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HER2 Testing in Canada: A Passing Grade

Junior Scientist Award Lecture 2012: Understanding Antibody-Mediated Rejection of Organ Transplants

Canadian Journal of

Pathology

Publications Agreement Number 40025049 • ISSN 1918-915X

Official Publication of the Canadian Association of Pathologists

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Volume 5, Issue 1 • Spring 2013

R2 Testing in Canada: A Passing Grade

iafficiall PuPubbllicaicattionion ofof tthhee CanadianCanadian AssAssoociaciattionion ooff PPaattholholoogisgisttss

Mess

1

64th Annual Meeting of the Canadian Association of Pathologists (CAP-ACP) held jointly with Association des pathologistes de Québec

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Québec City ■ June 8–12, 2013

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VOLUME 5, ISSUE 1 2013

About the Cover

4 Editorial: HER2 Testing in Canada: A Passing Grade Anita Bane, MB BCh, PhD, FRCPath(UK)

6 Éditorial : L’analyse de l’expression de l’oncogène HER2 : la note de passage Anita Bane, MB BCh, PhD, FRCPath(UK)

5 Thank You to Reviewers

19 Changes to the Editorial Board

25 Message from the Nominating Committee

Original Articles8 Evolution of HER2 Testing in Canada Wedad M. Hanna, MD 15 False-Positive FISH for HER2 Amplification in a Breast Cancer Owing to Loss of CEP17: A Case Report and Literature Review Jie Xu, PhD, FCCMG, Helen Ettler, MD, FRCPC, Peter Engbers, MD, FRCPC 20 Pathology in the 1930s and 1940s: A Poetic Critique by “The PEI–Dalhousie–Mayo Clinic Connection”: Drs. Malcolm B. Dockerty and Lewis B. Woolner James R. Wright Jr., MD, PhD

23 Case Report: Congenital Cervical Cartilaginous Rest Lamiaa Rouas, MD, Rajae Tahri, MD, Najat Lamalmi, MD, Zaitouna Alhamany, MD, Nadia Cherradi, MD

Images in Pathology26 Perforated Appendiceal Mass in a Patient with Cystic Fibrosis Tao Wang, MD, Eric Labelle, MD, FRCSC, Rajkumar Vajpeyi, MD, FRCPC

CAP-ACP Junior Scientist Award Lecture 201227 Understanding Antibody-Mediated Rejection of Organ Transplants: Mechanisms, Morphology, Molecular Patterns, and Personalized Precision Diagnosis Banu Sis, MD, FRCPC

Book Review37 The Corporate Capture of Laboratory Services: A Review of False Positive. Private Profit in Canada’s Medical Laboratories Reviewed by J. Godfrey Heathcote, MA, MB BChir, PhD, FRCPC

Professional Development/Employment Opportunities38 Interior Health38 Northern Health

EDITOR-IN-CHIEFJ. Godfrey Heathcote, MA, MB BChir, PhD, FRCPC

EDITORIAL BOARDManon Auger, MD, FRCPC, Cytopathology;

Calvino Cheng, BSc, MD, FRCPC, Pathology Informatics and Quality Management;

David K. Driman, MB ChB, FRCPC, Anatomical Pathology;

Todd F. Hatchette, BSc, MD, FRCPC, Medical Microbiology; Michael J. Shkrum, MD, FRCPC, Forensic Pathology;

MANAGING EDITORSusan Harrison

COPY EDITOR Scott Bryant

PROOFREADERScott Bryant

ART DIRECTORAndrea Brierley, [email protected]

TR ANSL ATORMarie Dumont

SALE S AND CIRCUL ATION COORDINATORBrenda Robinson, [email protected]

ACCOUNTINGSusan McClung

GROUP PUBLISHERJohn D. Birkby, [email protected]

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Canadian Journal of Pathology • Volume 5, Issue 1, 2013

Contents

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The cover image shows interphase FISH of paraffin sections of the breast carcinoma, showing one green/CEP17 and one to three orange/HER2 signals in the cells.

FOUNDING EDITORJagdish Butany, MBBS, MS, FRCPC

Pierre Douville, MD, FRCPC, Medical Biochemistry;

Lawrence Haley, MD, FRCPC, Hematopathology;

39 Sultan Qaboos University Hospital39 Dalhousie University

Canadian Journal of P athology 3Spring 2013

Spring 20134 Canadian Journal of P athology

Competing interests: None declared

EDITORIAL

The HER2 oncogene encodes for a receptor tyrosine

kinase, which is amplified in approximately 15–20% of

breast cancers.1 Patients with a breast cancer that over-

expresses and/or is amplified for HER2 have a worse

prognosis than those without such change and are eligible to

receive the therapeutic agent trastuzumab, which specifically

targets the gene.2 Given the prognostic importance and

predictive utility of HER2, it is routinely evaluated on all

newly diagnosed breast cancers and is recommended for

evaluation in the metastatic setting and in specimens post

neo-adjuvant therapy (NAT).3 Given the importance of this

assay, the College of American Pathologists (CAP) and the

American Society of Clinical Oncology (ASCO) have jointly

issued guidelines for the performance and interpretation of

this assay.4 Central to these guidelines is an algorithmic

approach to the evaluation of HER2, commencing with an

immunohistochemical (IHC) evaluation of the HER2 protein

with an additional assay for the evaluation of the HER2 gene

by in situ hybridization (ISH) for those cases with equivocal

results (2+) by IHC.4 Canadian guidelines have also been

published that mirror in large part the CAP/ASCO guidelines,

with emphasis on the pre-analytical, analytical, and quality

assurance (QA) procedures recommended for Canadian

laboratories assessing HER2.5 In this issue of the journal,

Dr. Hanna presents the findings of a national survey

conducted to evaluate the compliance of Canadian

laboratories with the above guidelines, the results of which

are both interesting and informative.6

Thirty-six pathologists from four regions of Canada with a

special interest in breast pathology and self-identified to have

a leading role in HER2 testing in their respective laboratories

were invited to participate in an online survey developed

jointly by Dr. Hanna, F. Hoffmann–La Roche and Summit

Strategy. Approximately two thirds of the invitees,

representing 22 different laboratories, participated in the

survey.

Many of the results are very encouraging: 82% of centres

reported testing all newly diagnosed invasive breast cancers,

all centres used neutral buffered formalin as the fixative of

choice, 82% of centres met the recommend cold ischemic

time (defined as the time from specimen excision to the

initiation of tissue fixation) of ≤1 hour,7 and a similar number

of centres reported fixing large excision specimens for the

optimal time period (24–48 hours). The majority of the

testing was reported to be performed on definitive surgical

specimens, with core-needle biopsy specimens used in cases

of locally advanced disease, patients being considered for NAT

and for metastatic biopsy specimens or when the definitive

surgical specimen was too small or considered too poorly

fixed for optimal IHC evaluation. All laboratories were

accredited to perform the tests, with 20 meeting the

recommended volume of cases. In addition, the average rates

of reporting of negative, equivocal, and positive cases by IHC

were broadly in accordance with those published in the

literature.4 Equivocal (2+) cases were re-evaluated with an

alternative ISH method in the majority of laboratories.

Encouragingly, all centres reported participation in a QA

program (provincial, national, or international) for HER2

testing, and the concordance rates between local and reference

laboratories for all sites were ≥95%.

However, the results were not uniformly positive and

highlight specific needs that should be addressed. The fact that

50% of the respondents were from one province suggests that

large swaths of the country are under-represented or not

represented in this survey, introducing a potential regional

reporting bias. In almost one fifth (18%) of laboratories

polled, not all newly diagnosed invasive breast cancers were

tested for HER2, despite its proven prognostic and predictive

utility. In two centres, cases identified as being 2+ or

indeterminate by IHC were not subject to any further

evaluation and were apparently left unresolved; clearly this is

suboptimal. While the average proportion of cases reported

as negative, indeterminate, and positive are in line with the

published guidelines alluded to above, the actual variance is

HER2 Testing in Canada: A Passing Grade


BANE

huge, with at least one centre reporting that 52% of cases

evaluated by IHC fell into the 2+ or indeterminate category,

thus requiring ISH for assessment of the HER2 gene status.

Such reportedly high rates of indeterminate results suggest

either analytical issues with the IHC assay in use or observer

uncertainty. The former may result in part from the lack of a

single standard antibody in use across all centres or as a result

of the potential confusion and discrepancy added when more

than one antibody is employed (64% of responding centres

reported using more than one antibody in their initial

evaluation of HER2).8 Observer uncertainty and over-reliance

on the 2+ category may speak to the need for more pathology

education around the interpretation and reporting of HER2

IHC, or fewer pathologists at any one site reading and

interpreting the result. Currently, there is no accepted

“minimum” number of HER2 samples an individual

pathologist must report annually to be considered proficient.

These high rates of 2+ IHC are not without consequences,

leading to reflex evaluation of a case by ISH and, hence, added

expense and increase in the turnaround time, which in 45%

of on-site and 68% of off-site centres was greater than 10

working days. A delay in turnaround time such as this may

hinder clinical management or patient eligibility for clinical

trials.9 Other factors hindering or limiting the delivery of a

quality service for both IHC and ISH HER2 testing are fiscal

constraints and manpower restrictions.

In summary, a select review of HER2 testing across Canada

suggests good compliance with national and international

guidelines, while simultaneously pointing to areas that require

improvement and increased resource allocation.

Anita Bane, MB BCh, PhD, FRCPath(UK)Staff PathologistJuravinski Hospital and Cancer CentreMcMaster University, Hamilton, Ontario

References1. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu

proto-oncogene in human breast and ovarian cancer. Science 1989;244:707–12.

2. Goldenberg MM. Trastuzumab, a recombinant DNA-derived humanized

monoclonal antibody, a novel agent for the treatment of metastatic breast cancer.

Clin Ther 1999;21:309–18.

3. Lester SC, Bose S, Chen YY, et al. Protocol for the examination of specimens

from patients with invasive carcinoma of the breast. Arch Pathol Lab Med

2009;133:1515–38.

4. Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical

Oncology/College of American Pathologists guideline recommendations for

human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol

Lab Med 2007;131:18–43.

5. Hanna W, O’Malley FP, Barnes P, et al. Updated recommendations from the

Canadian National Consensus Meeting on HER2/neu testing in breast cancer.

Curr Oncol 2007;14:149–53.

6. Hanna WM. Evolution of HER2 testing in Canada. Can J Pathol 2013;5:8–14.

7. Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical


immunohistochemical testing of estrogen and progesterone receptors in breast

cancer. J Clin Oncol 2010;28:2784–95.

8. Bilous M, Dowsett M, Hanna W, et al. Current perspectives on HER2 testing: a

review of national testing guidelines. Mod Pathol 2003;16:173–82.

9. Bartlett JM, Starczynski J, Atkey N, et al. HER2 testing in the UK:

recommendations for breast and gastric in-situ hybridisation methods. J Clin

Pathol 2011;64:649–53.

Thank You to ReviewersTHE CJP EDITORIAL BOARD WISHES TO ACKNOWLEDGE THE FOLLOWING PATHOLOGISTS

AND SCIENTISTS WHO REVIEWED ARTICLES SUBMITTED TO THE JOURNAL IN 2012:

S. AsaA. BaneP. J. BarnesF. BrimoM. J. Bullock Y. Carber

B. CarterS. ChakrabartiT. DaleyB. DicksonL. GeldenhuysW. Hanna

M. G. JosephL. KapustaD. LeBrunR. Liwski N. MillerD. Pilavdzic

H. ReesC. RickerH. SappM. TrotterN. van der Westhuizen

L’oncoprotéine HER2 est un récepteur membranaire

appartenant à la famille des facteurs de croissance

épidermique de type tyrosine kinase; le gène qui code pour

cette protéine est amplifié dans 15 % à 20 % des cancers du

sein1. Cette amplification qui conduit à la surexpression de

la protéine HER2 est associée à un pronostic clinique

défavorable; la patiente est admissible au traitement par le

trastuzumab, dirigé précisément contre le gène en question2.

Au vu de l’importance pronostique et de l’utilité

prévisionnelle de la protéine HER2, il est désormais d’usage

d’évaluer son expression dans le cas de cancer du sein

nouvellement diagnostiqué, et cette évaluation est

recommandée en présence de métastases et après la

chimiothérapie néoadjuvante3. Étant donné l’importance de

l’analyse de l’expression de l’oncoprotéine, le College of

American Pathologists (CAP) et l’American Society of

Clinical Oncology (ASCO) ont publié ensemble des lignes

directrices sur l’exécution et l’interprétation de ce test4. La

démarche préconisée par les lignes directrices repose sur un

algorithme; elle commence par l’analyse en

immunohistochimie de la protéine HER2 et se poursuit par

l’hybridation in situ quand le résultat de l’analyse

immunohistochimique est équivoque (2+)4. Les lignes

directrices canadiennes reprennent l’essentiel des lignes

directrices américaines tout en insistant sur les normes et

méthodes s’appliquant à tous les aspects de l’analyse de

l’expression de l’oncogène HER2 et à l’assurance de la

qualité de la procédure dans les laboratoires canadiens5.

Dans le présent numéro, le Dr Hanna présente les résultats

d’un sondage pancanadien sur la conformité des

laboratoires du pays à ces lignes directrices, des résultats ma

foi instructifs6.

Le Dr Hanna, qui a conçu le sondage en ligne de concert avec

le groupe pharmaceutique F. Hoffman-La Roche et le groupe

Summit Strategy, a invité 36 pathologistes de quatre régions

du pays œuvrant en pathologie mammaire et dirigeant le

volet de la détermination du statut HER2 dans leur

laboratoire respectif à participer au sondage. Les deux tiers,

représentant 22 laboratoires, ont accepté l’invitation.

À bien des égards, les résultats sont très encourageants : 82 %

des laboratoires exécutent le test dans tous les cas de cancer

du sein invasif nouvellement diagnostiqués, le formaldéhyde

neutre tamponné est le fixatif de choix dans tous les centres,

82 % de ceux-ci se conforment au délai d’ischémie froide

recommandé (qui s’étend du prélèvement à la fixation), soit

une heure au maximum7, et la même proportion prépare le

prélèvement volumineux en vue de la période optimale

(24 à 48 heures). Dans la majorité des cas, le tissu analysé est

prélevé à l’intervention chirurgicale, la biopsie au trocart

étant réservée à la maladie localement avancée, au cas pour

lequel on envisage une chimiothérapie néoadjuvante, à la

maladie métastatique ou lorsque le prélèvement à la

chirurgie est jugé trop petit ou mal fixé en vue de l’analyse

immunohistochimique. Les laboratoires sont tous agréés

dans l’exécution des tests, et 20 traitent le volume de cas

recommandé. En outre, on note une concordance entre le

taux moyen de résultat négatif, de résultat équivoque et de

résultat positif à l’immunohistochimie et les taux publiés4.

La majorité des laboratoires réévaluent le cas au résultat

équivoque (2+) selon la méthode d’hybridation in situ. De

plus, il est réjouissant de constater que tous les laboratoires

adhèrent à un programme d’assurance de la qualité de

l’analyse de l’expression de l’oncogène HER2, provincial,

canadien ou étranger, et que le taux de concordance entre le

laboratoire et la norme de référence est de 95 % à tout le

moins.

Toutefois, les résultats ne sont pas positifs sur tous les plans,

ils font ressortir des lacunes qui devraient être comblées.

