Acta Ophthalmologica
Thesis
https://www.doczj.com/doc/3315743708.html,
Doctoral Thesis
Tumours of the Lacrimal Gland. Epidemiological,Clinical and Genetic Characteristics
Sarah Line a von Holstein
Eye Pathology Institute,Department of Neuroscience and Pharmacology,Faculty of Health and Medical Sciences, University of Copenhagen,Denmark
P hD Thesis
Tumours of the lacrimal gland Epidemiological,Clinical and Genetic Characteristics
Sarah Lin e a von Holstein
Faculty of Health and Medical Sciences,University of Copenhagen,Denmark
ABSTRACT
Tumours of the lacrimal gland are rare,but the prognosis may be grave.To date,no population-based incidence and distribution data on lacrimal gland tumours exist.In addition,almost nothing is known about the genetic pro?le of epithelial tumours of the lacrimal gland.
We collected specimens and clinical?les on all biopsied lacrimal gland lesions in Denmark over a34-year period and re-evaluated the diagnosis to provide updated population-based incidence rates and epidemiological charac-teristics.Clinical data regarding symptoms,clinical examinations,treatment and follow-up were collected for patients with adenoid cystic carcinoma(ACC), pleomorphic adenoma(PA),carcinoma ex pleomorphic adenoma(Ca-ex-PA) and mucoepidermoid carcinoma(MEC).Using RT-PCR,FISH,immunohis-tochemistry,Q-PCR and high-resolution array-based comparative genomic hybridization(arrayCGH)we explored the genetic characteristics including copy number alterations(CNA)in ACC,PA,Ca-ex-PA and MEC.
The incidence of biopsied lacrimal gland lesions was1.3/1000000/year,and ~50%were neoplastic lesions.Of these,55%were malignant tumours with epithelial tumours as the most frequent.The overall incidence was increasing, and this was caused by an increase in biopsied non-neoplastic lesions.We found that10/14ACCs either expressed the MYB–NFIB fusion gene and/or had rearrangements of MYB.All ACCs expressed the MYB protein.ACC was characterized by recurrent copy number losses involving6q,12q and17q and gains involving19q,8q and11q.ArrayCGH revealed an apparently normal genomic pro?le in11/19PAs.The remaining8PAs had recurrent copy number losses involving1p,6q,8q and13q and gain involving9p.PA expressed PLAG1 in all tumours whereas only2/29tumours expressed HMGA2.Ca-ex-PA was characterized by recurrent copy number gain involving22q.PLAG1was expressed in3/5Ca-ex-PA whereas none of these tumours expressed HMGA2. MEC expressed the CRTC1–MAML2,and this fusion was found to be tumour-speci?c for lacrimal gland MEC.
In conclusion,lacrimal gland lesions that require pathological evaluation are rare in the Danish population,and the incidence rate of biopsied benign lesions is increasing.Epithelial tumours of the lacrimal gland are molecularly very similar to their salivary gland counterparts in the expression of the tumour-speci?c fusion genes and in their genomic imbalances as demonstrated by arrayCGH.MYB–NFIB is a useful biomarker for ACC and MYB,and its downstream target genes may be potential therapeutic targets for these tumours.
Acta Ophthalmol.2013:91,thesis6:1–28
a2013The Author
Acta Ophthalmologicaa2013Acta Ophthalmologica Scandinavica Foundation
doi:10.1111/aos.12271Introduction
Lacrimal gland tumours are rare,but the patients are relatively young and the prognosis may be grave.There is signif-icant variability in the reported series regarding frequency and distribution of lacrimal gland lesions,and most series comes from clinical referral centres which makes a certain selection and referral bias unavoidable.Furthermore, the pathological classi?cation of epithe-lial lacrimal gland tumours has changed over the last decade,and it is now generally accepted that these tumours should be classi?ed in the same way as the corresponding tumours of the sali-vary glands(Eveson et al.2005;Font et al.2006).This impacts the prognosis and treatment,and warrants a re-eval-uation of diagnoses made in the past before any clear conclusion about inci-dence and distribution of distinct tumour subtypes can be made.
Tumours of the lacrimal gland are morphologically identical to those of the salivary glands,and consequently, treatment regimes of advanced lacrimal tumours are extrapolated from clinical trials of patients with salivary gland tumours(Le Tourneau et al.2011). The rationale for this is the morpho-logical and embryological similarities between the lacrimal gland and the salivary glands.However,there are di?erences in frequency,distribution and in clinical behaviour between tumours arising in the di?erent glands. Molecularly,it remains to be estab-lished how similar tumours of the salivary and lacrimal glands are.
1 Acta Ophthalmologica2013
In this study,we evaluated the fre-quency and distribution of lacrimal gland tumours based on a whole nation (I).All tumours were reclassi?ed and we included all lacrimal gland lesions subjected to biopsy or surgical removal to provide perspective as to what lesions are diagnostic challenging and require histopathological evaluation. We reviewed the clinical characteristics and current treatment regimes of epi-thelial tumour of the lacrimal gland (II),and the most important points from this study are presented in the https://www.doczj.com/doc/3315743708.html,ing high-resolution array-based comparative genomic hybridization(arrayCGH),RT-PCR, Q-PCR and immunohistochemistry we investigated the molecular character-istics of epithelial lacrimal gland tumours.The purpose was to clarify how similar lacrimal and salivary gland
tumours are and to identify biomarkers that may serve as diagnostic and/or prognostic markers as well as targets for therapeutic intervention(III,IV and V).
Anatomy and function of the lacrimal gland
The lacrimal gland is a tubulo-acinar gland composed of the main lacrimal gland and several smaller accessory glands.The main lacrimal gland is located in the upper temporal part of the orbit(Fig.1)and is incompletely separated in pars orbitalis and pars palpebralis by the aponeurosis of mus-culus levator palpebrae superioris.The orbital part is located in the lacrimal fossa on the anterior lateral area of the orbit,whereas the palpebral lobe lies below the aponeurosis in contact with the superior and lateral fornix of conjunctiva.The gland is composed of many lobules that are separated by loose connective tissue.Each lobule contains many acini that are connected via a network of ductules that con-tinue into yet larger ducts and?nally gather into6–12excretory ducts that open into the fornix of conjunctiva (Fig.1)(Kivel€a1992).The acini are the secretory unit,composed of a basal myoepithelial cell layer and an inner part with acinar cells(Obata2006). The lacrimal gland produces water, electrolytes and protein,and the majority of this is secreted by the acinar cells and to a lesser extent by the duct cells.The gland thus provides the aqueous component to the three-
layered structure of the tear?lm.The
functions of the tear?lm include the
primary source of oxygen to the avas-
cular cornea,contributing to the
smooth optical properties of the cor-
neal surface and serve as a lubricant
between the eyelid and the ocular
surface.In addition,acinar cells pro-
duce various cytokines such as lyso-
zyme and lactoferrin both of which
inhibit bacterial growth.Plasma cells
in the connective tissue are responsible
for production of immunoglobulins
and in particular IgA(Franklin&
Montgomery1996).The lacrimal
gland hereby also plays an important
role in the immunity of the ocular
surface.
Epidemiology
Frequency and distribution
Lacrimal gland tumours represent6–
12%of all orbital space-occupying
lesions and approximately22–28%of
these are primary epithelial tumours
(Kennedy1984;Shields et al.1989,
2004a,b;Johansen et al.2000).The
most common tumour is the benign
pleomorphic adenoma(PA)that com-
prises around50%of epithelial
tumours(Shields et al.1989;Ni et al.
1992;Johansen et al.2000;Zeng et al.
2010).The remaining50%of the
primary epithelial tumours are malig-
nant.The most frequently encountered
is adenoid cystic carcinoma(ACC)
comprising20–30%,whereas carcinoma
ex pleomorphic adenoma(Ca-ex-PA)
constitutes~10%,adenocarcinoma not
otherwise speci?ed(NOS)5–10%and
mucoepidermoid carcinoma(MEC)1–
2%(Font&Gamel1978;Ni et al.1992;
Shields et al.2004a;Zeng et al.2010).
Other epithelial tumours have been
reported but they are exceptionally rare
with only a few single cases reported
(Font et al.2006;Weis et al.2009).
Lymphomas are the most common
non-epithelial tumours comprising
14%of lacrimal gland tumours(Shields
et al.2004b).The remaining non-epi-
thelial tumours are extremely rare.
Tumours arising from the surrounding
tissue may also invade the lacrimal
gland,but these are often excluded in
larger series,and consequently,the fre-
quency is largely unknown.
Demographics and etiology
Tumours of the lacrimal gland may
occur in patients of all ages,and this
largely depends on the type of tumour.
In contrast to lacrimal gland lympho-
mas that primarily occur in elderly
patients(Rasmussen et al.2012),epi-
thelial tumours are a disease of middle-
aged adults.PA typically presents in
patients with a mean age of40years;
however,it does occur in children as
well as in patients up to80years of age
(Font et al.2006).Patients with ACC
are usually diagnosed in their forties,
but a smaller proportion of patients are
diagnosed in the teenage years(Tellado
et al.1997).Ca-ex-PA,MEC and ade-
nocarcinoma NOS all have a mean age
at presentation of approximately
50years(Font&Gamel1978;Ni et al.
(A)
(B)(C)
Fig.1.Anatomy of the lacrimal gland.(A)Microphotograph illustrating the histology of the lacrimal gland.(B)Schematic drawing of the structure of the lacrimal gland duct system.(C)The lacrimal gland is located in the upper temporal part of the orbit.
2
Acta Ophthalmologica2013
1992;Zeng et al.2010).MEC is diag-nosed slightly more often in females (3:2),and adenocarcinoma more com-monly in males,whereas there is no apparent gender predilection for PA, ACC or Ca-ex-PA(Eviatar&Hornb-lass1993;Heaps et al.1993).
Neither race nor geography has been reported as predisposing factors for the development of epithelial tumours of the lacrimal gland(Font&Gamel 1978;Ni et al.1992;G€u nalp&G€u nd€u z 1994;Seregard&Sahlin1999;Zeng et al.2010).However,owing to the rarity of the tumours such di?erences might be undetected,and for the cor-responding tumours of the salivary gland,all but MEC and ACC are found more frequently among White people than Black people and Asians (Boukheris et al.2009).Similarly, studies demonstrating exogenous risk factors do not exist for lacrimal gland tumours but,although such reports also are scarce for tumours of the salivary gland,ionized radiation and tobacco have been implicated in the oncogenic process for some salivary gland carcinoma subtypes(Land et al. 1996;Sadetzki et al.2008).
