeune adolescente présentant une métrorragie hebdomadaire ayant commencé peu après son premier cycle menstruel. Un an plus tard, la patiente remarque une masse faisant saillie de manière intermittente dans son vagin. À cette époque, les examens d’imagerie n’avaient révélé aucune anomalie anatomique dans la zone. Au cours des 12 mois suivants, la patiente et sa mère ont remarqué que la bosse devenait plus proéminente. Un nouvel examen d’imagerie a révélé une grosse masse vasculaire exophytique dans le vagin englobant la partie inférieure du col de l’utérus et remplissant la partie intermédiaire du vagin sans invasion apparente de la paroi vaginale. Antécédents chirurgicaux : Aucune chirurgie antérieure.

Antécédents médicaux : Aucun antécédent de maladie. Son grand-père a reçu un diagnostic de cancer de la prostate et sa tante (du côté maternel) a reçu un diagnostic de cancer du sein. Examens d’imagerie : L’imagerie par résonance magnétique (IRM) a révélé une masse pédiculée exophytique de 5,6 cm émanant de la partie inférieure du col de l’utérus et se prolongeant dans les parties intermédiaire et inférieure du vagin sans invasion vaginale. Analyses sanguines/tests diagnostiques : L’examen pelvien a révélé une masse de 4 cm remplissant le vagin, émanant du col de l’utérus et ne s’attachant pas à la paroi vaginale. La paroi vaginale était lisse. Résultats de la biopsie : Rhabdomyosarcome de type embryonnaire/botryoïde.

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RubRiQue des assistants en pathologie caS Du MoiS rhaBDoMyoSarcoMe (suite) Intervention chirurgicale :

Résultats pathologiques :

Trachélectomie assistée par robot incluant le col de l’utérus, la partie supérieure du vagin et le tissu paramétrial bilatéral. L’approche initiale consistait en une trachélectomie élargie et un cerclage abdominal. Si, pendant l’intervention, la masse s’avérait non résécable, alors la décision serait prise d’effectuer une hystérectomie totale élargie ou d’administrer une chimiothérapie néoadjuvante.

Voir les figures 1 et 2.

cáÖìêÉ=NK=

Prélèvement de la trachélectomie cáÖìêÉ=OK=

Tumeur et marge cervicale la plus proche. 26

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Examen macroscopique Le prélèvement portant le nom de la patiente et la mention « trachélectomie élargie avec col de l’utérus, partie supérieure du vagin et tissu paramétrial bilatéral » consiste en une trachélectomie non orientée avec le dôme vaginal et le paramètre reçus non congelés. Le prélèvement complet pèse 62 grammes. La marge de résection cervicale supérieure est colorée à l’encre verte. Le tissu mou restant et les marges du tissu paramétrial sont colorés à l’encre bleue. Le col de l’utérus a un diamètre maximal de 3,5 cm et mesure 4,5 cm de longueur. L’orifice externe n’est pas identifié lors de la consultation intraopératoire. Lors de la préparation du prélèvement, la zone où le prélèvement est ouvert est désignée comme étant la position de 12 h. Le dôme vaginal mesure 9,0 cm de circonférence et 2,5 cm de longueur. Le paramètre droit mesure 2,0 cm x 1,0 cm. Le paramètre gauche mesure 2,0 cm x 1,0 cm. Il y a une masse exophytique presque circonférentielle située dans le col de l’utérus qui occupe la surface entière de l’horloge, sauf la position de 4 h à 5 h, qui mesure 5,5 cm x 5,0 cm. La masse provient de la partie postérieure du col de l’utérus, là où l’orifice se prolonge dans l’espace vaginal. La masse est charnue et brun clair et la coupe révèle une surface hémorragique brillante et d’un blanc pâle bigarré. La masse semble envahir à une profondeur de 3,1 cm, se trouve à 1,8 cm de la marge d’excision cervicale supérieure, à 0,8 cm de la marge du tissu mou marquée à l’encre et à 1,4 cm de la marge du dôme vaginal. La marge d’excision cervicale supérieure et une portion de la partie postérieure du col de l’utérus sont soumises à une consultation

RubRiQue des assistants en pathologie caS Du MoiS rhaBDoMyoSarcoMe (suite)

La marge d’excision cervicale supérieure et une portion de la partie postérieure du col de l’utérus sont soumises à une consultation intraopératoire.

intraopératoire. Des photographies sont prises. Du tissu est conservé congelé. Fragment soumis Sections représentatives : A - marge cervicale supérieure soumise de face B - section représentative de la partie postérieure du col de l’utérus

cáÖìêÉ=PK=

C - marge du dôme vaginal vue de face de 12 h à 3 h C - marge du dôme vaginal vue de face de 3 h à 6 h C - marge du dôme vaginal vue de face de 6 h à 9 h C - marge du dôme vaginal vue de face de 9 h à 12 h G - paramètre droit H - paramètre gauche I-K - coupe sagittale du prélèvement incluant le col de l’utérus, le dôme vaginal, la marge de tissu mou marquée à l’encre et la tumeur L-N - coupe sagittale du prélèvement incluant le col de l’utérus, le dôme vaginal, la marge de tissu mou marquée à l’encre et la tumeur O-Q - coupe sagittale du prélèvement incluant le col de l’utérus, le dôme vaginal, la marge de tissu mou marquée à l’encre et la tumeur Fragments additionnels : (veuillez noter que le prélèvement n’est pas orienté)

Tumeur et col de l’utérus. cáÖìêÉ=QK=

Tumeur et dôme vaginal.

R-AA - plus gros fragment du col de l’utérus, incluant le dôme vaginal et la tumeur, coupé de manière circonférentielle en 5 sections comme suit (coupes R, T, U, W, Y avec les marges marquées à l’encre) : R-S - tranche 1 bissectée T - tranche 2 bissectée U-V - tranche 3 bissectée W-X - tranche 4 bissectée Y-AA - tranche 5 bissectée (Y-Z - ½ du tissu bissecté) AB-AK - deuxième plus gros fragment du col de l’utérus, incluant le dôme vaginal et la tumeur, coupé de manière circonférentielle en 7 sections comme suit : (veuillez noter que toutes les coupes ont des marges marquées à l’encre) AB - tranche 1 AC - tranche 2 AD-AE - tranche 3 bissectée AF-AG - tranche 4 bissectée AH-AI - tranche 5 bissectée AJ - tranche 6 AK - tranche 7 AL-AN - troisième plus gros fragment du col de l’utérus, incluant le dôme Revue canadienne de pathologie | volume 10, numéro 2 | www.cap-acp.org

27

RubRiQue des assistants en pathologie caS Du MoiS rhaBDoMyoSarcoMe (suite)

Le RMSE répond bien à la chimiothérapie, qui est le traitement privilégié chez la plupart des enfants.

vaginal et la tumeur, divisé en trois (coupe AL avec marge marquée à l’encre) AO-AP - plus petit fragment du col de l’utérus, incluant le dôme vaginal et la tumeur, bissecté (coupe 3AO avec marge marquée à l’encre) Observations microscopiques Voir les figures 3, 4 et 5. Diagnostic • Rhabdomyosarcome embryonnaire avec caractéristiques anaplasiques diffuses; • Absence de tumeur sur les marges chirurgicales; • Présence de différenciation hétérologue (cartilage) • Absence d’invasion lymphovasculaire; • Absence de tumeur dans le tissu mou paramétrial bilatéral.

cáÖìêÉ=RK=

Discussion Durant le développement embryonnaire, des cellules rhabdomyoblastiques se développent pour former éventuellement le muscle squelettique. Le rhabdomyosarcome est un cancer formé de ces cellules qui constitueraient normalement le muscle squelettique. Ce type de cancer est beaucoup plus fréquent chez les enfants en raison de son origine embryonnaire. Le rhabdomyosarcome embryonnaire (RMSE) est le type le plus courant, survenant dans la région de la tête ou du cou, la vessie, le vagin et autour de la prostate ou des testicules. Le RMSE tend à avoir un bon pronostic. Ses soustypes sont le rhabdomyosarcome botryoïde et le rhabdomyosarcome à cellules fusiformes. Les rhabdomyosarcomes alvéolaires et anaplasiques sont les types les plus fréquents chez les jeunes adultes et les adultes. Ces malignités croissent rapidement et exigent un traitement d’intensité progressive. Le RMSE répond bien à la chimiothérapie, qui est le traitement privilégié chez la plupart des enfants. Une association de radiothérapie et de chirurgie sera envisagée en fonction de la taille et du site de la tumeur. Ressources Houghton JP, McCluggage WG. Embryonal rhabdomyosarcoma of the cervix with focal pleomorphic areas. J Clin Pathol 2007;60(1):88-9. (http://www.ncbi.nlm.nih.gov/pmc/artic les/PMC1860605/)

Tumeur et marge cervicale la plus proche. 28

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http://sarcomahelp.org/rhabdomyosarc oma.html

ReseaRCh aRtiCle Veuillez noter que seuls les résumés des articles sont offerts en français.

enquête Sur La SatiSfaction DeS MÉDecinS à L’ÉGarD DeS ServiceS D’anatoMie PathoLoGique en aLBerta auteurs :

Denise La Perle, technicienne de laboratoire médical (TLM)1; Angela Thompson, M.D., FRCPC2; Allie Moskalyk, TLM3; Reanne Cunningham, TLM4; Máire A. Duggan, M.D., FRCPC5.

affiliations : 1 Services de laboratoire, Alberta Health Services, Lethbridge, AB, Canada. 2 Département de pathologie et de médecine de laboratoire, Hôpital régional de Red Deer, Red Deer, AB, Canada. 3 Services de laboratoire, Hôpital Reine Elizabeth II, Grande Prairie, AB, Canada. 4 Département de pathologie, Hôpital de la miséricorde, Edmonton, AB, Canada. 5 Département de pathologie et de médecine de laboratoire, École de médecine Cumming, Université de Calgary, Calgary, AB, Canada.

RÉSUMÉ Objectif : Afin de déterminer le taux de satisfaction des Résultats : 998 exemplaires du questionnaire ont été envoyés, médecins ayant recours aux services d’anatomie pathologique et 257 médecins (26 %) ont répondu. Les résultats sommaires (AP) offerts par les Alberta Health Services (AHS), l’équipe étaient les suivants : provinciale responsable de la qualité de ces services a mis au point un questionnaire. Ce document a ensuite été envoyé à un groupe de q~ìñ=ÖäçÄ~ä=ÇÉ=ë~íáëÑ~Åíáçå nìÉëíáçåë médecins considérés comme de grands utilisateurs des services d’AP. 93 % Très élevée/élevée Ensemble des services fournis 94 % Très élevée/élevée Qualité et exactitude des rapports d’AP Méthodes : Nous avons passé en revue les 98 % Très élevée/élevée Qualité des interactions professionnelles avec articles publiés sur les méthodes

les pathologistes d’évaluation de la satisfaction des utilisateurs de services d’AP, mais avons 94 % Très élevée/élevée Niveau de courtoisie du personnel technique trouvé l’offre limitée. Puisqu’aucune Niveau de courtoisie du personnel de secrétariat 93 % Très élevée/élevée méthode d’enquête canadienne n’a été recensée, un sondage de type « QDans leurs commentaires, quelques répondants ont rapporté des problèmes concernant PROBES » du College of American la transmission en temps utile de certains rapports. Pathologists a servi à guider l’élaboration du questionnaire. Les 13 questions du sondage ont été regroupées Conclusions : De façon générale, le taux de satisfaction de ce comme suit : 1) interaction et communication; 2) administration; groupe de médecins à l’égard des services d’AP offerts en et 3) satisfaction globale à l’égard des services. Une section pour Alberta est élevé. Les résultats de l’enquête fournissent des les commentaires a également été incluse. Une copie papier du données de référence qui pourront servir au moment d’évaluer questionnaire a été envoyée aux médecins par la poste. Les le rendement futur du plan d’assurance de la qualité des répondants disposaient de six semaines pour répondre au sondage services d’AP et toute autre mesure d’amélioration de la et le renvoyer. Ils pouvaient également accéder à une version en qualité. ligne du questionnaire. Revue canadienne de pathologie | volume 10, numéro 2 | www.cap-acp.org

29

ReseaRCh aRtiCle This article was peer-reviewed.

KeywoRds: anatomical pathology physician satisfaction survey, quality assurance, alberta health services

Survey of PhySician SatiSfaction with anatoMicaL PathoLoGy ServiceS in aLBerta author:

Denise La Perle1 MLT, Angela Thompson2 MD, FRCPC, Allie Moskalyk3 MLT, Reanne Cunningham4 MLT, and Máire A. Duggan5 MD, FRCPC.

affiliations: 1Alberta Health Services; Laboratory Services, Lethbridge, AB, Canada. 2 Department of Pathology and Laboratory Medicine, Red Deer Regional Hospital, Red Deer, AB, Canada. 3 Laboratory Services, Queen Elizabeth II Hospital, Grande Prairie, AB, Canada. 4 Department of Pathology, Misericordia Hospital, Edmonton, AB, Canada. 5 Department of Pathology and Laboratory Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada. acknowledgement: Candace Spankie (Alberta Health Services, Primary Data Support, Analytics) and the AHS AP Quality Steering Committee are thanked for their contributions to this work. ^äÄÉêí~=eÉ~äíÜ=pÉêîáÅÉë=^å~íçãáÅ~ä=m~íÜçäçÖó= nì~äáíó=píÉÉêáåÖ=`çããáííÉÉ=jÉãÄÉêëÜáé `çJäÉ~ÇëW Denise La Perle, MLT, Máire A. Duggan MD, FRCPC

jÉãÄÉêëW Stephen Chow MD, FRCPC Reanne Cunningham, MLT Karim Khetani MD, FRCPC Irma Lapak, MLT Lisa DiFrancesco MD, FRCPC Sandra Eyton-Jones, MLT Pauline Alakija MD, FRCPC

Michael Greeff MD, FRCPC Diana Kristensen, PA Rita Leckie, MLT Allie Moskalyk, MLT Christopher Walker MD, FRCPC Angela Thompson MD, FRCPC Jenny Zadunayski, MLT Evelynn Blobel, MLT

The authors declare that there are no conflicts of interest regarding the publication of this paper. This research did not receive any specific grant from funding agencies in the public, commercial or not for profit sectors. All authors have provided CAP-ACP with non-exclusive rights to publish and otherwise deal with or make use of this article, and any photographs/images contained in it, in Canada and all other countries of the world. 30

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ReseaRCh aRtiCle anatoMicaL PathoLoGy uSer SatiSfaction Survey (cont.) ABSTRACT Objective: To determine the satisfaction rate of physician users of Anatomical Pathology (AP) services provided by Alberta Health Services (AHS), the provincial AP Quality team developed a survey that was distributed to a select physician group who were high volume users of the service. Methods: Published literature on methods to survey AP user satisfaction was reviewed and found to be scant. Since a Canadian based survey method was not found, a United States Q-probe survey from the College of American Pathologists was used to inform the development of the survey. The 13 survey questions were grouped as follows: 1) interaction/communication, 2) administration, and 3) overall satisfaction with services. A section for comments was also included. Physicians were mailed a paper copy with the option to use an online version, and were provided 6 weeks to complete and return the survey. Results: 998 copies of the survey were distributed and 257 physicians (26%) responded. Summary results were: nìÉëíáçåë

lîÉê~ää=ê~íÉ=çÑ=ë~íáëÑ~Åíáçå

Overall services Quality of AP report/accuracy Quality of professional interaction with pathologists Courtesy of technical staff Courtesy of secretarial staff

93% Very Good/Good 94% Very Good/Good 98% Very Good/Good 94% Very Good/Good 93% Very Good/Good

Some individual comments drew attention to problems with the timeliness of some reports.

Conclusions: Overall, there is a high level of satisfaction amongst this physician user group with AP services in Alberta. The results provide baseline data against which the future performance of the AP quality assurance plan and any quality improvement practices can be evaluated. INTRODUCTION In 2010, the Anatomical Pathology (AP) Quality Development and Implementation Team (AP Quality Team) was established by Alberta Health Services (AHS) to develop a provincial quality program that would measure, assess and identify opportunities to improve the quality of AP practice. This team reviewed the published literature, discussed current and best practice methodologies and developed and implemented the AHS AP Quality

Assurance (APQA) Plan.1 The plan identified key quality practices and generated 8 metrics to monitor and evaluate the quality of AP practice. One of the metrics (#17) was to survey physician satisfaction with AP services in Alberta. Monitoring stakeholder satisfaction with various aspects of AP services is also a requirement for laboratory accreditation by the College of Physicians and Surgeons of Alberta (CPSA).2 The purpose of this metric is to

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ReseaRCh aRtiCle anatoMicaL PathoLoGy uSer SatiSfaction Survey (cont.)

