s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6

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Major review

Diagnosis of orbital mass lesions: clinical,

radiological, and pathological recommendations

Ilse Mombaerts, MD, PhDa,*, Ingvild Ramberg, MDb,c,

Sarah E. Coupland, MD, PhDd,e, Steffen Heegaard, MD, DMScib,c
a
Department of Ophthalmology, University Hospitals Leuven, Leuven, Belgium
b
Department of Ophthalmology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
c
Section of Eye Pathology, Department of Pathology, Rigshospitalet, Copenhagen University Hospital,
Copenhagen, Denmark
d
Department of Cellular and Molecular Pathology, University of Liverpool, Liverpool, UK
e
Liverpool Clinical Laboratories, Royal Liverpool University Hospital, Liverpool, UK

article info abstract

Article history: The orbit can harbor mass lesions of various cellular origins. The symptoms vary consid-
Received 28 March 2019 erably according to the nature, location, and extent of the disease and include common
Received in revised form 21 June signs of proptosis, globe displacement, eyelid swelling, and restricted eye motility.
2019 Although radiological imaging tools are improving, with each imaging pattern having its
Available online 2 July 2019 own differential diagnosis, orbital mass lesions often pose a diagnostic challenge. To
provide an accurate, specific, and sufficiently comprehensive diagnosis, to optimize clinical
Keywords: management and estimate prognosis, pathological examination of a tissue biopsy is
orbital lesion essential. Diagnostic orbital tissue biopsy is obtained through a minimally invasive orbi-
orbital tumor totomy procedure or, in selected cases, fine needle aspiration. The outcome of successful
diagnosis biopsy, however, is centered on its representativeness, processing, and interpretation.
biopsy Owing to the often small volume of the orbital biopsies, artifacts in the specimens should
histology be limited by careful peroperative tissue handling, fixation, processing, and storage. Some
pathology orbital lesions can be characterized on the basis of cytomorphology alone, whereas others
morphology need ancillary molecular testing to render the most reliable diagnosis of therapeutic,
immunohistochemistry prognostic, and predictive value. Herein, we review the diagnostic algorithm for orbital
molecular analysis mass lesions, using clinical, radiological, and pathological recommendations, and discuss
cytology the methods and potential pitfalls in orbital tissue biopsy acquisition and analysis.
ª 2019 Elsevier Inc. All rights reserved.

1. Introduction 2.02 per million person-years.34 The causes of orbital masses

vary widely and can be categorized into 6 main pathologic
Orbital mass lesions, including lacrimal gland lesions, are processes: inflammatory, infectious, hemorrhagic, neoplastic,
extremely rare, occurring at an estimated incidence rate of metastatic, and developmental. Based on the cellular origin,