Comme 50 % des répondants sont de la même province, une

grande partie du pays est sous-représentée ou non

représentée dans le sondage, d’où le risque de distorsion

régionale des résultats. Près du cinquième (18 %) des

laboratoires interrogés ne procède pas à l’analyse de

l’expression de la protéine HER2 dans tous les cas de cancer

du sein invasif nouvellement diagnostiqués, en dépit de son

utilité pronostique et prévisionnelle avérée. Deux


Aucun conflit d’intérêts à déclarer

ÉDITORIAL

L’analyse de l’expression de l’oncogèneHER2 : la note de passage


BANE

laboratoires n’approfondissent pas l’évaluation du cas dont

le résultat immunohistochimique est 2+ ou de nature

incertaine; manifestement, cette façon de faire laisse à

désirer. Bien que les taux de résultat négatif, indéterminé et

positif concordent en moyenne avec les taux dont il est

question dans les lignes directrices mentionnées ici, en

réalité, l’écart est énorme parfois comme l’illustre ce centre

où 52 % des cas évalués à l’immunohistochimie se rangent

dans la catégorie du résultat 2+ ou indéterminé et font

l’objet de l’hybridation in situ. Un si grand taux de résultat

indéterminé laisse entrevoir des problèmes d’ordre

technique dans l’analyse immunohistochimique ou

l’incertitude de l’examinateur. Les premiers s’expliquent

peut-être par le fait que les laboratoires n’ont pas recours au

même anticorps standard ou par la confusion ou la

divergence potentielle découlant de l’utilisation de différents

anticorps (64 % des répondants s’en remettent à plus d’un

anticorps dans l’analyse initiale de l’expression de la protéine

HER2)8. L’incertitude de l’examinateur et la nette

propension à classer les résultats dans la catégorie 2+

soulignent sans doute la nécessité d’approfondir la

formation à propos de l’interprétation et de la présentation

des résultats de l’analyse immunohistochimique de

l’expression de l’oncogène HER2 ou de réserver

l’interprétation de ce test à un nombre restreint de

pathologistes du laboratoire. À l’heure actuelle, il n’y a pas

de norme quant au nombre « minimal » annuel convenu

d’analyses établissant la compétence professionnelle en la

matière. Les taux élevés de résultats 2+ ne vont pas sans

conséquence, puisqu’ils entraînent habituellement une

deuxième évaluation par hybridation in situ et de ce fait, des

dépenses supplémentaires et l’allongement du délai

d’exécution de l’analyse de l’expression de la protéine, lequel

excède les 10 jours ouvrables pour 45 % des tests effectués

sur place et 68 % des tests effectués à l’extérieur. Le délai de

cette longueur peut avoir des répercussions sur la prise en

charge clinique ou l’admissibilité de la patiente à un essai

clinique9. Enfin, il faut savoir que les compressions

budgétaires et la réduction de l’effectif sont d’autres facteurs

susceptibles d’entraver la prestation de services de qualité

dans l’analyse immunohistochimique ou par hybridation in

situ de l’expression de la protéine HER2.

En bref, le sondage sur l’analyse de l’expression de

l’oncogène HER2 au Canada révèle que la procédure se

conforme dans une bonne mesure aux lignes directrices

canadiennes et étrangères, mais il met en relief des aspects à

améliorer et pour lesquels il y aurait lieu d’accroître les

ressources.

Anita Bane, MB, BCh, PhD, FRCPath(R.-U.)Pathologiste membre du personnelHôpital et Centre de cancérologie Juravinski Université McMaster, Hamilton (Ontario)

Références 1. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-

oncogene in human breast and ovarian cancer. Science 1989;244:707–12.

2. Goldenberg MM. Trastuzumab, a recombinant DNA-derived humanized

monoclonal antibody, a novel agent for the treatment of metastatic breast

cancer. Clin Ther 1999;21:309–18.

3. Lester SC, Bose S, Chen YY, et al. Protocol for the examination of specimens

from patients with invasive carcinoma of the breast. Arch Pathol Lab Med

2009;133:1515–38.



human epidermal growth factor receptor 2 testing in breast cancer. Arch

Pathol Lab Med 2007;131:18–43.

5. Hanna W, O’Malley FP, Barnes P, et al. Updated recommendations from the


Curr Oncol 2007;14:149–53.

6. Hanna WM. Evolution of HER2 testing in Canada. Can J Pathol 2013;5:8–14.

7. Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical


immunohistochemical testing of estrogen and progesterone receptors in breast

cancer. J Clin Oncol 2010;28:2784–95.

8. Bilous M, Dowsett M, Hanna W, et al. Current perspectives on HER2 testing:

a review of national testing guidelines. Mod Pathol 2003;16:173–82.

9. Bartlett JM, Starczynski J, Atkey N, et al. HER2 testing in the UK:

recommendations for breast and gastric in-situ hybridisation methods. J Clin

Pathol 2011;64:649–53.


Evolution of HER2 Testing in Canada

Wedad M. Hanna, MD is a member of the Department of Laboratory Medicine and Pathobiology, University of Toronto, inToronto, Ontario. Correspondence may be directed to [email protected] article has been peer reviewed.Competing interests: Development of the survey and support for third-party writing assistance were funded by F. Hoffmann-La Roche Ltd. All participants received a small honorarium to compensate them for time taken to complete the survey.

Wedad M. Hanna, MD

ABSTRACTPurpose: The accuracy of HER2 testing plays a critical role in identifying patients who maybenefit from HER2-directed therapy. Canadian Consensus for HER2/neu Testing Guidelines in

Breast Cancer was published in 2007 with the aim of improving the accuracy of HER2 testing

across Canada.

Method: The authors surveyed Canadian pathologists with responsibility for HER2 testing ofbreast cancer samples to assess the impact of the Canadian consensus guidelines on current

HER2 testing procedures. Thirty-six expert pathologists were invited to participate in an online

survey between April 19 and May 28, 2010.

Results: Responses were received from 22 pathologists (61%). All but one of the respondentsperformed HER2 testing in accordance with American Society of Clinical

Oncology/CAP/Canadian consensus guidelines and used the same HER2 testing algorithm in

the adjuvant and metastatic settings. All respondents participated in a quality assurance

program, and concordance between HER2 testing results at local and central testing centres

was high (≥95%). In addition, the survey highlighted several practical considerations – including

sample collection and fixation issues – that may help to preserve, and improve upon, current

good practice.

Conclusions: Overall, survey responses indicate nationwide adherence to recommendedpractice guidelines for HER2 testing in breast cancer, resulting in excellent inter-laboratory

concordance.

RÉSUMÉ But : L’exactitude dans la détermination du statut HER2 revêt une importance primordialedans la sélection des patientes à qui offrir le traitement conçu pour s’opposer à l’oncogène. Les

groupes qui se sont alliés dans la publication en 2007 de lignes directrices consensuelles sur la

détermination du statut HER2 dans le cancer du sein avaient ainsi à cœur d’améliorer la

précision de l’analyse de l’expression de la protéine HER2 au Canada.

Méthode : Les auteurs ont sondé des pathologistes responsables de la détermination du statutHER2 afin d’évaluer l’influence de ces lignes directrices consensuelles sur les modalités d’analyse

de l’expression de l’oncogène HER2. Ils ont invité 36 pathologistes à participer à un sondage

en ligne dans la période allant du 19 avril au 28 mai 2010.

Résultats :Vingt-deux pathologistes (61 %) ont participé au sondage. Tous, à l’exception d’unseul, exécutent l’analyse de l’expression de la protéine HER2 conformément aux lignes

directrices de l’American Society of Clinical Oncology, de l’Association canadienne des

pathologistes et du consensus canadien, et ils appliquent le même algorithme d’analyse au

ORIGINAL ARTICLE


HANNA

Human epidermal growth factor 2 (HER2) is an

important prognostic and predictive biomarker in

breast cancer.1,2 Determination of HER2 status is

recommended for all patients with invasive breast cancer at

diagnosis.3 Consequently, the accuracy of HER2 testing is of

great importance in treatment decision making.4

The Canadian Consensus for HER2/neu Testing Guidelines in

Breast Cancerwas published in 2007,5 based on the experience

of a group of Canadian pathologists and on the American

Society of Clinical Oncology/College of American Pathologists

Guideline Recommendations for HER2/neu Testing in Breast

Cancer.6 The Canadian guidelines cover all aspects of HER2

testing, including pre-analytical steps (tissue handling and

fixation), assay validation, post-analytical interpretation

criteria, and training and quality assurance (QA)

requirements.5 It was anticipated that widespread adoption

of the Canadian guidelines would further improve the

accuracy of HER2 testing in Canada. It is important to note,

however, that HER2 testing policies and

funding/reimbursement criteria differ between Canadian

provinces.7

The Canadian National Breast Cancer Pathology Survey was

conducted to assess the current status of HER2 testing

practices and procedures across Canada as a follow-up to

publication of the Canadian HER2 testing guidelines. A

description of the survey and a summary of the results are

presented here.

MethodsWe identified approximately 1,200 pathologists currently

practising in Canada. From these we selected 36 pathologists,

representative of all four regions of Canada (Ontario,

Western, Quebec, and Atlantic), known to be involved in

breast cancer pathology. Selected pathologists were required

to hold responsibility for HER2 testing procedures, decisions,

and quality assurance (QA) at a specific centre, or have a lead

role in regional HER2 testing. The selected pathologists

received an invitation to participate in an online survey

between April 19 and May 28, 2010.

The content of the survey was developed by the author, with

survey questions drafted by F. Hoffmann-La Roche Ltd. and

Summit Strategy (a market research company). Prior to

distribution, the survey was reviewed by the author in order

to confirm the appropriateness and validity of the questions.

ResultsSurvey Respondents The response rate for the survey was 61% (22/36

pathologists). These respondents represented centres in the

Ontario (n = 11), Western (n = 7), Quebec (n = 3), and

Atlantic (n = 1) regions of Canada. All but two respondents

represented centres that provided a testing service for other

hospitals.

In accordance with the Canadian guidelines, the majority of

responding centres (91%) tested the required minimum of

250 samples in the 12 months preceding the survey (range:

250 to ≥2,500 samples per year).5 All other centres tested at

least 150 samples per year and fulfilled QA requirements.

Criteria for HER2 Testing at Diagnosis With the exception of patients diagnosed with ductal

carcinoma in situ – who would not normally be tested for

moment du traitement adjuvant et en présence de métastases. Ils adhèrent tous à un programme

d’assurance de la qualité et le taux de concordance entre les résultats de l’analyse de

l’établissement et la norme du laboratoire local ou central est élevé (≥ 95 %). En outre, le

sondage met en évidence plusieurs aspects techniques – qu’il s’agisse du mode de prélèvement

ou de la méthode de fixation – d’importance dans le maintien, voire l’amélioration, de la

pratique actuelle.

Conclusion : Somme toute, les résultats du sondage révèlent que les modalités de déterminationdu statut HER2 sont conformes aux lignes directrices sur ce sujet, qu’il y a donc une excellente

uniformité entre les laboratoires à ce chapitre.


EVOLUTION OF HER2 TESTING IN CANADA

HER2 status – most respondents (82%) tested the HER2

status of all breast tumours at the time of diagnosis. Samples

were excluded only if microinvasive carcinoma was present,

the patient was not eligible for adjuvant therapy, the clinician

did not request HER2 testing, or the specimen was poorly

fixed.

Are Core Needle Biopsies Used for HER2 Testing?Most centres (82%; 18/22) carried out HER2 testing using

core needle biopsies (CNBs) in addition to surgical excision

samples. The most commonly cited reasons for testing CNBs

included the patient being considered for neoadjuvant

therapy (56%; 10/18), the presence of metastases (22%; 4/18),

and the diagnosis of locally advanced breast cancer (17%;

3/18). Surgical specimens of insufficient size, inferior quality,

or with poor fixation also led to CNB testing. In the absence

of a CNB, 23% of respondents used a fine-needle aspirate

(FNA) for testing. For patients with metastatic disease, 59%

of respondents often or always used an archived tumour block

for testing in cases where a HER2 test result was not available

for the primary tumour. However, where an archived block

was unavailable, most respondents (59%) obtained a biopsy,

providing that the metastases were accessible. Other major

factors limiting HER2 testing of metastases were small size of

the biopsy, a compromised biopsy, and requirement for

decalcification of bone tissue.

Sample Collection and FixationFollowing surgical excision, 68% of respondents reported that

the breast specimens were sliced and fixed within 1 hour,

while 23% took between 1 and 2 hours (Figure 1).

All respondents used 10% neutral buffered formalin as a

fixative in the laboratory. Most respondents (82%) fixed large

excision specimens for 24–48 hours, with only 9% fixing

excision specimens for less than 24 hours prior to tissue

processing (Figure 2). As expected, the average fixation time

for CNBs was shorter: most respondents (74%) reported a

fixation time of at least 8 hours for CNBs, while 14% specified

a minimum fixation time of 24 hours.

Preferred Primary HER2 Testing Methodologies Immunohistochemistry (IHC) was used as the initial HER2

testing methodology by 91% (20/22) of respondents. Others

Figure 1. Average time from surgery to sample fixation, whensurgery takes place on-site.

A

B

Figure 2. Average fixation times for excised samples (A) and coreneedle biopsies (B).


HANNA

used silver-enhanced in situ hybridization

(SISH) (5%; 1/22) or another, unspecified

method (5%; 1/22). Most IHC testing was

automated (86%; 19/22), with only 14% (3/22)

favouring manual or mixed techniques.

Half of all respondents used the rabbit

monoclonal 4B5 HER2 antibody as their

primary antibody (Figure 3). A secondary

antibody was used by 64% (14/22) of

respondents; the most widely preferred were the

SP3 monoclonal antibody (NeoMarkers) and the

AO485 polyclonal antibody (DAKO), each used

by 43% (6/14) of respondents.

Confirmatory (Reflex) Testing Methodologies Following an IHC 2+ Test ResultIn the 20 centres that used IHC as their primary

testing methodology, samples scored as IHC 2+

were retested using fluorescence in situ

hybridization (FISH) (75%), SISH (20%), or

chromogenic in situ hybridization (CISH) (5%).

Automated ISH testing was used by 45% of

respondents, with others using a range of

manual and semi-automated procedures.

Respondents who did not perform confirmatory

testing for IHC 2+ samples cited sample

availability and funding restrictions as limiting

factors (Figure 4).

After FISH testing, 55% (12/22) of respondents

retested samples with an equivocal FISH ratio

(1.8–2.2), and 23% of respondents (5/22)

performed confirmatory testing for samples with

a HER2/chromosome 17 ratio of 2.2 exactly.

HER2 Status in Canadian Patients with Breast CancerOverall, the average proportion of IHC 0/1+,

IHC 2+, and IHC 3+ test results was 65% (range

38–80%), 23% (range: 2–52%), and 12% (range:

3–20%), respectively (Figure 5). When ISH

retesting of the IHC 2+ samples was taken into

account, the average overall HER2-positivity rate

(IHC 3+ and IHC 2+/ISH-positive) was 17.6%.

Figure 3. Preferred HER2 primary antibody used for immunohistochemistry testing.(Note: Some respondents selected more than one antibody.)

Figure 4. Identified restrictions to performing confirmatory HER2 tests. (Note:Respondents were asked to indicate all that apply.)

Figure 5. Stratification of initial HER2 test results.