The development of the AHS AP satisfaction survey commenced with a literature review to determine which survey methodologies/questions might be appropriate.

identify areas of deficiency or weakness within the system that could be This communication improved. describes the process of development, distribution, analysis, and interpretation of the first province-wide survey which was conducted in 2016. METHODS The development of the AHS AP satisfaction survey commenced with a literature review to determine which survey methodologies/questions might be appropriate. Key words/terms for the search were “physician satisfaction survey” and “Anatomical Pathology”, and the search was conducted by the AHS AP Quality team co-lead. The initial search identified 4 relevant articles that aligned with surgical pathology focus of the AHS AP survey. All articles were United States (U.S.) based and articles relating to AP user satisfaction of Canadian laboratories were not discovered. The 4 articles included a College of American Pathologists (CAP) Q-probe study on Customer Satisfaction,3 2 references that outlined quality practices in AP and reinforced the benefits of monitoring clinician satisfaction,4,5 and a commentary article by Zarbo6 advising on the implementation of the CAP Qprobe surveys over several iterations. Survey articles excluded from consideration included those that pertained only to clinical laboratories. In the absence of any published Canadian survey data and because of similarities in AP practices between some U.S. laboratories and those in Alberta, the CAP Q-probe study was used to inform the development of the AP satisfaction survey questions. Thirteen survey questions were selected by the AP Quality Team based on applicability and value to current AHS AP quality practices (Figure 1). Questions were grouped as follows: 1) interaction/communication with pathologist, technologist, 2) administration; quality of the report; timeliness of

32

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reporting; responsiveness to problems, and 3) overall satisfaction with services. A section for individualized comments was also included. The ratings of very good/good/average/poor/very poor/not applicable and a satisfactory benchmark of 80% or more were selected to mirror those used by the CAP Q-Probe survey.3 The AP Quality Team created a “Surgical Pathology, Health Care Provider Satisfaction Survey Plan” which was presented to and approved by the AHS provincial Anatomical Pathology Network and Laboratory Leaders group. A limited budget for the survey was approved by AHS and this restricted to 1,000 the number of surveys that could be distributed. For administrative purposes AHS has structured the province as 5 health zones: North, Edmonton, Central, Calgary and South. Each zone has its own organizational structure including a Quality Assurance committee. This allows for local level decision making and communication with stakeholders e.g., communities, patients and clients, physicians, and AHS employees. Each zone apart from Edmonton has a single laboratory organization: Edmonton has 3 laboratory entities. The sample size for each of the 5 zones was calculated based on the size of the previous year’s annual volume of surgical pathology samples. Each zone’s Quality Assurance Subcommittee was instructed to select a specified number of physician users from a provincial distribution list compiled from contact data filed with the CPSA. The list was to include high volume users of AP services such as surgeons, radiologists, and dermatologists. The AP Quality Team collaborated with the AP departments in all five AHS zones, encompassing 17 laboratory sites, to establish which physicians were to receive the survey. The AP Quality Team then submitted the physician list to AHS Primary Data Support, Analytics, who were responsible for the production, distribution, and analysis of the survey.

ReseaRCh aRtiCle anatoMicaL PathoLoGy uSer SatiSfaction Survey (cont.) cáÖìêÉ=NK==^m=pìêîÉó=J=èìÉëíáçåå~áêÉ

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ReseaRCh aRtiCle anatoMicaL PathoLoGy uSer SatiSfaction Survey (cont.) cáÖìêÉ=OK=oÉëéçåëÉ=o~íÉ

cáÖìêÉ=PK=^m=pìêîÉó=^ep=j~é

998 paper surveys were distributed on April 4th, 2016. Respondents had the option of completing either the paper or online version. The survey package included a cover letter, the survey form and a postage paid return envelope. The cover letter included an introduction outlining the purpose of the survey, and instructions for completion and return. A link to the webpage of the electronic version of the survey was included in the cover letter. The survey closed 6 weeks later. Respondents who completed the survey were polled as to their professional affiliations and all returned surveys remained confidential. Individuals could include their contact information in the comments if they wanted a direct response to their concerns. RESULTS There were 257 (26%) completed surveys and 52 comments. The majority of respondents completed the survey on the paper copy as opposed to electronic (241 versus 16, respectively). Physicians constituted 99.6% of respondents and 0.4% were Nurses. Response rates were consistent between zones with North Zone the highest at 31.8% and Calgary Zone the lowest at 19.6% (Figure 2). Overall satisfaction level of AP Services across the province was 93% rated as “good/very good”. 6% of respondents rated their overall experience as “average”, with only 1% rating service as “poor”. Some variation in overall satisfaction between the zones did occur (Figure 3). Even though the majority of responses were rated as either “very good” or “good”, some zones received a higher rating of “very good” while other zones received a higher rating of “good”. Three of 5 zones received a small percentage of “average” ratings and one zone received a rating of “poor”. A provincial summary of the other questionnaire results is shown in Table 1 and some highlights were as follows. Quality of accuracy in Anatomical

34

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ReseaRCh aRtiCle anatoMicaL PathoLoGy uSer SatiSfaction Survey (cont.) Pathology reporting was 93% and rated as “very good/good”. Quality of interaction with professional pathologists had the highest rating at 98% “very good/good”. Overall interaction with technical and secretarial staff were given ratings of 94% and 93% “very good/good” respectively. In addition to the provincial summary,

results were also stratified by zone. When comparing performance between the zones, the majority of questions received similar responses, scoring higher in either “very good” or “good”. Questions related to teaching conferences and education, as well as the question related to turn-around-time (TAT), appeared to have the largest

q~ÄäÉ=NK=pìãã~êó=çÑ=íÜÉ=nìÉëíáçåå~áêÉ=oÉëìäíë

Very Good

Good

Average

Poor

Very Poor

Quality of the report/diagnostic accuracy

52.4%

41.6%

5.6%

0.4%

0.0%

Relevant and meaningful information on surgical reports provided

46.3%

46.3%

7.3%

0.0%

0.0%

Turn-around-time

32.4%

37.3%

19.7%

8.6%

2.0%

Tumor board conferences/multidisciplinary rounds/consultation, etc.

48.8%

36.0%

12.8%

1.8%

0.6%

Teaching conferences, courses or presentations

42.9%

37.1%

13.3%

5.7%

1.0%

Pathologists' accessibility for intraoperative (frozen section) consultation

55.0%

34.9%

6.7%

3.4%

0.0%

Quality of professional interaction with pathologists

68.6%

29.0%

2.4%

0.0%

0.0%

Technical Staff

55.6%

37.2%

5.6%

1.7%

0.0%

Secretarial Staff

56.1%

37.8%

5.5%

0.6%

0.0%

Notification of urgent, critical, unusual results

53.1%

34.3%

9.6%

2.5%

0.4%

Anatomical Pathology Laboratory responsiveness to problems

46.5%

37.5%

13.0%

3.0%

0.0%

Question

Courtesy of technical and secretarial staff:

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ReseaRCh aRtiCle anatoMicaL PathoLoGy uSer SatiSfaction Survey (cont.) q~ÄäÉ=OK=`çããÉåíë=Å~íÉÖçêáòÉÇ=Äó=ïçêâÑäçï=éÜ~ëÉ=~åÇ=^äÄÉêí~=eÉ~äíÜ=pÉêîáÅÉë=^å~íçãáÅ~ä=m~íÜçäçÖó=nì~äáíó= ^ëëìê~åÅÉ=mä~å=jÉíêáÅ

Pre-examination Metric 1 – Rate of samples with proper identification (2 comments) Overly complicated request/consultation form Lost requisitions causing incomplete reports - process improvement needed Metric 2 – Rate of samples with clinical information provided (1 comment) Requirement of clinical history too demanding Examination Metric 8 – Intra-operative consult turnaround time (3 comments) Frozen section and interpretation of margins has improved slightly. Turn-around-time occasionally too long Too far away from OR Post-Examination Metrics 14 & 15 – Percentage of Histology Level 4, 5 and 6 samples that meet the defined Turn-Around-Time (TAT) (31 comments) Request to Improve TAT (x2 comments) Access to pathology services over holidays, etc. is limiting. TAT delays noted Access to particular pathologist limiting. Delay in TAT for specific cases/specimen types Communication of Rush/urgent poor. TAT is variable TAT very long. No conferences Suggest prioritization of inpatient samples to improve TAT TAT delays - specific sample type (x2 comments) Long TAT (x12) TAT often long. Preliminary results changed in final result without notification TAT variable - difficult to plan treatment. Diagnostic accuracy is variable from each individual. Long TAT for cancer diagnoses Long TAT for cancer diagnoses in specific sample types TAT delays - suggest re-assigning case to another pathologist when away Delay in TAT, unsure of expected/target TAT Slow TAT for specific specimen types TAT delays and variable quality in reporting between pathologists Services very good in general. Variability in quality of reporting and TAT 36

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ReseaRCh aRtiCle anatoMicaL PathoLoGy uSer SatiSfaction Survey (cont.) q~ÄäÉ=OK=`çåíáåìÉÇ

Long TAT for paper reports. TAT could be improved. Metric 13 – Communication of Results (including revised reports) Reports frequently indicate "insufficient info for copy to provider" – disrespectful Difficulty ensuring results get to correct physician and those receiving copy. Incomplete report - tissues not identified in original report Addendums not flagged - changes in pathology result missed Majority of services are fine. Recurring issue of incorrect physician receiving report (issues with delay in results, patient confidentiality) Language barriers Would like to know who the assigned pathologist is I have always had timely and accurate reports. Pathologists have always been very helpful when called to discuss a case. Miscellaneous Comments Have not seen or been invited to pathology CME event/course/presentation No or minimal urology The pathologists are fantastic Very responsive team. Excellent pathology services. Amazing TAT. Much appreciated by all my cancer patients. Hematopathology service provided is outstanding. On uterus specimens, weights are very useful. variation in satisfaction ratings between the zones. Two separate fields were added to the bottom of the form for clinicians to provide feedback. The first field requested specifics and/or suggestions for improvement for parameters rated “Poor” or “Very Poor”. 56/257 (21.8%) of the surveys included comments in this field. The second field was for general comments and/or suggestions for improving surgical pathology services. 95/257 (36.9%) of the surveys included comments in this field. Comments were grouped into those relating to the preexamination, examination and post examination phases of the practice

pathway and then assigned to a specific APQA Plan Metric (Table 2). The majority of negative comments related to delays in the TAT of testing. Others identified the need to evaluate current policies and processes pertaining to requests for relevant clinical history with submitted samples, and difficulties in accessing pathological conferences. Positive comments were also included and many were highly complementary of pathologists’ professionalism and skill. A stepwise approach to the communication of the survey results was decided upon by the AP Quality Steering Committee (which had by then replaced the AP Quality team). The committee

determined 2 versions of the report were needed in order to effectively present the findings. The first version included all collated comments. The comments were de-identified and this version was presented to all Medical and Leadership groups within AHS Laboratory Services. The second version of the report was specific to each zone. Comments from the respective zone were left as is, including identifying information. Each zonal Quality Assurance Subcommittee received their zone-specific report and determined whether or not additional actions were needed to improve services and respond to the survey comments. A formal communication to all laboratory physicians and staff informed them of

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ReseaRCh aRtiCle anatoMicaL PathoLoGy uSer SatiSfaction Survey (cont.) the survey process and the collated results. It also afforded an opportunity to provide feedback to the Quality Steering Committee. A similar communication was distributed to all Alberta physicians to express the committee’s appreciation of their participation, and to highlight proposed action items originating from the survey results. DISCUSSION The AHS AP Quality team developed and conducted a formal user survey of AP services provided by Alberta’s laboratories. The results of the survey demonstrated an overall high level of satisfaction with AP services. The satisfaction level was lowest (69%) for TAT of testing, and highest (97%) for quality of professional interactions with pathologists. The results provide baseline data against which the future performance of the APQA plan and any quality improvement practices can be evaluated. To the best of our knowledge, this is the first published study of user satisfaction of AP services provided by any Canadian laboratory and is the first provincially based survey. We share this experience so that other Canadian jurisdictions who may be developing user surveys of their own can benefit from our endeavors and wish to adopt or customize our methodology. The overall positive results can be partly attributed to the provincial APQA plan which has been in effect since 2010.1 Numerous process improvement initiatives have been implemented over the past 7 years, which could have affected the AP services in a positive manner. An organizational structure comprised of the AP Quality Steering Committee, the provincial AP Network, the provincial AP Special Interest Groups, and zonal AP Quality Assurance Subcommittees have established a functional network of expert resources in AP services in Alberta. This structure enables the 38

identification of quality issues, discussion and resolution at a provincial level, and a coordinated implementation of best quality practices. Based on the survey results and the comments received, provincial recommendations were developed to improve AP services. Recommendations included an in-depth review of metrics 14 & 15 which relate to TATs of level 4 and 5 specimens respectively, and additional education and communications to address the questions/concerns related to the laboratory request for clinical history. Zonal process improvement initiatives have been identified and are ongoing to decrease TAT. To address the questions about the submission of clinical history with AP samples, educational sessions are being provided to select clinics/programs/physicians along with a provincial bulletin, communicating what patient/clinical information is required and why it is important. Implementation of a new provincial AP requisition form will also be used as an opportunity to further educate clinicians about the need for provision of clinical history. This survey serves as a baseline for future surveys which will occur biannually. We accept that the format provided the opportunity to convey a lower degree of satisfaction in an area but did not allow for identification of the specific problem(s) that prompted a lower rating. We also acknowledge that the 5-scale rating system may have resulted in too many neutral responses. Development of the next AP survey for distribution is underway. A working group of the AP Quality Steering Committee has been established to revise the survey; questions and the rating system are being modified to obtain more meaningful and specific data on issues and problems. The next round of satisfaction surveys will also include questions related to the Cytopathology and Autopsy services since these are now part of the APQA plan. Some questions may be tailored

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more individually to address particular concerns. The method of distribution will be evaluated given that the more costly use of paper forms is surprisingly a preference in Alberta. Ideas to improve response rates will be explored and a broader spectrum of physicians will be selected so that responses can be normalized. REFERENCES 1. Duggan MA, Trotter T. Alberta Health Services: Anatomical Pathology Quality Assurance Plan. Can J Pathol. 2016; 8(3):10-21. 2. College of Physicians and Surgeons of Alberta. Western Canada Diagnostic Accreditation Alliance. CPSA Advisory Committee on Laboratory Medicine. Standards for Diagnostic Laboratory Accreditation: Anatomic Pathology, version: February 2017 - v5. 3. Zarbo RJ, Nakhleh RE, Walsh M; Quality Practices Committee, College of American Pathologists. Customer satisfaction in anatomic pathology. A College of American Pathologists Q-Probes study of 3065 physician surveys from 94 laboratories. Arch Pathol Lab Med. 2003; 127:23-9. 4. Nakhleh RE, Fitzgibbons PL. Quality management in anatomic pathology: promoting patient safety through systems improvement and error reduction. Northfield, IL:The College of American Pathologists; 2005. 5. Nakhleh RE. Core components of a comprehensive quality assurance program in anatomic pathology. Adv Anat Pathol. 2009;16:418-23. 6. Zarbo RJ. Determining customer satisfaction in anatomic pathology. Arch Pathol Lab Med. 2006;130:645-9.

ReseaRCh aRtiCle Veuillez noter que seuls les résumés des articles sont offerts en français.

SurexPreSSion et aMPLification De HER2 chez LeS PatientS atteintS D’un cancer GaStrique DiffuS à terreneuve-et-LaBraDor auteurs :

Robyn Ndikumana, M.D., B.Sc.Inf.1; Altaf Taher, M.D., FRCPC, FCAP1; Polycarp Erivwo, MBBS, FMCPath, FRCPC1; Melanie Seal, M.D., FRCPC2

affiliations : 1Département d’anatomie pathologique, Université Memorial, St. John’s, Terre-Neuve-et-Labrador, Canada 2 Département d’oncologie médicale, Université Memorial, St. John’s, Terre-Neuve-et-Labrador, Canada

RÉSUMÉ

La population de TerreNeuve-et-Labrador (T.-N.-L.) est unique. Elle compte en effet plusieurs familles porteuses d’une mutation du gène CDH1, qui sont prédisposées au cancer gastrique diffus.

