canaadian jtouhrnal ofolog y volume 5, issue 1 spring 2013canadianjournalofpathology.ca ›...
TRANSCRIPT
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
www.cap-acp.org
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
and the 27th World Congress of the World Association of Societies of Pathology and Laboratory Medicine (WASPaLM)
Québec City ■ June 8–12, 2013
Contact Us!CAP-ACP Conference [email protected] ■ 1 613 531 9210
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]
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Canadian Journal of Pathology is published four times annually by
Andrew John Publishing Inc., with offices at 115 King Street West, Dundas, ON, Canada L9H 1V1.
We welcome editorial submissions but cannot assume responsibility or commitment for unsolicited material. Any editorial material, including photographs that are accepted from an unsolicited
contributor, will become the property of Andrew John Publishing Inc.
FEEDBACKWe welcome your views and comments. Please send them to
Andrew John Publishing Inc., 115 King Street West, Dundas, ON, Canada L9H 1V1.
Copyright 2013 by Andrew John Publishing Inc. All rights reserved.Reprinting in part or in whole is forbidden without express
written consent from the publisher.
Publications Agreement Number 40025049 • ISSN 1918-915XReturn undeliverable Canadian Addresses to:
115 King Street West, Suite 220, Dundas, ON L9H 1V1
Canadian Journal of Pathology • Volume 5, Issue 1, 2013
Contents
For Instructions to Authors, please visit www.andrewjohnpublishing.com/CJP/instructionstoauthors.html
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
Canadian Journal of P athology 5Spring 2013
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
Oncology/College of American Pathologists guideline recommendations for
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
Spring 20136 Canadian Journal of P athology
Aucun conflit d’intérêts à déclarer
ÉDITORIAL
L’analyse de l’expression de l’oncogèneHER2 : la note de passage
Canadian Journal of P athology 7Spring 2013
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.
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
Oncology/College of American Pathologists guideline recommendations for
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.
Spring 20138 Canadian Journal of P athology
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
Canadian Journal of P athology 9Spring 2013
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.
Spring 201310 Canadian Journal of P athology
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).
Canadian Journal of P athology 11Spring 2013
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).
Spring 201312 Canadian Journal of P athology
EVOLUTION OF HER2 TESTING IN CANADA
A
B
Figure 6. Proportion of patients in whom HER2 status changedbetween the adjuvant and metastatic settings.
Canadian Journal of P athology 13Spring 2013
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.
Spring 201314 Canadian Journal of P athology
EVOLUTION OF HER2 TESTING IN CANADA
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
Canadian National Consensus Meeting on HER2/neu testing in breast cancer.
Curr Oncol 2007;14:149–53.
6. Wolff AC, Hammond MEH, 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. 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
Canadian Journal of P athology 15Spring 2013
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
Spring 201316 Canadian Journal of P athology
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.
Canadian Journal of P athology 17Spring 2013
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
Spring 201318 Canadian Journal of P athology
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.
Canadian Journal of P athology 19Spring 2013
XU ET AL.
molecular cytogenetics infrastructure used in this study was
funded by the Canadian Foundation for Innovation through
the Canadian Molecular Cytogenetics Platform.
References1. Masood S, Bui MM, Yung JF, et al. Reproducibility of LSI HER-2/neu
SpectrumOrange and CEP 17 SpectrumGreen Dual Color deoxyribonucleic acid
probe kit for enumeration of gene amplification in paraffin-embedded
specimens: a multicenter clinical validation study. Ann Clin Lab Sci 1998;28:215–
23.
2. Pauletti G, Dandekar S, Rong H, et al. Assessment of methods for tissue-based
detection of the HER-2/neu alteration in human breast cancer: a direct
comparison of fluorescence in situ hybridization and immunohistochemistry. J
Clin Oncol 2000;18:3651–4.
3. 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.
4. Wang S, Hossein Saboorian M, et al. Aneusomy 17 in breast cancer: its role in
HER2/neu protein expression and implications for clinical assessment of HER-
2/neu statues. Mod Pathol 2002;15:137–45.
5. Persons DL, Raymond R. Tubbs, et al. HER-2 fluorescence in situ hybridization:
results from the survey program of the College of American Pathologists. Arch
Pathol Lab Med 2006;130:325–31.
6. Gunn S, Yeh IT, Lytvak I, et al. Clinical array-based karyotyping of breast cancer
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
Spring 201320 Canadian Journal of P athology
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.
Canadian Journal of P athology 21Spring 2013
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).
Spring 201322 Canadian Journal of P athology
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.
Canadian Journal of P athology 23Spring 2013
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)
Spring 201324 Canadian Journal of P athology
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
Canadian Journal of P athology 25Spring 2013
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
Spring 201326 Canadian Journal of P athology
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
Canadian Journal of P athology 27Spring 2013
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
Spring 201328 Canadian Journal of P athology
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.
Canadian Journal of P athology 29Spring 2013
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.