(n=4)

A B C

Application of HER2 Testing Algorithm in Patients with Earlyand Metastatic Breast CancersAll but one of the respondents reported performing HER2

testing in accordance with American Society of Clinical

Oncology (ASCO)/College of American Pathologists

(CAP)/Canadian guidelines and used the same HER2 testing

algorithm in the adjuvant and metastatic settings.

Subsequent Retesting of HER2 StatusApproximately two thirds of respondents retested HER2

status upon completion of neoadjuvant treatment. The

majority of those who did not retest specified that this was

not requested by the clinician/oncologist. All centres retested

the HER2 status of patients who developed metastases

following adjuvant therapy for HER2-negative disease. The

rate of change in HER2 status between the adjuvant and

metastatic settings was reported to be 1–4% by most

pathologists (Figure 6).

Turnaround TimeIHC test results were provided within 3–5 working days in 55%

of on-site testing centres, with results available within 24 hours

at one centre. In a further 27% of on-site centres, IHC results

were available within 6–10 working days, with 14% reporting

an average turnaround time of more than 10 working days.

The minimum turnaround time for IHC HER2 tests

performed at off-site testing facilities (32% of respondents)

was 5–7 working days. Most (59%) off-site testing centres

provided results within 5–10 working days of surgery, but 23%

reported an average turnaround of more than 10 working

days.

Most centres (68%) performed only one batch of ISH tests per

week. Consequently, turnaround times for ISH-based HER2

testing were comparatively slower than for IHC testing: only

9% of on-site testing facilities and 5% of off-site testing centres

provided ISH test results within 5–7 working days of surgery.

An average turnaround time of more than 10 working days

was reported for 45% of on-site centres and 68% of off-site

centres.

Internal and External Monitoring of Testing Quality A variety of QA programs are in place across Canada, and all

respondents participated in a QA program. While national and

international programs were most popular in Western Canada,

Quebec, and Atlantic Canada, pathologists in Ontario favoured

the provincial program (Table 1).

Reported concordance rates for HER2-positivity and

negativity were ≥95% for all centres (local versus central

testing). The most commonly reported challenges to the

improvement of HER2 testing quality were a lack of

funding/resources and a lack of time/manpower (Table 2).



A

B

Figure 6. Proportion of patients in whom HER2 status changedbetween the adjuvant and metastatic settings.


HANNA

DiscussionOur survey of pathologists responsible for HER2 testing in

breast cancer indicates a high level of compliance with

recommended Canadian practice guidelines for HER2 testing.

HER2 testing was performed in accordance with

ASCO/CAP/Canadian consensus guidelines by all but one of

the 22 respondents, and a consistent HER2 testing algorithm

was applied in the adjuvant and metastatic settings. In

addition, high concordance between local and central HER2

testing centres in external QA programs indicate that, overall,

HER2 testing performed by Canadian centres appears

accurate and reliable.

In accordance with Canadian guidelines, most respondents

processed more than the recommended minimum of 250

samples during the preceding 12 months. However, some

centres report performing in excess of 2,500 tests per year, and

it is important that such centres have sufficient resources to

manage this demand for HER2 testing in order to preserve

test quality. In the majority of Canadian centres, HER2 status

is routinely tested at the time of diagnosis.

Our results suggest that pathologists are concerned about the

potential impact of delayed and inadequate tissue fixation on

the accuracy of HER2 testing. Respondents commented that

surgeons should be educated regarding the importance of

prompt receipt of large specimens in the histopathology

laboratory, following excision from patients, to allow for

appropriate handling and immediate fixation. In addition,

while a fixation time of 6–48 hours is recommended for both

excisions and CNBs,5,6 there appears to be no clear consensus

regarding the optimum fixation time for CNBs.

Automated IHC testing is the initial methodology in the

majority of Canadian centres. Conversely, about half of ISH

testing is performed manually. Confirmatory testing is

generally performed in accordance with HER2 testing

guidelines (i.e., samples with equivocal HER2 status either by

IHC or FISH). However, a small proportion of pathologists

identified restrictions, other than sample availability, to

confirmatory retesting in their laboratory. Confirmatory

retesting for equivocal cases is essential, and use of reference

centres is a clear choice where confirmatory retesting cannot

be performed on-site.

The accuracy and sensitivity of IHC testing can be affected by

pre-analytical and analytical procedures, as well as by

Table 1. Percentage of Respondents Participating in Various QA Programs

Percentage of RespondentsQA Program* All (n = 22) West (n = 7) Ontario (n = 11) Quebec (n = 3) Atlantic (n = 1)

National 68 100 36 100 100Provincial 59 29 91 33 –College of American Pathologists 59 71 36 100 100Local 45 43 55 33 –Nordic QC 27 43 18 – 100UKNEQAS 14 29 9 – –Others 13 – 18 33 –*Multiple responses accepted.QA = quality assurance.

Table 2. Top Challenges Faced by Pathologists in Terms ofQA Progress in HER2 Testing

Most Frequently Mentioned Challenges Percentage (n = 21)Lack of funding/budgets/resources 48Lack of time/manpower/HR 45Consistency of pre-analytical issues 25Pathology subjectivity/opinions/interpretation 20Quality/reliability of antibodies 19Time/limited time to develop QAmonitoring 14Ongoing assessment and competency 14Tumour heterogeneity 10Amplification of HER2 and CEP17 10External QC/ensuring external testing has QC in place 10Variable fixation rates 10No authority/support to implement changes 10Others (miscellaneous mentions) 30HR = human resources; QA = quality assurance; QC = quality control.



antibody sensitivity. Results should always be issued based on

an approved antibody. In the event of discordance between

antibodies, reflex testing should be performed using ISH. One

possible area of concern is the variability in specimen-

handling procedures and testing methodologies, which could

account for inter-laboratory discrepancies.

Stratification of initial IHC HER2 testing results revealed

substantial variation between centres in the number of

samples graded as IHC 0/1+ (38–80%), IHC 2+ (2–52%), and

IHC 3+ (3–20%). It seems unlikely that this variability reflects

true regional variations in HER2 status. Therefore, we may

assume that these results highlight technical difficulties in

some testing centres. Certainly, some centres reported relying

heavily on reflex testing for result confirmation.

Improvements in training and standardization of testing

methodologies may help to reduce the need for reflex testing.

All respondents to the survey retested HER2 status in patients

with metastases, an observation that reflects the importance

of high-quality HER2 testing in treatment decision making.

While it is unusual for HER2 status to change during disease

progression, this possibility should not be discounted. The

majority of respondents retested HER2 status in patients who

had undergone neoadjuvant therapy, although this is not a

requirement of the Canadian or ASCO/CAP guidelines. Such

retesting is important to further our understanding of the

clinical impact of HER2-directed therapy.

Over half of on-site testing centres were able to obtain HER2

test results within 3–5 working days. However, around a

quarter of IHC tests conducted at off-site centres took more

than 10 working days. Clearly, improvements in turnaround

time are desirable, particularly for off-site testing centres, as

longer turnaround times may have a clinical impact for

patients awaiting treatment.

In conclusion, accurate and standardized assessment of HER2

status is essential to identify patients who may benefit from

HER2-directed therapy. Overall, survey responses indicate

nationwide adherence to recommended practice guidelines

for HER2 testing in breast cancer, resulting in excellent inter-

laboratory concordance.

AcknowledgementsThe author would like to thank Dr. Penny Barnes, Dr. Richard

Berendt, Dr. Martin Chang, Dr. Anthony Magliocco, Dr.

Naomi Miller, and Dr. Henrike Rees for their invaluable input

during the development of the manuscript. In addition, Dr.

Hanna would like to thank Summit Strategy and F.

Hoffmann-La Roche Ltd. (Lisa Cesario and Laura Shields) for

their assistance in preparing the survey.

References1. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of

relapse and survival with amplification of the HER-2/neu oncogene. Science

1987;235:177–82.

2. Hynes NE. Amplification and overexpression of the erbB-2 gene in human

tumors: its involvement in tumor development, significance as a prognostic

factor, and potential as a target for cancer therapy. Semin Cancer Biol 1993;4:19–

26.

3. National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines

in Oncology (NCCN Guidelines™) Version 2.2011. Fort Washington (PA): The

Network, 2001; available at www.nccn.org. Accessed February 8, 2011.

4. O’Malley FP, Thomson T, Julian J, et al. HER2 testing in a population-based

study of patients with metastatic breast cancer treated with trastuzumab. Arch

Pathol Lab Med 2008;132:61–5.

5. Hanna W, O’Malley FP, Barnes B, et al. Updated recommendations from the


Curr Oncol 2007;14:149–53.

6. Wolff AC, Hammond MEH, Schwartz JN, et al. American Society of Clinical


human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol

2007;25:118–45.

7. Ragaz J. Research saves lives: can we get better value for our investment? Cancer

Care Can 2006–07:31–4; www.canceradvocacy.ca. Accessed February 8, 2011


False-Positive FISH for HER2 Amplification in a Breast Cancer Owing to Loss of CEP17:

A Case Report and Literature Review

Jie Xu, PhD, FCCMG, is with the Cytogenetics Laboratory, Victoria Hospital, London Health Sciences Centre and WesternUniversity, in London, Ontario. Helen Ettler, MD, FRCPC, is a member of the Department of Pathology, London Health Sci-ences Centre and Western University. Peter Engbers, MD, FRCPC, is a member of the Department of Pathology, WoodstockGeneral Hospital, in Woodstock, Ontario. Correspondence may be directed to [email protected] article has been peer reviewed.Competing interests: None declared

Jie Xu, PhD, FCCMG, Helen Ettler, MD, FRCPC, Peter Engbers, MD, FRCPC

ABSTRACTThe authors present a case of breast cancer with equivocal status for HER2 protein

overexpression by initial immunohistochemistry (IHC). Dual-colour fluorescence in situ

hybridization (FISH) of paraffin sections of the tumour showed a HER2/CEP17 ratio of 2.6:1,

indicative of HER2 gene amplification. However, this was interpreted as a false-positive test due

to a loss of CEP17 in 92% of the cells; this was supported by a negative result on repeat IHC

testing. Monosomy 17, as indicated by single CEP17, has been reported in 1–6% of patients

with breast cancer and can result in false-positive result on FISH. There have been no national

guidelines on how to manage and interpret such findings. The literature suggests that when a

possible false-positive result on FISH for HER2 amplification due to a loss of CEP17 is indicated,

a confirmation by repeat IHC testing, single-colour FISH, or array comparative genomic

hybridization (CGH) can help one to make an accurate interpretation and a final decision on

whether the patient is eligible for treatment with trastuzumab. More case reports and large

clinical studies would help to correlate FISH with clinicopathological findings and to determine

how patients with breast cancer with single CEP17, with or without HER2 gene amplification,

respond to treatment with trastuzumab or other drugs.

RÉSUMÉ Les auteurs présentent un cas de cancer du sein pour lequel l’analyse immunohistochimique

initiale ne parvient pas à se prononcer quant à la surexpression de la protéine HER2.

L’hybridation in situ en fluorescence à deux couleurs de la tumeur en coupes sur paraffine

illustre un rapport HER2 par centromère 17 (CEP17) de 2,6 pour 1, qui laisse entrevoir

l’amplification du gène HER2. Cependant, le résultat est interprété comme étant faussement

positif en raison de la perte de CEP17 dans 92 % des cellules. Cette interprétation se trouve

corroborée par le résultat négatif de l’analyse immunohistochimique subséquente. La

monosomie 17, soit la présence d’un seul CEP17, est le lot de 1 % à 6 % des cas de cancer du

sein; cet état peut entraîner un résultat faux positif à l’hybridation. Il n’y a pas de lignes

directrices applicables à l’échelle du pays qui balisent l’interprétation de cette constatation, ni

la conduite à tenir par la suite. La documentation propose de faire suivre l’hybridation in situ

ORIGINAL ARTICLE


FALSE-POSITIVE FISH FOR HER2 AMPLIFICATION IN A BREAST CANCER OWING TO LOSS OF CEP17

Amplification and overexpression of human epidermal

growth factor receptor 2 gene (HER2) occur in

approximately 18–20% of human breast cancers and indicate

a poor prognosis. Accurate assessment of HER2 status in

breast cancer patients is critical for decision making in

targeted therapy with trastuzumab (Herceptin, Genentech,

Inc., South San Francisco, California; Hoffmann-La, Roche

Ltd., Basel, Switzerland) and lapatinib (Tykerb,

GlaxoSmithKline, Philadelphia, Pennsylvania). Dual-colour

fluorescence in situ hybridization (FISH) using a probe for

HER2 (located at 17p11.2) and a control probe for

centromere 17 (D17Z1/CEP17) is now a standard test, and

HER2 amplification is indicated if the HER2/CEP17 signal

ratio is ≥2.00, as recommended by the US Food and Drug

Administration (FDA),1,2 or >2.2, according to the Guidelines

of the American Society of Clinical Oncology and College of

American Pathologists, (ASCO/CAP).3 Among the factors

affecting interpretation of HER2 status is the finding of a

single CEP17 signal. This is commonly referred to as

monosomy 17 and can lead to artificial skewing of the

HER2/CEP17 ratio and, thus, a false-positive result for HER2

gene amplification.4–6 There have been no national guidelines

on how to manage such cases. More case studies would help

to increase the understanding of mechanisms for loss of

CEP17, establish appropriate test algorithms, and correlate

FISH results with clinicopathological findings. Here we

present a case with a false-positive FISH result, and a review

of the literature.

Case ReportA 57-year-old woman was diagnosed with invasive mammary

carcinoma and underwent mastectomy with sentinel lymph

node biopsy. Grossly there was a firm 1.4 × 1.3 cm grey-white

subareolar mass with no connection to the nipple or skin. No

other abnormality was noted, and there were a total of six

lymph nodes, including two separately submitted sentinel

nodes. Sections were submitted after 24 hours’ fixation in

neutral buffered formalin. Microscopically the tumour was

an invasive mammary carcinoma with lobular features. The

combined histologic grade (Scarff-Bloom-Richardson) was

II/III (score 6/9: nuclear pleomorphism 2, tubule formation

3, and mitotic score 1 with a mitotic count of <5/10 high-

power fields [HPFs]).

Immunohistochemical analysis for HER2/neu protein

overexpression was performed on slides prepared from

paraffin-embedded sections of the tumour. In our laboratory,

two antibodies are used routinely to ensure appropriate

sensitivity and specificity. Initial tests showed no reaction with

antibody A0485 (rabbit polyclonal antibody, Dako, at a dilution

of 1:500) and equivocal (2+) reactivity for antibody CB11

(mouse monoclonal antibody, Vector Laboratories, at a dilution

of 1:500) with complete membrane staining of weak to

moderate intensity in 20% of the invasive carcinoma. Overall,

HER2 protein overexpression was interpreted to be equivocal.

Interphase FISH using Abbott Path HER2 kit with dual probes

for HER2 and CEP17 was also conducted on paraffin sections

with the target area marked by the pathologist. A total of 90

cells were scored by three cytogenetics technologists and the

results were interpreted by a certified cytogeneticist (J. X.) and

included in the final pathology report.