Clinical characteristics
Symptoms
Typical?rst presenting symptoms of all types of lacrimal gland tumours include facial asymmetry due to dis-placement of the eyeball(Figs2A and 3A),swelling of the lacrimal gland (Fig.4A),reduced eye motility,diplo-pia and ptosis(Rose&Wright1992; Wright et al.1992;Eviatar&Hornb-
lass1993;Heaps et al.1993).Pain is not common in patients with benign tumours and should raise suspicion of malignancy.However,pain is a cardinal symptom in patients with ACC,and up to80%have been reported to have pain as a?rst pre-senting symptom(Wright et al.1992; Font et al.2006).The mean duration of symptoms before the?rst ophthal-mic consultation is typical longer for patients with benign tumours than for patients with malignant tumours. Accordingly,patients with PA have an average of2years with symptoms before seeing a physician,whereas patients with ACC have had symptoms for approximately6months(Lee et al. 1985;Rose&Wright1992;Zeng et al. 2010).Diagnosis
The presurgical diagnosis is based on
the clinical history and image analysis.
On CT and MRI scans,PA typically
appears as a solid,well-de?ned,round
or oval space-occupying lesion
(Fig.2B)that occasionally shows signs
of calci?cation and bone remodelling.
This is in contrast to malignant
tumours that may have irregular mar-
gins and signs of bone erosion(Eviatar
&Hornblass1993;Vaidhyanath et al.
2008).In addition,ACC may appear
nodular(Fig.3B),in?ltrate adjacent
tissue and cause bone destruction(Va-
idhyanath et al.2008).
The?nal diagnosis of lacrimal
tumours is made after histopathologi-
cal evaluation of the lesion.For many
years,incisional biopsy of a lacrimal
gland mass caused much concern
among surgeons owing to the risk of
recurrence and malignant transforma-
tion of PA associated with the proce-
dure(Font&Gamel1978;Wright
et al.1979;Rose&Wright1992).
However,in recent years,it has become
generally accepted that incisional
biopsy of PA can be performed
although with caution,as long as the
biopsy tract is subsequently removed
(Lai et al.2009).Fine needle biopsy of
orbital tumours has been shown to be
useful and reliable in making diagnosis,
and some surgeons prefer this to an
open approach(Tani et al.2006);in
addition,the procedure does not seem
to be associated with an increased risk
of recurrence(Ka?enberger et al.
2010).
(B)
(A)
(D)
(C)
(F)
(E)
Fig.2.(IV).(A)A45-year-old woman with painless swelling progressing over2years and with downwards,medial displacement of the right eye(arrow)(case21,Table10).(B)Computed tomography scan of the same patient as in(A)demonstrating a well-de?ned ovoid tumour in the right orbit(arrow)originating in the lacrimal gland.(C)Microscopy showed ductal structures surrounded by a myxoid stroma consistent with a diagnosis of pleomorphic adenoma(case21, Table10)(HE staining,original magni?cation910).(D)Carcinoma ex pleomorphic adenoma (case18,Table10)with a carcinoma component reminiscent of a high-grade mucoepidermoid carcinoma(HE staining,original magni?cation920).(E)Pleomorphic adenoma(case28, Table10)with strong nuclear immunoreactivity for PLAG1(original magni?cation920).(F) Pleomorphic adenoma(case20,Table10)with strong nuclear immunoreactivity for HMGA2 (original magni?cation940).
3
Acta Ophthalmologica2013
Histopathological characteristics Pleomorphic adenoma is usually a well-de?ned tumour,characterized by epithelial and modi?ed myoepithelial elements that intermingle with mesen-chymal components(Fig.2C)(Barnes et al.2005).The epithelial component
manifests itself as a mixture of well-
formed ductal structures and non-duc-
tal cells that include spindle,round,
stellate,plasmacytoid,oncocytoid,
polygonal and clear cells.Occasional
squamous elements are present.The
mesenchymal component demonstrates
varying degrees of myxoid,hyaline,
cartilaginous or osseous di?erentiation.
Immunohistohemically,the epithelial
cells of PA and occasionally the myo-
epithelial cells express cytokeratin.The
myoepithelial cells may express muscle-
speci?c antigen and glial?brillary
acidic protein(Grossniklaus et al.
1990;Eveson et al.2005).
Ca-ex-PA is a carcinoma that shows
histological evidence of arising in or
from a PA.Histological analysis usu-
ally reveals residual benign PA with
zones of transition between the benign
and malignant parts.In the majority of
tumours,there is an obvious in?ltrative
growth pattern accompanied with sig-
ni?cant cytological atypia.The carci-
nomatous element can be any type of
carcinoma,but adenocarcinoma NOS
and MEC(Fig.2D)are the most
frequent forms(Font et al.2006).
Adenoid cystic carcinoma is a malig-
nancy of modi?ed myoepithelial and
ductal di?erentiated cells(Barnes et al.
2005;Font et al.2006).It is character-
ized by three histological growth pat-
terns:the cribriform(‘Swiss cheese’or
sieve-like),solid and tubular forms,
seen in varying combinations and
dominance(Fig.3D).The cribriform
pattern is most common,while the
solid pattern is least frequent.How-
ever,there is usually a mixture of
patterns within each.Within the cyst-
like structures of the cribriform type,
the connective tissue stroma contains
an accumulation of basophilic amor-
phous glycosaminoglycans and/or
eosinophilic and hyalinised basal lam-
ina.In the tubular and solid ACC,
basaloid myoepithelial cells dominate;
particularly,in the latter subtype,there
is a scarcity or absence of glycosami-
noglycan and basal lamina-containing
cyst-like spaces(Yamamoto et al.
1992).The perineural growth pattern
of ACC is a hallmark of these tumours
and contributes to the intractable
nature of this disease.The immunohis-
tochemical pro?le of ACC is not fully
clari?ed;however,Ki-67immunostain-
ing may be helpful in di?erentiating
ACC from the less aggressive polymor-
phous low-grade adenocarcinoma,but
there is no clear association between
Ki-67and clinical outcome(Eveson
et al.2005).Similarily,KIT,EGRF
and E-cadherin are positive in the
majority of cases,but the correlation
with prognosis is unclear(Eveson et al.
2005;Bell et al.2010).
(B)
(A)
(D)
(C)
Fig.3.(A)A41-year-old man(case10,Table9)with proptosis of the left eye(arrow)developing over6months.(B)MR scan of the same patient(case10,Table9)demonstrating a tumour in the upper lateral part of the orbit(arrow).(C)The tumour was removed by a lateral orbitotomy.(D) Microphotograph of the tumour(case10,Table9)showing a mainly cribriform adenoid cystic carcinoma composed of nests of cells with microcystic spaces?lled with basophilic mucoid and hyaline material(HE staining,original magni?cation910).
(B)
(A)
(D)
(C)
Fig.4.(V).(A)A73-year-old man with swelling of the left lacrimal gland(arrow).(B)Computed tomography scan showing the tumour at the site of the left lacrimal gland(asterisk).(C)Micro-photograph of the tumour demonstrating cystic spaces lined by epidermoid,intermediate and mucus-producing cells consistent with the diagnosis of mucoepidermoid carcinoma(HE staining, original magni?cation910).(D)Immunostaining of the CRTC1–MAML2fusion protein (original magni?cation920).Note the predominant nuclear staining of the tumour cells.
4
Acta Ophthalmologica2013
MEC is composed of three basic cellular elements:epidermoid-squa-mous,mucous-secreting and interme-diate cells(Fig.4C).In addition,there may be admixed columnar-,clear-and oncocytic cells(Font et al.2006). Mucous-secreting cells may be large, ovoid or goblet-shaped,possessing an abundant foamy cytoplasm.Intracyto-plasmic mucin can be visualized using periodic acid-Schi?(PAS),but in many tumours,the mucin is only evident with the use of special stains,such as muci-carmine or Alcian blue(Auclair et al. 1992;Font et al.2006).
Treatment
The primary treatment of lacrimal gland tumours is surgical removal. The classically advocated surgical approach for lacrimal gland masses is through a lateral orbitotomy with removal of the lateral wall of the orbit (Fig.3C).Orbital exenteration is the needed when the lacrimal carcinoma is extensive and the mass has in?ltrated beyond its capsule.The surgery will in most cases of malignant tumours be followed by radiotherapy or chemo-therapy(Garden et al.1995;Shields& Shields2008).PA may also bene?t from radiotherapy when not amenable to surgery(Mendenhall et al.2008). However,the treatment for malignant lacrimal gland tumours,and particular ACC is a subject of controversy.The type of surgery(e.g.globe-sparring versus exenteration)has not been dem-onstrated convincingly to correlate with the prognosis(Lee et al.1985; Wright et al.1992;Skinner et al.2011), and the type of adjuvant treatment and the combination of surgery with radi-ation and/or chemotherapy are highly debated.Some authorities advocate globe-sparring surgery followed by external beam(Wright et al.1992)or proton beam radiation(Pommier et al. 2006)and others suggest brachythera-py(Shields et al.2003).Meldrum et al. (1998)suggested neoadjuvant intracar-otid chemotherapy.This treatment regime includes pre-and postoperative intracarotid chemotherapy,orbital exenteration and postoperative radio-therapy and although the toxicity is high,the potential for cure is encour-aging(Meldrum et al.1998;Tse et al. 2006).The rarity of malignant lacrimal gland tumours and the tendency for late metastasis in ACC make evalua-tion of new treatment regimes chal-lenging.Accordingly,randomized
controlled clinical trials are non-exis-
tent,and most data on chemotherapy
and molecularly targeted agents are
derived from data obtained from sim-
ilar tumours of the salivary gland(Le
Tourneau et al.2011).
Prognosis
The prognosis of PA is good,unless
there has been disruption of the capsule
and this has lead to local recurrence
(Currie&Rose2007;Lai et al.2009).
Multiple local recurrences increase the
risk of malignant transformation as do
longstanding tumours and age>45
years(Font et al.2006;Suh et al.
2009).The clinical course of malignant
epithelial lacrimal tumours is often
aggressive.Most patients with Ca-ex-
PA die within few years following
diagnosis,the mean survival being only
3years(Wright et al.1992;Font et al.