Le cancer gastrique diffus est associé à un pronostic défavorable. Or, au stade avancé de la maladie, la surexpression du récepteur 2 du facteur de croissance épidermique humain (HER2) a des conséquences thérapeutiques importantes. La population de Terre-Neuve-et-Labrador (T.-N.-L.) est unique. Elle compte en effet plusieurs familles porteuses d’une mutation du gène CDH1, qui sont prédisposées au cancer gastrique diffus. Notre objectif consistait à mesurer le taux de cancers gastriques diffus HER2 positifs à T.-N.-L. et à déterminer le nombre de patients atteints d’une mutation du gène CDH1 au sein de cette population. Dans cette optique, nous avons mené une étude rétrospective descriptive sur l’ensemble des patients de l’est de Terre-Neuve ayant reçu un diagnostic de cancer gastrique diffus de 2004 à 2013. Les cas de cancer gastrique diffus ont été relevés grâce au registre provincial du cancer, et ceux auxquels étaient associés des tissus archivés ont été inclus dans l’étude. Des échantillons pathologiques prélevés par biopsie ou par résection chirurgicale ont été obtenus, puis classés en fonction des catégories « diffus » ou « peu différencié ». Une immunohistochimie (IHC) a ensuite été réalisée pour déterminer le statut HER2. L’hybridation in situ en fluorescence (FISH) a été utilisée pour définir le statut HER2 des cas présentant des résultats équivoques (2+) à l’IHC. Nous avons établi que parmi les 100 cas de cancer gastrique diffus recensés, 17 (17 %) présentaient une mutation connue du gène CDH1. Dans la cohorte associée à une mutation du gène CDH1, 1 cas s’est avéré HER2 positif. Par ailleurs, 3 des 83 cas de cancer gastrique diffus restants étaient HER2 positifs. Notre étude nous a permis de conclure que le taux de positivité HER2 chez les patients atteints d’un cancer gastrique diffus dans l’est de Terre-Neuve est d’environ 4 %, un résultat comparable à ceux déjà publiés. Revue canadienne de pathologie | volume 10, numéro 2 | www.cap-acp.org

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ReseaRCh aRtiCle This article was peer-reviewed.

KeywoRds: diffuse gastric cancer, heR2, Cdh1

her2 overexPreSSion anD aMPLification in DiffuSe GaStric cancer in newfounDLanD anD LaBraDor authors:

Robyn Ndikumana1 MD, BScN, Altaf Taher1 MD, FRCPC, FCAP, Polycarp Erivwo1 MBBS, FMCPath, FRCPC, Melanie Seal2 MD, FRCPC.

affiliations: 1Department of Anatomical Pathology, Memorial University of Newfoundland, St. John’s, NL, Canada. 2 Department of Medical Oncology, Memorial University of Newfoundland, St. John’s, NL, Canada.

The authors declare that there are no conflicts of interest regarding the publication of this paper. All authors have provided CAP-ACP with non-exclusive rights to publish and otherwise deal with or make use of this article, and any photographs/images contained in it, in Canada and all other countries of the world.

ABSTRACT

The population of Newfoundland and Labrador (NL) is unique and has multiple families with a CDH1 gene mutation, and thus a predisposition to developing diffuse gastric cancer. 40

Diffuse gastric cancer is associated with poor outcomes, however, human epidermal growth factor receptor 2 (HER2) overexpression, in the setting of advanced disease, has significant therapeutic implications. The population of Newfoundland and Labrador (NL) is unique and has multiple families with a CDH1 gene mutation, and thus a predisposition to developing diffuse gastric cancer. Our objectives were to determine the rates of HER2 positive diffuse gastric cancer in NL, and to determine the number of patients with a CDH1 gene mutation in this population. A retrospective descriptive study of all patients diagnosed with diffuse gastric cancer from 2004-2013 within eastern NL was conducted. Patients were identified using the provincial cancer registry and included if archival tissue was available. Pathological samples from biopsy or surgical resection were obtained, classified as diffuse or poorly differentiated, then assessed using immunohistochemistry (IHC) to determine HER2 status. Fluorescence in-situ hybridization (FISH) was used as gold standard to characterize HER2 status in the cases of equivocal (2+) results on IHC. We determined that of the 100 cases of diffuse gastric cancer, 17 (17%) have a known positive CDH1 gene mutation. In the cohort with a CDH1 gene mutation, 1 patient was found to be HER2 positive. Of the remaining 83 cases of diffuse gastric cancer, 3 were HER2 positive. Our study concluded that the rate of HER2 positivity among patients with diffuse gastric cancer in eastern Newfoundland is approximately 4%; this is comparable with the published literature.

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ReseaRCh aRtiCle her2 in DGc (cont.)

Gastric cancer (GC) remains a leading cause of cancer-related deaths worldwide.

INTRODUCTION

METHODS

Gastric cancer (GC) remains a leading cause of cancer-related deaths worldwide. The World Health Organization (WHO) places GC as the third most common cause of cancer deaths globally.1 The Canadian Cancer Statistics report projects that 2200 new cases of gastric cancer will be diagnosed in 2017.2

This is a retrospective descriptive study of all patients diagnosed with diffuse gastric cancer between 2004 and 2013 within the eastern region of NL. Patients were identified using the provincial cancer registry and included if archived tissue was available. An initial broad search of the provincial cancer database was performed to obtain all patients in NL who had been diagnosed with any type of gastric cancer between 20002013. From this data of 1059 patients, each anatomical pathology report was assessed to determine the specific type of gastric cancer diagnosis for each patient. Patients were included if they were 18 years of age or older, were diagnosed with diffuse or poorly differentiated gastric cancer between 2004-2013 within Eastern Health, NL, and if archived tissue and health records were available.

Human epidermal growth factor receptor (HER2) is an oncoprotein that is a known predictive biomarker for GC, as the addition of trastuzumab to chemotherapy has shown a survival benefit in advanced HER2 positive GC.3,4 Trastuzumab is a recombinant humanized monoclonal antibody directed against the human epidermal growth factor receptor 2.5 The most common histologic subtype of GC is intestinal type, which is related to environmental factors like Helicobacter pylori infection, diet, and lifestyle.4 Intestinal type GC is well described in the literature, with HER2 positive rates ranging from 3.7-34.8%.6-13 Diffuse gastric cancer is less common, with HER2 positive rates in recent studies ranging from 2.8-6.6%.6-11,14 Newfoundland has four large kindreds that exhibit a CDH1 gene mutation and thus a predisposition to hereditary diffuse gastric cancer (HDGC).15 Many individuals with a CDH1 positive mutation choose to undergo a prophylactic gastrectomy as their cumulative lifetime risk of developing diffuse GC is approximately 70% in males and 56% for females.16 Currently, little is known regarding the relationship between a positive CDH1 mutation and rates of HER2 positive GC. This study was designed to help further understand this relationship, therefore the objectives of this study were to determine the rates of HER2 positive diffuse gastric cancer in the NL population; and to determine the number of patients with a CDH1 gene mutation who have HER2 positive diffuse gastric cancer.

Pathology samples from biopsy or surgical resection were individually obtained and stained with hematoxylin and eosin (H&E) to confirm the presence of diffuse gastric cancer. In cases where both biopsy and gastrectomy specimens were available, biopsies were used for further testing due to the increased efficiency of searching a smaller tissue sample. Each sample was reviewed and interpreted by a Royal College of Physicians and Surgeons of Canada certified Anatomical Pathologist (AT & PE). Samples were assessed using the Lauren classification14,17 to confirm a diagnosis of diffuse gastric adenocarcinoma (Figure 1). Those determined to be intestinal adenocarcinoma were not included in this study. All samples were assessed by immunohistochemistry (IHC) to determine HER2 status using the Ventana PATHWAY kit (Primary antibody 4B5). Samples were scored on a scale of 0 to 3+. A positive score (3+) is defined as strong complete, basolateral, or lateral membranous reactivity in ≥10% of tumour cells.18 In the cases of equivocal (2+) results

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ReseaRCh aRtiCle her2 in DGc (cont.) cáÖìêÉ=NK= A

B

Diffuse gastric carcinoma with signet ring cells. Hematoxylin and eosin. A (10x) and B (20x).

(weak to moderate complete, basolateral, or lateral membranous reactivity in ≥10% of tumour cells), fluorescence in-situ hybridization (FISH) was used as the gold standard to determine the final HER2 status. The Abbott PathVysion FISH kit was used for this test. HER2 positivity using FISH is defined as HER2:chromosome 17 ratio of ≥2.0.18 IHC testing was completed at the Health Sciences Center laboratory in St. John’s, NL for 56 samples. 44 samples had previously received IHC testing at a central laboratory, as during that time period IHC testing was not offered at the Health Sciences Center in St. John’s, NL. All FISH testing was completed at a central laboratory as this test is not currently available at our laboratory. CDH1 mutation testing was completed on available samples from consenting patients who had been previously referred to the provincial genetics program. Target Mutation analysis was

CDH1 mutation testing was completed on available samples from consenting patients who had been previously referred to the provincial genetics program. 42

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completed using TaqMan SNP Genotyping technique (Real-time PCR) to test for c.2398delC mutation of the CDH1 gene. This testing was performed at the Health Sciences Center, St. John’s, NL for 12 patients; the remaining 8 were tested at a central laboratory. Ethics approval was obtained through the Health Research Ethics Board (HREB) prior to the commencement of this study. RESULTS Of the 100 samples, 88 were biopsy specimens, and 12 were gastrectomy specimens (8 of which were prophylactic gastrectomy specimens for a positive CDH1 mutation). This study determined that of the 100 cases of diffuse gastric cancer that were found through the search of the provincial cancer database, 20 underwent testing for CDH1 gene mutation, and 17 of these had a positive CDH1 gene mutation. The remaining patients were either not referred (78), refused testing (1), or died before their appointment (1) with a genetic counsellor. In the cohort with a CDH1 gene mutation, 1 patient was HER2 positive. Of the remaining 83 cases of diffuse

ReseaRCh aRtiCle her2 in DGc (cont.)

A total of 4 patients out of 100 (4%) ultimately had HER2 positive diffuse gastric carcinoma.

gastric cancer, 3 were HER2 positive (Figure 2). 18 specimens had equivocal (2+) results on IHC testing and were subsequently tested using FISH for confirmation of HER2 status. Of the 18 equivocal (2+) samples that underwent FISH, 2 had amplified HER2 scores (Figure 3). A total of 4 patients out of 100 (4%) ultimately had HER2 positive diffuse gastric carcinoma. DISCUSSION Our study of patients in NL with diffuse gastric cancer over a ten-year period (2004-2013) found that the rate of HER2 positivity in diffuse gastric was 4%. The results of this study are comparable with other studies found in the literature in which HER positive rates in diffuse gastric cancer have been cited ranging from 2.8-6.6%.6,-11,14 Aditi et al. (2016)8 used a very similar approach in their study, first using Lauren’s classification to distinguish between intestinal and diffuse adenocarcinoma, then using IHC, and subsequently FISH in equivocal cases, to

determine HER2 status. This study had a total of 58 specimens, of which only 15 were diffuse type adenocarcinoma. While their results demonstrated diffuse GC with a HER2 positive rate of 6.2%, this sample size is small. Silva et al. (2015)11 found that 5% (2/38) of diffuse gastric cancers showed HER2 positivity, but again this was a rather small sample size. Koopman et al. (2015) demonstrated a HER2 positive rate of 3.5% among their diffuse gastric adenocarcinoma specimens which is similar to our finding of 4%.9 This study used chromogenic in situ hybridization (CISH) rather than FISH. While the latter has been established as the gold standard, CISH has been found to be a suitable alternative with strong concordance.19 Baykara et al. (2015) demonstrated that 6.6% of diffuse gastric carcinomas in their study were HER2 positive.10 They also completed FISH testing on all samples, regardless of IHC score, and evaluated concordance. They found that concordance was 87.5% between IHC 3+ and FISH, and it was 94.9% between

cáÖìêÉ=OK=

53

27 18 2

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ReseaRCh aRtiCle her2 in DGc (cont.) cáÖìêÉ=PK=

2014), as the study only involved 60 total cases of which 11 were diffuse carcinomas, so this may be a limiting factor in their results.12

16

2 HER2 FISH results for 18 patients with equivocal (2+) IHC scores

While intestinal type gastric adenocarcinoma is known to have increased HER2 positivity rates ranging from 3.7-32.7%, the literature remains limited regarding diffuse gastric adenocarcinoma and HER2 positivity.6,7,9-13 44

IHC 0 and 1+ with FISH.10 This study examined 598 total patient specimens, comprised of a combination of biopsy and surgical resection specimens, of which the exact breakdown was not indicated. The results indicated that 69.9% had IHC scores of 0, 9.7% had IHC scores of 1+, 8.4% had IHC scores of 2+, and 12% had IHC scores of 3+. Of the 50 equivocal (2+) IHC results, 18(38.2%) were found to be HER2 positive via FISH. Interestingly, both Cruz-Reyes & Gamboa-Dominguez (2013) and Rajagopal (2015) determined that none of their cases of diffuse gastric adenocarcinoma demonstrated HER2 positivity (0/120 and 0/11, respectively).12,13 Unfortunately, neither the criteria used to classify cases as intestinal versus diffuse carcinoma nor the HER2 IHC classification system used were described, so it is difficult to compare these results with ours. Rajagopal (2015) used a small number of cases from a five-year period (2009-

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While intestinal type gastric adenocarcinoma is known to have increased HER2 positivity rates ranging from 3.7-32.7%, the literature remains limited regarding diffuse gastric adenocarcinoma and HER2 positivity.6,7,9-13 The genetics of the population of NL is unique, particularly as it relates to hereditary diffuse gastric carcinoma, adding to the importance of further exploration of this topic. Due to the limited knowledge of HER2 positivity in diffuse gastric cancer and its relationship to CDH1 gene mutation, our study is an important step in further understanding this relationship. Our results demonstrate that 1 out of 17 patients with a CDH1 gene mutation was HER2 positive. To our knowledge, no other studies to date have compared HER2 positivity with CDH1 gene mutation status. A major strength of our study was the thorough and inclusive review of all cases of diagnosed gastric cancer within eastern NL during a 10-year period. Furthermore, all tissue samples were examined and confirmed to have diffuse gastric cancer using H&E staining prior to completing HER2 testing. Three main limitations were noted during this study. First is the small sample size, however, Eastern NL contains over 50% of the population of the province and was therefore an ideal starting point for this study. Second, some tissues were not available for testing during the time of this study because of limited initial diagnostic tissue or because the tissue was being examined for another research study. Third, not all patients in our study underwent genetic testing for CDH1 gene mutations (20%) which was expected given the retrospective nature of the study. The remaining patients either declined testing, were not referred for testing, or died before their referral appointment date. As a result, the true number of patients with a CDH1

ReseaRCh aRtiCle her2 in DGc (cont.)