Spring 201330 Canadian Journal of P athology
UNDERSTANDING ANTIBODY-MEDIATED REJECTION OF ORGAN TRANSPLANTS
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
Canadian Journal of P athology 31Spring 2013
SIS
• 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
Spring 201332 Canadian Journal of P athology
UNDERSTANDING ANTIBODY-MEDIATED REJECTION OF ORGAN TRANSPLANTS
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.
Canadian Journal of P athology 33Spring 2013
SIS
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
Spring 201334 Canadian Journal of P athology
UNDERSTANDING ANTIBODY-MEDIATED REJECTION OF ORGAN TRANSPLANTS
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.
References1. Canadian Institute for Health Information. Canadian Organ Replacement
Register Annual Report: Treatment of End-Stage Organ Failure in Canada,
2001 to 2010. Toronto (ON): The Institute; 2010.
2. Organ Procurement and Transplantation Network. Donors Recovered in the
U.S. by Donor Type, Donors Recovered: January 1, 1988 – January 31. Vienna
(VA): The Network; 2012.
3. Meier-Kriesche HU, Schold JD, Srinivas TR, Kaplan B. Lack of improvement
in renal allograft survival despite a marked decrease in acute rejection rates
over the most recent era. Am J Transplant 2004;4(3):378–83.
4. Sis B. Endothelial molecules decipher the mechanisms and functional
pathways in antibody-mediated rejection. Hum Immunol 2012;73(12):1218–
25.
5. Gaston RS, Cecka JM, Kasiske BL, et al. Evidence for antibody-mediated injury
as a major determinant of late kidney allograft failure. Transplant
2010;90(1):68–74.
6. Einecke G, Halloran PF. Antibody-mediated microcirculation injury is the
major cause of late kidney transplant failure: response to Dr. Loupy et al. Am
J Transplant 2010;10(4):953.
7. Sellares J, De Freitas D, Mengel M, Reeve J, Einecke G, Sis B, et al.
Understanding the causes of kidney transplant failure: the dominant role of
antibody-mediated rejection and non-adherence. Am J Transplant
2012;12(2):388–99.
8. El Zoghby ZM, Stegall MD, Lager DJ, et al. Identifying specific causes of kidney
allograft loss. Am J Transplant 2009;9(3):527–35.
9. Hidalgo LG, Campbell PM, Sis B, et al. De novo donor specific antibody at the
time of kidney transplant biopsy associates with microvascular pathology and
late graft failure. Am J Transplant 2009;9(11):2532–41.
10. Kissmeyer-Nielsen F, Olsen S, Peterson VP, Fjeldborg O. Hyperacute rejection
of kidney allografts associated with pre-existing humoral antibodies against
donor cells. Lancet 1966;2:662–5.
11. Jeannet M, Pinn VW, Flax MH, et al. Humoral antibodies in renal
allotransplantation in man. N Engl J Med 1970;282:111–7.
12. Halloran PF, Wadgymar A, Ritchie S, et al. The significance of the anti-class I
antibody response. I. Clinical and pathologic features of anti-class I-mediated
rejection. Transplant 1990;49(1):85–91.
13. Halloran PF, Schlaut J, Solez K, Srinivasa NS. The significance of the anti-class
I response II. Clinical and pathologic features of renal transplants with anti-
class I-like antibody. Transplant 1992;53:550–5.
14. Feucht HE, Felber E, Gokel MJ, et al. Vascular deposition of complement-split
products in kidney allografts with cell-mediated rejection. Clin Exp Immunol
1991;86(3):464–70.
Canadian Journal of P athology 35Spring 2013
SIS
15. Feucht HE, Schneeberger H, Hillebrand G, et al. Capillary deposition of C4d
complement fragment and early renal graft loss. Kidney Int 1993;43:1333–8.
16. Colvin RB, Smith RN. Antibody-mediated organ-allograft rejection. Nat Rev
Immunol 2005;5(10):807–17.
17. Mauiyyedi S, Colvin RB. Humoral rejection in kidney transplantation: new
concepts in diagnosis and treatment. Current Opin Nephrol Hyperten
2002;11(6):609–18.
18. Haas M, Rahman MH, Racusen LC, et al. C4d and C3d staining in biopsies of
ABO- and HLA-incompatible renal allografts: correlation with histologic
findings. Am J Transplant 2006;6(8):1829–40.
19. Kraus ES, Parekh RS, Oberai P, et al. Subclinical rejection in stable positive
crossmatch kidney transplant patients: incidence and correlations. Am J
Transplant 2009;9(8):1826–34.
20. Mauiyyedi S, Pelle PD, Saidman S, et al. Chronic humoral rejection:
identification of antibody-mediated chronic renal allograft rejection by C4d
deposits in peritubular capillaries. J Am Soc Nephrol 2001;12(3):574–82.
21. Regele H, Bohmig GA, Habicht A, et al. Capillary deposition of complement
split product C4d in renal allografts is associated with basement membrane
injury in peritubular and glomerular capillaries: a contribution of humoral
immunity to chronic allograft rejection. J Am Soc Nephrol 2002;13(9):2371–
80.