1998).In salivary gland Ca-ex-PA,the
prognosis is known to correlate with
the invasiveness(extension beyond the
capsule or not)and the histological
grade of the carcinoma component
(Seethala2009).ACC is often referred
to as the slow killer as recurrences and/
or metastases may occur several years
after the initial treatment.Patients with
ACC die from distant metastases and
intracranial spread,and the chances of
surviving10years after diagnosis are
only20–30%(Wright et al.1992;Font
et al.2006).‘Solid’histology and
tumour size correlate with a worse
prognosis for patients with ACC
(Hamper et al.1990;Ahmad et al.
2009).MEC is graded in low-,inter-
mediate-and high-grade types accord-
ing to a scoring system de?ned by the
WHO(Eveson et al.2005),and
whereas the low-and intermediate-
grade MECs have a favourable prog-
nosis,the high-grade tumours are asso-
ciated with poor survival(Eviatar&
Hornblass1993).
Cancer genomics
Genes and cancer
‘Cancer is,in essence,a genetic disease
at the cellular level’(Vogelstein&
Kinzler2004).A normal cell is con-
verted into a neoplastic cell by a series
of genetic events that liberates it from
its normal proliferation and growth
control mechanisms(Klein&Klein
1985;Weinberg1989;Bishop1991).A
varying number of genetic alterations
are needed for this to happen,depend-
ing on the cell and tumour type(Hahn
et al.1999;Hanahan&Weinberg
2000).The majority of all neoplasms
are sporadic lesions that occur when a
somatic cell has accumulated a su?-
cient number of genetic alterations for
a tumour to develop.Approximately
10%of all cancers are hereditary neo-
plasms caused by predisposing muta-
tions in germ-line cells.
Two types of cancer-initiating
genetic events have been identi?ed:
(i)inactivation of genes by deletions,
mutations or epigenetic mechanisms,
and(ii)activation/deregulation of
genes by point mutations,ampli?ca-
tions or gene fusions caused by bal-
anced chromosome translocations
(Mitelman et al.2007).In order for
these genetic events to lead to cancer,
they must a?ect genes that play impor-
tant roles in for example transcrip-
tional regulation,signal transduction
and growth control.These cancer genes
can be divided into oncogenes,tumour
suppressor genes and DNA repair
genes.
Oncogenes are called proto-oncoge-
nes in the normal cell where their
function is to regulate for example cell
proliferation,gene transcription and
di?erentiation(Weinberg1994).
Proto-oncogenes are dominant genes,
and gain-of-function mutations in one
allele are,therefore,enough to disrupt
and/or deregulate the expression and
convert proto-oncogenes into oncoge-
nes.Examples of well-known oncoge-
nes are MYC(Whit?eld&Soucek
2012)and MYB(Ramsay&Gonda
2008),both of which encode transcrip-
tion factors.Fusion oncogenes are a
special type of oncogene that consists
of the full length and/or parts of two
genes that form novel functional tran-
scription units( Aman2005).They are
formed as a result of chromosome
rearrangements,in particular translo-
cations,inversions and insertions.
There are two major types of fusion
genes.In the?rst type,the breaks occur
within the coding regions of one or
both genes,resulting in formation of a
new chimeric gene encoding a chimeric
fusion protein.In the second type,the
breaks occur within the non-coding
regions of both genes,resulting in
exchange of5′-regulatory elements
and consequently overexpression of a
normal but ectopically expressed pro-
tein.This mechanism is also known as
5
Acta Ophthalmologica2013
promoter swapping (Kas et al.1997; Aman 1999),and an example of this is the t(3;8)(p21;q12)translocation in PA that results in activation of the PLAG1gene due to promoter swapping with the ubiquitously expressed CTNNB1gene (Fig.5).Recurrent translocations often result in formation of tumour-type-speci?c fusion oncogenes that may be used as diagnostic biomarkers (Rabbitts 1994;Lengauer et al.1998; Aman 1999;Rowley 2001;Scandura et al.2002;Mitelman et al.2004;Stenman 2005).
Tumour suppressor genes are growth-constraining and counterbal-ance the growth promoting proto-onc-ogenes.Inactivating loss-of-function mutations in tumour suppressor genes liberates the cell from the constrains imposed by these genes and contributes to the uncontrolled growth behaviour of cancer cells (Weinberg 1994).Tumour suppressor genes are recessive in nature,and consequently,both alleles must be inactivated in tumour cells.This can be achieved by deletions,mutations or DNA methylation (Jones &Laird 1999;Sherr 2004;Weinberg 2007).Proteins encoded by tumour suppressor genes are involved in cellu-lar functions,such as transcriptional regulation,inhibition of proliferation,apoptosis and genetic stability.Exam-ples of well-known tumour suppressor genes are TP53(Muller &Vousden 2013)and BRCA1(Roy et al.2012).DNA repair genes are also referred to as caretaker genes as they are responsible for the repair of mistakes during normal DNA replication and for the repair of damaged DNA due to
exposure to mutagenes (Peltomaki &de la Chapelle 1997;Gupta &Lutz 1999;Vogelstein &Kinzler 2004).Mutation and inactivation of DNA repair genes result in increased genomic instability and accumulation of addi-tional mutations that may a?ect proto-oncogenes and tumour suppressor genes (Vogelstein &Kinzler 2004).In addition to the above-mentioned genes,genes encoding microRNAs (miRNA)may also play an important role in the neoplastic process.They can function as tumour suppressor genes or oncogenes by post-transcriptional reg-ulation of the expression of target genes involved in oncogenesis.miR-NAs are evolutionarily conserved,small (~22nucleotides)non-coding,endogenous RNAs that play an impor-tant role in the regulation of a variety of biological processes,such as cell di?erentiation,proliferation,organ development,maintenance of stem cell potency and apoptosis (Reinhart et al.2000;Ambros 2004;Chan et al.2005;Cheng et al.2005).
Genetic alterations in the above-mentioned genes disrupt the balance between cell proliferation and cell death and through a succession of genetic changes leads to cancer devel-opment.Although every cancer has a distinct phenotype,all cancer cells must acquire eight functional capabilities in order for the cancer cells to survive,proliferate and disseminate (Hanahan &Weinberg 2000,2011).The eight hallmarks of cancer are as follows:(i)sustaining proliferative signalling,(ii)evading growth suppressors,(iii)evading apoptosis,(iv)limitless replicative potential,(v)sustained angiogenesis,(vi)tissue invasion and metastasis,(vii)reprogramming of energy metabolism and (viii)evading immune destruction.Underlying the hallmarks of cancer is genomic insta-bility and in?ammation (Hanahan &Weinberg 2011).The genomic instabil-ity leads to mutant genotypes that have selective growth advantages over the adjacent cells,enabling their outgrowth in the surrounding tissue.The in?am-mation contributes to multiple hall-mark capabilities by supplying bioactive molecules to the microenvi-ronment as well as inductive signals that activates hallmark-facilitating cel-lular programs.
Understanding which genes and pathways that are activated in speci?c
types of cancer is essential when searching for new diagnostic/prognos-tic biomarkers and therapeutic targets.For epithelial tumours of the lacrimal gland,and particularly ACC,the need for understanding the underlying mechanisms is urgent as current treat-ment regimes do not seem to prevent patients form developing recurrencies and/or metastases.
Genetic characteristics of epithelial tumours of the lacrimal and salivary gland
To date,only little has been published about the genetics of lacrimal gland tumours (Hrynchak et al.1994;Pers-son et al.2009b;Brill et al.2011),but numerous studies have been published about the genetics of the corresponding tumours of the salivary glands.Table 1gives an overview of the most impor-tant genes deregulated as a result of gene fusions in salivary gland tumours.Tumours originating in the salivary and lacrimal glands are thought to be very similar genetically because they have identical morphology and embry-ology (Le Tourneau et al.2011).There are,however,di?erences in frequency and distribution of tumour subtypes and patients with ACC of the lacrimal gland are younger and have a worse prognosis than patients with ACC of the salivary glands (Li et al.2012).
Pleomorphic adenoma and carcinoma ex pleomorphic adenoma.Extensive cytoge-
netic studies of salivary gland PA have revealed that they are characterized by a highly speci?c pattern of chromo-some rearrangements including at least four major subgroups (Stenman 2005).One subgroup has rearrangements of 8q12,involving the oncogene gene PLAG1(pleomorphic adenoma gene 1).The proto-oncogene PLAG1is a transcription factor gene (Kas et al.1998)that is developmentally regulated and expressed mainly in fetal lung,liver and kidney tissues but not in adult tissues (Kas et al.1997).When PLAG1is overexpressed,the protein has onco-genic properties demonstrated by its ability to transform NIH3T3cells and produce tumours in nude mice (Hensen et al.2002).This oncogenic e?ect seems to be mediated by induction of growth factors and in particularly by activation of the IGF2(Insulin-like Growth Factor 2)signalling pathway (Voz et al.2000;Stenman 2005).IGF2is a growth factor that plays a
role
Fig.5.Fusion oncogenes in pleomorphic ade-noma,adenoid cystic carcinoma and mucoep-idermoid carcinoma of the salivary gland.The lines have the same colour as the names of the tumours in which the speci?c fusions occur.
6
Acta Ophthalmologica 2013
in embryogenesis and also in many
cancers including Wilm’s tumour,
rhabdomyosarcoma and neuroblas-
toma(Toretsky&Helman1996;Pollak
2008).Overexpression of PLAG1has
been demonstrated in tumours such as
PAs,lipoblastomas,uterine leiomyo-
mas and leiomyosarcomas( Astr€o m et al.1999;Hibbard et al.2000).In
salivary gland PAs,the8q12-rear-
rangements commonly result in the
formation of fusion oncogenes in
which PLAG1is overexpressed owing
to promoter swapping with the ubiqui-
tously expressed genes CTNNB1,
LIFR,TCEA1,CHCHD7or FGFR1
(Fig.5)(Kas et al.1997;Voz et al.
1998; Astr€o m et al.1999;Asp et al. 2006;Persson et al.2008).
The second subgroup has rearrange-
ments of12q14–15,involving the
HMGA2gene(high motility group
protein gene).The12q14–15rearrange-
ments commonly result in fusions in
which the3′-end of HMGA2is replaced
by the3′-end of a variety of other genes
leading to activation of HMGA2(Sten-
man2005).In salivary gland PA,at
least three fusion partners have been
identi?ed namely WIF1,FHIT and
NFIB(Fig.5)(Geurts et al.1997,
1998;Stenman2005).HMGA2is an
architectural transcription factor
involved in the assembly of nucleopro-
tein complexes(Grosschedl et al.