ResultsOnly one CEP17 signal was seen in 92% (83/90) of the cells

(Figure 1A), and 60% of the cells had 1 CEP17 and 3 HER2

signals. A small number of cells (6%) had 4 CEP17 signals,

but none had >4 CEP17 signals.

en fluorescence ayant abouti à un résultat faux positif en raison de la perte d’un CEP17 d’une

autre analyse immunohistochimique, de l’hybridation in situ en fluorescence à une couleur ou

de l’hybridation génomique comparative afin d’interpréter avec exactitude le résultat quant à

l’amplification de HER2 et de déterminer l’admissibilité au traitement par le trastuzumab. Des

exposés de cas et de vastes études cliniques seraient certes utiles dans l’évaluation de la

corrélation entre les résultats de l’hybridation in situ en fluorescence et les constatations

clinicopathologiques, et de la réponse thérapeutique des patientes ayant un seul CEP17 au

trastuzumab ou à d’autres médicaments, sans égard à l’amplification du gène HER2.


XU ET AL.

The FISH score showed a HER2/CEP17 ratio of 2.6, indicative

of HER2 gene amplification according to the criteria of the

FDA (≥2.00)1,2 or ASCO/CAP (>2.2).3 However, this was

interpreted as likely a false-positive caused by loss of the

centromeric signal in the vast majority of the cells and

skewing of the HER2/CEP17 ratio.

Routine G-banding and FISH of 10 metaphase cells and 200

interphase cells of peripheral blood of the patient showed a

normal constitutional female karyotype (46,XX) and, more

specifically, a normal HER2 and CEP17 pattern with the 2

CEP17 signals being of approximately the same size (see

Figure 1B–D). This confirmed that the loss of the CEP 17 seen

in the breast carcinoma was an acquired aberration.

In light of the FISH finding in the breast cancer, repeat IHC

was performed using antibody A0485 and a third antibody

SP3 (rabbit monoclonal antibody, Thermo Scientific, dilution

1:100) because the performance of

antibody CB11 was considered

suboptimal. The result was clearly

negative for overall HER2 protein

overexpression: 1+ for both SP3 and

A0485 with complete membrane staining

of weak to moderate intensity in <5% of

the invasive carcinoma. This negative

result was consistent with the

interpretation of the FISH test as a false-

positive.

DiscussionIt is difficult to obtain an accurate

estimate of the prevalence of monosomy

17 (indicated by single CEP17) in breast

cancer patients because reports vary in

patient selection criteria, number of cells

scored, testing methods, sample size, and

even definition of monosomy 17. For

example, in one study,4 monosomy 17

was based on 1.11 CEP17 signals per cell,

but 1 copy of CEP17 in >60% of nuclei

of invasive cells was required in another.7

Nevertheless, reported rates of

monosomy 17 from large studies range

from ~1% (2/189)4 to ~5% (56/1,170)8 to

~6% (102/1,783).7 Another term, “hypodisomy 17,” is widely

used for cases with ≤2 centromere 17s, including monosomy

17, and is defined as ≤1.754,8 or <1.59 CEP17 signals per cell.

Hypodisomy is reported in 6% (5/89)4 to 22.7% (265/1,170)9

of breast cancer patients. Monosomy and/or hypodisomy 17

can result in skewing of a HER2/CEP17 ratio and a false-

positive FISH result for HER2 amplification.

How does one determine a FISH test to be a false-positive?

Table 1 shows several reported cases that had HER2/CEP17

ratios of ≥2.00 or >2.2 but were interpreted as false-positive

based on negative findings by IHC and/or array comparative

genomic hybridization (CGH), or ≤2 HER2 signals/cell by

dual-colour FISH, or <4 HER2 signals/cell by single-colour

FISH (INFORM HER2/neu probe; Ventana). For example,

Wang et al.4 reported 2/189 cases with monosomy 17 (with

1.11 and 1.03 CEP17 signals per cell) but no HER2 protein

A

B C D Figure 1. A, Interphase fluorescence in situ hybridization (FISH) of paraffin sections of thebreast carcinoma showing 1 green/CEP17 and 1–3 orange/HER2 signals in the cells. B–D,FISH of the peripheral blood showing 2 green/CEP 17 and 2 orange/HER2 signals of equalsize on the metaphase chromosome 17s (B) and in interphase cells (C and D).

B C D


FALSE-POSITIVE FISH FOR HER2 AMPLIFICATION IN A BREAST CANCER OWING TO LOSS OF CEP17

overexpression with scores of 1.8 and 1.9. Both cases had

HER2 amplification by dual-colour FISH (with HER2/CEP17

ratios of 2.98 and 2.02) but no amplification by single-colour

FISH (HER2 copy number 3.29 and 2.08 signals per cell).

Corzo et al.10 reported a case with a HER2/CEP17 ratio of 2

resulting from 2 copies of HER2 and 1 copy of CEP17. This

was not considered as amplification because of monosomy

17; the interpretation was supported by negative IHC.

Recently Gunn et al.6 reported two false-positive cases: one

case had a HER2/CEP17 of 2.86 but IHC of 1+, and another

with HER2/CEP17 ratio of 2.7 and monosomy 17. In light of

the questionable HER2 status by FISH and/or IHC, array

CGH using probes for HER2 and the pericentric regions and

123 additional loci covering chromosome 17 were performed.

Array CGH identified that both cases had loss of the complete

p arm but no loss of 17q.

Studies by Gunn et al.6 demonstrate that array CGH has

advantages over other methods in determining if a finding of

a single CEP 17 signal is the result of loss of the centromeric

probe, a chromosome segment with the probe, or the entire

chromosome (monosomy 17). Therefore, CGH can be

potentially used to classify so-called monosomy 17 patients

into at least 3 cytogenomic subgroups, which will permit

study of the clinical implications of each subgroup. It is

possible, although rare, that breast cancer patients may have

a constitutional reduction of centromeric copy number for

D17Z1. In addition, the presence of constitutional mosaic

markers involving centromere 17 has been well documented.11

Such constitutional cytogenomic rearrangements could

contribute to a finding of aberrant interphase FISH patterns,

including single CEP 17, in breast cancer cases. Therefore, for

cases with all or the vast majority of interphase cells with a

single CEP17, karyotyping and FISH of peripheral blood can

be used to rule out the possibility of a constitutional basis.

Some monosomy 17 cases are interpreted as HER2-positive.

For example, Perez et al.7 reported that 100 of the 1,783 (6%)

cases with monosomy 17 had FISH ≥2 or IHC 3+ and were

interpreted as HER2-positive. Risio et al.12 suggested that

HER2 amplified breast cancers with monosomy 17 were

poorly responsive to trastuzumab-based treatment. The

response rate was 53% in the patients with monosomy 17 in

comparison with 92% in those with >2 centromeric signals

(chromosome 17 eusomy/polysomy). This finding raises an

interesting question as to whether monosomy 17 can serve as

a biomarker for a subclassification of patients for diagnosis

and treatment.

In conclusion, when dual-colour FISH indicates a possible

false-positive result for HER2 amplification owing to loss of

CEP17, confirmation by repeat IHC, array CGH, or single-

colour FISH and consultation with the pathologists should

be considered. This would help the interpretation of the

HER2 status and a final decision on whether or not the patient

is eligible for treatment with trastuzumab and lapatinib. Large

clinical studies would help correlate the FISH results and

clinicopathological findings and establish how breast cancer

patients with single CEP17, with or without HER2

amplification, respond to treatment with trastuzumab or

other drugs.

AcknowledgementsWe thank the staff of the Cytogenetics Laboratory, London

Health Sciences Centre, for technical assistance. The

Table 1. Reported Cases of False-Positive FISH Results from Loss of CEP17

FISHReference Dual-Colour, HER2/CEP17 Single-Colour, HER2/cell IHC aCGHWang et al.4

Case 1 2.98 (1.11 CEP17/cell) 3.29 HER2/cell ACIS 1.8Case 2 2.2 (1.03 CEP17/cell) 2.08 HER2/cell ACIS 1.9Corzo et al.10 2.00 NegGunn et al.6

Case EQ16 2.86 1+ Neg (FIR 0.97)Case EQ17 2.7 Neg (FIR 1.15)Present case 2.6 (1CEP17 in 93% cells) 1+aCGH = array comparative genome hybridization; ACIS = an image analyzer (ChromaVision Medical System, Inc. San Juan Capistrano, California) – assisted IHC quantitation;CEP17 = centromere 17 sequence D17Z1; FIR = fluorescence intensity ratio; IHC = immunohistochemistry; Neg = negative.


XU ET AL.

molecular cytogenetics infrastructure used in this study was

funded by the Canadian Foundation for Innovation through

the Canadian Molecular Cytogenetics Platform.

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with equivocal HER2 status resolves gene copy number and reveals chromosome

17 complexity. BMC Cancer 2010;10:396.

7. Perez EA, Reinholz MM, Hillman DW, et al. HER2 and chromosome 17 effect

on patient outcome in the n9831 adjuvant trastuzumab trial. J Clin Oncol

2010;28(28):4307–15.

8. Zeng X, Liang ZY, Wu SF, et al. HER2 status in breast cancer of Chinese women:

a study of 1170 cases using fluorescence in-situ hybridization. Zhonghua Bing

Li Xue Za Zhi 2008;37:594–8.

9. Dal Lago L, Durbecq V, Desmedt C, et al. Correction for chromosome-17 is

critical for the determination of true Her-2/neu gene amplification status in

breast cancer. Mol Cancer Ther 2006;5:2572–9.

10. Corzo C, Tusquets I, Suarez M, et al. Intratumoral heterogeneity of HER2/neu

and topoisomerase IIalpha in breast cancer: a case with clonal monosomy 17.

Cancer Genet Cytogenet 2004;154:89–90.

11. Lieher T. FISH. Jena, Germany: University of Jena; http://www.med.uni-

jena.de/fish/sSMC/17.htm#Start17.

12. Risio M, Casorzo L, Redana S, Montemurro F. HER2 gene-amplified breast

cancers with monosomy of chromosome 17 are poorly responsive to

trastuzumab-based treatment. Oncol Rep 2005;13:305–9.

On December 31, 2012, two members of the Editorial Board,

Dr. Louis Wadsworth, section editor for hematopathology,

and Dr. Eleftherios Diamandis, section editor for medical

biochemistry, stepped down. I would like to thank them for

their support over the past 4 years as we have worked to

establish the Canadian Journal of Pathology. This is an

opportunity to welcome their replacements on the Editorial

Board.

Dr. Lawrence Haley, MD, FRCPC, is head of pathology and

laboratory medicine at the Royal Columbian Hospital and

head of hematopathology for the Fraser Health Authority in

British Columbia, where he is also a clinical associate professor

in the Department of Pathology at the University of British

Columbia. His principal professional interests are malignant

hematopathology, transfusion medicine, and teaching.

Dr. Pierre Douville, MD, FRCPC, is a senior medical

biochemist at the Centre hospitalier universitaire de Quebec

and a professor at Laval University. He serves on the Executive

of the Canadian Association of Medical Biochemists and on

the Education Committee of the Canadian Society of

Atherosclerosis, Thrombosis and Vascular Biology. His

professional interests lie in renal disease, dyslipidemias, and

cancer biomarkers.

I look forward to working with them to promote the

Canadian Journal of Pathology as a vehicle for the

dissemination of advances in pathology, laboratory medicine,

and science in Canada.

J. Godfrey HeathcoteEditor-in-Chief

Changes to the Editorial Board


Pathology in the 1930s and 1940s: A Poetic Critique by “the PEI–Dalhousie–Mayo

Clinic Connection”: Drs. Malcolm B. Dockerty and Lewis B. Woolner

James R. Wright Jr., MD, PhD, is professor and head of the Department of Pathology and Laboratory Medicine at the Universityof Calgary/Alberta Health Services Diagnostic and Scientific Centre, in Calgary, Alberta. Correspondence may be directedto [email protected] article was peer reviewed.Competing interests: None declared

James R. Wright Jr., MD, PhD

ABSTRACTMalcolm B. Dockerty and Lewis B. Woolner were Prince Edward Islanders who were raised on

potato farms during the Great Depression. From humble beginnings, they worked their way

through Dalhousie Medical School; both became world-renowned surgical pathologists,

spending their entire careers at the Mayo Clinic. Both were raised loving poetry. One also wrote

his own verse throughout his life and used his poetry as his memoir. Dockerty and Woolner,

based upon their experiences in medical school compared with those while in practice at the

Mayo Clinic, were highly critical of the state of pathology at Dalhousie in the 1930s and 1940s.

Comparing pathology practice at Dalhousie and the Mayo Clinic during that time perhaps

seems unfair. However, times have changed, and all pathology laboratories are now expected to

meet uniform, high standards. It is useful to reflect upon how far we have come toward closing

the quality gap with the very best laboratories in the world.

RÉSUMÉ Natifs de l’Île-du-Prince-Édouard, Malcolm B. Dockerty et Lewis B. Woolner ont grandi dans

une ferme de pommes de terre durant la Crise de 1929. Des origines modestes qui ne les ont

pas empêchés de poursuivre des études à la faculté de médecine de l’Université Dalhousie et de

devenir des pathologistes qui allaient acquérir une renommée mondiale en pathologie

chirurgicale. Toute leur carrière s’est déroulée à la clinique Mayo. La poésie les berce depuis leur

enfance. L’un d’eux en a même écrit tout au long de sa vie, des poèmes qui forment un recueil

de mémoires. Comparant leur formation médicale et celle de leurs collègues à la clinique Mayo,

l’un comme l’autre ont eu des mots très durs à propos de la discipline de la pathologie à

l’Université Dalhousie dans les années 1930 et 1940. Qui sait, peut-être était-il injuste de

comparer l’exercice de la pathologie dans ces deux institutions à cette époque? Toujours est-il

que les temps ont changé et que tous les laboratoires de pathologie doivent désormais se

conformer aux mêmes normes de qualité strictes. L’histoire de ces deux éminents pathologistes

nous permet de constater le chemin parcouru par les laboratoires de pathologie du pays qui se

mesurent favorablement aux meilleurs du monde.

ORIGINAL ARTICLE

Two of the pre-eminent “American” pathologists of the

20th century, both spending their entire careers at the

Mayo Clinic, were Prince Edward Islanders who were raised

in poverty on potato farms. They both attended one-room

country schools followed by Prince of Wales College in

Charlottetown and Dalhousie Medical School (Figure 1).

Both became world-renowned surgical pathologists,

publishing over 750 peer-reviewed papers and book chapters

between them. Each trained literally hundreds of Mayo

residents and fellows in pathology and surgery.1 Both had

strong interests in poetry and could quote the classics at

length from memory. One also wrote his own verse

throughout his entire life and even used his poetry as his

memoir2; the other loved the classics and called his colleague

“a (minor) poet.” In their retirement years, both received

honorary doctorates from Dalhousie. In a future paper, I will

present historical vignettes of Malcolm B. Dockerty and

Lewis B. Woolner, since, except for a brief mention in a 1964

CMAJ article titled “The Mayo Clinic and the Canadians,”3

little has been written about them. However, here I report

their independent analyses of the state of pathology practice

at Dalhousie in the 1930s and 1940s.

I first met Lew Woolner after I nominated him for an

honorary doctorate in 2001 while I was a faculty member at

Dalhousie. We met again at his home in Rochester,

Minnesota, in 2010 while I was conducting research on

Mayo pathologist Albert C. Broders.4 Lew, at 96 years old,

was the only living Mayo pathologist whose practice

overlapped with that of Broders. At one point in the

interview, he spontaneously volunteered his thoughts on the

state of the practice of pathology when he was at Dalhousie.