While the incidence of intestinal type gastric cancer has been found to be on the decline, it appears that the incidence of diffuse gastric cancer is rising.20

mutation may not be reflected in the final results. Future directions will be to compare our findings with an analysis of the rest of the province of Newfoundland and Labrador, as this may allow for a more accurate representation of the rate of HER2 positive diffuse GC. While the incidence of intestinal type gastric cancer has been found to be on the decline, it appears that the incidence of diffuse gastric cancer is rising.20 Overall prognosis from all types of gastric cancer is poor. Diffuse gastric cancer, particularly when it presents as linitis plastica, is highly aggressive, invasive, and commonly metastasizes early.20 A further understanding of this disease process and its characteristics is crucial to help have more effective, targeted therapies. An expert panel from the American Society for Clinical Oncology (ASCO), American Society for Clinical Pathology (ASCP), and the College of American Pathologists (CAP) developed guidelines regarding decision-making for HER2 testing and clinical management.21 They currently recommend that HER2 testing be performed on tumour tissue in the biopsy or surgical resection specimen in all patients who are potential candidates for HER2-targeted therapy, prior to commencing therapy. Pathologists should use the Ruschoff/Hofmann method for evaluating HER2 IHC and ISH results. These guidelines also recommend combination chemotherapy with trastuzumab as the initial choice of treatment for all patients with advanced HER2-positive gastric carcinoma as there is an overall survival benefit. It is well documented that the membrane staining of gastric carcinoma can often

be incomplete, resulting in heterogeneity of HER2 status reporting.22,23 Park et al. demonstrated that primary tumours that were initially reported as being HER2 negative, showed HER2 positive disease on re-biopsy in as many as 8.7% of patients.22 Metastatic and recurrent sites were also sampled in that study, and although the initial primary tumour was reported as HER2 negative, as many as 5.7% of patients were HER2 positive disease at metastatic or recurrent sites.22 Our study did not compare concordance rates between the biopsy and surgical resection specimens for HER2 positivity however, this is planned in future studies. CONCLUSION The rate of HER2 positivity among patients with diffuse gastric cancer in eastern Newfoundland is 4% which is comparable to rates quoted in the literature. Newfoundland is known to have many cases of CDH1 mutation carriers and thus a predisposition to HDGC. Our study demonstrated that 17% of patients with diffuse GC had a CDH1 gene mutation, although not all patients were tested. Within this cohort, one patient with a CDH1 gene mutation was also found to have a HER2 positive GC. This study adds to the growing body of literature on diffuse GC, particularly as it relates to HER2 status. REFERENCES 1. Stewart BW, Wild CP (eds). World Cancer Report 2014. World Health Organization:Geneva. [Cited February 1 2018.] Available from http://publications.iarc.fr/NonSeries-Publications/World-CancerReports/World-Cancer-Report2014. 2. Canadian Cancer Society. Canada Cancer Statistics (2017). [Cited January 15 2018.] Available from http://www.cancer.ca/~/media/

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ReseaRCh aRtiCle her2 in DGc (cont.) cancer.ca/CW/publications/Canadian %20Cancer%20Statistics/CanadianCancer-Statistics-2017-EN.pdf 3. Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): A phase 3, openlabel, randomised controlled trial. Lancet. 2010;376:687-97. 4. Hu B, El Hajj N, Sittler S, Lammert N, Barnes R, Meloni-Ehrig A. Gastric cancer: classification, histology and application of molecular pathology. J Gastrointest Oncol, 2012:3:251-61. 5. National Cancer Institute (2015). National Cancer Institute Drug Dictionary: Trastuzumab. [Cited January 10, 2018.] Available from https://www.cancer.gov/publications/ dictionaries/cancer-drug/def/ trastuzumab 6. Koopman T, Smits MM, Louwen M, Hage M, Boot H, Imholz AL. HER2 positivity in gastric and esophageal adenocarcinoma: clinicopathological analysis and comparison. J Cancer Res Clin Oncol. 2015;141:1343-51. 7. Van Cutsem E, Bang YJ, Feng-Yi F, Xu JM, Lee KW, Jiao SC, et al. HER2 screening data from ToGA: targeting HER2 in gastric and gastroesophageal junction cancer. Gastric Cancer. 2015;18:476-84. 8. Aditi R, Aarathi R, Pradeep R, Hemalatha L, Akshatha C, Amar K. HER2 expression in gastric adenocarcinoma- A study in a tertiary care center in South India. Indian J Surg Oncol. 2016;7:18-24. 9. Koopman T, Louwen M, Hage M, Smits MM, Imholz AL. Pathologic diagnostics of HER2 positivity in 46

gastroesophageal adenocarcinoma. Am J Clin Pathol. 2015;143:257-64. 10. Baykara M, Benekli M, Ekinci O, Irkkan SC, Karaca H, Demirci U, et a. Clinical significance of HER2 overexpression in gastric and gastroesophageal junction cancers. J Gastrointest Surg, 2015;19: 1565-71. 11. Silva MR, Alarcão A, Ferreira T, d'Aguiar M, Ladeirinha A, Balseiro S, et al. Evaluation of HER2 by automated FISH and IHC in gastric carcinoma biopsies. Int J Biol Markers. 2016;31(1):e38-e43. 12. Rajagopal I, Niveditha SR, Sahadev R, Nagappa PK, Rajendra SG. HER2 expression in gastric and gastro-esophageal junction (GEJ) adenocarcinomas. J Clin Diagn Res. 2015;9(3):EC06-10. 13. Cruz-Reyes C, Gamboa-Dominguez A. HER2 amplification in gastric cancer is a rare event restricted to the intestinal phenotype. Int J Surg Pathol. 2013:21:240-6. 14. Xu R, Oiu MZ, Zhou Y, Zhang X, Wang F, Shao JY, et al. The relationship between HER2 expression and Lauren classification in Chinese gastric cancer patients [Abstract]. J Clin Oncol. 2014; 32, no. 15 suppl (May 2014):4065. 15. Kaurah P, MacMillan A, Boyd N, Senz J, De Luca A, Chun N, et al. Founder and recurrent CDH1 mutations in families with hereditary diffuse gastric cancer. JAMA. 2007;297:2360-72. 16. van der Post RS, Vogelaar IP, Carneiro F, Guilford P, Huntsman D, Hoogerbrugge N, et al. Hereditary diffuse gastric cancer: updated clinical guidelines with an emphasis on germline CDH1 mutation carriers. J Med Genet. 2015;52:361-74.

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17. Lauren P. The two histological types of gastric carcinoma: diffuse and socalled intestinal-type carcinoma. An attempt at a histo-clinical classification. Acta Pathol Microbiol Scand. 1965;64:31-49. 18. Rüschoff J, Hanna W, Bilous M, Hofmann M, Osamura RY, PenaultLlorca F, et al. HER2 testing in gastric cancer: a practical approach. Mod Pathol. 2012;25:637-50. 19. Kiyose S, Igarashi H, Nagura K, Kamo T, Kawane K, Mori H, et al. Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: Comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Pathol Int. 2012;62:728-34. 20. He Z, Li B. Recent progress in genetic and epigenetic profile of diffuse gastric cancer. Cancer Transl Med. 2015;1:80-93. 21. Bartley AN, Washington MK, Colasacco C, Ventura CB, Ismaila N, Benson AB 3rd, et al. HER2 testing and clinical decision making in gastroesophageal adenocarcinoma: guideline from the College of American Pathologists, American Society for Clinical Pathology, and American Society of Clinical Oncology. J Clin Oncol. 2017;35:446-64. 22. Albarello L, Pecciarini L, Doglioni C. HER2 testing in gastric cancer. Adv Anat Pathol. 2011;18:53-9. 23. Park SR, Park YS, Ryu MH, Ryoo BY, Woo CG, Jung HY, et al. Extragain of HER2-postive cases through HER2 reassessment in primary and metastatic sites in advanced gastric cancer with initially HER2-negative primary tumours: Results of GASTric cancer HER2 reassessment study 1 (GASTHER1). Eur J Cancer. 2016;53:42-50.

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ReView Veuillez noter que seuls les résumés des articles sont offerts en français.

MiSe à jour Sur La PathoLoGie MoLÉcuLaire DeS GLioMeS DiffuS infiLtrantS auteurs :

Andrew F. Gao1,2 M.D. et David G. Munoz1,2 M.D., M. Sc.

affiliations : 1Département de médecine de laboratoire, Hôpital St. Michael, Toronto, Ontario, Canada 2 Département de médecine de laboratoire et de pathobiologie, Université de Toronto, Toronto, Ontario, Canada

RÉSUMÉ Notre compréhension de la pathologie moléculaire des néoplasmes du système nerveux central (SNC) s’est accrue à un rythme effarant au cours des dernières années. Ces changements ont rapidement été intégrés à la pratique courante de la neuropathologie et de la neurooncologie, à des fins diagnostiques, pronostiques et prédictives. De plus, les paramètres moléculaires font désormais partie intégrante de la plus récente classification des tumeurs du système nerveux central publiée en 2016 par l’OMS. Dans cet article, nous proposons une mise à jour sur les plus importantes modifications qui touchent le diagnostic et la classification des gliomes diffus infiltrants. L’un des principaux progrès accomplis dans ce domaine est la découverte des mutations récurrentes de l’isocitrate-déshydrogénase (IDH) dans la plupart des gliomes de bas grade et dans certains glioblastomes. Combinées à des paramètres moléculaires tels que la codélétion des chromosomes 1p et 19q et les mutations des gènes ATRX et p53, ces nouvelles connaissances ont permis d’améliorer et de raffiner nos méthodes diagnostiques et notre classification des astrocytomes et des oligodendrogliomes. La méthylation du promoteur de MGMT est un marqueur prédictif important de réponse des glioblastomes à la chimiothérapie, particulièrement chez les patients plus âgés. Enfin, la découverte des mutations de l’histone a permis d’approfondir considérablement notre compréhension des gliomes de haut grade qui touchent les enfants et les jeunes adultes. Perspective : Le présent article fournit une mise à jour sur les avancées moléculaires les plus récentes en lien avec les gliomes diffus infiltrants. Les pathologistes et les internes doivent être conscients de ces changements, qui ont rapidement été intégrés à la pratique courante de la neuropathologie et de la neurooncologie.

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ReView This article was peer-reviewed.

KeywoRds: Cns tumour, astrocytoma, oligodendroglioma, glioblastoma, idh, 1p/19q codeletion, atRX, histone h3, K27M, g34R/V

uPDate on the MoLecuLar PathoLoGy of DiffuSeLy infiLtratinG GLioMaS authors:

Andrew F. Gao1,2 MD and David G. Munoz1,2 MD, MSc

affiliations:

1 2

Department of Laboratory Medicine, St. Michael’s Hospital, Toronto, ON, Canada. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.

The authors declare that there are no undisclosed conflicts of interest regarding the publication of this paper. All authors have provided CAP-ACP with non-exclusive rights to publish and otherwise deal with or make use of this article, and any photographs/images contained in it, in Canada and all other countries of the world.

ABSTRACT Our understanding of the molecular pathology of central nervous system (CNS) neoplasms has increased at a dramatic pace in the last few years. These changes have become rapidly incorporated into routine neuropathology and neuro-oncology practice for diagnostic, prognostic, and predictive purposes, and molecular parameters now form an essential component of the most recent 2016 WHO Classification of Tumours of the Central Nervous System. Here we provide a focused update on the major changes in the diagnosis and classification of diffusely infiltrating gliomas. The main advance has been the discovery of recurrent isocitrate dehydrogenase (IDH) mutations in most lower-grade gliomas and some glioblastomas. In combination with molecular parameters such as 1p/19q-codeletion and mutations in ATRX and p53, this has improved and refined our diagnostic approach and classification of astrocytomas and oligodendrogliomas. MGMT promoter methylation is an important marker in predicting response to chemotherapy in glioblastoma, especially in elderly patients. Finally, the discovery of histone mutations has significantly advanced our understanding of pediatric and young adult high-grade gliomas. Perspective: This article provides an update on the most recent molecular advances in the diffusely infiltrating gliomas. Pathologists and trainees should be aware of these changes as they have become rapidly integrated into routine neuropathology and neuro-oncology practice. Revue canadienne de pathologie | volume 10, numéro 2 | www.cap-acp.org

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ReView MoLecuLar PathoLoGy of DiffuSe GLioMaS (cont.)

Lower-grade diffuse gliomas are characterized by mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2), most commonly IDH1 R132H...

INTRODUCTION Our understanding of the molecular pathology of central nervous system (CNS) neoplasms has increased at a dramatic pace in the last few years. These changes have become rapidly incorporated into routine neuropathology and neuro-oncology practice for diagnostic, prognostic, and predictive purposes, and molecular parameters now form an essential component of the most recent 2016 WHO Classification of Tumours of the Central Nervous System.1 This edition made a significant break from tradition by incorporating molecular parameters into the very definitions of certain tumour entities.2 Here, we provide a focused update on several of the most significant discoveries that have altered our understanding and approach to the diffusely infiltrating gliomas. A full discussion of advances in medulloblastomas, ependymomas, and circumscribed low grade glial or glioneuronal neoplasms is outside the scope of this review. ADULT LOWER-GRADE DIFFUSE GLIOMAS

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Lower-grade (WHO grade II or III) diffuse gliomas typically occur in

younger adults.3 Diffuse gliomas were previously classified based on histologic features as astrocytoma, oligodendroglioma, or mixed oligoastrocytoma. Tumour classification based on morphology alone resulted in high interobserver variability and thus potentially heterogeneous enrolment in clinical trials.4 Molecular advances described below are summarized in Figure 1. Lower-grade diffuse gliomas are characterized by mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2), most commonly IDH1 R132H which comprises approximately 90% of cases.5 IDH1/2 mutations result in neomorphic (i.e. leading to new action and independent of wildtype gene dosage) enzyme activity and the production of 2hydroxyglutarate, which is ultimately believed to effect aberrant methylation of multiple CpG islands across the genome, predisposing progenitor cells to neoplastic transformation.6 In oligodendrogliomas, IDH mutation is accompanied by codeletion of entire chromosome arms 1p and 19q, thought to result from an unbalanced pericentromeric translocation between

A simplified algorithm for the classification and workup of the diffuse gliomas. See text for detailed description. Modified from Louis et al.2 *Diffuse midline glioma does not necessarily exhibit high grade histology and may range from grade II to grade IV. ‡High grade glioma, H3 G34R/V-mutant has not yet been officially recognized by the WHO. 50

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ReView MoLecuLar PathoLoGy of DiffuSe GLioMaS (cont.) chromosomes 1 and 19 with subsequent loss of the derivative chromosome composed of 1p and 19q.7 Demonstrating the presence of these 2 alterations is required to diagnose the 2016 entity of “oligodendroglioma, IDH-mutant and 1p/19q-codeleted” (grade II; or with the modifier “anaplastic” for grade III based on the presence of increased mitotic activity, necrosis, or microvascular proliferation),2 most commonly by IDH1 R132H immunohistochemistry with a commercially available antibody and by fluorescence in situ hybridization (FISH) using probes targeting 1p and 19q. Methods for detecting noncanonical IDH mutations are increasingly available, such as PCRbased assays.8 Alternatives to FISH, which may not distinguish between partial- and whole-arm deletion,9 include multiplex ligation-dependent probe amplification10 and genome-wide methylation and copy-number profiling (e.g. Illumina Infinium HumanMethylation450 BeadChip array).11 The presence of 1p/19qcodeletion is associated with superior prognosis and response to procarbazine/CCNU/vincristine chemotherapy and temozolomide.12 In fact, it represents the most favourable prognostic molecular profile among the diffuse gliomas, with median survival of approximately 15-20 years.13

should nevertheless be specified in the diagnostic line. Instead of 1p/19qcodeletion, accompanying mutations in IDH-mutant astrocytoma include ATRX (causing loss of protein expression) and TP53.16 ATRX loss and 1p/19q-codeletion are essentially mutually exclusive.17 Therefore, molecular testing for IDH, ATRX, p53 (all of which can be done via immunohistochemistry with commercially available antibodies), and 1p/19q-codeletion can segregate oligodendroglioma from astrocytoma in virtually all cases.18 Hence, the most recent WHO classification strongly discourages the use of “oligoastrocytoma” as a diagnosis, which is reserved for cases where there is ambiguous morphology and molecular testing could not be performed.2 IDH mutation in lowergrade astrocytoma is also associated with superior prognosis although not quite as favourable as molecular oligodendroglioma, with median survival of approximately 10-15 years.13 IDH mutation in GBM is also associated with superior prognosis compared to IDH-wildtype glioblastoma and, in some studies, superior or similar to grade III IDHwildtype anaplastic astrocytoma.19 In other words, some astrocytomas that are lower-grade but IDH-wildtype may behave similarly to GBM.20

In astrocytomas, IDH mutation is not definitional but is present in the vast majority of lower-grade astrocytomas (diffuse astrocytoma, grade II, and anaplastic astrocytoma, grade III).14,15 Only 10% of high-grade astrocytomas, i.e. glioblastoma (GBM, grade IV), have IDH mutation and typically arise from a low-grade precursor.5 IDH status, i.e. “IDH-mutant” or “IDH-wildtype”,

ADULT GLIOBLASTOMA

IDH-wildtype GBMs make up the vast majority of all GBMs (90%) and in fact are the most common malignant primary CNS tumour in adults

As discussed above, IDH-mutant GBMs are relatively uncommon, especially with increasing age. There are no official guidelines on when to test for IDH mutation in glioblastoma or if IDH status can be settled through IDH1 R132H immunohistochemistry alone, although some authors have developed a prediction tool,21 available online (http://www.kcr.uky.edu/webapps/IDH/a pp.html). Generally, if the patient is 55 years of age or younger, testing for noncanonical IDH mutations should be sought if R132H immunohistochemistry is negative.2

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ReView MoLecuLar PathoLoGy of DiffuSe GLioMaS (cont.)