1994),and the expression is restricted
to embryonic tissue(Rogalla et al.1996;Rommel et al.1997;Gattas et al.
1999).HMGA2is involved in growth
regulation,and cell proliferation
(Ashar et al.2010)which is illustrated
by the induction of the pygmy pheno-
type and reduced overall body adipose
tissue in HMGA2knockout mice
(Zhou et al.1995).The oncogenic
properties of HMGA2have been dem-
onstrated by the gene’s ability in trun-
cated form to transform NIH3T3cells
(Fedele et al.1998)and by the forma-
tion of tumours in transgenic mice
overexpressing or misexpressing
HMGA2(Fedele et al.2002;Zaidi
et al.2006).The down-stream targets
of HMGA2include the cell cycle reg-
ulators CCNA and CCNB2(Tessari
et al.2003;De Martino et al.2009).
Overexpression of HMGA2is found in
many tumours such as salivary gland
PAs,lipomas,uterine leiomyomas,?-
broadenomas of the breast,pituitary
adenomas,pancreatic carcinoma,lung
carcinoma and squamous cell carci-
noma of the oral cavity(Sreekantaiah
et al.1991;Staats et al.1996;Hennig
et al.1997;Abe et al.2003;Miyazawa
et al.2004;Pierantoni et al.2005;
Meyer et al.2007).
The last two subgroups of PAs con-
tain tumours with non-recurrent clonal
changes and tumours with an appar-
ently normal karyotype(Stenman
2005).
Ca-ex-PA is the result of malignant
transformation of a benign PA.Con-
sequently,many of the genetic altera-
tions found in PA may also be present
in Ca-ex-PA such as alterations involv-
ing PLAG1and HMGA2(Hrynchak
et al.1994;Persson et al.2009a).In
addition,several alterations have been
suggested to be of importance for
malignant transformation such as
ampli?cation of ERBB2and12q genes
(MDM2in particular),deletions of
5q23.2–q31.2,gains of8q12.1(PLAG1)
and8q22.1–q24.1(MYC)(Persson
et al.2009a).Loss of17p has also been
suggested to play a role in the late
event of the malignant transformation
(El-Naggar et al.2000).The tumour
suppressor gene TP53is located at
17p13and has been reported by several
authors to be implicated in the malig-
nant transformation of PA(Nordkvist
et al.2000;Fowler et al.2006;Ihrler
et al.2007).To date,however,the
key oncogenic events remain to be
elucidated.
Adenoid cystic carcinoma.In1994Hryn-
chak et al.described one lacrimal ACC
with the t(6;9)(q23;p22)translocation.
Interestingly,at about the same time,
Nordkvist and co-workers showed,
that the t(6;9)(q22–23;p23–24)chromo-
somal translocation was a recurrent
and tumour-type-speci?c event in ACC
of the head and neck(Nordkvist et al.
1994).More recently,Persson and co-
workers demonstrated that the trans-
location results in a MYB–NFIB fusion
oncogene in ACC of the breast and
head and neck(Fig.5)(Persson et al.
2009b).Subsequent studies have shown
that MYB–NFIB is expressed in more
than85%of ACCs and that the major
consequence of the fusion is activation
of the MYB oncogene(Brill et al.2011;
Mitani et al.2011;West et al.2011;
Persson et al.2012).MYB encodes a
transcription factor that regulates a
large number of target genes including
such as MYC,KIT,BCL2,CCNE1
and CCNB1(Ramsay&Gonda2008).
Knockout studies have demonstrated
that MYB plays an important role in
haematopoiesis(Mucenski et al.1991;
Emambokus et al.2003),as regulator
for progenitor cells in colonic crypts
(Malaterre et al.2007)and in neuro-
genic niches in the adult brain(i.e.
specialized compartments in the adult
brain in which ongoing production of
neurons take place)(Malaterre et al.
2008).MYB is highly expressed in
immature,proliferating cells and is
Table1.Important genes involved in fusion oncogenes in salivary gland epithelial tumours. Gene Function Involvement in cancer
PLAG1Transcription factor expressed
mainly in fetal lung,liver and
kidney tissue Pleomorphic adenoma of the salivary glands Lipoblastoma
Uterine leyimyoma
Leiomyosarcoma
HMGA2Architectural transcription factor
involved in the assembly of
nucleoprotein complexes Pleomorphic adenoma of the salivary glands Lipomas
Uterine leiomyomas
Fibroadenomas of the breast
Pituitary adenomas
Pancreatic carcinoma
Lung carcinoma
Squamous cell carcinoma of the oral cavity
MYB Transcription factor that plays an
important role in haematopoiesis,
as regulator for progenitor cells in
colonic crypts and in neurogenic
niches in the adult brain Adenoid cystic carcinoma of the head and neck Colon cancer
Oestrogen receptor-a positive breast cancer Leukaemia
CRTC1CREB co-activator.CREB is a
transcription factor that regulates
numerous of genes involved in cell
proliferation,di?erentiation and
apoptosis Mucoepidermoid carcinoma of the salivary
glands,thyroid,cervix and lung
Warthins tumour
Clear cell hidradenoma of the skin
7
Acta Ophthalmologica2013
down-regulated as cells become di?er-entiated(Ramsay&Gonda2008). MYB may be activated by mutations in a regulatory sequence in intron1or by structural alterations and/or geno-mic duplications and has been impli-cated in colon cancer,oestrogen receptor-a positive breast cancer and certain leukaemias(Anfossi et al.1989; Guerin et al.1990;Calabretta et al. 1991;Ramsay et al.1992).In MYB–NFIB fusion-positive salivary ACCs,a possible mechanism for MYB activa-tion is loss of binding sites for nega-tively regulating miRNAs in the3′-UTR as a result of the fusion of MYB to NFIB(Persson et al.2009b).Recent studies have demonstrated that in ACC MYB may also be activated by other complex rearrangements than gene fusion(Mitani et al.2011;Persson et al.2012),thus further emphasizing the signi?cance of MYB activation in ACCs.In addition to activation of MYB,salivary gland ACCs are char-acterized by recurrent genomic imbal-ances,involving segmental losses of1p, 5q,6q,11q,12q and14q and segmental gains involving1q and22q(Persson et al.2012).The target genes of most of these rearrangements are,however,still unknown.
Mucoepidermoid carcinoma.MEC of the salivary glands is characterized cytoge-netically by a high percentage of tumours having a t(11;19)(q21;p13) translocation(Stenman2005).The translocation results in formation of the fusion oncogene CRTC1–MAML2 (Fig.5)(Tonon et al.2003;Behboudi et al.2006).CRTC1encodes a cAMP response element-binding protein (CREB)co-activator,whereas MAML2(mastermind-like2)encodes a nuclear protein that functions as a co-activator for NOTCH receptors.As a result of the fusion,the N-terminal NOTCH-binding domain of MAML2 is replaced by the CREB-binding domain of CRTC1.The fusion protein is believed to activate or interact with target genes mainly in the cAMP/ CREB-pathway but also in the NOTCH-pathway(Tonon et al.2003; Enlund et al.2004;Coxon et al.2005; Wu et al.2005;Komiya et al.2006). CREB is a transcription factor that regulates numerous of genes involved in cell proliferation,di?erentiation and apoptosis,and deregulation of CREB has been implicated in a number of cancers(Mayr&Montminy2001).
Studies have demonstrated that sus-
tained expression of CRTC1–MAML2
protein is necessary for tumour cell
growth in CRTC1–MAML2positive
MEC cells(Komiya et al.2006),and
the fusion has been identi?ed in MECs
of a number of di?erent anatomical
locations in addition to the salivary
gland including the lung,thyroid and
cervix(Tonon et al.2003;Enlund et al.
2004;Behboudi et al.2006;Tirado
et al.2007;Lennerz et al.2009).Taken
together,these?ndings indicate that
CRTC1–MAML2is an early genetic
event and a driver mutation in
MEC regardless of anatomical loca-
tion.Expression of CRTC1–MAML2
is preferentially found in low-
grade MECs,and there seems to be
an association between expression of
the fusion and less aggressive
tumour behaviour(Behboudi et al.
2006;Okabe et al.2006;Miyabe et al.
2009).
Hypotheses and aims of
the study
Hypothesis
The following hypotheses formed the
basis for this study of lacrimal gland
tumours in the Danish population:
The incidence of lacrimal gland
tumours in the Danish population
is low and has not increased during
the last35years.
The distribution of di?erent types of
lacrimal gland tumours as well as
the demographic and clinical char-
acteristics of the patients in Den-
mark is similar to what is reported
from other parts of the world.
New treatment strategies for
patients with lacrimal gland carci-
nomas are currently under develop-
ment.
Epithelial tumours of the lacrimal
gland have similar genetic altera-
tions as the corresponding tumours
of the salivary glands:
PAs overexpresses PLAG1and/or
HMGA2have rearrangements
involving8q and12q,but many
cases will have no or very few
genetic changes
Ca-ex-PA has the same genetic
alterations as PA,and in addition,
other alterations that are of impor-
tance for malignant transformation.
ACC expresses the MYB–NFIB
fusion oncogene has high expression
of MYB and has genetic imbalances
including segmental losses involving
1p,5q,6q,11q,12q,14q and
segmental gains involving1q and
22q.
MEC expresses the CRTC1–
MAML2fusion oncogene.
Genetic changes identi?ed in the
tumours can be linked to the onco-
genic process and serve as diagnostic
biomarkers and potential therapeu-
tic targets.
Aims
To address these hypotheses,this thesis
is composed of?ve papers with the
following aims:
To determine the incidence,time-
trends,distribution and epidemio-
logical characteristics of all biopsied
lacrimal gland lesions in Denmark
between1974and2007(Paper I).
To review the current state of the art
regarding diagnosis,treatment and
prognosis for epithelial tumours of
the lacrimal gland(Paper II).
To characterize ACC of the lacrimal
gland clinically and genetically with
special emphasis on MYB–NFIB,
MYB and other genomic imbalances
(Paper III).
To study genomic imbalances and
the expression of the translocation
target genes PLAG1and HMGA2in
PA and Ca-ex-PA(Paper IV).