I did not have the same opportunity to know Dockerty, as

he died in 1987. To become better acquainted, I spent a day

in the Mayo Historical Unit reviewing his file and reading

all of his poetry; then I interviewed his friends, colleagues,

and son. In Dockerty’s Rhymed Reminiscences of a

Pathologist: His Life’s Story,2 there is a section called “Medical

School Days.” Interestingly, his descriptions of pathology at

Dalhousie confirmed everything Woolner had told me.

Suffice it to say, neither Maritimer seriously considered a

career in pathology at Dalhousie. Both agreed that pathology

at the time each had graduated from medical school was

very weak. Ralph Paterson Smith (MD, Glasgow, and

Diploma of Public Health, Edinburgh), was the chair of

pathology from 19275 to 1949 (see Figure 1) and his

pathology laboratory at the Victoria General Hospital was

hugely problematic, as Dockerty documented in the poem

“Our Lab (V.G. Hospital – 1934),” written while a medical

student2:

Our lab is in a sorry mess.

Mahaney is to blame, I guess;

Each day he’s doing less and less

To keep the place supplied.

The centrifuge is out of true,

The Bunsen burner needs a screw,

The Benedict’s is nearly through,

There’s not one single slide!

The desk is slithered o’er with grime,

The floor is slippery with slime,

Test tubes are hard as H to find,

The litmus paper’s done.

Bottles in wild confusion thrown

Assume an order all their own.


WRIGHT

Figure 1. Dalhousie graduation photos of Drs.Malcolm B. Dockerty, left, and Lewis B. Woolner,right, as well as a photograph of Dr. Ralph PatersonSmith, centre, Dalhousie professor of pathology(courtesy of Dr. Allan Marble).


PATHOLOGY IN THE 1930s AND 1940s

Pipettes and graduates have flown;

The water tap won’t run.

Mel McIntosh is soon to find

The place is fifty years behind.

I hope the beggar turns his mind

To bring it up to date,

And that the present government

With ample funds will circumvent

The grim expense of the event

And clean the dirty slate.

Dockerty’s poem was prefaced in his book by, “Had I not

known in 1934 that Mayo’s Laboratories were a far cry from

the Victoria General’s counterpart, operated by interns, I

never would have considered pathology as a possible future

field.”

Not only were the laboratory facilities substandard,

apparently so was the chief of service. According to Woolner,

Ralph Smith was “a second rate microscopist” but did

concede that he “might have been OK at autopsies” (verbal

communication, June 2, 2010). Dockerty is a little more

charitable in his medical school poem “Ode to Pathology

(V.G. Hospital – 1934),” but possibly he was swayed by the

observation that “R.P. Smith gave me the highest marks ever

awarded by him in pathology!”5 However, that did not stop

Dockerty from many years later composing the following

lines about Smith’s alleged tendency to write lengthy

descriptive reports without actual definitive diagnoses6:

The tissue section sent to me

Revealed diffuse hypertrophy.

With here and there a giant cell.

Some epithelial pearls as well.

In one small corner of the slide

Some coarse mitoses I have spied.

But on a whole, I’m not content.

I’d like to have more tissue sent.

Clearly, during Dockerty’s and Woolner’s time at Dalhousie,

the clinical pathology laboratories were substandard, under-

resourced, and run by unsupervised interns. Facilities were

in a constant state of disrepair, and the practice of surgical

pathology was characterized by equivocation rather than

definitive diagnosis as required for definitive treatment.

Comparing pathology practice at Dalhousie and the Mayo

Clinic in the 1930s and 1940s perhaps seems unfair.

However, times have changed and all pathology laboratories

now are expected to meet uniform, high standards. Having

trained at a top American pathology laboratory and then

immediately having worked as a pathologist at Dalhousie

from 1988 to 2005, it is apparent to me that the quality gap

between the practice of laboratory medicine at the “Mayo

Clinics of the world” versus that in “the Dalhousies of the

World” has substantially narrowed. In a time period in

which the practice of pathology in Canadian laboratories

has been repeatedly scrutinized in the news,7 it is important

to take an historical perspective and reflect upon how far we

have come toward closing the quality gap with the very best

laboratories in the world. Although “lab errors” still occur,

standardized residency training followed by board

certification of pathologists, standardized training of

laboratory technologists, and formalized laboratory

accreditation programs have made the diagnostic world

much better and safer for Canadian patients today.

AcknowledgementsThe following individuals and institutions are thanked for

help with obtaining articles, historical materials, or historical

insights: Lewis B. Woolner, MD (interview 6/2/2010, and

phone interview 3/7/2012); Elizabeth Anne Woolner; Dottie

Hawthorne, Mayo Clinic Libraries; Renee E. Ziemer and

Kristen Van Hoven, Mayo Historical Unit; Sherry Mount;

Docs for Docs.

References1. Woolner LB. Surgical pathology at the Mayo Clinic. In: Rosai J, ed. Guiding

the Surgeon’s Hand: The History of American Surgical Pathology. Washington

(DC): Armed Forces Institute of Pathology; 1997:145–79.

2. Dockerty MB. Rhymed Reminiscences of a Pathologist: His Life’s Story.

Privately printed 1980:54. (Dockerty papers, Mayo Historical Unit, Rochester,

MN)

3. Hewitt RM, Eckman JR, Miller RD. The Mayo Clinic and the Canadians.

CMAJ 1964;91:1161–72.

4. Wright JR Jr. Albert C. Broders’ paradigm shifts involving the prognostication

and definition of cancer. Arch Pathol Lab Med 2012;136(11):1437–46.

5. University and Educational Notes. Science 1927;66(1717):506.

6. Woolner L. Reminiscences of a retired pathologist. MeDal 1997;Fall/Winter:7–

12. (Woolner papers, Mayo Historical Unit, Rochester, MN).

7. Chorneyko K, Butany J Hebert PC, et al. Canada’s pathology. CMAJ

2008;178(12):1523–6.


Case Report: Congenital Cervical Cartilaginous Rest

Lamiaa Rouas, MD, is an assistant professor of pathology; Rajae Tahri, MD, is a pathology resident; and Najat Lamalmi, MD,Zaitouna Alhamany, MD, and Nadia Cherradi, MD, are professors of pathology; Department of Pathology, Children’s Hospitalof Rabat, Faculty of Medicine and Pharmacy, Mohamed V. University Souissi, in Rabat, Morocco. Correspondence may bedirected to [email protected] article has been peer reviewed.Competing interests: None declared

Lamiaa Rouas, MD, Rajae Tahri, MD, Najat Lamalmi, MD, Zaitouna Alhamany, MD, Nadia Cherradi, MD

ABSTRACTCongenital cartilaginous rests are branchial arch remnants. They are anatomical curiosities and

are of interest to the pediatric pathologist. Histopathological examination is required to confirm

the diagnosis. The authors report a case of a cervical chondrocutaneous remnant in a 13-month-

old girl without other apparent abnormalities. This article reviews the clinical features,

histopathology, and differential diagnosis of this extremely rare condition.

RÉSUMÉ La formation cartilagineuse congénitale est un vestige embryonnaire de l’arc branchial.

Essentiellement une curiosité anatomique, elle revêt de l’intérêt pour le pathologiste en

pédiatrie. L’examen histopathologique est nécessaire pour confirmer le diagnostic. Les auteurs

se penchent sur le cas d’une fillette de 13 mois présentant un vestige chondrocutané cervical

sans autres anomalies. Ils passent en revue les caractéristiques cliniques, les aspects

histopathologiques et le diagnostic différentiel de ce trouble extrêmement rare.

ORIGINAL ARTICLE

Congenital cartilaginous rests (CCRs), first described by

Berry in 1890, are branchial arch remnants.1 They may

be single or multiple, unilateral or bilateral, polypoid

elevations, and soft or firm on palpation because of their

underlying core of cartilage. Histopathological examination

is required for diagnosis because of the rarity of this

condition and the typically non-specific clinical picture.

Case ReportA 13-month-old girl presented with a solitary lesion of the

lateral neck noticed since birth. Physical examination

revealed a firm, sessile, mobile mass in the lower third of the

neck. The overlying skin was normal. No fistula, tenderness,

or other sign of inflammation was seen. No other

abnormalities were noted. The lesion was surgically excised.

Histopathological examination revealed skin with cutaneousFigure 1. Cervical cartilaginous remnant with subcutaneous core ofcartilage. (Hematoxylin and eosin)


CONGENITAL CERVICAL CARTILAGINOUS REST

adnexae (hair follicles), dilated blood vessels, and a central

core of mature cartilage (Figure 1) that was attached to some

muscle fibres on the deeper side of the specimen. These

findings led to a diagnosis of cervical chondrocutaneous

branchial remnants.

DiscussionCongenital developmental and inflammatory lesions

represent the majority of the masses in the neck of the

pediatric population. Among congenital lesions, cysts and

sinuses are frequently seen, whereas CCRs are extremely

rare. These lesions are known by a number of names: skin

tags, cervical auricles, branchial appendages, branchiomas,

papillomata, and chondromata. They also appear in the

literature as cervical accessory tragi and wattles. The term

“cervical chondrocutaneous branchial arch remnants,”

proposed by Atlan et al. in 1997,2 is most appropriate since

it describes the origin of the lesion. During embryonic

development, the branchial apparatus is reshaped and

structures may disappear or form vestigial remnants by the

end of the embryonic period. Errors in the development of

the second arch are thought to give rise to

chondrocutaneous branchial remnants, although some

authors believe that the cartilage arises through metaplasia

of undifferentiated mesenchymal cells.3

CCRs may present as dome-shaped papules or nodules on the

lateral neck along the lower half or one third of the anterior

border of the sternocleidomastoid muscle. These lesions are

always present at birth and more commonly seen in males.2

Associated anomalies may be found in the auditory,

respiratory, genitourinary, and cardiovascular systems.2,4

Thus, a complete physical examination of the patient and

ultrasound examination of the genitourinary tract are

recommended. In our case, the lesion formed a cutaneous

nodule with a subcutaneous core of cartilage that extended

into the deeper tissue of the neck. Muscle tissue, possibly

arising from the platysma or the sternocleidomastoid muscle,

was seen attached to the cartilage. The overlying dermis

Table 1. Differential Diagnosis of Cervical Congenital Cartilaginous Rests

Lesion Location Age Pathological FeaturesBranchial cleft cyst Lateral neck Infants, children, adolescents, adults Cystic mass containing fluid, up to 6 cm

Midline Lined by stratified squamous epithelium, respiratory epithelium, or both; lymphoid tissue; keratinous debris

Bronchogenic cyst Suprasternal notch At, or soon after, birth Asymptomatic nodules or sinusesor manubrium sterni; exuding mucoid materialrarely in the anterior Cysts range from 0.3 to 6 cm neck or shoulder Lined by ciliated, pseudostratified

columnar epitheliumCyst wall contains smooth muscle, elastic fibres, and seromucous glands

Dermoid cyst Midline or near-midline, Infants, children, adolescents Cystic – solid or multilocular lobulatedupper neck, near the mass up to 12 cm thyroid cartilage, and Lined by stratified squamous epitheliumnear the suprasternal supported by a fibrous tissue wallnotch Ectodermal derivatives present, such as

hair follicles, sebaceous glands, and sweatglands

Cutaneous inclusion Presternal Early embryonic life Cystic, echolucent, filiform capsulecystChondrocutaneous Always at birth Lower half or one third Never cystic or fistulous vestige of the anterior aspect Polypoid lesions

of sternocleidomastoid Fibrovascular tissue with hair folliclesmuscle and small sebaceous glands and

underlying fat and cartilage in the coreSome follicles are vellus typeMay be associated with malformations


ROUAS ET AL.

contained skin appendages.

The clinical differential diagnosis includes lymphadenopathy

and other polypoid lesions such as fibroepithelial polyps,

polypoid melanocytic nevi, and pedunculated hemangiomas.

Histopathological evaluation is necessary for definitive

diagnosis, but the location on the anterolateral neck of a

congenital nodule or polypoid lesion is a clue. A summary of

the differential diagnosis is given in Table 1. The management

of CCRs involves complete surgical excision.

References1. Matsushita K, Tahara M, Sato H, et al. Cartilaginous choristoma deep in the

upper midline oral vestibule. Br J Oral Maxillofac Surg 2004;42:436–38.

2. Atlan G, Egerszegi EP, Brochu P, et al. Cervical chrondrocutaneous branchial

remnants. Plast Reconstr Surg 1997;100:32–9.

3. Chou LS, Hansen LS, Daniels TE. Choristomas of the oral cavity: a review.

Oral Surg Oral Med Oral Pathol 1991;72:584–93.

4. Gilboa Y, Achiron R, Zalel Y, Bronshtein M. Prenatal diagnosis of cervical

chondrocutaneous vestige. Ultrasound in obstetrics & gynecology : the

official journal of the International Society of Ultrasound in Obstetrics and

Gynecology." Ultrasound Obstet Gynecol 2007;30:1010–2.

Dear CAP-ACP Members,

The Nominating Committee proposes the following Slate of

Officers for June 2013; the only new nominations are for

vice-president and members-at-large:

Executive:

• President – Martin Trotter

• Vice-president – Victor Tron

• Past president – Vina Alexopoulou

• Secretary treasurer – Brian Cummings

• Continuing professional development chair – Jason Ford

• Annual meeting chair – Avrum Gotlieb

• Resource development chair – Alan Spatz

• Website editor – Tadaki Hiruki

• Journal editor-in-chief (ex-officio) – Godfrey Heathcote

• Patient safety and quality assurance chair (ex-officio) –

Laurette Geldenhuys

• Member-at-large – Marciano Reis

• Member-at-large – Julianne Klein

Other:

• Membership chair – Bernard Tetu

If you would like to make an alternative nomination for the

positions of vice-president or member-at-large, please submit

your nomination to [email protected].

The nomination

• must be signed by five qualified ordinary members, with

membership dues paid;

• must be agreed to by the proposed nominee, who must

be a qualified ordinary member; and

• must be received at least 30 days prior to the Annual

General Meeting.

Laurette GeldenhuysPast PresidentChair, Nominating Committee

CAP-ACP NEWS

Message from the Nominating Committee


Perforated Appendiceal Mass in a Patient with Cystic Fibrosis

Tao Wang, MD, is a member of the Department of Laboratory Medicine and Pathobiology, University of Toronto, in Toronto,Ontario. Eric Labelle, MD, FRCSC, is with the Timmins and District General Hospital, in Timmins, Ontario. Rajkumar Vajpeyi,MD, FRCPC, is a member of the Department of Laboratory Medicine and Pathobiology, University of Toronto, and the De-partment of Anatomical Pathology, University Health Network, in Toronto. Correspondence may be directed to [email protected] article has been peer reviewed.Competing interests: None declared

Tao Wang, MD, Eric Labelle, MD, FRCSC, Rajkumar Vajpeyi, MD, FRCPC

A25-year-old male with cystic fibrosis (CF)

developed acute abdominal pain with peritoneal

signs over 24 hours. On examination, there was a 3

cm tender, mobile mass in the right lower quadrant.

Ultrasonography demonstrated a prominent

appendix, but the radiologist was uncertain whether

this represented appendicitis or mucous impaction.

An appendectomy was performed (Figure 1).

On macroscopic examination, the appendix had a

diameter of 2.7 cm and was distended by mucoid

material. A 1.0 cm mural defect was noted, and there

was purulent exudate on the external surface.

Histologically, there was luminal mucus with partial

compression of the mucosa and acute and chronic

inflammation. The final diagnosis was perforated

appendicitis in the context of CF.