... histone H3.3 (H3F3A) or H3.1 (HIST1H3B or HIST1H3C) mutations are found in infiltrative highgrade gliomas that mainly affect children.29 However, they are not uncommon in young adults... IDH-wildtype GBMs make up the vast majority of all GBMs (90%) and in fact are the most common malignant primary CNS tumour in adults, with median survival of approximately 12-18 months.3 They are molecularly complex: alterations include TERT promoter mutation (80%), homozygous deletion of CDKN2A/CDKN2B (60%), loss of chromosomes 10p (50%) or 10q (70%), EGFR alterations (55%), PTEN mutation/deletion (40%), TP53 mutation (25-30%), and PI3K mutations (25%).22 Despite the myriad molecular alterations, they do not play a significant role in routine neuropathology practice. Instead, O-6methylguanine-DNA methyltransferase (MGMT, involved in DNA repair) promoter methylation is a strong predictive marker of response to alkylating and methylating chemotherapeutic agents such as temozolomide. It is important to note that immunohistochemical evaluation of the protein product is not a substitute for the more expensive promoter methylation testing.12 In elderly GBM patients, MGMT promoter methylation status may be used to assign patients to either radiotherapy alone (unmethylated) or chemotherapy alone (methylated). Although combined therapy results in better survival, radiation in the elderly is avoided if possible because of its adverse effects, including white matter damage and sommolence.23 Two variants of glioblastoma have distinct molecular features. Giant cell glioblastoma is characterized by the presence of multinucleated and sometimes very bizarre giant cells. They commonly show TP53 mutations, like lower-grade astrocytomas, but are IDH52

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wildtype and retain ATRX expression.24 Epithelioid glioblastoma is characterized by epithelioid morphology mimicking metastatic carcinoma or melanoma. Approximately half of epithelioid GBMs have BRAF V600E mutation.25 In this regard, epithelioid GBM shares some morphologic and molecular features with pleomorphic xanthoastrocytoma, a low-grade epilepsy-associated circumscribed glioma predominantly of childhood, leading some authors to question if they are related entities.26 Despite the existence of BRAF inhibitors, there is only anecdotal evidence of efficacy in epithelioid GBM27 and no large-scale studies have been published. PEDIATRIC HIGH-GRADE GLIOMAS The most recent discovery in the molecular pathology of gliomas is mutations in histones, proteins that package DNA into nucleosomes and regulate DNA transcription.28 Specifically, histone H3.3 (H3F3A) or H3.1 (HIST1H3B or HIST1H3C) mutations are found in infiltrative highgrade gliomas that mainly affect children.29 However, they are not uncommon in young adults, and so may be encountered in adult neuropathology practice. Histone H3 K27M mutations characterize a new entity, “diffuse midline glioma, H3 K27M–mutant”, which subsumes previous terms such as brainstem glioma or diffuse intrinsic pontine glioma.2 These tumours are restricted to midline locations, such as the thalamus (where they comprise 50% of high-grade gliomas in this location), pons (80%), cerebellum, or spinal cord (60%) and have predominantly astrocytic differentiation.30 On histology, there are no particularly distinguishing features, although the presence of giant cells can be suggestive.31 An antibody to histone H3 K27M is commercially available and positive nuclear staining is diagnostic. ATRX mutations (loss of protein expression) and TP53 mutations are

ReView MoLecuLar PathoLoGy of DiffuSe GLioMaS (cont.) sometimes associated with H3 K27M mutation, more commonly in the thalamus and spinal cord.32 H3 K27M mutation confers a worse prognosis than wildtype tumours. In contrast, histone H3.3 G34R/V mutations characterize pediatric highgrade gliomas (astrocytomas) that are hemispheric in location; these too may affect young adults and can occur in patients as late as their 50s.33 On histology, these tumours may have the typical appearance of glioblastoma but a subset have a prominent primitive neuronal component that expresses neuronal markers. Currently, there is no commercially available H3 G34R/V antibody validated for routine clinical use and diagnosis may require direct sequencing. Immunohistochemical clues to this diagnosis include a lack of Olig2 expression,33 which is generally not seen in other types of astrocytomas. ATRX mutations (loss of protein expression) and TP53 mutations are strongly associated with H3 G34R/V mutant tumours, more so than H3 K27M and even IDH-mutant gliomas. Prognosis of G34R/V tumours is intermediate between IDH-wildtype and IDH-mutant GBM. CONCLUSION In summary, the molecular advances in our understanding of diffusely infiltrating gliomas have led to significant changes in diagnostic practice. The presence or absence of IDH mutations is of crucial importance in adult gliomas, as is determination of 1p/19q status in adult gliomas to separate oligodendrogliomas from astrocytomas. MGMT promoter methylation status helps to guide clinical therapy for glioblastoma, and the discovery of histone mutations has led to new categories of pediatric high-grade gliomas. Pathologists and trainees must be aware of these changes and the way they are now integrated into routine neuropathology and neuro-oncology practice.

REFERENCES 1. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Ellison DW, FigarellaBranger D, et al. WHO Classification of Tumours of the Central Nervous System. Revised 4th ed. Lyon: International Agency for Research on Cancer; 2016. 2. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, et al. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol. 2016;131(6):803-20. 3. Ostrom QT, Gittleman H, Liao P, Rouse C, Chen Y, Dowling J, et al. CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2007-2011. Neuro Oncol. 2014;16 Suppl 4:iv1-63. 4. van den Bent MJ. Interobserver variation of the histopathological diagnosis in clinical trials on glioma: a clinician's perspective. Acta Neuropathol. 2010;120(3):297-304. 5. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med. 2009;360(8):765-73. 6. Ichimura K. Molecular pathogenesis of IDH mutations in gliomas. Brain Tumor Pathol. 2012;29(3):131-9. 7. Griffin CA, Burger P, Morsberger L, Yonescu R, Swierczynski S, Weingart JD, et al. Identification of der(1;19)(q10;p10) in five oligodendrogliomas suggests mechanism of concurrent 1p and 19q loss. J Neuropathol Exp Neurol. 2006;65(10):988-94. 8. Catteau A, Girardi H, Monville F, Poggionovo C, Carpentier S,

Frayssinet V, et al. A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma. Acta Neuropathol Commun. 2014;2:58. 9. Vogazianou AP, Chan R, Backlund LM, Pearson DM, Liu L, Langford CF, et al. Distinct patterns of 1p and 19q alterations identify subtypes of human gliomas that have different prognoses. Neuro Oncol. 2010;12(7):664-78. 10. Natte R, van Eijk R, Eilers P, Cleton-Jansen AM, Oosting J, Kouwenhove M, et al. Multiplex ligation-dependent probe amplification for the detection of 1p and 19q chromosomal loss in oligodendroglial tumors. Brain Pathol. 2005;15(3):192-7. 11. Wiestler B, Capper D, Hovestadt V, Sill M, Jones DT, Hartmann C, et al. Assessing CpG island methylator phenotype, 1p/19q codeletion, and MGMT promoter methylation from epigenome-wide data in the biomarker cohort of the NOA-04 trial. Neuro Oncol. 2014;16(12):1630-8. 12. Weller M, Stupp R, Hegi ME, van den Bent M, Tonn JC, Sanson M, et al. Personalized care in neurooncology coming of age: why we need MGMT and 1p/19q testing for malignant glioma patients in clinical practice. Neuro Oncol. 2012;14 Suppl 4:iv100-8. 13. Leeper HE, Caron AA, Decker PA, Jenkins RB, Lachance DH, Giannini C. IDH mutation, 1p19q codeletion and ATRX loss in WHO grade II gliomas. Oncotarget. 2015;6(30):30295-305. 14. Von Deimling A, Huse JT, Yan H, Brat D, Ohgaki H, Kleihues P, et al. Anaplastic astrocytoma, IDHmutant. In: Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Ellison DW, Figarella-Branger D, et al.,

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ReView MoLecuLar PathoLoGy of DiffuSe GLioMaS (cont.) editors. WHO Classification of Tumours of the Central Nervous System. Lyon: International Agency for Research on Cancer; 2016. p. 24-7. 15. Von Deimling A, Huse JT, Yan H, Brat DJ, Reifenberger G, Ohgaki H, et al. Diffuse astrocytoma, IDHmutant. In: Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Ellison DW, Figarella-Branger D, et al, editors. WHO Classification of Tumours of the Central Nervous System. Lyon: International Agency for Research on Cancer; 2016. p. 18-23. 16. Haberler C, Wohrer A. Clinical Neuropathology practice news 22014: ATRX, a new candidate biomarker in gliomas. Clin Neuropathol. 2014;33(2):108-11. 17. Sahm F, Reuss D, Koelsche C, Capper D, Schittenhelm J, Heim S, et al. Farewell to oligoastrocytoma: in situ molecular genetics favor classification as either oligodendroglioma or astrocytoma. Acta Neuropathol. 2014;128(4):551-9. 18. Wiestler B, Capper D, Holland-Letz T, Korshunov A, von Deimling A, Pfister SM, et al. ATRX loss refines the classification of anaplastic gliomas and identifies a subgroup of IDH mutant astrocytic tumors with better prognosis. Acta Neuropathol. 2013;126(3):443-51. 19. Sanson M, Marie Y, Paris S, Idbaih A, Laffaire J, Ducray F, et al. Isocitrate dehydrogenase 1 codon 132 mutation is an important prognostic biomarker in gliomas. J Clin Oncol. 2009;27(25):4150-4. 20. Cancer Genome Atlas Research Network, Brat DJ, Verhaak RG, Aldape KD, Yung WK, Salama SR, et al. Comprehensive, integrative genomic analysis of diffuse lower54

grade gliomas. N Engl J Med. 2015;372(26):2481-98. 21. Chen L, Voronovich Z, Clark K, Hands I, Mannas J, Walsh M, et al. Predicting the likelihood of an isocitrate dehydrogenase 1 or 2 mutation in diagnoses of infiltrative glioma. Neuro Oncol. 2014;16(11):1478-83. 22. Louis DN, Suva ML, Burger PC, Perry A, Kleihues P, Aldape KD, et al. Glioblastoma, IDH-wildtype. In: Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Ellison DW, FigarellaBranger ., et al., editors. WHO Classification of Tumours of the Central Nervous System. Lyon: International Agency for Research on Cancer; 2016. p. 28-45. 23. Arvold ND, Reardon DA. Treatment options and outcomes for glioblastoma in the elderly patient. Clin Interv Aging. 2014;9:357-67. 24. Oh JE, Ohta T, Nonoguchi N, Satomi K, Capper D, Pierscianek D, et al. Genetic alterations in gliosarcoma and giant cell glioblastoma. Brain Pathol. 2016;26(4):517-22. 25. Kleinschmidt-DeMasters BK, Aisner DL, Birks DK, Foreman NK. Epithelioid GBMs show a high percentage of BRAF V600E mutation. Am J Surg Pathol. 2013;37(5):685-98. 26. Alexandrescu S, Korshunov A, Lai SH, Dabiri S, Patil S, Li R, et al. Epithelioid glioblastomas and anaplastic epithelioid pleomorphic xanthoastrocytomas--same entity or first cousins? Brain Pathol. 2016;26(2):215-23. 27. Robinson GW, Orr BA, Gajjar A. Complete clinical regression of a BRAF V600E-mutant pediatric

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glioblastoma multiforme after BRAF inhibitor therapy. BMC Cancer. 2014;14:258. 28. Kallappagoudar S, Yadav RK, Lowe BR, Partridge JF. Histone H3 mutations--a special role for H3.3 in tumorigenesis? Chromosoma. 2015;124(2):177-89. 29. Schwartzentruber J, Korshunov A, Liu XY, Jones DT, Pfaff E, Jacob K, et al. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature. 2012;482(7384):226-31. 30. Hawkins C, Ellison DW, Sturm D. Diffuse midline glioma, H3 K27Mmutant. In: Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Ellison DW, Figarella-Branger D, et al., editors. WHO Classification of Tumours of the Central Nervous System. Lyon: International Agency for Research on Cancer; 2016. p. 57-9. 31. Neumann JE, Dorostkar MM, Korshunov A, Mawrin C, Koch A, Giese A, et al. Distinct histomorphology in molecular subgroups of glioblastomas in young patients. J Neuropathol Exp Neurol. 2016;75(5):408-14. 32. Solomon DA, Wood MD, Tihan T, Bollen AW, Gupta N, Phillips JJ, et al. Diffuse midline gliomas with histone H3-K27M mutation: a series of 47 Cases sssessing the spectrum of morphologic variation and associated genetic alterations. Brain Pathol. 2016;26(5):569-80. 33. Korshunov A, Capper D, Reuss D, Schrimpf D, Ryzhova M, Hovestadt V, et al. Histologically distinct neuroepithelial tumors with histone 3 G34 mutation are molecularly similar and comprise a single nosologic entity. Acta Neuropathol. 2016;131(1):137-46.

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All peer-reviewed articles appearing in this publication have undergone a double blind peer-review process. The views or opinions expressed in this Journal are those of the authors and contributors, and do not necessarily reflect those of this Journal, the editors, the editorial board, the publisher of this Journal, or the Canadian Association of Pathologists (CAP-ACP). Although the CAP-ACP has made reasonable efforts to ensure accuracy the articles herein, the Journal, the editors, the editorial board, the publisher of this Journal, or the CAP-ACP, take no responsibility whatsoever for any errors, omissions, or any consequences of reliance on any material or the accuracy of any information contained in this publication. In the event of a discrepancy, between the original and translated versions of the texts, the original version shall take precedence. The mention of trade names, commercial products or organizations, and the inclusion of advertisements in the Journal does not imply endorsement by this Journal, the editors, the editorial board, the publisher of this Journal or the CAP-ACP. This publication is copyright in its entirety. Material may not be reprinted, stored in a retrieval system, or copied without the written permission of CAP-ACP. Contact through www.cap-acp.org.

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Canadian Association of Pathologists
Association canadienne des pathologistes

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TIVSTEBZ JanVBSZ
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to SunEBZ JanVBSZ
1 201 | Hilton Garden Inn - Toronto Airport West

COURSE DIRECTORS Penny J. Barnes, MD, FRCP(C) (Halifax, NS) Jagdish Butany, MBBS, MD, FRCPC (Toronto, ON)

WHO SHOULD ATTEND? This course is open to all Canadian trainees, US trainees, CAP-ACP resident members as well as non-members and pathologists. Priority will be given to PGY5 and PGY4 residents who are members of the CAP-ACP. Limited availability is also extended to non MD Laboratory Professionals at a fee per day.

OBJECTIVES An experienced faculty team has been assembled to collaborate on the topics and presentation of the course material to provide attendees with a practical review of major topics in the specialty of Anatomical Pathology. Your

faculty of skilled pathologists will focus on what you need to know in preparation for the examination. At the end of the meeting participants will be able to: 1.

Review and update core medical knowledge in preparation for the Royal College of Physicians and Surgeons of Canada certification examinations in Anatomic Pathology, General Pathology, and Hematological Pathology.

2.

Develop strategies for optimal preparation and exam performance in the written, practical, and oral components of the examination, and understand examination formats

3.

Discuss and assess the significance of new findings and observations in the context of current literature

4.

Demonstrate competence in the non-medical expert CanMEDS roles, in particular laboratory management and quality assurance

ACCREDITATION The Canadian Association of Pathologists’/Association canadienne des pathologistes Residents Review Course is accredited by the Canadian Association of Pathologists. This event is an Accredited Group Learning Activity (Section 1) as defined by the Maintenance of Certification program of The Royal College of Physicians and Surgeons of Canada.

For more information and to register: www.cap-acp.org/rrc.php

Case RepoRts & ReViews Veuillez noter que seuls les résumés des articles sont offerts en français.

anGioSarcoMe PriMitif De L’oreiLLette Droite exPriMant c-Kit : ÉtuDe De caS et revue De La LittÉrature auteurs :

Mubarak Al-Shraim, M.D., FRCPC1; Howadah Elhakeem, M.D.2; Ahmed Rezk, M.D.3; Mahmoud Rezk Abdelwahed Hussein, M.D., Ph. D., FRCPath, FRCPC2; Jagdish W. Butany, M.D., FRCPC4.

affiliations : 1Département de pathologie, Faculté de médecine, Université King Khalid, Abha, Arabie saoudite. 2 Département de pathologie, hôpital militaire King Fahd, région du sud, Khamis Mushyat, Arabie saoudite. 3 Département de chirurgie cardiaque, hôpital militaire King Fahd, région du sud, Khamis Mushyat, Arabie saoudite. 4 Département de pathologie, University Health Network, Toronto, ON, Canada.