To investigate whether lacrimal
gland MEC expresses the tumour-
type-speci?c gene fusion CRTC1–
MAML2(Paper V).
Material and methods
Material
Patients and tumour material
The thesis is based on tumours identi-
?ed by reviewing pathology reports
?led at the Eye Pathology Section,
University of Copenhagen.In addition,
we searched the National Danish
Pathology Databank using SNOMED
(Systemised Nomenclature of MEDi-
cine)codes.Topographical codes for
the lacrimal gland,lacrimal apparatus
and orbit were used,to minimize the
number of cases missed because of
unspeci?c coding and coding errors.
All lacrimal gland lesions that were
8
Acta Ophthalmologica2013
either biopsied or surgically removed in Denmark during the period1974–2007 were included(I).The time period was chosen to determine incidence and time-trends of lacrimal gland lesions in Denmark.We included non-neoplas-tic lesions as well as neoplastic lesions because we wanted to investigate which non-neoplastic lesions that where biop-sied and why(I).We collected the original histological sections and/or
tumour blocs as well as the original
pathology reports in all cases plausible
to have originated in or secondarily
have invaded the lacrimal gland.All
histopathological diagnoses of the non-
lymphoma neoplasias were re-evalu-
ated according to the latest WHO
classi?cation(Eveson et al.2005).The
diagnosis of the lymphomas was
recently re-evaluated by Sj?et al.
(2008)and Rasmussen et al.(2012).
Only lesions that could be con?rmed to
have originated in or secondarily have
invaded the lacrimal gland were
included.Specimens were classi?ed as
shown in Table2(I).
In the genetic investigations,we
included patients with con?rmed
lacrimal gland ACC(III),PA(IV),
Table2.Distribution of lacrimal gland lesions(I).
Category%*Lesions(n)?Patients(n)Recurrence/
Patients Gender(M/F)
Median age
years(range)
Dacryops10.32424010/1451(14–73)
In?ammation26.762602/220/4048(9–91) Non-granulomatous in?ammation22.853512/217/3446(10–91) Acute dacryoadenitis 3.48804/449(14–79) Chronic dacryoadenitis19.044422/212/3046(10–91) Fistula0.41101/071 Granulomatous in?ammation 3.99903/653(9–64) Non-necrotizing granulomatous in?ammation 3.48803/554(26–64) Necrotizing granulomatous in?ammation0.41100/19
Normal tissue11.627252/26/1949(8–77) Benign tumours22.8534211/719/2348(2–81) Benign epithelial tumours13.431256/412/1344(14–81) Pleomorphic adenoma13.431256/412/1344(14–81) Benign lymphoid tumours 4.3101004/654(11–74) Benign lymphoid hyperplasia 3.99904/553(11–74) Benign lymphoepithelial lesion0.41100/155
Benign vascular tumours 4.31055/32/329(22–53) Angiolymphoid hyperplasia with eosinophilia 3.9945/32/227(22–51) Vascular malformation0.41100/153 Neuroepithelial tumours0.41100/12
Neuro?broma0.41100/12 Mesenchymal Tumours0.41101/038
Granular cell tumour?0.41101/038 Malignant tumours28.466597/726/3363(18–83) Malignant epithelial tumours14.632257/713/1245(18–73) Adenoid cystic carcinoma8.620155/58/745(23–71) Carcinoma ex pleomorphic adenoma 2.2541/11/343(31–65) Adenocarcinoma0.92200/230(18–42) Mucoepidermoid carcinoma 1.3321/12/069(64–73) Ductal carcinoma0.41101/053
Squamous cell carcinoma0.41101/069 Malignant lymphoid tumours10.8252508/1768(42–83) Extranodal marginal zone lymphoma 3.48802/666(45–83) Di?use large B–cell lymphoma
1.74401/376(71–80)
Follicular lymphoma 1.74402/262(49–63) Mantle cell lymphoma 1.74402/270(42–83) B-cell lymphoma,unclassi?ed 1.33300/363(55–68) Small lymphocytic lymphoma0.92201/171(68–74) Secondary invading tumours 3.07705/258(38–66) Adenoid cystic carcinoma 1.74403/161(38–66) Haemangiopericytoma0.41101/039
Meibomian carcinoma0.41100/164
Squamous cell carcinoma0.41101/054 Metastasis0.92200/273(69–76) Primary mamma carcinoma0.92200/273(69–76) Total100.023221022/1881/12953(2–91)
*Percentage of lesions from the total number of lesions included in the study.
?Total number of lesions including primary tumours and local recurrences.
?Previously published by von Holstein et al.(2009).
9
Acta Ophthalmologica2013
Ca-ex-PA(IV)and MEC(V)from whom su?cient tumour material and/ or clinical data was available.This added up to a total of14patients with ACC from whom we had access to formalin-?xed para?n-embedded (FFPE)tumour material in all cases and additional fresh frozen(FF)tissue from two cases(III).We included a total of32patients with PA and/or Ca-ex-PA(including two cases from Sweden and three cases diagnosed after 2007)from whom36tumour specimens (31PAs,?ve Ca-ex-PAs)were available in the form of FFPE in34cases and/or FF tissue in?ve cases(IV).One patient with MEC was included in the study, and from this case,we had only access to FFPE tumour material(V).We included positive control tissue from salivary gland tumours previously demonstrated to harbour the MYB–NFIB fusion(III)(Persson et al. 2009b)and the CRTC1–MAML2 fusion(V)(Behboudi et al.2006).In the immunohistochemical experiments, we used the tumours as positive con-trols expect in the case of HMGA2, where we used a uterine leiomyoma previously tested positive for HMGA2 (III,IV,V).Normal salivary gland tissue was used for comparison of MYB expression in the Q-PCR exper-iment(IV).
Methods
Clinical data
Data regarding time of diagnoses,age and gender were collected for all patients(I).The reason for taking a biopsy was the indication for surgery given on the pathology requisition.If the information on the requisition was incomplete or missing,the clinical diagnosis and reason for biopsy were categorized as‘not speci?ed’.
Complete clinical?les were col-lected and reviewed for all patients with epithelial tumours(III,IV and V) with special references to patient char-acteristics,symptoms,treatment and follow-up.TNM-staging of the carci-nomas was performed according to the latest classi?cation system(Edge et al.2009;Rootman&White2009) based on information from clinical ?les.Follow-up data were updated in November2011(III)and in July2012 (IV)using each patient’s personal identi?cation number(CPR)as entry for a search in Patobank and in the National Patient Administrative Sys-
tem that holds registries of all hospital
contacts.Death certi?cates were col-
lected for patients that died outside
the hospital or where the cause of
death was indecisive.Tumour-related
death was registered for patients
that died due to tumour progression
or as a complication of the cancer
treatment.
Statistics
A yearly(1974–2007)Danish popula-
tion count strati?ed by gender,age was
obtained from Statistics Denmark
(www.statistikbanken.dk2010).Age
was hereafter expressed as8age
groups,and time period was expressed
as?ve6-year groups and one4-year
group.Crude incidence rates(overall
and period-,age-and gender-speci?c)
were calculated as the number of cases
divided by the number of person years
in the(relevant subset of the)data,and
expressed in cases per1000000per-
son-years;con?dence intervals were
calculated by assuming a Poisson dis-
tribution.A multivariable Poisson
regression model including period,age
and gender was used to investigate the
independent e?ects of period,age and
gender on the incidence,that is,the
e?ect of period adjusted for age and
gender,etc.(Frome1983).These e?ects
are expressed for each of the subgroups
as rate ratios(RR)relative to the?rst
period(1974–1979),the lowest age
group(0–20years)or female,respec-
tively,and the signi?cance of the vari-
ables,that is,whether for all subgroups
of a speci?c variable RR=1,is tested
with likelihood ratio(LR)tests.Di?er-
ences in patient characteristics were
tested using an unpaired-samples t test
(I)or Mann–Whitney’s U test(III,IV)
(continuous variables)and Fisher’s
exact test(binary variables)(I,III and
IV).Overall survival was counted from
time of diagnosis to last follow-up or
death.Univariate analyses of survival
were performed using the Kaplan–
Meier method and compared by the
log-rank test.All statistical analyses
were performed using SPSS statistical
software version19(IBM,Armonk,
New York,USA).
Histopathology and immunohistochemistry
The morphology of all epithelial
tumours of the lacrimal gland where
evaluated on haematoxylin and eosin
(HE),periodic acid-Schi?(PAS),
PAS+diastase and Alcian-Van Gie-
sen-stained sections.A primary panel
of antibodies was applied when neces-
sary with antibodies directed against
cytokeratin(AE1/AE3+8/18,Biocare
Birmingham,United Kingdom),vi-
mentin(clone3B4,Dako,Glostrup,
Denmark),epithelial membrane anti-
gen(clone E29,Dako),glial?brillary
acidic protein(Dako),S-100(Dako)
and smooth muscle actin(clone1A4,
Dako).All these antibodies are well-
known monoclonal or polyclonal anti-
bodies with high speci?city(Gatter
et al.1985;Azumi&Battifora1987;
Sorensen et al.1987;Viale et al.1991;
Ilg et al.1996;Brennan et al.2000).
Details regarding the antibodies
against MYB,KIT,BCL2,MYC,
CCNB1,CCNE1,PLAG1,HMGA2
and CRTC1–MAML2including which
protocol we used(I,III,IV and V)are
summarized in Table3.For the immu-
nohistochemistry,FFPE tissue samples
were cut at4-m-thick sections and
mounted on coated slides(Dako Flex
IHC microscope slides).The sections
were thereafter treated using either the
Dako or Ventana autostainer system
(Table3).When the Dako autostainer
was used,the following protocol was
applied:Antigens were depara?nised
and retrieved with Dako Target Retrie-
val Solutions High pH or Low pH for
20min at97°(Code K8002)using the
Dako pretreatment(PT)link.After Table3.Antibodies,manufacturer,clone,dilution and staining system.