Appendiceal disease in CF ranges from sterile mucous

impaction to abscess. Notably, acute appendicitis has

a lower prevalence (1–2%) in CF patients than in the

general population (7%).1 The reason for this is

unknown. One suggestion is that mucous distension

helps to maintain luminal patency and prevents acute

inflammation.2 The slow sterile distension may lead

to higher frequencies of asymptomatic perforations

that escape detection.2 The frequent use of

prophylactic antibiotics may also decrease bacterial

load or mask inflammatory symptoms. Regardless,

with increasing life expectancies in the CF population,

pathologists will likely encounter more cases of

appendiceal disease in these patients.

IMAGES IN PATHOLOGY

Figure 1. Ultrasonography demonstrated a dilated appendix ofapproximately 3 cm (A). Macroscopically, there was luminal distension withmucus (B), confirmed on microscopic examination (C). At highermagnification, there was compression of the mucosa by mucus, as well asacute and chronic inflammation (D and E). (Hematoxylin and eosin)

References1. Lardenoye S, Puylaert J, Smit M, Holscher H. Appendix in children with cystic

fibrosis: US features. Radiology 1994;232:187–9.

2. McCarthy VP, Mischler EH, Hubbard VS, et al. Appendiceal abscess in cystic

fibrosis: a diagnostic challenge. Gastroenterology 1984;86:564–8


Understanding Antibody-Mediated Rejectionof Organ Transplants: Mechanisms,

Morphology, Molecular Patterns, and Personalized Precision Diagnosis

Banu Sis, MD, FRCPC, is a member of the Department of Laboratory Medicine and Pathology, University of Alberta, in Edmonton, Alberta. Correspondence may be directed to [email protected] article has been peer reviewed.Competing interests: None declared

Banu Sis, MD, FRCPC

ABSTRACTTransplantation is a life-saving treatment for patients with end-stage organ failure. While

modern drugs have significantly improved 1-year transplant survival, the rate of organ

transplant failure after the first year remains substantial. The lack of improvement in long-term

transplant survival can be explained, at least partially, by the production of antibodies against

donor antigens, which causes organ transplant damage (rejection) and failure. Clinical studies

show that antibody-mediated rejection is a major problem in organ transplantation because of

its negative impact on transplant outcomes and function, with no effective treatments.

Microvascular endothelium is the main target of injury in antibody-mediated rejection of organ

transplants. This group previously observed upregulation of several endothelial genes in kidney

transplant biopsies from patients with alloantibody, indicating active antibody-mediated

rejection and poor graft survival. Furthermore, endothelial molecular signals discovered a

previously unknown clinical phenotype: C4d-negative antibody-mediated rejection. This article

reviews the current understanding of effector mechanisms of antibody-mediated rejection, its

morphological and molecular patterns in allograft tissues, and current insensitive diagnostic

criteria, and discusses a new diagnostic approach that will bring pathologists closer to precision

diagnosis.

RÉSUMÉ En cas d’insuffisance organique terminale, la transplantation sauve la vie. Bien que les

médicaments modernes aient amélioré grandement le taux de survie la première année de

l’intervention, le taux de rejet de la greffe par la suite demeure notable. La stagnation du taux

de survie au long cours s’explique, du moins en partie, par la production d’anticorps dirigés

contre les antigènes du donneur, qui provoque à terme la défaillance et le rejet de l’organe

transplanté. Des études cliniques démontrent que le rejet par ce mécanisme immunologique

représente un problème majeur en transplantation en raison de ses répercussions sur l’issue de

la transplantation et le fonctionnement de l’organe transplanté en l’absence de traitements

efficaces.

CAP-ACP JUNIOR SCIENTIST AWARD LECTURE 2012


UNDERSTANDING ANTIBODY-MEDIATED REJECTION OF ORGAN TRANSPLANTS

Transplantation is a life-saving treatment for patients

with end-stage organ failure. In the past decade, more

than 25,000 Canadians with end-stage organ failure received

organ transplants, including approximately 11,000 kidney

transplants.1 Each year in the United States, more than

28,000 solid organ transplants are performed, with 116,000

patients on waiting lists, including >91,000 needing kidney

transplantation.2 While modern immunosuppression

therapies significantly lowered the incidence of acute T-cell-

mediated rejection and improved 1-year graft survival, the

rate of late graft loss after the first year remains substantial.3

The lack of improvement in long-term graft survival is

primarily due to B-cell responses and alloantibodies, and

recurrent diseases.4–8

Antibody-Mediated Rejection Is a Major Problem in KidneyTransplantationAntibody-mediated organ rejection (ABMR) is a major

problem in transplantation because it has a huge negative

impact on transplant outcomes and function, and

unfortunately there are no effective treatments. ABMR is

caused by donor-specific antibodies (DSA) against human

leukocyte antigens (HLA), ABO blood group antigens, and

(possibly) non-HLA antigens. The cross-match testing (a

test that determines if the recipient has antibody to the

potential donor) and blood group matching pre-

transplantation virtually eliminated the hyperacute ABMR

phenotype from the clinic by avoiding transplantation

across positive cross-matches or by use of desensitization

protocols. However, transplanting cross-match negative

organs cannot prevent delayed ABMR phenotypes in non-

pre-sensitized or not highly pre-sensitized patients at later

time points after transplantation. This is because many

patients develop clinical problems due to de novo DSA or

increased activity of pre-existing low-titre DSA after

transplantation (22% and 15%, respectively, of kidney

transplant recipients who underwent indicated biopsy).9

After the historical recognition of the destructive potential

of alloantibodies causing hyperacute rejection in the early

1960s, the deleterious effects of alloantibodies after

transplantation remained unrecognized for three

decades.10,11 This ignorance ended in the early 1990s with a

couple of concomitant clinical observations that recognized

delayed acute rejection of kidney allografts associated with

anti-HLA DSA.12–14 These clinical observations coincided

with careful studies of graft pathology, which led to the

discovery of C4d (a breakdown product of activated

complement protein C4) staining in allograft tissues as a

diagnostic biomarker for active ABMR.14,15 Acute C4d+

ABMR has since been reported to occur in about 7% of

conventional (cross-match negative) kidney transplant

recipients, 20–30% of biopsies with acute rejection, and

20–48% of positive cross-match patients after

desensitization treatments.16–19 A decade after the discovery

of C4d as a biomarker for ABMR, late graft losses due to

antibody and morphological features of chronic ABMR were

recognized.20–23 ABMR has been demonstrated in kidney,

heart, pancreas, lung, and, less often, liver allografts and is

not restricted to pre-sensitized patients.17,24–34 In fact, the

transplant community now recognizes that de novo

alloantibody production (sensitization) after transplantation

is a common etiology underlying chronic graft injury.9,26,35

ABMR is a major cause of late kidney transplant loss. Data

from two large transplant centres (Mayo Clinic and

Dans le rejet médié par les anticorps, l’endothélium microvasculaire est le principal siège des

lésions. Ce groupe ont déjà constaté la régulation à la hausse de plusieurs gènes endothéliaux

chez des receveurs d’un rein ayant formé des isoanticorps, signe d’un rejet actif médié par les

anticorps et de survie abrégée du greffon. De plus, la signalisation moléculaire endothéliale a

permis de découvrir un phénotype clinique inconnu jusque-là : le rejet humoral C4d négatif.

L’article passe en revue les mécanismes effecteurs du rejet médié par les anticorps, ses

caractéristiques morphologiques et moléculaires dans l’allogreffe et les critères diagnostiques

actuels dénués de sensibilité, et il examine une nouvelle méthode qui saura fort probablement

rehausser la précision diagnostique.


SIS

Edmonton) and the multicentre Long-Term Deterioration

of Kidney Allograft Function (DEKAF) study independently

indicated that most late kidney transplant losses have a

specific etiology, with ABMR being the leading cause, while

idiopathic fibrosis or drug toxicity were rarely responsible

for renal allograft loss.5,8,36

Morphological Phenotypes of ABMRThe following are the two major phenotypes of ABMR in

human kidney transplants:

1. Early/acute ABMR developing in pre-sensitized patients

early after transplantation (first weeks or months)

2. Late/chronic ABMR with de novo DSA or low titres of

preformed DSA developing late post-transplantation

(usually after 1 year)37

According to the international Banff classification of allograft

pathology, diagnosis of ABMR requires the simultaneous

presence of C4d staining along allograft capillaries, detection

of DSA, and histopathological evidence of allograft tissue

injury.38,39

In acute ABMR, microvascular endothelial injury and

inflammation are the predominant morphological changes

observed in organ allografts. Microvascular inflammation is

seen as aggregates of leukocytes (monocytes/macrophages,

natural killer [NK] cells, neutrophils, and, less often,

T lymphocytes) in the lumina of capillaries,40–43 and this

feature is common to all organ transplants experiencing

ABMR: peritubular capillaritis and glomerulitis in kidney

transplants, myocardial capillaritis in heart transplants,

interacinar capillaritis in pancreas transplants, and alveolar

capillaritis in lung transplants.25,37,38,42,44–46 Active antibody-

mediated injury, recognizable as C4d staining and/or

microvascular inflammation, is a harbinger of subsequent

chronic graft damage, especially if left untreated.21,47,48 Other

pathological features of acute ABMR in kidney transplants

include acute tubular injury,38 vascular thrombi, capillary

dilatation and congestion, interstitial inflammation,49 and

rarely intimal arteritis.37

Smouldering or episodic alloantibody-mediated injury causes

chronic structural changes in kidney allograft microvessels

and arteries, interstitial fibrosis (often with inflammation in

scarred areas), and tubular atrophy. Chronic ABMR of kidney

EC

Complement activation via fixing C1q

Harmful effects of ongoing inflammation in the tissueDegranulation (reactive O2 species), phagocytosis, cell death

Microvascular thrombosis

C1q

Endothelial lysis

C5a and C3a split products

Endothelial cell activation Leukocyte recruitment, adhesion, and activation (Monocytes and neutrophils)

IgG Fc FcR interactions(NK, macrophages, neutrophils)

Complement Dependent Mechanismsin Antibody-Mediated Rejection

Complement Independent Mechanismsin Antibody-Mediated Rejection

C5b-9

Ligation of Antibody on Endothelium

Figure 1. Pathogenesis of antibody-mediated transplant rejection. Ig = immunoglobulin; NK = natural killer.



allografts is thus dominated by microvascular structural

remodelling, which is seen as duplication and/or

multilamination of basement membranes of glomerular and

peritubular capillaries: transplant glomerulopathy and

peritubular capillary basement membrane multilayering,

respectively.37,50–52 Chronic ABMR in other organ allografts

(non-renal) is not well defined.

Pathogenesis of ABMRAlthough alloantibodies play major roles in transplant

rejection, the precise mechanisms by which alloantibodies

cause allograft injury, dysfunction, and loss are not well

understood. However, it is well recognized that donor

endothelium is the principal target of ABMR.53–56

Microvascular endothelial cells are at the interface between

the allograft parenchyma and recipient blood and thus suffer

as the main targets of DSA.

Effector mechanisms of ABMR (Figure 1) include

downstream effects of (1) complement activation (endothelial

cell lysis, chemotaxis, leukocyte recruitment, and endothelial

activation); and (2) complement-independent immuno-

globulin (Ig) Fc-Fc receptor-mediated tissue injury (apoptotic

cell death, degranulation, phagocytosis); (3) endothelial

activation that facilitates recruitment of leukocytes, platelet

activation, and vascular thrombosis; and (4) influx of

leukocytes promoting further tissue injury.16,57–60 Sustained or

episodic antibody-endothelial interactions and associated

intravascular inflammation result in active endothelial injury,

which over time induces structural remodelling with loss of

fenestrations between the endothelial cells and increased

capillary basement membrane deposition, and probable

downstream chronic ischemic effects of capillary remodelling

on parenchymal cells of the allografts.

Complement-Dependent Mechanisms of ABMRAntibodies activate the classical pathway of complement

initiated by binding of C1q to a pair of Fc domains of IgG

or IgM ligated to tissues,16,61,62 While IgM, IgG1, and IgG3

antibodies are effective in activating complement, IgG2 and

IgG4 antibodies are less effective; thus, not all antibodies

activate complement. Complement-dependent mechanisms

underlying microcirculation injury involve the following:

• Split products mediate chemotaxis (C3a and C5a) and

activate monocytes/macrophages and neutrophils

through complement receptors (CR1, CR3), leading to

microcirculation inflammation (capillaritis).

• Binding of receptors on endothelium to C3a and C5a

induces endothelial activation, leading to

expression/secretion of proinflammatory molecules

(adhesion molecules, cytokines, chemokines), which

further increase leukocyte recruitment, platelet

adherence, vascular permeability, and

coagulability.16,60,63,64

• Terminal complement components (C5b-9, the

membrane attack complex) trigger endothelial cell lysis

by forming pores in the target cell membrane.16,57–59

• Sub-lytic concentrations of C5b-C9 can also induce

endothelial activation with increased expression of

adhesion molecules and tissue factor, leading to

microcirculation inflammation and pro-coagulant

state.65

Complement-Independent Mechanisms of ABMRAlthough complement activation is crucial for ABMR,

several effector mechanisms triggered by tissue-bound

alloantibody, that is, endothelial activation and Fc receptor-

mediated leukocyte recruitment and activation, do not

require complement (see Figure 1). In support of this view,

there is growing evidence suggesting that complement-

independent mechanisms predominate particularly in

chronic ABMR, causing ongoing endothelial injury and

remodelling:

• Alloantibody can cause endothelial activation in the

absence of complement with increased release of von

Willebrand factor (VWF) and externalization of P-

selectin on endothelial cells upon stimulation by anti-

HLA class I.64

• Activated endothelial cells express/secrete

proinflammatory molecules (i.e., E-selectin, P-selectin,

ICAM-1, VCAM-1, CX3CL1) that increase leukocyte

recruitment. Recruited and activated leukocytes secrete

cytokines such as TNF-α and IL-1β, which further

increase endothelial activation by inducing expression

of tissue factor, adhesion molecules, and chemokines.66


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• Leukocytes can kill/damage endothelium via antibody-

dependent cellular cytotoxicity by NK cells or

monocytes/macrophages through IgG Fc/Fc receptors.

• Recent clinical trial data suggest that complement-

independent mechanisms may predominate in chronic

ABMR. Terminal complement inhibition by eculizumab

(humanized anti-C5 antibody) has been found to

decrease the frequency of biopsy-proven acute ABMR

in highly pre-sensitized kidney transplant recipients.67

However, the initial results of an eculizumab trial to

prevent chronic kidney transplant damage are

discouraging: anti-C5 blockade did not decrease the

prevalence of chronic ABMR after 1 year in highly pre-

sensitized kidney transplant recipients.68 In a series of

positive cross-match kidney transplant recipients who

were treated with eculizumab for 3–12 months, the

prevalence of transplant glomerulopathy and

peritubular capillaritis at 1 year was high in both

eculizumab-treated and control patients (transplant

glomerulopathy: 30% versus 36%, respectively;

peritubular capillaritis: 56% versus 57%, respectively).68

Endothelial Molecules Have Taught Us a Frightening Lesson: About 50% of Patients with Active Late ABMR Are Misdiagnosed in the ClinicDetecting which renal transplants are at risk for antibody-

mediated deterioration is an unmet need. Anti-HLA

antibodies develop in 25% of renal transplant recipients and

are associated with increased graft failure,69 but have low

predictive value for rejection in individual patients. In a

retrospective study of biopsies from 1,320 patients, we

observed that approximately 50% of patients with HLA

alloantibodies and transplant glomerulopathy were C4d

negative.23 This earlier observation suggested that C4d may

be an insensitive biomarker for chronic ABMR.