RÉSUMÉ L’angiosarcome primitif du cœur est une tumeur très rare associée à une présentation clinique non spécifique et à un pronostic sombre. Son diagnostic repose sur la combinaison d’un examen pathologique et d’analyses immunohistochimiques. Le proto-oncogène C-KIT est un récepteur à activité tyrosine kinase qui code la protéine CD117 exprimée dans certains sarcomes. Dans le présent article, nous décrivons le cas d’un patient âgé de 33 ans qui présentait des symptômes associés à une tamponnade cardiaque (dyspnée, douleur thoracique et évanouissements). Une échocardiographie, une échocardiographie transœsophagienne et une tomographie assistée par ordinateur ont révélé une masse dans l’oreillette droite et un important épanchement péricardique. Une intervention chirurgicale d’urgence, comprenant une résection de la masse auriculaire, a été pratiquée. D’un point de vue histologique, la masse consistait en une tumeur de haut grade composée de cellules fusiformes formant des canaux vasculaires anastomosés et exprimant des marqueurs vasculaires (CD34 et facteur VIII) et CKIT (CD117). Un diagnostic d’angiosarcome exprimant C-KIT (CD117) a été établi. Finalement, dans cet article, nous examinons également le rôle que pourraient jouer C-KIT, les facteurs angiogéniques, les mastocytes et leur facteur de croissance (facteur de croissance des cellules souches) dans le développement des angiosarcomes.

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ReView

Case RepoRts & ReViews

This article was peer-reviewed.

KeywoRds: heart, angiosarcoma, cardiac tamponade, immunohistochemical stain, c-kit

PriMary riGht atriaL carDiac anGioSarcoMa with c-Kit exPreSSion: a caSe rePort anD review of Literature Mubarak Al-Shraim1 MD, FRCPC, Howadah Elhakeem2 MD, Ahmed Rezk3 MD, Mahmoud Rezk Abdelwahed Hussein2 MD, PhD, FRCPath, FRCPC, Jagdish W. Butany4 MD, FRCPC.

authors:

affiliations:

1

Department of Pathology, College of Medicine, King Khalid University, Abha, Saudi Arabia. Department of Pathology, King Fahad Military Hospital, Southern Region, Khamis Mushyat, Saudi Arabia. 3 Department of Cardiac Surgery, King Fahad Military Hospital, Southern Region, Khamis Mushyat, Saudi Arabia. 4 Department of Pathology, University Health Network, Toronto, Ontario, Canada. 2

authorship contribution: All authors were involved in the generation of the data and writing of this manuscript.

The authors declare that there are no conflicts of interest regarding the publication of this paper. All authors have provided CAP-ACP with non-exclusive rights to publish and otherwise deal with or make use of this article, and any photographs/images contained in it, in Canada and all other countries of the world. ABSTRACT Primary cardiac angiosarcoma is an exceptionally rare tumour that has a nonspecific clinical presentation and dismal prognosis. Its diagnosis is achieved by combined pathological examination and immunohistochemical studies. C-KIT proto-oncogene is a tyrosine kinase receptor that encodes CD117 protein which is expressed in some sarcomas. Here we present a case of a 33-year-old male patient who presented with features of cardiac tamponade (dyspnea, chest pain and fainting). Echocardiography, transesophageal echocardiography and computed tomography revealed a right atrial mass and massive pericardial effusion. Emergency surgery with resection of the atrial mass was done. On histology, the mass consisted of a high-grade spindle cell tumour forming anastomosing vascular channels and expressing vascular markers (CD34 and factor VIII) and C-KIT (CD117). The diagnosis of C-KIT (CD117)-expressing angiosarcoma was established. The possible roles of C-KIT, angiogenic factors, mast cells and their growth factor (stem cell factor) in the development of angiosarcomas are discussed. 58

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Case RepoRts & ReViews carDiac anGioSarcoMa with c-Kit exPreSSion (cont.)

Primary cardiac tumours are extremely rare with an incidence of 0.2%.1 About 70% of these tumours are benign, with myxomas being the most frequent.1

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INTRODUCTION Primary cardiac tumours are extremely rare with an incidence of 0.2%.1 About 70% of these tumours are benign, with myxomas being the most frequent.1 The primary malignancies represent 25% of cardiac tumours with sarcomas being the most common. Angiosarcoma of the heart represents about 2% of all primary cardiac neoplasms2 and it usually involves the right atrium. It typically affects adult males in their third through fifth decades and presents clinically with dyspnea, chest pain, cough, cardiac tamponade, hemopericardium, pulmonary hypertension, and pulmonary embolism.3 Angiosarcomas usually arise from the myocardium or less frequently from the pericardium.4 Although the pathogenesis of angiosarcoma is poorly understood, several pathways seem to be implicated.

The Rb, PI3K/Akt, and C-KIT pathways are involved in angiosarcomas of bone and soft tissue.5 C-KIT encodes CD117, which is a transmembrane type III receptor tyrosine kinase involved in cell signal transduction pathways in many cells types. Normally C-KIT protooncogene is activated (phosphorylated) by binding of its ligand (the stem cell factor) leading to a phosphorylation cascade involving several transcription factors that regulate6 cell differentiation, proliferation and apoptosis. KIT-dependent cell types include mast cells, hematopoietic stem cells, germ cells, melanocytes, and Cajal cells of the gastrointestinal tract. CD117 is expressed in several sarcomas such as malignant fibrous histiocytoma, synovial sarcoma, leiomyosarcoma and angiosarcoma.7 To date, several reports indicated the expression of C-KIT in noncardiac angiosarcoma8-10 but reports of C-KIT expression in cardiac angiosarcoma are limited.2 Here we present the clinicopathologic and immunohistological features of a fatal primary cardiac angiosarcoma. CASE REPORT

A: Post-contrast axial CT scan showed soft tissue density (arrow) in the right atrium with marked pericardial effusion. B: Histological features showing a high grade malignant tumour forming anastomosing vascular channels with cytologic atyia and atypical mitoses. (Hematoxylin and eosin stain, original magnification x100) C: CD34 strong membranous staining of tumour cells (x100). D: Factor VIII strong membranous and cytoplasmic staining of tumour cells (x100). E: CD117 moderate membranous staining of tumour cells (x100). F: MIB-1 strong nuclear staining of 60-70 % of tumour cells (x100).

A 33-year-old male patient presented to the Emergency department with severe dyspnea, dizziness and chest pain (features of cardiac tamponade). Transthoracic echocardiography and computed tomography revealed a huge pericardial effusion and a right atrial mass (Figure 1-A). Emergency median sternotomy showed firmly adherent fibrous adhesions over the thickened pericardium. Cardiopulmonary bypass was established and cardiac chambers explored. There was a tumour mass infiltrating through lateral wall of the right atrium into the atrial cavity. The right atrial mass was removed with the adjacent right atrial wall with safety margins and sent for histopathology. The inter-atrial septum, tricuspid valve, superior vena cava and inferior vena cava openings were free of tumour. The patient died one day after surgery and

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Case RepoRts & ReViews carDiac anGioSarcoMa with c-Kit exPreSSion (cont.)

Angiogenic cytokines (regulators of vascularization) are released by mast cells and play roles in the progression of angiosarcoma. unfortunately his family autopsy examination.

declined

Gross examination revealed a 2.0 x 1.0 x 0.5 cm ill-defined mass and 1.0 x 0.5 x 0.5 cm (aggregate) multiple tissue pieces (pericardial adhesions), both with hemorrhagic cut section and soft consistencies. Histologically, both the tumour and the pericardial adhesions consisted of malignant spindle cells forming anastomosing vascular channels. The tumour cells had pleomorphic mitotically active nuclei with dispersed nuclear chromatin, small nucleoli and scant eosinophilic cytoplasm. Areas of necrosis were seen throughout the tumour. The tumour cells were reactive for vimentin, CD34, Factor VIII, and C-KIT (CD117). MIB1 proliferative index was up to 60-70 % (Figure 1: B-F). Negative stains included pancytokeratins AE1/AE3, desmin, S100 and HMB-45. The diagnosis of high-grade angiosarcoma was established. DISCUSSION Angiosarcomas are rare vascular sarcomas that arise in several anatomical sites11 such as liver, skin, spleen, bone, breast, soft tissue, mesentery and pelvis.3,4 In the case presented here, the clinical presentation is similar to that described in previous studies.4 The radiological studies (transthoracic echocardiography and computed tomography) were not able to establish the final diagnosis. The initial intraoperative clinical impressions included metastatic malignant tumours 60

and mesothelioma. The bulk of the tumourous mass involved the right atrium with a focal invasion into the pericardium and therefore, we considered the diagnosis of a primary cardiac malignant tumour. Further immunohistochemical studies were consistent with angiosarcoma Review of literature suggest possible roles for C-KIT, angiogenic factors, mast cells and their growth factor (stem cell factor) in the progression of angiosarcomas. 2,12-18 Yonemori et al examined angiogenic markers and CKIT in patients with angiosarcoma. In some angiosarcomas, the tumour cells are reactive for C-KIT, plateletderived growth factor receptor (PDGFR)-alpha, PDGFR-beta, and vascular endothelial growth factor receptors. 17 Angiogenic cytokines (regulators of vascularization) are released by mast cells and play roles in the progression of angiosarcoma. In support, the number of mast cells and their growth factor (stem cell factor) is high in cutaneous angiosarcomas when compared to normal skin. This growth factor helps recruit C-KIT receptor of the mast cells and therefore contributes to the proliferation of the angiosarcomatous cells.18 Our finding of CD117 reactivity in the cardiac angiosarcoma presented here is not only in agreement with previous studies2,12-17 but also suggests possible roles in the pathogenesis of this aggressive neoplasm. CD117 may also have some therapeutic ramifications. Batzios et al reported a primary cardiac angiosarcoma in a 32 year-old-patient with lung and bone secondaries. The sarcomatous cells were reactive for CD117 and the patient received Imatinib (Gleevec) (anti-CD117) with a good clinical response.2 Similarly, Bellitti et al reported a case of cardiac angiosarcoma in a 25-year-old male patient who achieved clinical remission and long-term free survival following treatment with CD117 (C-KIT)

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inhibitors.15 Adhikari et al reported a collision tumour composed of angiosarcoma and gastric gastrointestinal stromal tumour in an 81-year-old male patient with a gastric mass. Histology revealed vasoformative malignant spindle cell neoplasm reactive for vascular markers and CD117.12 Chahbouni, et al reported a case angiosarcoma of the small bowel positive for CD31 and Factor VIII and CD117 but without underlying KIT mutations.14 Similarly, Sherid, et al reported two cases of colonic angiosarcoma histologically reactive for CD31, CD34 and CD117.16 Some investigators have studied canine hemangiosarcomas cell lines as a model to explore the molecular features of these endothelial tumours.19 CD117 is expressed by most primitive hematopoietic stem cells.19 In fact, CD117 is a sensitive maker to distinguish between benign canine angioma (negative) from malignant canine angiosarcoma (positive).19 Miettinen et al have showed that CD117 is expressed in human fetal capillaries, but not in adult endothelial cells.8 The same group reported CD117 expression in 56% of soft tissue non-cardiac angiosarcomas, but the protein was not identified in 31 hemangiomas and 10 epithelioid hemangioendotheliomas. This may indicate that malignant cells in angiosarcoma originate from poorly differentiated, primitive endothelial cells that can be identified by expression of CD117, whereas hemangiomas originate from differentiated mature endothelial cells. This observation has both diagnostic and therapeutic ramifications. It is possible that an understanding of CKIT expression in cardiac angiosarcoma can be utilized as a basis for targeted angiosarcoma therapy. Imatinib (Gleevec) is a tyrosine kinase inhibitor which is been used in treatment of gastrointestinal stromal tumours.20 Some studies addressed the use of imatinib for cardiac angiosarcoma with clinical improvement.2

Case RepoRts & ReViews carDiac anGioSarcoMa with c-Kit exPreSSion (cont.) To conclude, here we present a case of primary cardiac angiosarcoma with CD117 protein expression. The expression of CD117 suggests that the stem cell factor/C-KIT pathway may be implicated in the tumourigenesis of angiosarcoma. Whether CD117 protein expression reflects underlying C-KIT mutations awaits examination of a larger series of cardiac angiosarcomas. There may be therapeutic value of anti C-KIT (CD117) as a potential targeted therapy in this rare and aggressive tumour.

from its soft tissue counterpart. Mod Pathol. 2013;26:1211-21. 6.

Hussein MR. Expression of KIT receptor tyrosine kinase protein in normal human skin: preliminary observations. Cell Biol Int. 2007;31:748-51.

7.

Miettinen M, Lasota J. KIT (CD117): a review on expression in normal and neoplastic tissues, and mutations and their clinicopathologic correlation. Appl Immunohistochem Mol Morphol. 2005;13:205-20.

8.

Miettinen M, Sarlomo-Rikala M, Lasota J. KIT expression in angiosarcomas and fetal endothelial cells: lack of mutations of exon 11 and exon 17 of C-kit. Mod Pathol. 2000;13:536-41.

9.

Komdeur R, Hoekstra HJ, Molenaar WM, Van Den Berg E, Zwart N, Pras E, et al. Clinicopathologic assessment of postradiation sarcomas: KIT as a potential treatment target. Clin Cancer Res. 2003;9:2926-32.

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Fernandes CP, Oliveira FA, Costa FW, Patrocínio RM, Mota MR, Nunes Alves AP, et al. Clinical, histological, and immunohistochemical features of a mandibular metastasis from a primary cardiac angiosarcoma. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;116:e121-7. Batzios S, Michalopoulos A, Kaklamanis L, Stathopoulos J, Christopoulou M, Koutantos J, et al. Angiosarcoma of the heart: case report and review of the literature. Anticancer Res. 2006;26:4837-42. Herrmann MA, Shankerman RA, Edwards WD, Shub C, Schaff HV. Primary cardiac angiosarcoma: a clinicopathologic study of six cases. J Thorac Cardiovasc Surg. 1992;103:655-64. Yao H, Miyamoto T, Mukai S, Yamamura M, Nakagawa T, Ryomoto M. Angiosarcoma of the pericardium. Review of 9 reports from Japan. Jpn J Thorac Cardiovasc Surg. 2003;51:65-7. Verbeke SL, Bertoni F, Bacchini P, Oosting J, Sciot R, Krenács T,, et al. Active TGF-beta signaling and decreased expression of PTEN separates angiosarcoma of bone

10. Hornick JL, Fletcher CD. Immunohistochemical staining for KIT (CD117) in soft tissue sarcomas is very limited in distribution. Am J Clin Pathol. 2002;117: 188-93. 11. Fedok FG, Levin RJ, Maloney ME, Tipirneni K. Angiosarcoma: current review. Am J Otolaryngol. 1999;20:223-31. 12. Adhikari M, Wu ML, Zhao X. Gastrointestinal stromal tumor colliding with angiosarcoma. Int J Surg Pathol. 2006;14:252-6. 13. Zhou Y, Pan P, Yao L, Su M, He P, Niu N, et al. CD117-positive cells of the heart: progenitor cells or mast cells? J Histochem Cytochem. 2010;58:309-16.

14. Chahbouni S, Barnoud R, Watkin E, Devouassoux-Shisheboran M. High-grade small bowel angiosarcoma associated with angiosarcomatosis: a case report[article in French]. Ann Pathol. 2011;31:303-6. 15. Bellitti R, Buonocore M, De Rosa N, Covino FE, Casale B, Sante P. Primary cardiac angiosarcoma in a 25-year-old man: excision, adjuvant chemotherapy, and multikinase inhibitor therapy. Tex Heart Inst J. 2013;40:186-8. 16. Sherid M, Sifuentes H, Brasky J, Shah DA, Ehrenpreis ED. Clinical and endoscopic features of angiosarcoma of the colon: two case reports and a review of the literature. J Gastrointest Cancer. 2013;44:12-21. 17. Yonemori K, Tsuta K, Ando M, Hirakawa A, Hatanaka Y, Matsuno Y, et al. Contrasting prognostic implications of platelet-derived growth factor receptor-beta and vascular endothelial growth factor receptor-2 in patients with angiosarcoma. Ann Surg Oncol. 2011;18:2841-50. 18. Yamamoto T, Umeda T, Nishioka K. Immunohistological distribution of stem cell factor and kit receptor in angiosarcoma. Acta Derm Venereol. 2000;80:443-5. 19. Fosmire SP, Dickerson EB, Scott AM, Bianco SR, Pettengill MJ, Meylemans H, et al. Canine malignant hemangiosarcoma as a model of primitive angiogenic endothelium. Lab Invest. 2004;84:562-72. 20. Heinrich MC, Corless CL, Demetri GD. Defining the impact of adjuvant therapy in molecularly defined subsets of gastrointestinal stromal tumor : from lumping to splitting. JAMA Oncol. 2017;3:597-9.