Antibody(study no)Manufacturer/Reference Clone/catalogue no Dilution Auto-stainer MYB(III)Genetex GTX178511:25Ventana MYC(III)Sigma-Aldrich9E101:200Dako
BCL2(III)Dako1241:100Dako
KIT(III)Dako CD117,A42021:100Dako CCNB1(III)Invitrogen V1521:400Dako CCNE1(III)Novus HE121:25Dako PLAG1(IV) Astr€o m et al.(2000)1:400Ventana HMGA2(IV)Cell Signalling D1A71:50Ventana CRTC1–MAML2(V)Behboudi et al.(2006)1:1000Dako
10
Acta Ophthalmologica2013
blocking of endogenous peroxidase activity with Dako EnVision Flex+ (Dako K8002)for5min,tissue sec-tions were incubated with primary antibody diluted in Dako antibodydil-uent(Dako K8006)for20min at room temperature.The reaction was visualized using Dako EnVisionFlex+ mouselink for15min followed by Dako EnVision Flex+/HRP for 20min.,and?nally,Dako EnVision Flex+diaminobenzidine(DAB)for 10min.The sections were counter-stained with haematoxylin for5min.
When the Ventana system was used, the following protocol was used:The sections were depara?nised using the EZ-prep followed by pretreatment with Ventana CC1(cell conditioning pH 8.5)for64min and incubated with the antibody diluted in Dako antibody-diluent S2022for32min at36°C in Ventana Benchmark Ultra.The reac-tion was visualized using Ventana Ultra View DAB-kit.The sections were counterstained with haematoxylin for 8min.
Negative and positive controls were included in all stainings,and internal controls were evaluated in all speci-mens.Immunostainings with antibod-ies against MYB,KIT,BCL2,MYC, CCNB1,CCNE1,PLAG1,HMGA2 and CRTC1–MAML2were considered positive if greater than25%of the tumour cells in a given specimen showed positive immunoreactivity (III,IV and V).
Quantitative RT-PCR analysis
To investigate MYB expression in lac-rimal ACC,we performed quantitative real-time PCR(qPCR)analysis on two frozen tumour samples using the AB TM 7500Fast Real-time PCR system (Applied Biosystems,Foster City,Cali-fornia,USA)as previously described (Persson et al.2009b).MYB expression was analysed using the TaqMan TM Gene Expression assay for MYB exons 1–2(Hs00920554_m1)(Applied Bio-systems).All samples were assayed in triplicate.The relative expression levels of MYB in tumour and normal salivary gland samples were calculated with the SDS Software v2.0.1(Applied Bio-systems)using the comparative Ct method(Livak&Schmittgen2001) with the housekeeping gene GAPDH (Hs99999905-m1)as endogenous con-trols and cDNA from normal salivary gland tissue as calibrator.RT-PCR and nucleotide sequence analysis
Total RNA was extracted from?ve10-
l m sections of FFPE blocks(ACC,
MEC,PA and Ca-ex-PA)and from FF
tissue(ACC and PA)using the RNeasy
FFPE kit(Qiagen Hilden,Germany)
and RNeasy Mini kit(Qiagen),respec-
tively(III,IV and V).DNase-treated
(DNA-free TM;Ambion Austen,Texas,
USA)total RNA was subsequently
converted to cDNA using the iScript TM
cDNA Synthesis Kit(Bio-Rad
Munich,Germany)according to the
manufacturer′s manual.As a control
for intact RNA and cDNA,RT-PCR
reactions for expression of ACTB(b-
actin)were performed on all samples
using the following primers:forward
5′-ATCA CCATTGGCAATGAGCG-
3′and reverse5′-TTGAAGGTAGT
TTCGTGGAT-3′(ampli?cation of a
98-bp product).
All ACCs as well as19non-ACC
lacrimal epithelial neoplasms were
screened for expression of the most
common MYB–NFIB fusion transcript
variants,including MYB exon12or14
fused to either of NFIB exons8a,8c or
9.The MYB–NFIB fusion transcripts
were ampli?ed by direct or touchdown
PCR as previously described(Fehr
et al.2011).The primers used are
shown in Table4.As positive control,
a salivary ACC with known MYB–
NFIB fusion was used(Persson et al.
2009b).
The MEC case and12non-MEC
lacrimal epithelial neoplasms were
screened for the CRTC1–MAML2
fusion.The CRTC1–MAML2tran-
script was ampli?ed by nested PCR.
The?rst-round PCR was performed
using the primers CRTC1-54F5′-GA-
GAAGATCGCGCTGCAC-3′and
MAML2-1855R5′-CTTGCTGTTGG
CAGGAGA-3′(ampli?cation of a150-
bp product),and the second-round
PCR was performed using the primers
CRTC1-99F5′-GCCTTCGAGGAGG
TCA TGA-3′and MAML2-1834R
5′-GGTTAACTACCTGTTTTCTTTT
CAAGG-3′(ampli?cation of a85-bp
product).As positive control,we used
a salivary MEC known to express
CRTC1–MAML2(Behboudi et al.
2006).
All PCRs(III and V)were per-
formed in triplicate.PCR products
were gel-puri?ed and sequenced using
an ABI PRISM310Genetic Analyzer
(Applied Biosystems).The resulting
sequences were analysed using the
BLAST tool provided by the National
Center for Biotechnology Information
(https://www.doczj.com/doc/3315743708.html,).
Fluorescence In Situ Hybridization
(FISH)
We performed FISH analysis on FFPE
sections of the ACCs using a dual-
colour MYB split apart probe
(Abnova,Taipei City,Taiwan)(III).
MAML2rearrangements were investi-
gated using the Zyto LightaSPEC
MAML2Dual Color Break-Apart
Probe(ZytoVision Bremerhaven,
Germany)(V).The protocols for pre-
treatment,hybridization and posthy-
bridization washes were in all cases as
recommended by the manufacturer.
Fluorescence signals were digitized,
processed and analysed using the
CytoVision TM image analysis system
(Applied Imaging International Ltd.
Newcastle-Upon-Tyne,United King-
dom).Thirty to100nuclei were scored
from each case.
ArrayCGH
Genomic DNA was isolated from
FFPE tumour specimens using the
Qiagen DNeasy Blood and Tissue Kit
(Qiagen)as described for FFPE mate-
rial(Agilent Technologies Inc.Palo
Alto,California,USA).ArrayCGH
analysis was subsequently performed
using Human Genome CGH Micro-
array244K(III and IV),400K(IV)or
Table4.PCR primers used for ampli?cation of MYB–NFIB fusion transcripts in adenoid cystic carcinoma(III).
Primer Localization(exon)Primer sequence
MYB-1693F125′-GCAGGATGTGATCAAACAGG-3′NFIB-1197R95′-CCGGTAAGATGGGTGTCCTA-3′MYB-1925F145′-GCACCAGCATCAGAAGATGA-3′NFIB-862R8a5′-GCCAGGCACTTTCCCTACTAC-3′MYB-1910F145′-AGCTCCGTTTTAATGGCACC-3′NFIB-1096R8c5′-GGGTATAAATGCCTGCCGTT-3′MYB-1925F145′-GCACCAGCATCAGAAGATGA-3′NFIB-1197R95′-CCGGTAAGATGGGTGTCCTA-3′
11
Acta Ophthalmologica2013
180K(IV)oligonucleotide arrays from Agilent Technologies.The experiments were performed as recommended by the manufacturer(Barret et al.2004; Persson et al.2008).Slides were scanned on an Agilent High-Resolu-tion C Microarray Scanner,followed by data extraction and normalization using Feature Extraction v.10.7.1(Ag-ilent Technologies)with linear normal-ization(protocol CGH_107_Sep09). Data analysis was carried out using NEXUS Copy Number v.6.0Discov-ery Edition(BioDiscovery Inc.El Segundo,California,USA).The signif-icance threshold for segmentation was set to p=1.0E-6(III)and p=1.0E-7 (IV),and the settings for aberration calls for all were1.0for ampli?cation, 0.4for gain,à0.4for loss andà1.0for homozygous deletion(III).For case 17a,b and c(IV),the setting for the signi?cance threshold was p=1.0E-7. Each aberration was checked manually to con?rm the accuracy of the call.A copy number alteration was considered recurrent if two or more tumour sam-ples carried the same alteration with a p-value of less than0.05.Sex chromo-somes were excluded from the analysis as was regions partially or completely covered by a previously reported copy number variation(Database of Geno-mic Variants;hhtp://dgvbeta.tcag/dgv/ app/news?ref=NCBI36/hg18)(Iafrate et al.2004).
Review
The review(II)was primarily based on literature identi?ed searching PubMed, and the most important points from this study are presented in the discussion. Ethics
The study adheres to the tenets of the Declaration of Helsinki and has been approved by the Local Scienti?c Ethics Committee(journal no.H-C-2007-0065)and the Danish Data Protection Agency(journal no.2007-41-0690)as well as by the Regional Ethics Com-mittee in Gothenburg,Sweden(D-no: 178-08).
Results
Epidemiology
Incidence
A total of232lesions from210patients were con?rmed to have either origi-nated in or invaded the lacrimal gland
(Table2).
The overall incidence of lacrimal
gland lesions was1.3lesions/1000000/
year,and the incidence of neoplastic
lesions was0.7lesions/1000000/year
(Table5).The incidence increased over
the study period,from0.9lesions/
1000000/year(in1974–1979)to 2.1
lesions/1000000/year during the last
period(2004–2007,Table5).Adjusted
for age and gender(rate ratio,RR),the
incidence more than doubled during the
study period:RR=2.3for the last time
period versus the?rst time period
(p<0.0001,Table5).This increase
was driven by a threefold increase in
non-neoplastic lesions(RR=2.8for the
last time period compared with the?rst
period,p<0.0001)and a fourfold
increase in benign tumours(RR=4.0for
the last time period compared with the
?rst time period,p=0.0486),while no
signi?cant increase was observed for
malignant tumours.
Distribution
The distribution of lacrimal gland
lesions is summarized in Table2(I).
Demographics
Age and gender characteristics are
summarized in Table2(I).Overall,
patients with malignant neoplasms of
the lacrimal gland were signi?cantly
older than patients with benign neo-
plasms(63versus48years,p<0.001),
and patients with malignant epithelial
tumours were signi?cantly younger
than patients with lymphoma(45ver-
sus68years,p<0.001)(I).
Indications for surgical intervention
are presented in Table6(I).
Clinical characteristics
Patient characteristics
A summary of the clinical characteris-
tics of patients with ACC,PA,Ca-ex-
PA and MEC is given in Table7(III,
IV and V).We found no association
between survival and T designation for
the ACC patients;however,patients
with tumours less than2.5cm in great-
est dimension had a median survival of
15.0years compared with8.6years for
patients with larger tumours(p=0.28)
(III).There was a signi?cant(p<0.03)
association between presence of
tumour cells in the resection margins
and local recurrence of PA(IV).