We then pursued this finding by new studies using gene

microarray assessment of renal allograft biopsy tissues. We

identified a literature-based “endothelial cell-associated

transcript” (ENDAT) set (323 probe sets; 119 unique genes),

in which genes were identified based on their selective

expression in cultured human endothelial cells when

compared with non-endothelial cells or by serial analysis

gene expression (SAGE) libraries comparing tag frequencies

in endothelial versus non-endothelial libraries.70,71 In a large

number of kidney transplants biopsied for clinical

indications, the biopsies with high expression of ENDATs

and HLA antibodies showed histopathological lesions of

ABMR (transplant glomerulopathy, capillaritis, glomerulitis,

peritubular capillary basement membrane multilayering,

and fibrosis/atrophy) and poor outcomes in comparison to

biopsies with HLA antibodies and no ENDAT

upregulation.72 Surprisingly, many of these active ABMR

cases were missed. Only 40% of kidneys with high ENDAT

expression and chronic ABMR morphology or graft loss

were diagnosed by C4d positivity, an observation that was

validated in an independent set of 82 kidneys. In this way,

we recognized the cases of C4d-negative active ABMR and

the importance of a full phenotype of patients incorporating

all clinical, pathological, and outcome data.

The key message is that endothelial molecular stress plus the

presence of alloantibody is a high-risk state for ABMR and

graft failure. Importantly, the presence of DSA without

upregulation of intragraft ENDATs is not associated with

graft damage or worse outcomes compared to patients with

no DSA. It should also be noted that high ENDAT

expression per se is not an indicator of graft damage or

eventual graft loss in patients who lack HLA antibodies.

Therefore, ENDAT changes in renal transplants occur in

rejection and in other forms of renal injury (infections,

ischemic injury), but their impact on progressive graft

deterioration and loss is principally in patients with HLA

antibodies. In an extended cohort of 329 biopsies with

complete DSA testing, including cases of T-cell-mediated

rejection, borderline changes, glomerulonephritis, polyoma

virus nephropathy, and acute tubular necrosis, some biopsies

showed ENDAT expression in the absence of DSA.42

However, high ENDAT scores plus DSA positivity increased

the specificity for ABMR (97%), capturing both C4d-

positive and C4d-negative active ABMR cases. Therefore, the

specificity of ENDAT scores is high in patients with DSA,

detecting both active injury and ABMR histology. We

recommend use of ENDAT scores together with DSA testing

results.

High ENDAT expression with the presence of antibodies

predicts graft loss with higher sensitivity (77% versus 31%)

and slightly lower specificity (71% versus 94%) than the



presence of C4d. Thus, high intragraft endothelial transcript

expression in patients with circulating alloantibody is a

proven indicator of active antibody-mediated allograft

damage and poor graft outcome.72 Graft survival did not

differ significantly between patients with antibodies and

high ENDAT with C4d or without C4d staining; thus, C4d

positivity did not have an additional adverse prognostic

impact over high ENDAT with HLA antibodies.

We also analyzed the diagnostic value of individual

endothelial transcripts. In a training set (n = 201 biopsies),

we selected a cut-off signal for each gene for detecting

ABMR (histology/serology) with at least 80% sensitivity by

receiver operating characteristic (ROC) curve analysis.

Applying the same cut-offs in an independent validation set

(n = 202 biopsies), Duffy antigen receptor for chemokines

(DARC) upregulation plus DSA showed 81% sensitivity and

93% specificity for ABMR, whereas C4d plus DSA showed

48% sensitivity and 97% specificity.73 Figure 2 shows the

poor prognosis of kidney allografts with molecularly

diagnosed ABMR (DARC plus DSA, C4d positive or

negative) when compared to allografts without molecular

ABMR (DSA present or absent). Thus, measuring a few

endothelial transcripts in biopsies from kidneys with

alloantibody is a highly sensitive and specific method to

detect ABMR, and is more sensitive than C4d staining.

Endothelial molecular parameters should be incorporated

into the histopathology-based Banff classification to

improve diagnosis of ABMR.

In our experience, most C4d-negative ABMR biopsies

demonstrated chronic ABMR morphology (transplant

glomerulopathy, capillaritis, peritubular capillary basement

membrane multilayering, fibrosis/atrophy). Whether there

is a form of acute ABMR independent of complement effects

is an open question. If so, the answer will likely come from

data from highly sensitized cross-match positive transplant

populations. There is still much debate on whether or not

C4d-negative acute ABMR truly exists in patients presenting

with clinical problems. In fact, C4d staining may have a

higher sensitivity for acute ABMR than it has for chronic

ABMR.

Microarrays of Kidney Allograft Biopsies Dissected: TheMolecular Phenotype of ABMRThe biopsies analyzed by gene expression microarrays

displayed three intragraft molecular phenotypes during

ABMR: upregulation of endothelial genes,72 NK cell–

associated genes,43 and effects of interferon-gamma

(IFN-γ).74

Endothelial Molecular SignalsMany individual ENDATs were increased in ABMR and

predicted future graft loss. The expression of VWF, EDN1

(endothelin 1), CAV1 (caveolin 1), CDH5 (cadherin 5),

CDH13 (cadherin 13), PALMD (palmdelphin), PECAM1

(platelet/endothelial cell adhesion molecule 1), SELE

(selectin E), CD34, DARC, RHOJ (ras homolog family

member J), SOX7 (SRY-box containing gene 7), SOX18

(SRY-box containing gene 18), THBD (thrombomodulin),

and MALL (BENE; interacts with CAV1) were higher in

ABMR than in T-cell-mediated rejection biopsies.72 The

Training set 112 patients

Validation set 154 patients

Cum

ulat

ive

surv

ival

Post-biopsy time (days)

No DSA

DSA+ with no DARC

DSA+ with DARC+, C4d-

DSA+ with DARC, C4d+

p = .32

p < .001

p = .004

Post-biopsy time (days)

No DSA

DSA+ with no DARC

DSA+ with DARC+, C4d-

DSA+ with DARC, C4d+

p = .34

p = .004

p = .004

Figure 2. Prediction of kidneyallograft survival by molecularlydiagnosed antibody-mediatedtransplant rejection. DARC = Duffyantigen receptor for chemokines;DSA = donor-specific antibodies.


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increased expression of endothelial genes in ABMR by

microarrays was confirmed by reverse transcription

polymerase chain reaction (RT-PCR).72 However,

immunohistochemical staining of endothelial proteins such

as VWF and PECAM1 could not be quantified for

upregulation because of their high baseline staining in renal

endothelium.75 A functional annotation analysis on

endothelial transcripts that were increased in biopsies with

ABMR compared to normal renal cortex showed a number

of significantly enriched functional pathways operating in

human ABMR biopsy tissues.

Endothelial Cells Gain Adhesive Properties and Thereby

Facilitate Leukocyte Trafficking

Functional annotation cluster analysis using the Gene

Ontology database on a list of endothelial genes that were

upregulated in ABMR biopsies showed a number of

significantly enriched categories4: regulation of cell

migration and motility, facilitation of leukocyte rolling,

adhesion, and transmigration via endothelial selectins (E-

selectin, P-selection), CD34, integrin ligands (VCAM1,

ICAM2), PECAM1, and interaction with basement

membrane components, and an increased endothelial

procoagulant state (VWF) that enables platelet activation.

We observed upregulation of DARC as one of the top genes

increased in human ABMR samples.72 DARC mediates

incomplete chemokine transcytosis, leading to a retention

of intact chemokines on the endothelial apical surface and

more leukocyte migration. Mice overexpressing DARC on

vascular endothelium were reported to have enhanced

leukocyte recruitment and extravasation.76 Thus,

upregulated DARC expression on endothelium seems to be

contributing to sustained microvascular inflammation and

injury during ABMR in humans.

Endothelial Cells Gain Pro-coagulant Properties and Thereby

Facilitate Platelet Aggregation and Coagulation

Functional annotation analysis on ENDATs increased in

ABMR biopsies also showed significant enrichment of

clusters related to coagulation and platelet activation.4

Interestingly, we observed VWF as the top transcript

increased in biopsy tissues with ABMR. The VWF protein

plays a major role in hemostasis by carrying the blood

coagulation factor VIII and by linking endothelial activation

to platelet adhesion and aggregation at sites of vascular

injury. Endothelial cells store VWF protein as large

multimers together with P-selectin in cytoplasmic Weibel-

Palade bodies.77 Upon endothelial cell activation, immediate

endothelial exocytosis facilitates the release of preformed

VWF and externalization of P-selectin from the Weibel-

Palade bodies to the endothelial surface.65,77 The large

multimeric bundles of VWF released onto endothelial

surface binds to glycoprotein Ib on platelets, leading to

effective platelet activation and aggregation.78

Analogous to our findings in human ABMR biopsy samples,

Morrell et al.60 observed in a mouse skin transplantation

model that repeated injections of alloantibodies caused

VWF release and sustained endothelial-platelet interactions

mediating platelet activation and rolling in vivo. The

researchers also showed that alloantibody-endothelial-

platelet interactions in this model were associated with

vascular inflammation, thrombi, and complement

component C4d deposition in vessels.

Increased IFN-γ Effects IFN-γ, a soluble cytokine and a type II interferon, which is

predominantly released by antigen-triggered effector T cells

and NK cells, plays major roles in allograft rejection. Both

ABMR and T-cell-mediated rejection biopsies show high

expression of IFN-γ induced transcripts such as CXCL11,

CXCL9, and CCL5 (RANTES).72,74 IFN-γ induces expression

of many genes in the donor tissue and in host infiltrating

cells, inducing chemokines such as CXCL9, CXCL10,

CXCL11 and major histocompatibility complex class Ia

(HLA-A, HLA-B), class Ib (e.g., HLA-E), and class II (HLA-

DP, -DQ, and -DR).79,80

IFN-γ secreted by T cells and/or NK cells is essential for

induction of class I and class II antigens on endothelium.80

IFN-γ increases antibody ligation to endothelium by

increasing the density of major histocompatibility complex

antigens on the endothelium and thus making antibodies

more effective in inducing graft inflammation and injury.

Innate Leukocytes, Especially NK Cells, Emerge as EffectorCells in ABMRMicrovascular inflammation is a typical histopathological



feature of ABMR.37,42 Leukocyte recruitment to the allograft

capillaries can be mediated by complement split products

(C3a, C5a), activated endothelial cells, and activated

platelets. There are also in vitro data that suggest

alloantibody alone, independent of the complement, may

trigger endothelial activation and thereby may induce

microvascular inflammation.64

Antibody-dependent cellular cytotoxicity (ADCC) is a

powerful way to damage/kill endothelial cells coated by

alloantibodies. NK cells, monocytes/macrophages, and other

leukocytes have surface receptors for the Fc portion of IgG.

In particular, Fc-γ receptor 3 (CD16) binding to IgG causes

release of cytotoxic granules (perforin, granzymes) from NK

cells or macrophages, which in turn triggers apoptosis of the

target cell.16,81 Supporting the contribution of this

mechanism to ABMR, we identified increased NK cell

transcripts and CD56+ NK cells in biopsies with ABMR.43

Recently, Hirohashi et al. reported that NK cells were

increased in DSA-induced obliterative arteriopathy lesions

in heart allografts transplanted into immunodeficient mice

that lack T and B cells and complement protein C3. They

also showed that obliterative arteriopathy lesions were

decreased upon depletion of NK cells with anti-NK1.1

antibodies, suggesting that NK cells in the presence of DSA

mediate chronic ABMR, independently of complement

activation.82

Conclusion Effector mechanisms of ABMR include downstream effects

of complement activation and complement-independent Ig

Fc-Fc receptor-mediated tissue injury, the involvement of

activated endothelial cells that facilitates recruitment of

leukocytes, platelets, and vascular thrombosis, and an influx

of leukocytes promoting further tissue injury. Sustained or

episodic antibody-endothelial interactions and associated

intravascular inflammation result in active endothelial

injury, which over time induces structural remodelling with

loss of fenestrations between the endothelial cells and

increased capillary basement membrane deposition, as well

as their probable downstream chronic ischemic effects on

the parenchymal cells of the allografts. Many of these

effector mechanisms do not require complement. There is

growing evidence that complement-independent

mechanisms predominate in chronic ABMR causing

endothelial injury and remodelling. To improve diagnosis

and quantify the degree of antibody-mediated tissue injury

in clinical transplants, endothelial cells provide a useful

readout.

AcknowledgementsThe author’s work has been supported by research grants

from Canadian Institutes of Health Research (MOP-102705),

Roche Organ Transplantation Research Foundation,

University of Alberta Hospital Foundation, and Kidney &

Urology Foundation of America – Renal Pathology Society.

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nephropathy (‘CAN’). Am J Transplant 2007;7(3):518–26.

40. Magil AB, Tinckam K. Monocytes and peritubular capillary C4d deposition

in acute renal allograft rejection. Kidney Int 2003;63(5):1888–93.

41. Fahim T, Bohmig GA, Exner M, et al. The cellular lesion of humoral rejection:

predominant recruitment of monocytes to peritubular and glomerular

capillaries. Am J Transplant 2007;7(2):385–93.

42. Sis B, Jhangri GS, Riopel J, et al. A new diagnostic algorithm for antibody-

mediated microcirculation inflammation in kidney transplants. Am J

Transplant 2012;12(5):1168–79.

43. Hidalgo LG, Sis B, Sellares J, et al. NK cell transcripts and NK cells in kidney

biopsies from patients with donor-specific antibodies: evidence for NK cell

involvement in antibody-mediated rejection. Am J Transplant

2010;10(8):1812–22.

44. Loupy A, Cazes A, Guillemain R, et al. Very late heart transplant rejection is

associated with microvascular injury, complement deposition and progression

to cardiac allograft vasculopathy. Am J Transplant 2011;11(7):1478–87.

45. Drachenberg CB, Torrealba JR, Nankivell BJ, et al. Guidelines for the diagnosis

of antibody-mediated rejection in pancreas allografts – updated Banff grading

schema. Am J Transplant 2011;11(9):1792–802.

46. Girnita AL, McCurry KR, Yousem SA, et al. Antibody-mediated rejection in

lung transplantation: case reports. Clin Transpl 2006;508–10.

47. Lerut E, Naesens M, Kuypers DR, et al. Subclinical peritubular capillaritis at 3

months is associated with chronic rejection at 1 year. Transplant

2007;83(11):1416–22.

48. Loupy A, Suberbielle-Boissel C, Hill GS, et al. Outcome of subclinical

antibody-mediated rejection in kidney transplant recipients with preformed

donor-specific antibodies. Am J Transplant 2009;9(11):2561–70.

49. Mauiyyedi S, Crespo M, Collins AB, et al. Acute humoral rejection in kidney

transplantation: II. Morphology, immunopathology, and pathologic

classification. J Am Soc Nephrol 2002;13(3):779–87.

50. Wavamunno MD, O’Connell PJ, Vitalone M, et al. Transplant glomerulopathy:

ultrastructural abnormalities occur early in longitudinal analysis of protocol

biopsies. Am J Transplant 2007;7(12):2757–68.