* Corresponding author: Ilse Mombaerts, MD, PhD, Department of Ophthalmology, University Hospitals Leuven, Herestraat 49, 3000
Leuven, Belgium.
E-mail address: Ilse.Mombaerts@uzleuven.be (I. Mombaerts).
0039-6257/$ e see front matter ª 2019 Elsevier Inc. All rights reserved.
https://doi.org/10.1016/j.survophthal.2019.06.006
742 s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6
they can further be classified as either (1) primary lesions
originating from the orbit including the lacrimal gland, which
encompass 82% to 87% of orbital mass lesions; (2) secondary
lesions, which, by contiguity, extend to involve the orbit from
neighboring structures such as the paranasal sinus, conjunc-
tiva, eye, eyelid, lacrimal sac, face, anterior and middle cranial
fossae, orbital bone, nasopharynx, palate, and parotid gland,
representing 9% to 11% of cases; and (3) metastatic tumors,
which account for 4% to 8% of cases.11,43,77
Although thyroid orbitopathy is a frequent cause of orbital
disease, with a diagnosis usually being obtained and based on
typical clinical features, a substantial number of orbital mass
lesions cannot be determined or classified based on clinical
and radiological findings alone.4,46 With a prevalence of
25e36%, malignant neoplasms represent a large disease group
among the orbital lesions and are usually only disclosed upon
tissue biopsy.8,77 The most commonly and increasingly
occurring orbital malignancy in the adult population is lym-
phoproliferative disease, diagnosed through histomorpho-
logical examination of an orbital biopsy.34,42,65,78 In addition,
38e55% of lacrimal gland lesions are a manifestation of a
previously unknown systemic disease revealed through
biopsy.51,54,88
There are no current consensus guidelines concerning
orbital tissue sampling. Specimens removed from the orbit are
usually small, which imposes challenges on tissue processing
and histopathological interpretation. A sample showing
nonspecific inflammation, for instance, may compound these
challenges as it can be indicative of the common lesion of
idiopathic orbital inflammation (IOI) or may represent sam-
pling error and thus miss the true nature of the disease.27,59
2. Clinical signs and symptoms
The symptomatology of orbital tumors varies according to the
nature, focus, and extent of disease. With known tumor pro-
pensities for agedcancer being more common in older
patientsd, gender, and race, a differential diagnosis is
generated based on the orbital clinical assessment.25,42,70,77
Using the mnemonic of the 7 P’s, the assessment includes
the 3 P’s of orbital history (i.e., pain, progression, and past
medical history) and the 4 P’s of physical examination (i.e.,
proptosis, periocular changes, palpation, and pulsation).64 The
key sign suggestive of an orbital tumor is proptosis or globe
displacement; however, proptosis of less than 4 mm might go
undetected, obscuring occult pathology.49 Other signs suspi-
cious of an orbital mass include palpable mass, eyelid
swelling, edema, ptosis and erythema, conjunctival swelling,
limitation of eye motility, diplopia, increased retropulsion,
visual disturbance from optic nerve compression, choroidal
folds and induced hyperopia, and periocular paresthesia and
anesthesia; however, these signs are not specific for the origin
of the mass lesion.
The differential diagnosis can be narrowed down by fitting
into one of the pathologic processes through detailed medical
and orbital history and physical examination. When both or-
bits are involved, underlying systemic neoplastic and in-
flammatory diseases become more likely.61,86,96 Investigation
of systemic disease may be negative, however, because the
orbit can be the first presentation of a condition or represent a
limited manifestation of the disease.54 Furthermore, serology
may be inconclusive because inflammatory markers are
generally nonspecific and do not necessarily reflect the un-
derlying etiology.80,81 Importantly, a clinical response to sys-
temic corticosteroids is not diagnostic for mass lesions
originating from IOI because it confers a low positive predic-
tive value and poor specificity as a diagnostic test.57
3. Imaging modalities
The imaging modalities commonly used to evaluate the
location and extent of orbital tumors are computed tomog-
raphy and magnetic resonance (MR) imaging. Dedicated
multiplanar thin MR images with fat signal intensity sup-
pression and contrast enhancement delineate orbital soft-
tissue lesions and depict the orbital apex and optic pathway.
The combined use of diffusion-weighted and dynamic
contrast-enhanced magnetic resonance imaging can aid dis-
tinguishing malignant from benign lesions, in particular high-
grade lymphoma from inflammation and reactive lymphoid
hyperplasia.38,72,83,84 On the other hand, computed tomogra-
phy is the preferred mode for imaging of the orbital bones,
sinuses, and osseous lesions and can detect calcifications
within lesions that are suggestive of malignant disease.
In general, 5 anatomical imaging patterns of orbital tu-
mors can be identified: intraconal, extraconal, extraocular
muscle, optic nerve sheath complex, and infiltrative
(involving any orbital structure), with each imaging pattern
having its own indicative diagnostic possibilities (Fig. 1).55
Some distinct lesions carry characteristic diagnostic fea-
tures on imaging (Table 1).50 In general, benign orbital lesions
are more often oval shaped and associated with encroaching
and deforming the vicinity, hyperostosis, and hyperintensity
on T2 weighted magnetic resonance imaging, whereas ma-
lignant tumors are characterized by an irregular shape,
molding around neighboring orbital structures, diffuse in
nature, perineural involvement, and bony erosion.7 None of
the imaging features have a high sensitivity to distinguish
malignant and benign tumors, however, with the lowest
sensitivity and positive predictive value for the inflammatory
lesions.7,46,91
4. Obtaining orbital specimens
Diagnostic yield and accuracy of the biopsy are dependent on
the size, site, timing, and technique of procurement. Details of
the clinicoradiological context must be shared with the
pathologist, precluding any diagnosis other than nonspecific,
and hence preventing the need for a repeat biopsy and delay
in diagnosis.
4.1. Representative orbital biopsy
A biopsy should not be attempted in those cases that lack an
identifiable mass or enlarged structure on orbital imaging.
 s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6 743