Histopathological characteristics
Six of the232lesions were rediagnosed
(I).Five lesions originally diagnosed as
adenocarcinoma were rediagnosed as
ductal carcinoma,squamous cell carci-
noma,ACC,Ca-ex-PA and metastasis
from a breast carcinoma.One case of
benign lymphoid hyperplasia was redi-
agnosed as benign lymphoepithelial
lesion.
The histopathological features of
ACC included two(14%)cases with a
Table5.Changes in incidence rate of biopsied lacrimal gland lesions in Denmark between1974 and2007(I).
Lesions(n)
Incidence per
1000000(95%CI)
Rate Ratio
adjusted(95%CI)p-value Overall232 1.311(1.153–1.491)
Period
1974–1979260.854(0.581–1.254) 1.000.0000 1980–1985270.879(0.603–1.282) 1.01(0.59–1.73)
1986–199136 1.170(0.844–1.621) 1.32(0.80–2.18)
1992–1997260.831(0.566–1.221)0.93(0.54–1.60)
1998–200372 2.247(1.784–2.831) 2.50(1.59–3.92)
2004–200745 2.075(1.549–2.780) 2.28(1.41–3.70)
Age
0–20200.418(0.269–0.647) 1.000.0000 21–30240.934(0.626–1.394) 2.25(1.24–4.07)
31–4033 1.269(0.902–1.785) 2.95(1.69–5.14)
41–5036 1.544(1.114–2.141) 3.55(2.05–6.13)
51–6039 1.889(1.380–2.586) 4.23(2.47–7.25)
61–7044 2.647(1.970–3.557) 6.15(3.63–10.44)
71–8029 2.524(1.754–3.633) 5.76(3.26–10.19)
81+7 1.326(0.632–2.781) 2.79(1.18–6.60)
Gender
Female140 1.563(1.325–1.845) 1.000.0068 Male92 1.053(0.858–1.292)0.70(0.54–0.91)
12
Acta Ophthalmologica2013
mainly solid growth pattern and 12(86%)cases with a predominant tubu-lo-cribriform growth pattern (Fig.3D)(III).There was no association between survival and histopathological growth pattern for ACC patients (III).The carcinoma part of the four Ca-ex-PA was MEC (grade I and II)in two cases and myoepithelial carcinoma and ade-nocarcinoma NOS in one case each (IV).The MEC was grade I (low-grade,Fig.4C)according to the scoring sys-tem de?ned by WHO (V)(Eveson et al.2005).
Genomic imbalances
The recurrent copy number alterations identi?ed in ACC (III),PA and Ca-ex-PA (IV)are presented in Table 8.
Adenoid cystic carcinoma
Using RT-PCR and nucleotide sequence analysis,we detected one or more chimeric MYB –NFIB fusion tran-script variants in 7/14ACCs (Fig.6,Table 9).The composition of all fusion transcripts was in agreement with pre-viously published data (Persson et al.
2009b).In contrast,none of the 19non-ACC lacrimal neoplasms expressed MYB –NFIB fusion transcripts.
Using a dual-colour MYB break-apart FISH probe (Fig.7A),we could detect rearrangements of MYB in 8of 13cases (Table 9).The patterns of MYB rearrangements observed included split signals (Fig.7B,C)in six tumours and gain of one or more copies of MYB in two cases (Fig.7D).In one case,the quality of the FFPE material prevented analysis of the FISH results.
The expression of MYB mRNA was studied using qPCR on fresh frozen tumour tissue from two cases.Both cases overexpressed MYB mRNA com-pared with normal salivary gland tis-sue.In addition,immunohistochemical staining revealed strong nuclear stain-ing of the MYB protein (Fig.8A)in all 13lacrimal gland ACCs tested (in one case,the quality of the FFPE sections prevented analysis of the immunohis-tochemical results).All 13tumours stained positive for the MYB targets KIT and BCL2,12tumours were positive for MYC and CCNE1and nine tumours were positive for CCNB1(Fig.8).
In 10/14ACCs,we were able to get su?cient DNA for arrayCGH analy-sis.A total of 44partial genomic imbalances and nine gains of whole chromosomes were detected in the 10ACCs (Table 9,Fig.9).The average number of copy number alterations per tumour was 5.1(range 1–15).Eleven recurrent copy number changes,de?ned as minimal common regions of deletions and gains,were identi?ed (Table 8).The only recurrent whole chromosome alterations were gains of chromosomes 19and 22seen in 3tumours each.We found no associa-tion between fusion status or any of the recurrent copy number alterations and survival.
Table 7.Clinical characteristics of the patients with pleomorphic adenoma,carcinoma ex pleomorphic adenoma,adenoid cystic carcinoma and mucoepidermoid carcinoma (III,IV,V).
Pleomorphic adenoma*
Carcinoma ex pleomorphic adenoma*Adenoid cystic
carcinoma Mucoepidermoid carcinoma No.of patients 28
4
14
1Median age (range)
44(13–81)43(31–65)43(23–67)73Gender distribution (M:F)14:14
1:3
7:7
1:0Median duration of
symptoms in years (range)2(0.1–25)
1(0.1–2)
0.5(0.1–2)
0.5
Symptoms (%):
Displacement of eye 18(64)2(50)9(64)0Ptosis
4(14)03(21)0Tumour mass 16(57)2(50)6(43)1Pain 2(14)1(25)10(71)0Diplopia
10(36)05(36)0Decreased motility 6(21)08(57)0T-stage (TNM)(%)T1–1(25)01T2–1(25)5(36)0T3–02(14)0T4
–
2(50)9(64)0Treatment (initial)(%)Tumour excision 28(100)3(75)11(79)1Orbital exenteration 01(25)3(21)0Adjuvant radiotherapy 0
3(75)11(79)0Median time of follow-up in years (range)
15.5(1–51)12(4–20)
7(1–27)
5No.patients with local recurrence
7(25)1(25)5(36)0Tumour-related death 1(4)?1(25)?8(57)0Alive
22(79)3(75)5(36)0Dead of other causes
5(18)
1(7)
1
*Two patients initially presented with pleomorphic adenoma,but later developed carcinoma ex pleomorphic adenoma,and they are present in both columns.?
The same patient and he died of causes related to the Ca-ex-PA.
Table 6.Indications for surgical intervention and histopathological diagnosis (I).
Clinical diagnosis
Histopathological diagnosis Tumour suspicion n (%)Dacryops n (%)Prolapse n (%)In?ammation n (%)Not speci?ed n (%)Total n (%)Dacryops 5(21)15(63)––4(17)24(100)In?ammation 26(42)–5(8)10(16)21(34)62(100)Normal tissue 10(37)1(4)6(22)4(15)6(22)27(100)Benign tumours 47(89)1(2)–2(4)3(6)53(100)Malignant tumours 64(97)1(2)––1(2)66(100)Total
152
(66)
18(8)
11(5)
16
(7)
35(15)
232
(100)
13
Acta Ophthalmologica 2013
Pleomorphic adenoma and carcinoma ex pleomorphic adenoma
We observed positive nuclear staining for PLAG1(Fig.2E)in all29PAs and in3/5(60%)of the Ca-ex-PAs (Table10).Only2/29(7%)PAs showed nuclear positivity for HMGA2 (Fig.2F).None of the Ca-ex-PAs were positive for HMGA2.
We were able to obtain su?cient DNA for arrayCGH in19PAs and three Ca-ex-PAs.Eleven of19PAs had normal genomic pro?le(Table10).The remaining8PA and three Ca-ex-PA
samples contained a total of47par-
tial genomic imbalances and two gains
of whole chromosomes(Table10,
Fig.10).The average number of geno-
mic imbalances per tumour was 1.3
(range0–7)in PAs compared with7.7
(range4–12)in Ca-ex-PAs.Five recur-
rent copy number changes,de?ned as
minimal common regions of deletions/
gains found in two cases or more,were
identi?ed in PAs(Table8).Gain of
9p23–p22.3was seen in two PAs and in
one Ca-ex-PA(Fig.10D).Two PAs
had losses involving8q11–q13,but in
none of the cases,the PLAG1gene was
altered.Similarly,one PA had a gain of
12q13.13but without involvement of
the HMGA2gene.We identi?ed one
recurrent copy number alteration in the
Ca-ex-PAs and that is gain of22q12.3-
qter(Fig.10E).
Mucoepidermoid carcinoma
We detected the CRTC1–MAML2
fusion transcript in the lacrimal gland
MEC(Fig.11)but not in15non-MEC
lacrimal gland neoplasms.FISH anal-
ysis revealed rearrangements of
MAML2,consistent with a CRTC1–
MAML2gene fusion in the majority of
tumour cells https://www.doczj.com/doc/3315743708.html,ing a custom-
made polyclonal CRTC1–MAML2
antibody,we observed distinct nuclear
staining of the fusion protein in the
majority of tumour cells,including
epidermoid,intermediate and mucous-
producing cells(Fig.4D).
Discussion
Epidemiological characteristics
We searched two population-based
registries and re-evaluated the diagno-
ses of all the identi?ed lacrimal gland
lesions.The average annual incidence
of biopsied lacrimal gland lesions in
Denmark between1974and2007was
Table8.Recurrent copy number alterations(minimal common regions of gains and losses)in adenoid cystic carcinomas,pleomorphic adenomas and carcinoma ex pleomorphic adenomas of the lacrimal gland.