22. Vongwiwatana A, Gourishankar S, Campbell PM, et al. Peritubular capillary
changes and C4d deposits are associated with transplant glomerulopathy but
not IgA nephropathy. Am J Transplant 2004;4(1):124–9.
23. Sis B, Campbell PM, Mueller T, et al. Transplant glomerulopathy, late
antibody-mediated rejection and the ABCD tetrad in kidney allograft biopsies
for cause. Am J Transplant 2007;7(7):1743–52.
24. Colvin RB. Antibody-mediated renal allograft rejection: diagnosis and
pathogenesis. J Am Soc Nephrol 2007;18(4):1046–56.
25. Reed EF, Demetris AJ, Hammond E, et al. Acute antibody-mediated rejection
of cardiac transplants. J Heart Lung Transplant 2006;25(2):153–9.
26. McKenna RM, Takemoto SK, Terasaki PI. Anti-HLA antibodies after solid
organ transplantation. Transplant 2000;69(3):319–26.
27. Miller GG, Destarac L, Zeevi A, et al. Acute humoral rejection of human lung
allografts and elevation of C4d in bronchoalveolar lavage fluid. Am J
Transplant 2004;4(8):1323–30.
28. 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.
29. Drachenberg CB, Odorico J, Demetris AJ, et al. Banff schema for grading
pancreas allograft rejection: working proposal by a multi-disciplinary
international consensus panel. Am J Transplant 2008;8(6):1237–49.
30. Girnita AL, Lee TM, McCurry KR, et al. Anti-human leukocyte antigen
antibodies, vascular C4d deposition and increased soluble C4d in broncho-
alveolar lavage of lung allografts. Transplant 2008;86(2):342–7.
31. Musat AI, Agni RM, Wai PY, et al. The significance of donor-specific HLA
antibodies in rejection and ductopenia development in ABO compatible liver
transplantation. Am J Transplant 2011;11(3):500–10.
32. Bellamy CO, Herriot MM, Harrison DJ, Bathgate AJ. C4d immunopositivity
is uncommon in ABO-compatible liver allografts, but correlates partially with
lymphocytotoxic antibody status. Histopathology 2007;50(6):739–49.
33. Kozlowski T, Rubinas T, Nickeleit V, et al. Liver allograft antibody-mediated
rejection with demonstration of sinusoidal C4d staining and circulating
donor-specific antibodies. Liver Transpl 2011;17(4):357–68.
34. Watson R, Kozlowski T, Nickeleit V, et al. Isolated donor specific alloantibody-
mediated rejection after ABO compatible liver transplantation. Am J
Transplant 2006;6(12):3022–9.
35. Wiebe C, Gibson IW, Blydt-Hansen T, et al. Evolution and clinical pathologic
correlations of de novo donor-specific HLA antibody post kidney transplant.
Am J Transplant 2012;12(5):1157–67.
36. Einecke G, Sis B, Reeve J, et al. Antibody-mediated microcirculation injury is
the major cause of late kidney transplant failure. Am J Transplant
2009;9(11):2520–31.
37. Mengel M, Husain S, Hidalgo L, Sis B. Phenotypes of antibody-mediated
rejection in organ transplants. Transpl Int 2012;25(6):611–22.
38. Racusen LC, Colvin RB, Solez K, et al. Antibody-mediated rejection criteria –
an addition to the Banff 97 classification of renal allograft rejection. Am J
Transplant 2003;3(6):708–14.
39. Solez K, Colvin RB, Racusen LC, et al. Banff ʼ05 Meeting report: differential
diagnosis of chronic allograft injury and elimination of chronic allograft
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
Spring 201336 Canadian Journal of P athology
UNDERSTANDING ANTIBODY-MEDIATED REJECTION OF ORGAN TRANSPLANTS
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
Canadian Journal of P athology 37Spring 2013
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.
Our main referral centre, University Hospital of Northern BC, includes a catchment population of 350,000 and is
articipation in medical student and resident teaching through the Northern Medical P
Our main referral centre, University Hospital of Northern BC, includes a catchment population of 350,000 and is
rogram, University ofarticipation in medical student and resident teaching through the Northern Medical P
Our main referral centre, University Hospital of Northern BC, includes a catchment population of 350,000 and is
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
rogram, University of
rthernhealth.ca careers.no
rogram, University of
the northern way of caring
the northern way of caring
HER2/neu. . ....now part of Gamma-Dynacare’s test menu offering.
• Cytology
• Histology
• Immunohistochemistry
• Fluorecent In Situ Hybridisation (FISH)
• Allergy testing
• Flow Cytometry
• Esoteric testing
• Toxicology (SAMSHA accredited)
We provide customized and flexible
services, a comprehensive menu of
immunohistochemistry markers,
slide digitization and access to
industry experts and consultation.
www.gamma-dynacare.com
Make our laboratoriesan extension of your hospital,our pathologistsa part of your team!
Partner with us to help you make definitive diagnoses.
• Cytology
• Histology
• Immunohistochemistry
• Fluorescent In Situ Hybridization (FISH)
• Allergy testing
• Flow Cytometry
• Esoteric testing
• Toxicology (SAMHSA accredited)