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Case RepoRts & ReViews Veuillez noter que seuls les résumés des articles sont offerts en français.

vaScuLarite aPPenDicuLaire Suivie D’un SynDroMe D’activation MacroPhaGique auteurs :

Jolanta Jedrzkiewicz, M.D.1; Liane Heale, M.D.2; Robert H. Riddell, MBBS3; William Dubinski, M.D.4; Ronald M. Laxer, M.D.2; Iram Siddiqui, MBBS5

affiliations : 1Université de l’Utah, Salt Lake City, Utah, États-Unis 2 Hôpital pour enfants, médecine pédiatrique, Toronto, Ontario, Canada 3 Hôpital Mount Sinaï, Toronto, Ontario, Canada 4 Hôpital Humber River, Toronto, Ontario, Canada 5 Hôpital pour enfants, médecine de laboratoire pédiatrique, Toronto, Ontario, Canada

RÉSUMÉ Un adolescent de 14 ans s’est présenté à l’hôpital avec une forte fièvre et des douleurs abdominales. Comme les signes cliniques pointaient vers un diagnostic d’appendicite aiguë, une appendicectomie a été réalisée. Or, l’examen histologique de l’appendice a révélé une inflammation aiguë, focale et superficielle des muqueuses, et une absence de caractéristiques typiques de l’appendicite aiguë, comme une inflammation aiguë sous-muqueuse ou transmurale. Par ailleurs, de nombreux vaisseaux de taille moyenne présentaient une vascularite accompagnée d’une nécrose fibrinoïde et d’une inflammation lymphohistiocytaire. Une coloration des fibres élastiques a permis de confirmer l’implication des artères et une interruption de la limitante élastique. Peu après l’intervention chirurgicale, le patient a été hospitalisé de nouveau, car on soupçonnait une septicémie. L’adolescent a été traité à l’aide d’antibiotiques. L’état clinique du patient s’est ensuite détérioré; une pancytopénie et une hyperferritinémie, deux symptômes associés au syndrome d’activation macrophagique, sont apparues. Bien que les résultats des analyses par chimioluminescence se soient avérés anticorps antiADNdb positifs, le patient ne présentait pas d’auto-anticorps antinucléaires. Qui plus est, son évolution clinique ne concordait pas avec un diagnostic de lupus érythémateux systémique. L’adolescent a développé une neuropathie périphérique et, compte tenu de son exposition à l’hépatite B et des signes pathologiques observés lors de la biopsie, un diagnostic de polyartérite noueuse a été établi. Le patient a bien réagi à l’immunosuppression et au traitement antiviral, et n’a connu aucune récidive durant la période de suivi d’un an. En conclusion, si une vascularite nécrosante peut apparaître dans l’appendice de manière isolée, elle peut également indiquer la présence d’une maladie systémique, comme le démontre le cas décrit dans le présent article.

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Case RepoRts & ReViews This article was peer-reviewed.

KeywoRds: vasculitis, appendix, macrophage activation syndrome.

aPPenDiceaL vaScuLitiS with SuBSequent MacroPhaGe activation SynDroMe authors:

Jolanta Jedrzkiewicz1 MD, Liane Heale2 MD, Robert H. Riddell3 MBBS, William Dubinski4 MD, Ronald M. Laxer2 MD, Iram Siddiqui5 MBBS.

affiliations: 1University of Utah, Salt Lake City, UT, USA. 2 Hospital for Sick Children, Pediatric Medicine, Toronto, ON, Canada. 3 Mount Sinai Hospital, Toronto, ON, Canada. 4 Humber River Hospital, Toronto, ON, Canada. 5 Hospital for Sick Children, Pediatric Laboratory Medicine, Toronto, ON, Canada.

The authors declare that there are no conflicts of interest regarding the publication of this paper. All authors have provided CAP-ACP with non-exclusive rights to publish and otherwise deal with or make use of this article, and any photographs/images contained in it, in Canada and all other countries of the world.

ABSTRACT A 14-year-old male presented acutely with fever and abdominal pain. Appendectomy was performed for clinically suspected acute appendicitis. Histologic examination of the appendix showed focal superficial mucosal acute inflammation without typical features of acute appendicitis, such as submucosal and transmural acute inflammation. In addition, multiple medium sized vessels showed vasculitis with associated fibrinoid necrosis and lymphohistiocytic inflammation. The elastic stain confirmed involvement of arteries and disruption of the elastic lamina. Soon after surgical intervention, the patient was readmitted due to suspected sepsis and was treated with antibiotics. His clinical status deteriorated with the development of pancytopenia and hyperferritinemia, consistent with macrophage activation syndrome. While he tested positive for dsDNA antibodies by chemiluminescence, his ANA was negative and his clinical course was not consistent with a diagnosis of systemic lupus erythematosus. The patient developed a peripheral neuropathy and, given his hepatitis B exposure and the pathological findings on biopsy, polyarteritis nodosa became the working diagnosis. He responded well to immunosuppression and antiviral therapy and has not had a disease recurrence during one year of follow up. Although necrotizing vasculitis can be an isolated finding in the appendix, it may also indicate a systemic illness as seen in the current case. Revue canadienne de pathologie | volume 10, numéro 2 | www.cap-acp.org

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Case RepoRts & ReViews aPPenDiceaL vaScuLitiS (cont.) INTRODUCTION Diseases affecting the appendix frequently present acutely with abdominal pain localized to the right lower quadrant, fever and malaise. Subsequent imaging typically shows induration of the appendiceal wall, confirming the clinical impression of acute appendicitis. Invariably, appendectomy is the treatment of choice, although a subset of patients can be treated empirically with observation and antibiotics.1

Other incidental findings such as vasculitis have been reported in both adult and pediatric cases.6,7 The presence of vasculitis within an accompanying acute appendicitis likely indicates that the vascular injury is a secondary process. Conversely, an isolated vasculitis may cause concern for impending systemic illness.

Histopathological findings in cases of acute appendicitis include a submucosal and/or transmural acute inflammatory infiltrate, variable amounts of tissue necrosis, perforation and necroinflammatory process involving periappendiceal fibroadipose tissue. It is generally accepted that the main pathophysiology of appendicitis is obstruction and entrapment of bacteria within the appendiceal lumen. This combination of events leads to tissue necrosis, inflammation and perforation. Bacterial overgrowth may be observed in the appendiceal lumen but is not one of the criteria for histopathologic diagnosis. The most frequently isolated bacteria from acute appendicitis are Escherchia coli, Bacteriodes spp. Klebsiella pneumoniae and Pseudomonas aeruginosa.1 Additionally, 16S ribosomal gene sequence analysis of the luminal fluid of appendix identified Fusobacterium spp. as a common pathogen in cases of acute appendicitis.3,4 Other microorganisms that may be observed during the morphologic assessment include viral inclusions of adenovirus or parasites, particularly Enterobius vermicularis.5

A 14-year-old previously healthy male presented with fever, vomiting and abdominal pain of one-week duration. He was suspected to have acute appendicitis and underwent an uncomplicated appendectomy and was discharged home the following day. Four days later, he was readmitted with fever and recurrent syncope. He was treated for presumed sepsis but his fever persisted despite broad spectrum antibiotics. He developed pancytopenia and hyperferritinemia and was transferred to the Hospital for Sick Children for further management.

Rarely, pathologic examination of the appendix will reveal unexpected findings. These findings include neoplasms, with the most common being neuroendocrine neoplasms, serrated and mucinous neoplasms or colonic type adenocarcinomas.5 All are extremely rare in pediatric patients. 64

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PATIENT PRESENTATION AND METHODS

Following transfer to the tertiary care hospital, the patient’s status continued to deteriorate. He developed delirium and nephrotic-range proteinuria. His bloodwork revealed a progressive pancytopenia, extreme hyperferritinemia (67341.0 ug/L), hypofibrinogemia (1.5 g/L), transaminitis (ALT 385U/L, AST 769U/L and GGT 74U/L), an elevated LDH (7109 U/L) and coagulopathy (PTT of 56sec and INR of 1.4), all consistent with macrophage activation syndrome (MAS). His bone marrow biopsy showed hemophagocytosis, which supported the clinical impression. He was initially treated with intravenous immunoglobulin and intravenous pulse methylprednisolone but required the addition of anakinra - an interleukin 1 (IL1) receptor antagonist - to control his disease. With this therapy, he had a rapid resolution of the fever and renal

Case RepoRts & ReViews aPPenDiceaL vaScuLitiS (cont.) disease and a more gradual improvement in the cognitive dysfunction. Prior to discharge, he was transitioned to cyclosporine for maintenance therapy. Subsequent histopathological evaluation of the entirely submitted previously resected appendix revealed prominent inflammation centered on medium sized vessels with accompanying fibrinoid necrosis (Figure 1 A and B). Elastic trichrome stain showed disruption of the internal and external elastic lamina of the involved arteries (Figure 1 C). There was only focal involvement of a medium sized vein. The small vessels appeared uninvolved. The inflammation was mostly composed of lymphocytes with an approximate CD4 to CD8 ratio of 3:1, histiocytes highlighted by CD163 cáÖìêÉ=NK=^ééÉåÇáñ=ëÜçïáåÖ=ÑáÄêçìë=çÄäáíÉê~íáçå=çÑ=äìãÉå=~åÇ=ëé~êëÉ éÉêáî~ëÅìä~ê=áåÑä~ãã~íáçåK

Hematoxylin and eosin (H&E) stained sections (A) 1X and (B) 20X demonstrated vasculitis in the appendix involving medium-sized vessels. The vascular wall showed histiocytic and lymphocytic cell infiltrate with associated fibrinoid necrosis of the vessel wall; (C) The 20X elastic special stain showed medium sized arteries with damaged internal elastic lamina; The inflammatory infiltrate included (D) CD4-positive T cells, (E) CD8-positive T cells, and (F) scattered CD163-positive histocytes.

immunohistochemical stain, plasma cells and rare eosinophils (Figure 1 D-F). Hemophagocytosis was not observed. The hepatitis B surface and core antigen immunohistochemical stains were also performed and were negative. The inflamed vessels were localized within the appendix as well as the periappendiceal fibroadipose tissue. Otherwise the appendix showed fibrous obliteration of the lumen but only focal mucosal acute inflammation. Focal bacterial overgrowth was noted within the lumen but no fungal organisms or viral inclusions were seen (negative GMS and Ziehl-Neelsen stains). Microscopic findings were communicated to the clinical team to help guide further workup. He had extensive investigation to look for the underlying disease trigger for his MAS that could present with vasculitis of the appendix. While his positive antidouble stranded DNA antibody of 106 IU/ml, and hypocomplementemia (C3 0.51g/L and C4 0.12 g/L), suggested a diagnosis of systemic lupus erythematosus (SLE), the negative antinuclear antibody (ANA) and extractable nuclear antigens along with the rapid resolution of the renal involvement and complement levels made lupus much less likely. His negative anti-neutrophilic antibody (ANCA) and pathological findings of medium-size vessel disease made a diagnosis of ANCA-associated vasculitis very unlikely. Interestingly, he was found to have Hepatitis B core antibody and Hepatitis B viral DNA positive for which he received antiviral therapy. Subsequently, he also developed a peripheral neuropathy, and therefore hepatitis B-induced polyarteritis nodosa became the working diagnosis. His CT angiography did not show any other large or medium vessel disease. The remainder of the infectious disease work up was unremarkable and the bone marrow biopsy was negative for malignancy. The patient had genetic testing for primary hemophagocytic lymphohistiocytosis that was negative.

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Case RepoRts & ReViews aPPenDiceaL vaScuLitiS (cont.)

The unexpected finding of vasculitis on appendiceal pathology may help in diagnosing an underlying systemic vasculitis. DISCUSSION The unexpected finding of vasculitis on appendiceal pathology may help in diagnosing an underlying systemic vasculitis. The differential diagnosis includes systemic inflammatory vascular disease manifesting with a localized disease or a single organ vasculitis that is typically limited to the resected organ.8 Gastrointestinal tract involvement has been reported to occur in a number of systemic inflammatory diseases including polyarteritis nodosa, as well as granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, Henoch-Schonlein purpura (also known as IgA vasculitis), Behçet’s disease, giant cell arteritis and Takayasu arteritis.9-11 Although the definitive diagnosis relies on clinical and radiographic features, microscopic examination can provide valuable information that narrows down the differential diagnosis. Helpful microscopic observations include size and type of involved vessels and the description of the inflammatory infiltrate, particularly presence or absence of granulomas. A medium sized arteritis with fibrinoid necrosis as seen in the current case has been linked to the diagnosis of polyarteritis nodosa in a review of 12 cases.12 In this review, nine patients had vasculitis that appeared limited to a single organ without a systemic disease. However three patients developed complications of polyarteritis nodosa on follow up and died due to the disease. The same three patients also carried a diagnosis of another autoimmune disease including 66

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rheumatoid arteritis, Sjogren’s syndrome, scleroderma, and SLE.12 Overall, 7% of polyarteritis nodosa patients have a concurrent hepatitis B infection.13 During the course of the disease there is inflammatory injury to medium sized arteries causing fibrinoid necrosis and vascular aneurysms. Subsequently, the clotting cascade is activated and thrombosis occurs, leading to occlusion of the vessel and tissue infarction.13 The most commonly involved organs are the kidneys, heart, nervous system and gut with typical sparing of the pulmonary circulation. Other diseases that have been described to cause vasculitis in the appendix are connective tissue diseases such as SLE and rheumatoid arthritis.6 A diagnosis of SLE was unlikely in this patient given the negative ANA result but was considered clinically because of the positive dsDNA antibodies, proteinuria with hypocomplementemia and CNS involvement.14 Additionally, macrophage activation syndrome is more commonly associated with SLE than polyarteritis nodosa.15 However, the histopathological observations reported in cases of SLE include inflammation of much smaller vessels than was seen in this case.16 Additionally, his negative ANA and extractable nuclear antigens and disease course made SLE less likely. Of note, many of the symptoms of polyarteritis nodosa and SLE overlap clinically making a definitive diagnosis challenging especially when the presentation is acute.17 Although macrophage activation syndrome is most frequently associated with -SLE, other autoimmune disorders including polyarteritis nodosa can also have this association.18,19 In this patient, the unexpected finding of isolated vasculitis mainly involving the medium sized arteries in the appendix that appeared to have polyarteritis nodosa-like features, was the initial presentation of a severe

ReView aPPenDiceaL vaScuLitiS (cont.)

Other diseases that have been described to cause vasculitis in the appendix are connective tissue diseases such as SLE and rheumatoid arthritis.6

systemic illness in an otherwise healthy child. Following appendectomy, the patient developed macrophage activation syndrome which responded well to immune suppression. Considering his hepatitis B exposure, pathologic findings and peripheral neuropathy, polyarteritis nodosa is the working diagnosis. He is currently clinically well off immunosuppression and will continue his antiviral medication for a total of 6 months post-immunosuppresive therapy. There was no disease recurrence during the one year of follow up. This case highlights the importance of taking a multidisciplinary approach to investigate for underlying systemic inflammatory diseases when the pathology reveals an unexpected isolated organ vasculitis.

with an abundance of bacteria from the phylum Fusobacteria. J Pediatr Surg. 2014;49:441-6. 4.

Swidsinski A, Dörffel Y, LoeningBaucke V, Theissig F, Rückert JC, Ismail M, et al. Acute appendicitis is characterised by local invasion with Fusobacterium nucleatum necrophorum. Gut. 2011;60:34-40.

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Akbulut S, Tas M, Sogutcu N, Arikanoglu Z, Basbug M, Ulku A, et al. Unusual histopathological findings in appendectomy specimens: a retrospective analysis and literature review. World J Gastroenterol. 2011;17:1961-70.

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Misdraji J, Graeme-Cook FM. Miscellaneous conditions of the appendix. Semin Diagn Pathol. 2004;21:151-63.