Cytoband Location Chromosome Region Region
Length(bp)Event
No.of
Tumors
No.of
Genes Potential Target Genes
Adenoid cystic carcinoma
6q23.3Chr6:135,377,625–135,614,043236418Gain22MYB
6q23.3Chr6:137,085,474–137,872,891787417Loss312
6q24.2–25.1Chr6:143,638,308–150,305,8146667506Loss365LATS1,PLAGL1
6q25.1–q26Chr6:151,436,540–161,876,24610436706Loss396PARK2,WTAP
6q27Chr6:164,714,653–167,867,1123152459Loss331RPS6KA2,RNASET2
8q24.13–q24.21Chr8:124,624,995–129,751,4175126421Gain350MYC,PTV1
11q12.3–q14.1Chr11:62,989,903–77,828,132********Gain2419CCND1,ESRRA,FOSL1,RELA,RIN1 12q12–q14.1Chr12:37,941,004–60,662,39022721386Loss3452LIMA1,NR4A1
17p13.3–p12Chr17:3,039,487–11,800,9088761421Loss2290TP53,MYBBP1A
19q12Chr19:34,305,176–35,950,7631645587Gain414CCNE1
19q13.31-qter Chr19:48,617,748–63,811,65115193903Gain4867FOSB,RELB,RRAS,BCL3 Pleomorphic adenoma
1pter-p31.3Chr1:0–61,817,31161817311Loss21199TP73,CHD5,CASP9,CTNNB1,UBE4B,
RUNX3,SFN,NBL1,PRDM2
6q22.1–q24.3Chr6:122,836,233–146,803,02623966793Loss2207BCLAF,PLAGL1,MED23
8q24.22–q24.3Chr8:134,124,105–142,077,6887953583Loss231NDRG1
9p23–p22.3Chr9:13,883,571–14,393,919510348Gain22NFIB
13q21.31–q21.33Chr13:67,448,634–69,957,0302508396Loss25
Carcinoma ex pleomorphic adenoma
22q12.3-qter Chr22:33,946,622–49,691,43215744810Gain2400
PDGFB
Fig.6.(III).Reverse transcription–polymerase chain reaction(RT-PCR)analysis of MYB–NFIB fusion transcripts in14lacrimal gland adenoid cystic carcinomas(ACCs).(A)Detection of MYB–NFIB fusion transcripts in FFPE tumor tissues from7/14ACCs.ACTB was used as an internal control to test for intact RNA and cDNA.The sizes of the ampli?ed fragments are indicated.
(B)Nucleotide sequence analysis of the PCR product generated from case3con?rmed that it indeed corresponded to a chimeric transcript consisting of MYB exon14fused to NFIB exon8c. 14
Acta Ophthalmologica2013
~1.3per1000000and we observed a doubling of the incidence rate during the study period.The registries from which the lesions were identi?ed included the archive at the Eye Pathol-ogy Section that serves as a nationwide ophthalmic pathology institute receiv-ing tissue specimens from private ophthalmologists and departments of ophthalmology throughout Denmark. We also searched Patobank because some ophthalmology departments send their specimens to local pathology departments.Patobank was founded with the purpose of registering all pathology diagnoses in Denmark,and by the1980s,all pathology depart-ments in Denmark registered their diagnoses in this Databank.The hos-pitals that registered diagnoses from the1960s were the major specialized hospitals in Denmark.As the present study period begins in1974,a few cases in the beginning of the time period might have been missed.However, because the hospitals that registered diagnoses from the beginning are the same in which patients with lacrimal
gland tumours where treated,the cases
missed are more likely to be non-
neoplastic than neoplastic of origin.
Taken together with the use of two
population-based registries,the inci-
dence of lacrimal gland neoplasia is
considered reliable.
The doubling of incidence rate that
we observed during the study period
was driven by an increase in non-
neoplastic lesions,primarily in?amma-
tory conditions,and benign tumours
and was independent of changes in
gender and age composition of the
Danish population.The increase was
observed after the1990s(Table5)and
it can therefore be safely assumed that
the increase is not caused by lack of
registering diagnoses in the early time-
periods.More than40%of the in?am-
matory lesions were biopsied because
of suspicion of tumour(Table6),sug-
gesting that increasing awareness or
fear of overlooking a malignant lesion
might explain the increased incidence
of biopsied lacrimal gland lesions.The
increase in benign tumours was pri-
marily caused by a combined increase
in benign lymphoid hyperplasia and
angiolymphoid hyperplasia with eosin-
ophilia.These lesions are di?cult to
distinguish clinically from in?amma-
tory lesions,and the increase might
therefore be explained by the increase
in in?ammatory lesions.Furthermore,
benign lymphoid hyperplasia may
undergo malignant transformation
(Polito et al.1996)and the increase in
this lesion may therefore be associated
with the increased incidence of oph-
thalmic lymphoma in Denmark(Sj?
et al.2008).In contrast,the incidence
of malignant tumours was stable dur-
ing the study period.
We did not include lacrimal gland
lesions treated conservatively and/or
where the clinical diagnosis did not
require histopathological evaluation.
Consequently,the incidence of lacrimal
gland lesions re?ects lesions that are
diagnostic challenging or requires
surgery.Accordingly,we found the
frequency of chronic dacryoadenitis to
be higher than acute dacryoadenitis(44
lesions vs.8lesions).This di?erence is
for the most part is explained by the
ease of which acute dacryoadenitis is
diagnosed clinically without the use of
histopathological evaluation(Snebold
1994).Furthermore,the indolent
course of chronic dacryoadenitis may
resemble tumour formation and neces-
sitate biopsy.This notion is supported
by the fact that the most common
indication for biopsy in chronic
dacryoadenitis was tumour suspicion
(Table6).
Approximately half of the lacrimal
gland lesions were neoplastic and of
these malignant tumours constituted
55%.The present series is not prone to
selection-or referral bias because the
material covers a whole nation,how-
ever,the inclusion of secondary invad-
ing tumours results in a higher
proportion of malignant tumours.
Although secondary invading tumours
often are excluded in the majority of
series published,we believe they are
important as di?erential diagnosis to
primary lacrimal gland tumours for the
reason that they may present with the
same symptoms.
The distribution of the diagnoses of
the tumours in general was in line with
other reported series in that epithelial
tumours were the most frequent
tumour types with PA as the most
common followed by ACC(Kennedy
1984;Shields et al.1989;Ni et al.1992;
(B)
(A)
(D)
(C)
Fig.7.(III).Fluorescence in situ hybridization(FISH)analyses of MYB rearrangements in
lacrimal gland adenoid cystic carcinomas(ACC).(A)Schematic illustration of the MYB break-
apart probe consisting of a centromeric(green)and a telomeric(red)probe covering the MYB
locus and its?anking sequences.(B)interphase nuclei from a MYB–NFIB fusion positive ACC
(case10,Table9),showing an intact signal(fused red/green signals)and a split signal(separated
red and green signals),indicating a breakpoint within the MYB gene.(C)interphase nuclei from a
MYB–NFIB fusion-negative ACC(case6,Table9),showing an identical FISH pattern as in B
with rearrangement of the MYB locus.(D)Interphase nuclei from a MYB–NFIB positive ACC
(case7,Table9),showing two fused red/green signals and1-2additional green signals indicating a
selective gain of the MYB locus.
15
Acta Ophthalmologica2013
Zeng et al.2010).The demographic characteristics of patients with lacrimal gland lesions in Denmark were not di?erent from patients from other parts of the world(Font&Gamel1978; Shields et al.1989;Ni et al.1992; Wright et al.1992;G€u nalp&G€u nd€u z 1994).Accordingly,we observed more women than men with in?ammatory lesions(67%,p=0.014),whereas the gender distribution was equal in all other diagnostic categories.Patients with benign tumours where signi?-cantly younger than patients with malignant tumours(48versus63years,p<0.001),and patients with malig-
nant epithelial tumours were younger
than patients with lymphomas(45ver-
sus68years,p<0.001).
Clinical characteristics
The clinical characteristics of the
patients with PA,Ca-ex-PA,ACC
and MEC(III,IV and V)were based
on the original data recorded by local
physicians.The Central Population
Registry(CPR)provides all persons
living in Denmark with an individual
identi?cation number,and with this
CPR number,it is possible to link
patient data from clinical?les at the
Eye Pathology Section,Patobank,hos-
pital registries and the Danish National
Death Registry.We thus had no
patients lost to follow-up.The risk of
miscoding dates and cause of death is
likewise minimized by cross-linking
information from clinical?les and the
Danish National Death Registry and it
may be safely assumed that any per-
son not registered as dead were alive
(Jensen et al.1985).
Clinically,we found that the patients
with PA,Ca-ex-PA,ACC and MEC
(III,IV and V)were similar in regard to
age at diagnosis,gender distribution,
symptoms,treatment and survival to
previous reports(Rose&Wright1992;
Wright et al.1992;Eviatar&Horn-
blass1993;Esmaeli et al.2004;Font
et al.2006;Ahmad et al.2009).
Accordingly,patients with PA had a
wide age range at diagnosis(13–81),
and they had symptoms for a median
time of2years before?rst ophthalmic
consultation.The rate of local recur-
rences was25%for patients with PA,
and recurrences were signi?cantly
(p<0.03)associated with the presence
of tumour cells in the resection margins
but not with incisional biopsy prior to
surgery.This?nding emphasizes the
importance of clear resection margins
and suggests that biopsy of PA may be
performed as long as the subsequent
tumour removal includes excision of
the biopsy tract,in agreement with the
report by Lai et al.(2009).
Two of28(7%)patients with PA
developed Ca-ex-PA which is similar to
what previously has been reported for
salivary gland PAs(Gnepp1993).Only
one patient with Ca-ex-PA died of
tumour-related causes;the three others
were still alive without evidence of
recurrences or metastases at the end
of follow-up(follow-up:6–20years).
Traditionally,the prognosis of patients
with lacrimal gland Ca-ex-PA is poor
with tumour-related deaths occurring
within a few years after diagnosis
(Wright et al.1992;Font et al.1998).
However,for patients with Ca-ex-PA
of the salivary gland,the prognosis is
known to be dependent on whether the
carcinoma part extends beyond the
capsule and with the histological grade
of the carcinoma(Seethala2009).
In the present cases of lacrimal gland
Ca-ex-PA,all the three patients that
were still alive had tumours that were (B)
(A)
(D)
(C)
(F)
(E)
Fig.8.Immunohistochemical staining of MYB and downstream targets.(A)Strong nuclear MYB immunoreactivity was observed in this classical example of a tubulo-cribriform adenoid cystic carcinoma(original magni?cation910,insert940).(B)Nuclear MYC immunoreactivity in the histopathologically solid part of a grade3tumor(original magni?cation925).(C)Strong cytoplasmatic immunoreactivity of BCL2(original magni?cation940).(D)Strong cytoplasmatic immunoreactivity of KIT(original magni?cation910).(E)Strong nuclear immunoreactivity of CCNB1in the cystic area of tumor(original magni?cation940).(F)Immunoreactivity of CCNE1in the tubular area of a tumor.Observe that the staining is strongest in the non-luminal tumor cells(original magni?cation940).
16
Acta Ophthalmologica2013