51. Haas M, Mirocha J. Early ultrastructural changes in renal allografts: correlation

with antibody-mediated rejection and transplant glomerulopathy. Am J

Transplant 2011;11(10):2123–31.

52. Sis B, Mengel M, Haas M, et al. Banff ʼ09 Meeting report: antibody mediated

graft deterioration and implementation of Banff working groups. Am J

Transplant 2010;10(3):464–71.

53. Rodriguez ER, Skojec DV, Tan CD, et al. Antibody-mediated rejection in

human cardiac allografts: evaluation of immunoglobulins and complement

activation products c4d and c3d as markers. Am J Transplant 2005;5(11):2778–

85.

54. Jin YP, Jindra PT, Gong KW, et al. Anti-HLA class I antibodies activate

endothelial cells and promote chronic rejection. Transplant 2005;79(3

Suppl):S19–21.

55. Regele H, Bohmig GA. Tissue injury and repair in allografts: novel

perspectives. Curr Opin Nephrol Hypertens 2003;12(3):259–66.

56. Minami K, Murata K, Lee CY, et al. C4d deposition and clearance in cardiac



transplants correlates with alloantibody levels and rejection in rats. Am J

Transplant 2006;6(5 Pt 1):923–32.

57. Valujskikh A, Heeger PS. Emerging roles of endothelial cells in transplant

rejection. Curr Opin Immunol 2003;15(5):493–8.

58. Baldwin WM III, Kasper EK, Zachary AA, et al. Beyond C4d: other

complement-related diagnostic approaches to antibody-mediated rejection.

Am J Transplant 2004;4(3):311–8.

59. Baldwin WM III, Larsen CP, Fairchild RL. Innate immune responses to

transplants: a significant variable with cadaver donors. Immunity

2001;14:369–76.

60. Morrell CN, Murata K, Swaim AM, et al. In vivo platelet-endothelial cell

interactions in response to major histocompatibility complex alloantibody.

Circ Res 2008;102(7):777–85.

61. Murata K, Baldwin WM III. Mechanisms of complement activation, C4d

deposition, and their contribution to the pathogenesis of antibody-mediated

rejection. Transplant Rev (Orlando) 2009;23(3):139–50.

62. Wehner JR, Morrell CN, Rene RE, et al. Immunological challenges of cardiac

transplantation: the need for better animal models to answer current clinical

questions. J Clin Immunol 2009;29(6):722–9.

63. Pober JS, Min W, Bradley JR. Mechanisms of endothelial dysfunction, injury,

and death. Annu Rev Pathol 2009;4:71–95.

64. Yamakuchi M, Kirkiles-Smith NC, Ferlito M, et al. Antibody to human

leukocyte antigen triggers endothelial exocytosis. Proc Natl Acad Sci U S A

2007;104(4):1301–6.

65. Saadi S, Holzknecht RA, Patte CP, Platt JL. Endothelial cell activation by pore-

forming structures – pivotal role for interleukin-1 alpha. Circulation

2000;101(15):1867–73.

66. Saadi S, Takahashi T, Holzknecht RA, Platt JL. Pathways to acute humoral

rejection. Am J Pathol 2004;164(3):1073–80.

67. Stegall MD, Diwan T, Raghavaiah S, et al. Terminal complement inhibition

decreases antibody-mediated rejection in sensitized renal transplant recipients.

Am J Transplant 2011;11(11):2405–13.

68. Cornell LD, Gloor JM, Raghavaiah S, et al. Chronic antibody mediated

histologic changes in positive-crossmatch kidney transplants (+XMKTx)

receiving early C5 blockade. Am J Transplant 2012;12(s3):450.

69. Terasaki PI, Ozawa M. Predicting kidney graft failure by HLA antibodies: a

prospective trial. Am J Transplant 2004;4(3):438–43.

70. Ho M, Yang E, Matcuk G, et al. Identification of endothelial cell genes by

combined database mining and microarray analysis. Physiol Genomics

2003;13(3):249–62.

71. Sengoelge G, Luo W, Fine D, et al. A SAGE-based comparison between

glomerular and aortic endothelial cells. Am J Physiol Renal Physiol

2005;288(6):F1290–300.

72. Sis B, Jhangri G, Bunnag S, et al. Endothelial gene expression in kidney

transplants with alloantibody indicates antibody-mediated damage despite

lack of C4d staining. Am J Transplant 2009;9(10):2312–23.

73. Sis B, Hidalgo LG, Einecke G, et al. Developing a molecular definition of

antibody mediated rejection using endothelial transcripts: can molecular

assessments replace C4d? Am J Transplant 2010;10(s4):188.

74. Mueller TF, Einecke G, Reeve J, et al. Microarray analysis of rejection in human

kidney transplants using pathogenesis-based transcript sets. Am J Transplant

2007;7(12):2712–22.

75. Sis B, unpublished data, 2013

76. Pruenster M, Mudde L, Bombosi P, et al. The Duffy antigen receptor for

chemokines transports chemokines and supports their promigratory activity.

Nat Immunol 2009;10(1):101–8.

77. Wasowska BA. Mechanisms involved in antibody- and complement-mediated

allograft rejection. Immunol Res 2010;47(1–3):25–44.

78. Dayananda KM, Singh I, Mondal N, Neelamegham S. von Willebrand factor

self-association on platelet GpIbalpha under hydrodynamic shear: effect on

shear-induced platelet activation. Blood 2010;116(19):3990–8.

79. Famulski KS, Einecke G, Reeve J, et al. Changes in the transcriptome in

allograft rejection: IFN-gamma-induced transcripts in mouse kidney

allografts. Am J Transplant 2006;6(6):1342–54.

80. Goes N, Urmson J, Hobart M, Halloran PF. The unique role of interferon-

gamma in the regulation of MHC expression on arterial endothelium.

Transplant 1996;62(12):1889–94.

81. Lee CY, Lotfi-Emran S, Erdinc M, et al. The involvement of FcR mechanisms

in antibody-mediated rejection. Transplant 2007;84(10):1324–34.

82. Hirohashi T, Chase CM, Della PP, et al. A novel pathway of chronic allograft

rejection mediated by NK cells and alloantibody. Am J Transplant

2012;12(2):313–21.

DISPLAY CLASSIFIED

Cytopathology Review Course Montréal, Québec, Canada

www.muhc-cme.mcgill.ca

Course Director: Dr. Manon Auger

For further information contact:Email: [email protected] University Health Centre, Continuing Education OfficeTel: (514) 934-8253 • Fax: (514) 934-1779


The Corporate Capture of Laboratory ServicesA review of False Positive. Private Profit in Canada’s Medical Laboratories

Dr. Heathcote is editor-in-chief of CJP. For a period of approximately 1 year in the mid-1990s, he undertook remuneratedwork for a private medical laboratory in Ontario.

The state is charged with advancing the welfare of its citizens; a company’s purpose is to

increase its owners’ wealth.

– Mindell et al., “The Corporate Capture of Public Health”

In False Positive, Ross Sutherland presents a series of arguments as to why “the practice of using

for-profit laboratory corporations is not acceptable in the provision of an essential service.” He

begins with a description of how pathologists promoted the establishment of private

laboratories at a time when universal medical insurance was being introduced in the 1960s. As

might be expected, this is a complicated story, which might well not be familiar to younger

pathologists.

Ontario is at the centre of the author’s story, and in subsequent chapters he covers the growth

of for-profit laboratory corporations despite half-hearted attempts by government to control

their activities through licensing, quality management, and the limitation of overuse. It is not

an edifying story, and there is little to suggest that pathologists, private corporations, or

government itself really acted in the public interest.

Across the country, there is widespread belief in government in the advantages of greater

integration of laboratory services. In chapter four, the author dissects the fiasco of laboratory

reorganization in Ontario in the 1990s, a move resisted by private laboratories, and how

government policies actually undermined its own initiatives. Even for someone who worked

as a laboratory medical director during that turbulent decade in Ontario, this chapter provides

new insight into the seemingly endless stream of futile attempts at reorganization and

integration. The book continues with a review of the current participation of laboratory

corporations in the laboratory services of each province.

In the closing chapters, the author examines the belief that “the private sector does it better” as

it applies to laboratory services and comes to the conclusion that there is little or no evidence

to support this. The book appears well researched, although it would have been of interest to

hear more from laboratory physicians who are currently engaged with for-profit laboratory

corporations.

Any discussion of laboratory corporations is likely to provoke an ideological reaction among

pathologists and laboratory physicians and, after reading this book, some may still believe that

the private sector is more efficient in the provision of laboratory services. These physicians

presumably also believe that their hospital laboratories of today are much cleaner than they

were before janitorial services were contracted out to private companies.

J. Godfrey Heathcote, MA, MB BChir, PhD, FRCPCHead, Department of Pathology and Laboratory MedicineQueen Elizabeth II Health Sciences Centre and Capital District Health AuthorityHalifax, Nova Scotia

BOOK REVIEW

Ross SutherlandFernwood Publishing, Halifaxand Winnipeg, 2011ISBN: 978-1-55266-409-4135 pagesList price: $17.95

DISPLAY CLASSIFIED

SULTAN QABOOS UNIVERSITYSultanate of Oman

Sultan Qaboos University Hospital is a government institution and a teaching hospital situated within the campus of the Sultan Qaboos University and has 550 beds capacity.

DEPARTMENT OF PATHOLOGY

SENIOR CONSULTANT1. Qualifications: Medical Graduate with postgraduate degree in

Pathology (M.D/FRCPath or equivalent). Must have been working in the grade applied, for at least 5 years.

2. Teaching experience: Must have certified experience of teaching undergraduates and postgraduates in a medical institution of repute where an integrated curriculum is taught for more than 10 years. Exposure to Medical Education technology and practices is desirable.

3. Professional experience a. Candidate must have Surgical Pathology diagnostic experience for

> 10 years, preferably in a tertiary level sub-speciality hospital with expertise in histopathology, cytology and ancillary techniques like immunofluorescence, immunohistochemistry and electron microscopy. Experience in molecular pathology is desirable.

b. Candidates must have subspeciality interest and experience.

4. Research contributions must be demonstrated through a. Research grants b. Publications in international peer-reviewed journals c. Conference presentations d. Reviewer/Editor of Medical Journals

BENEFITS:Tax-free salary in Oman and in addition to a good remuneration package, free furnished accommodation, electricity and water allowance, 60 days paid annual leave with return air ticket, end of contracts service gratuity and free medical treatment in the hospital. Contracts are for 2 years, renewable by mutual agreement.

Oman is a modern and peaceful country situated on the shores of the Arabian Sea. Its many unspoiled beaches and dramatic mountains and valleys make Oman a haven for those who enjoy the outdoor life.

All interested applicants who meets the selection criteria may submit their application letter and indicate the position applying for along with their detailed CV, names & addresses of three referees, copies of academic & professional certificates, relevant pages of passport and a recent passport size photo to :

Director of Personnel Affairs DepartmentSultan Qaboos University HospitalP.O. Box 38 Al-Khod, PC 123Sultanate of Omanor send the above at e-mail address:[email protected]: www.squ.edu.om

Pediatric / PerinatalAnatomical Pathologist

Department of Pathology and Laboratory MedicineThe IWK Health Centre and Dalhousie University Medical School

Halifax, Nova Scotia, Canada

The Department of Pathology and Laboratory Medicine of the IWK Health Centre and the Department of Pathology at Dalhousie University located in Halifax, Nova Scotia, Canada, have a vacancy for a full-time Academic Pediatric/Perinatal Pathologist.

The IWK Health Centre is a full service pediatric and high-risk obstetrical tertiary referral center for the three Maritime provinces. It comprises a 150-bed children’s wing and 197-bed maternity wing providing care for high risk pregnant patients and approximately 5,000 deliveries per year. The Dalhousie University Pathology Department offers residency training programs in Anatomical, General and Hematopathology. The Research Department of the IWK Health Centre has programs and facilities specifically tailored to assist faculty who are developing their research careers. Also, protected academic time, commensurate with the candidate’s prospects for research success, can be provided.

We seek a candidate who is academically oriented and possesses certification from the Royal College of Physicians and Surgeons of Canada or equivalent. The candidate should be Board certified, or Board eligible, in Pediatric Pathology or have extensive experience and expertise in Pediatric Pathology. Candidates should possess strong diagnostic skills which can be directed to the evaluation, diagnosis and sign out of surgical pathology, placentas and autopsies, including complex perinatal cases. Academic responsibilities will include teaching of both house staff and students.

The successful candidate will join a group of three other Pediatric Pathologists, a Hematopathologist, Medical Microbiologist, Clinical Biochemist, two Clinical Cytogeneticists and two Clinical Molecular Geneticists. Salary and Dalhousie Medical School academic rank are commensurate with experience and level of academic activity.

Send curriculum vitae, a statement describing previous experience/career goals and names of three references to:

Dr. Timothy L. MailmanDepartment of Pathology & Laboratory Medicine

The IWK Health Centre, P.O. Box 9700Halifax, Nova Scotia B3K 6R8

E-Mail: [email protected]

All qualified persons are encouraged to apply although Canadian citizens and permanent residents will be given priority.

DISPLAY CLASSIFIED

Interior Health is one of six health authorities responsible for publicly funded health services in British Columbia. With over 18,000 employees and approximately 1,800 physicians on our team, we are developing the future of health care through innovation and professional development.

Kelowna – These opportunities are situated in a breathtaking "four seasons" playground with a wide range of activities to enjoy. Kelowna is the largest city in British Columbia’s Okanagan Valley and is known for its hot summer and temperate winters. Ranked as one of the most livable cities in Canada, Kelowna attracts visitors and residents from around the world to explore what the city has to offer.

Kelowna General Hospital (KGH) –

Penticton Regional Hospital, Vernon Jubilee Hospital, and Kootenay Boundary Hospital. The Pathology department

Hematopathologist – The successful candidate will work with four General Pathologists and report to the

Hematology Laboratory at KGH, determining test menus offered in coordination with clinical departments and other sites through the regional Hematology Working Group, overseeing transfusion medicine and taking responsibility for quality assurance, ranking capital equipment needs, and teaching medical and laboratory students

Anatomic and/or General Pathologists X2 –

surgical pathology, cytopathology and autopsy pathology, quality assurance activities, and teaching of medical and -

Life’s better here. Find out [email protected]

or view us online at our website www.betterhere.ca

Looking for a Hematopathologist, Anatomic and/or General Pathologists

the northern way of caring

ortunity pathologist OpPLife style matters. . .

ortunity

Life style matters. . .

Northern Health is situated in the heart of British Columbia.

Our main referral centre, University Hospital of Northern BC, includes a catchment population of 350,000 and is

specialties

articipation in medical student and resident teaching through the Northern Medical PP

Northern Health is situated in the heart of British Columbia.


articipation in medical student and resident teaching through the Northern Medical P


rogram, University ofarticipation in medical student and resident teaching through the Northern Medical P


rogram, University of

articipation in medical student and resident teaching through the Northern Medical PNorthern British Columbia, and University of British Columbia

.of this exciting opportunity y.

For more information contact:Sheilagh Wilson hysician R, PPhone:Email:

articipation in medical student and resident teaching through the Northern Medical PNorthern British Columbia, and University of British Columbia

For more information contact:tment Coordinatorecruihysician R

isit our website:V careers.no


rthernhealth.ca careers.no




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canaadian jtouhrnal ofolog y volume 5, issue 1 spring 2013canadianjournalofpathology.ca ›...

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