Paent with suspected orbital tumor

Consider thyroid orbitopathy

History and physical examinaon when typical features are present

MRI or CT showing orbital mass

Intraconal Extraconal Infiltrave

Extraocular muscle Opc nerve sheath complex (involving several orbital structures)
Consider: Consider: Consider: Consider:
Cavernous venous malformaon Thyroid orbitopathy Opc nerve sheath meningioma Malignancy
Lymphac malformaon Idiopathic orbital myosis Opc nerve glioma Idiopathic orbital inflammaon
Venous varix IgG4-related disease Lymphoproliferave disorder IgG4-related disease
Lymphoproliferave disorder Lymphoproliferave disorder Sarcoidosis Lymphoproliferave disorder
Metastasis Metastasis Opc neuris Metastasis
Schwannoma of the III, IV and VIth nerve Orbital venous congeson Opc perineuris Sarcoidosis
Giant cell myosis Malignant glioma Granulomatosis with polyangiis
Amyloidosis Glioblastoma Plexiform neurofibroma
Paraneoplasc orbital myopathy Rhabdomyosarcoma
Neuroblastoma
Orbital cellulis
Periorbital Vascular lesion
Lacrimal gland Bony lesion Other extraconal Solitary fibrous tumor
(originang from sinonasal, facial, or intracranial area) Retained organic foreign body
Consider: Consider: Consider: Consider:
Idiopathic dacryoadenis Dermoid cyst Malignancy Lymphoproliferave disorder
Lymphoproliferave disorder Hematopoec and hisocyc lesion Mucocele Schwannoma of the V1 and V2th nerve
IgG4-related disease Cholesterol granuloma Midline destrucve disease Cavernous venous malformaon
Sarcoidosis Giant cell granuloma Fungal infecon Solitary rous tumor
Sjögren syndrome Aneurysmal bone cyst Encephalocele Malignant peripheral nerve sheath tumor
Granulomatosis with polyangiis Fibrous dysplasia Capillary haemangioma
Infecous dacryoadenis Osteoma Orbital cellulis - abscess
Dacryops Osteoblastoma
Benign epithelial neoplasm Neoplasm
Malignant epithelial neoplasm Sphenoid wing meningioma
Infecous dacryoadenis Metastasis
Metastasis

Fig. 1 e Diagnostic algorithm of orbital mass lesions. The diagnostic possibilities for each imaging pattern are based on
likelihood and are not fully comprehensive.

Corticosteroids and other immunosuppressants may alter heterogeneity, multiple specimens are recommended to be
pathological features; hence, any systemic or intralesional taken from different sites of the lesion and, where fibrous
administration should be avoided or suspended at least tissue predominates, adjunctive samples from the periphery
10e14 days before performing the biopsy.13 To be represen- of the tumor bed. If the biopsy does not yield sufficient in-
tative of the entire lesion, samples should be obtained from formation for diagnosis, a second biopsy from a different site
the largest cross-section. In the case of macroscopic of the lesion should be considered.

Table 1 e Characteristic features on magnetic resonance and computed tomography imaging to distinguish among orbital
lesions
Imaging features Possible diagnosis

Coca-cola bottle sign (spindle-shaped enlargement of the muscle) Thyroid orbitopathy

Hockey stick sign (engorged ophthalmic vein) Carotid-cavernous fistula

Tram track sign (sparing of the optic nerve within a mass) Optic nerve sheath meningioma

Tubular, tortuous, or spindle-shaped enlargement of the optic nerve Optic nerve glioma

Anterior tendon enlargement of the muscle Orbital myositis; lymphoproliferative disorder

Internal septations/fluid-fluid level Lymphatic malformation/recent intralesional hemorrhage