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Plaut A. Asymptomatic focal arteritis of the appendix; 88 cases. Am J Pathol. 1951;27:247-63.

8.

Kint N, De Haes P, Blockmans D. Comparison between classical polyarteritis nodosa and single organ vasculitis of medium-sized vessels: a retrospective study of 25 patients and review of the literature. Acta Clin Belg. 2016;71:26-31.

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Ahn E, Luk A, Chetty R, Butany J. Vasculitides of the gastrointestinal tract. Semin Diagn Pathol. 2009;26:77-88.

12. Moyana TN. Necrotizing arteritis of the vermiform appendix. A clinicopathologic study of 12 cases. Arch Pathol Lab Med.1988;112:738-41. 13. Ebert EC, Hagspiel KD, Nagar M, Schlesinger N. Gastrointestinal involvement in polyarteritis nodosa. Clin Gastroenterol Hepatol 2008;6:960-6. 14. Tian X-P, Zhang X: Gastrointestinal involvement in systemic lupus erythematosus: Insight into pathogenesis, diagnosis and treatment. World J Gastroenterol. 2010;16:2971-7. 15. Bennett TD, Fluchel M, Hersh AO, Hayward KN, Hersh AL, Brogan TV, et al. Macrophage activation syndrome in children with systemic lupus erythematosus and children with juvenile idiopathic arthritis. Arthritis Rheum. 2012;64:4135-42. 16. Cellini C, Hoda SA, Spigland N. Lupus-associated vasculitis manifesting as acute appendicitis in a 16 year old girl. Pediatr Rheumatol Online J. 2008;6:10. 17. Marques VL, Guariento A, Simoes MS, Blay G, Lotito AP, Silva CA. Childhood-onset systemic polyarteritis nodosa and systemic lupus erythematosus: an overlap syndrome? Rev Bras Reumatol. 2016;56:551-3.

10. Zamani F, Shakeri R, Modiramani O, Malekzadeh R. Abdominal pain as presentation of Takayasu's arteritis in an adolescent male patient. MedGenMed. 2006;8:69.

18. Atteritano M, David A, Bagnato G, Beninati C, Frisina A, Iaria C, et al. Haemophagocytic syndrome in rheumatic patients. A systematic review. Eur Rev Med Pharmacol Sci. 2012,16:1414-24.

11. Scola CJ, Li C, Upchurch KS. Mesenteric involvement in giant cell arteritis. An underrecognized complication? Analysis of a case series with clinicoanatomic correlation. Medicine (Baltimore). 2008;87:45-51.

19. Dhote R, Simon J, Papo T, Detournay B, Sailler L, Andre MH, et al. Reactive hemophagocytic syndrome in adult systemic disease: report of twenty-six cases and literature review. Arthritis Rheum. 2003;49:633-9.

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Case RepoRts & ReViews Veuillez noter que seuls les résumés des articles sont offerts en français.

ParaGanGLioMe Du corDon SPerMatique : ÉtuDe De caS et revue De La LittÉrature auteurs :

Ryan DeCoste, M.D.1; Jonathan Moore, M.D.2; Padraic O’Malley, M.D.2; Jennifer Merrimen, M.D.1,2

affiliations : 1Département de pathologie, Université Dalhousie, Halifax, Nouvelle-Écosse, Canada 2 Département d’urologie, Université Dalhousie, Halifax, Nouvelle-Écosse, Canada

RÉSUMÉ Les paragangliomes sont des tumeurs chromaffines extrasurrénaliennes dérivées des paraganglions présents dans l’organisme. Bien que des sites courants aient été recensés, la formation de paragangliomes dans des zones inusitées est largement reconnue. Les paragangliomes du cordon spermatique sont rares et, jusqu’à présent, ont seulement été décrits dans un petit nombre d’études de cas. Cet article porte sur le cas d’un homme de 43 ans chez qui un paragangliome du cordon spermatique se présentant sous forme de masse paratesticulaire indolore a été diagnostiqué. Le patient a ensuite subi une excision chirurgicale conservatrice du testicule. On y traite également des articles publiés à ce jour sur cette rare entité.

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Case RepoRts & ReViews This article was peer-reviewed.

KeywoRds: paraganglioma; spermatic cord; paratesticular.

SPerMatic corD ParaGanGLioMa: a caSe rePort anD Literature review authors:

Ryan DeCoste1 MD, Jonathan Moore2 MD, Padraic O’Malley2 MD, Jennifer Merrimen1,2 MD.

affiliations: 1Department of Pathology, Dalhousie University, Halifax, NS, Canada. 2 Department of Urology, Dalhousie University, Halifax, NS, Canada.

This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors declare that there are no conflicts of interest regarding the publication of this paper. All authors have provided CAP-ACP with non-exclusive rights to publish and otherwise deal with or make use of this article, and any photographs/images contained in it, in Canada and all other countries of the world.

ABSTRACT Paragangliomas are tumours of extra-adrenal chromaffin cells derived from paraganglia distributed throughout the body. While common sites exist, their occurrence in unusual sites is well-recognized. Spermatic cord paragangliomas are rare and described thus far in a small number of case reports. We describe a case of spermatic cord paraganglioma presenting as painless paratesticular mass in a 43-year-old man, treated with testis-sparing surgical excision. In addition, we review the available literature regarding this rare entity.

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Case RepoRts & ReViews SPerMatic corD ParaGanGLioMa (cont.) INTRODUCTION Paragangliomas are tumours arising from extra-adrenal chromaffin cells. Although common sites are recognized, cases in rare locations throughout the body have been reported. One such location is the spermatic cord/paratesticular region, with a small number of cases reported in the literature to date. Herein, we describe a case of spermatic cord paraganglioma and review the available literature on this topic. CASE REPORT A 43-year-old man presented with an asymptomatic right-sided scrotal mass. His past medical history included chronic back pain, acid reflux, left ACL repair, bilateral quadriceps ruptures, and a tonsillectomy. There was no personal history of malignancy. His medications included omeprazole and over-thecounter analgesics. He has two children. His mother, a smoker, had lung cancer, while his father had a malignancy, the origin of which was unknown to the patient.

Physical exam revealed normal testis and epididymis bilaterally. On the right side, superior to the testicle was a distinct mass separate from the epididymis and testis. It was spherical, non-tender and non-fluctuant. There were no overlying skin changes. Ultrasound showed a 1.6 cm solid, hypervascular mass superior to right testis and associated with the spermatic cord. He was brought to the operating room, where a suprascrotal incision was made parallel to the spermatic cord. The mass was dissected away from the overlying skin and adjacent cord structures. Intraoperative Doppler was used to confirm vascular flow to the testis. Separate proximal and distal margins were excised. An ilioinguinal nerve block was performed for post-operative pain control. Gross pathologic examination revealed a well-circumscribed 1.3 cm tan-yellow ovoid tumour (Figure 1). Microscopy showed an encapsulated

cáÖìêÉ=NK=

Gross examination showed a well-circumscribed tan-yellow ovoid tumour (arrow). 70

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Case RepoRts & ReViews SPerMatic corD ParaGanGLioMa (cont.) lesion composed of variably-sized nests of polygonal cells separated by a fibrovascular network (“Zellballen” appearance) (Figure 2). Cells contained variable granular to vacuolated pink, gray, or clear cytoplasm. Nuclei contained vesicular chromatin with occasionally prominent nucleoli. Pleomorphism was evident in the form of variably sized and bizarre nuclei. Cell nests cáÖìêÉ=OK=

were smaller peripherally, with larger nests containing necrosis, and increased and atypical mitoses seen centrally (Figure 3). There was moderate to focal high cellularity with spindling (3%.

Low-power photomicrograph demonstrating an encapsulated tumour surrounded by spermatic cord vasculature and adipose tissue (arrow) (Hematoxylin and Eosin, 20x magnification). cáÖìêÉ=PK=

To date, our patient has had follow up imaging with a repeat scrotal ultrasound demonstrating benign, postsurgical changes and an abdominopelvic CT demonstrating no retroperitoneal lymphadenopathy, additional suspicious lesions, or visceral metastasis. Chest x-ray has been normal. The patient is disease free at 6 months post-surgery. DISCUSSION

Large nests of cells with pleomorphism and central necrosis were concentrated near the centre of the tumour (Hematoxylin and Eosin, 100x magnification).

The paraganglionic system comprises collections of neuroepithelial cells throughout the body, characterized by intracytoplasmic secretory granules containing catecholamines or precursors, with much of this tissue concentrated in the adrenal glands.1 1-10% of paraganglion-derived tumours originate outside the adrenals, with sites including head and neck, retroperitoneum, pelvis and thorax.1-5 However, they have been described more rarely in other locations throughout the body.6

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Case RepoRts & ReViews SPerMatic corD ParaGanGLioMa (cont.)

The differential diagnosis of spermatic cord tumours includes benign lipomas, adenomatoid tumours, leiomyomas and other less common entities. In addition, 20% of spermatic cord tumours are malignant, most being sarcomas and metastases In the 1970s, two cases of spermatic cord “pheochromocytoma” were described by Eusebi and Massarelli, and Soejima et al., which would be classified today as paragangliomas.2-3 Since then, we identify 8 additional reported cases in the English literature of paraganglioma in the spermatic cord or paratesticular region, including one case reported as pheochromocytoma,1,4-10 indicating the rarity of this entity. Additionally, a case reported as spermatic cord pheochromocytoma has been described in a patient with previous history of bilateral carotid body paragangliomas and bilateral adrenal pheochromocytomas. This lesion may also have represented a metastasis, and the possibility of a syndromic association in this patient is raised by the authors.11 One additional case of spermatic cord paraganglioma is identified in the French literature by its published English abstract,12 and large testicular paraganglioma has been described as well.13 Cases are reported in men ages 18-69, often presenting as a non-functional, painless unilateral scrotal mass. The majority of paragangliomas produce, but do not secrete catecholamines, allowing them to avoid detection for long periods.9 Most cases have occurred as solitary lesions, ranging from 1.3-10.0 cm in size, and treatment has been through transscrotal resection or radical inguinal orchiectomy. The differential diagnosis of spermatic cord tumours includes benign lipomas, adenomatoid tumours, leiomyomas and other less common 72

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entities. In addition, 20% of spermatic cord tumours are malignant, most being sarcomas and metastases.5-6 Microscopically, paragangliomas are comprised of “Zellballen” nests of polygonal cells with cytoplasmic neuroendocrine granules, separated by delicate, vascularized fibrous septae and containing a network of S100positive sustentacular cells.4 Endocrine-type atypia may be present. Helpful IHC markers include tumour cell expression of vimentin, synaptophysin, chromogranin, neuronspecific enolase and CD56.8-9 The origin of spermatic cord paraganglioma remains unconfirmed. However, it has been hypothesized that fetal extra-adrenal chromaffin tissue, present in the retroperitoneum in association with sympathetic ganglia and plexuses, may accompany the testicular descent into the scrotum and persist. Rarely, this tissue may later give rise to a paraganglioma.2 A subgroup of paragangliomas may have a hereditary basis involving Rearranged during Transfection (RET), von Hippel-Lindau (VHL), or succinate dehydrogenase subunit (SDH-B, C, D) genes. Alataki et al. have reported a case associated with SDCD mutation identified with IHC, and confirmed by mutation analysis, and suggest this approach may be considered even in absence of family history.9 Paragangliomas are benign in approximately 90% of cases and, as in pheochromocytoma, the only reliable indicator of malignancy is metastasis.6 However, multiple histologic grading/scoring schemes have been proposed to evaluate risk of malignancy in pheochromocytoma and paraganglioma, two of which have recently been applied to a case of spermatic cord paraganglioma.10 The PASS score evaluates tumours based on the presence or absence of large nests/diffuse growth, central or

Case RepoRts & ReViews SPerMatic corD ParaGanGLioMa (cont.) confluent tumour-type necrosis, high cellularity, cellular monotony, tumour cell spindling (even if focal), mitoses >3 per 10 high power fields, atypical mitoses, extension into adipose tissue, vascular invasion, capsular invasion, profound nuclear pleomorphism and nuclear hyperchromasia.14 The GAPP grade categorizes differentiation based on scores in categories of histological pattern, cellularity, comedo necrosis, vascular or capsular invasion, Ki67 index and catecholamine type.15 Aside from the documented case of large testicular paraganglioma, which occurred with possible lung metastases,13 and the spermatic cord paraganglioma occurring in the context of previous pheochromocytomas and paragangliomas,11 no reported cases have been associated with additional tumours. Lymphovascular invasion has been reported rarely.8-10 To our knowledge, no case of recurrence has been reported. To date, our patient has had follow up imaging with a repeat scrotal ultrasound demonstrating benign, postsurgical changes and an abdominopelvic CT demonstrating no retroperitoneal lymphadenopathy, additional suspicious lesions, or visceral metastasis. Chest x-ray has been normal. The patient is disease free at 6 months post-surgery.

REFERENCES 1.

Attaran SY, Shakeri S, Sobhani AR. Paraganglioma of the spermatic cord: report of a case. J Urol. 1996;155:651.

2.

Eusebi V, Massarelli G. Phaeochromocytoma of the spermatic cord: report of a case. J Pathol. 1971;105:283-4.

3.

Soejima H, Ogawa O, Nomura Y, Ogata J. Pheochromocytoma of the spermatic cord: a case report. J Urol. 1977;118:495-6.

4.

Bacchi CE, Schmidt RA, Brandão M, Scapulatempo R, Costa JC, Schmitt FC. Paraganglioma of the spermatic cord: report of a case with immunohistochemical and ultrastructural studies. Arch Pathol Lab Med. 1990;114:899-901.

5.

Mashat F, Meccawi A, Garg S, Christian E. Paraganglioma of the spermatic cord. Annals of Saudi Medicine. 1993;13:208-10.

6.

Garaffa G, Muneer A, Freeman A, Abdel Raheem AM, Ralph DJ, Minhas S, et al. Paraganglioma of the spermatic cord: case report and review of the literature. Scientific World Journal. 2008;8:1256-8.

7.

Young IE, Nawroz IM,Aitken RJ. Phaeochromocytoma of the spermatic cord. J Clin Pathol. 1999;52:305-6.

8.

Gupta R, Howell RS, Amin MB. Paratesticular paraganglioma: a rare cause of an intrascrotal mass. Arch Pathol Lab Med. 2009; 133:811-3.

9.

Alataki D, Triantafyllidis A, Gaal J, Rodiou C, Vouros J, Papathanasiou A, et al. A non-catecholamineproducing sympathetic paraganglioma of the spermatic cord: the importance of performing

CONCLUSION We have presented a rare case of spermatic cord paraganglioma in a 43year-old man treated with testis-sparing excision. Pathologic examination included PASS score indicative of possible malignant potential and GAPP grade indicating moderate differentiation. These rare lesions are most often sporadic and benign but may occur in association with hereditary genetic mutations. Recurrence has not been described in reported cases and surgical management is expected to be curative.

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Case RepoRts & ReViews SPerMatic corD ParaGanGLioMa (cont.) candidate gene mutation analysis. Virchows Arch. 2010;457:619-22. 10. Kwon AY, Kang H, An HJ, Kim G, Kim TH, Heo JH, et al. Spermatic cord paraganglioma with histologically malignant features. Urology. 2016;93:e7-e8. 11. Abe T, Matsuda H, Shindo J, Nonomura K, Koyanagi T. Ectopic pheochromocytoma arising in the spermatic cord 5 years after removal of bilateral carotid body tumors and adrenal pheochromocytomas. Int J Urol. 2000;7:110-1. 12. Majdoub W, Nfoussi H, Rhouma SB, Chelly I, Zehani A, Haouet S, et al. Paragangliome du cordon spermatique. Prog Urol. 2013;23:486-8.

13. Makris MC, Koumarelas KC, Mitrousias AS, Psathas GG, Mantzioros A, Sakellariou SP, et al. A ‘giant’ paraganglioma in the testis. Endocrinol, Diabetes and Metab Case Rep. 2014;2014:140055. 14. Thompson LD. Pheochromocytoma of the Adrenal Gland Scaled Score (PASS) to separate benign from malignant neoplasms: a clinicopathologic and immunophenotypic study of 100 Cases. Am J Surg Pathol. 2002;26:551-66. 15. Kimura N, Takayanagi R, Takizawa N, Itagaki E, Katabami T, Kakoi N, et al. Pathological grading for predicting metastasis in phaeochromocytoma and paraganglioma. Endocr Relat Cancer. 2014;21:405-14.

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