Fat-soft tissue-fluid component Dermoid cyst

Air-fluid level in rim-enhanced lesion Abscess

Orbital nerve enlargement Immunoglobulin G4-related disease; reactive lymphoid hyperplasia;

perineural or intraneural spread of malignancy

Calcifications Phleboliths in venous malformation; malignancy;

meningioma; dermoid cyst

Wedge sign of enlarged lacrimal gland (triangle of tissue Lacrimal gland epithelial or nonepithelial malignancy
between lateral orbital wall and lateral rectus muscle)
Almond-shaped enlarged lacrimal gland Dacryoadenitis

Ground-glass bone density Fibrous dysplasia; ossifying fibroma

744 s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6
4.2. Technique of orbital biopsy
The tissue sampling can be procured via open surgery, fine
needle aspiration, or as one or several core needle biopsies. In
the case of local anesthesia, the injections should be given at a
distance from the area to be biopsied to prevent injection ar-
tifacts in the specimen. Principally, to limit morbidity due to
the biopsy technique, the lesion should be approached via a
route not crossing critical orbital structures such as the optic
nerve. When confining the biopsy to the lesion, the function of
involved structures such as the lacrimal gland or extraocular
muscle will not be additionally affected.58,60 By contrast,
removal of a piece of the optic nerve, orbital nerves, and
orbital apex causes significant visual, sensory, and motility
deficit and must be avoided where possible.10 In lesions of the
posterior part of the extraocular muscle, damage to the motor
nerves can be prevented by obtaining tissue only from the
orbitaldand not from the globaldlayer of the muscle.60
4.2.1. Open biopsy
For detailed pathological analysis including immunohisto-
chemistry (IHC) and molecular testing, the specimens should
preferably be medium sized. Although samples as small as 2 
2  2 mm can be used for analysis, for best practice and based
on experience, the biopsy should yield a sample of at least 6 
6  6 mm in size. The specimen should not be crushed, torn, or
cauterized during surgical dissection and removal, and hem-
orrhage should be avoided. For lesions of the anterior part of
the extraocular muscle, a disposable dermatological biopsy
punch may be used to minimize trauma to both the muscle
and the specimen, placing the punch perpendicular to the
muscle with the aid of a malleable retractor to lift the muscle
and protect the globe.45 When indicated, marking sutures are
used to orient the surgical margins of the specimen.
The contemporary orbitotomy for open biopsy purposes is
a minimally invasive and safe procedure associated with low
morbidity and can often be performed under local anes-
thesia.31,41,89 Biopsies from the lacrimal gland, extraocular
muscles, and other orbital soft tissues, deep in the apex, can
be obtained through anterior, lateral, or medial orbitotomy.
The framework of surgical decision is governed by the
location of the lesion in the orbit in relation to the optic nerve
and the periosteum (Fig. 2). Lesions located in the upper sector
of the orbit, which includes the lacrimal gland, are reached via
anterior or lateral orbitotomy through an upper eyelid skin-
crease incision.33 Alternative approaches to the lateral orbit
include the lateral canthotomy and lateral retrocanthal orbi-
totomy.32,56 A bone-swinging lateral orbitotomy is used to
increase access to the orbital apex. The lower sector of the
orbit is accessed with a transconjunctival lower anterior
orbitotomy, using the lower fornix postseptal approach for
intraperiosteal exploration of extraconal and intraconal le-
sions, and the subtarsal preseptal approach for entry to the
extraperiosteal space.75 When increased exposure is required,
a lower lateral canthotomy and cantholysis (swinging eyelid)
can be used, or the conjunctival incision can be extended into
a retrocaruncular incision. A medial orbitotomy exposes le-
sions of the orbit located medial to the optic nerve and uses a
conjunctival peritomy approach that is combined with
desinsertion of the medial rectus muscle to gain access to the
deep intraconal space.75 The retrocaruncular route allows
access to the extraconal and extraperiosteal space of the
medial orbit. In addition, the medial orbit can be approached
with an anterior orbitotomy through a superomedial lid crease
incision or, for the deeper intraconal and extraconal spaces, a
vertical upper lid split.68,79 Alternatively, an upper fornix
incision can be used, combined with a vertical lid split or su-
perior lateral cantholysis to gain field of exposure.76
Where the lesion is not entirely confined to the orbit, a
combined biopsy of both the orbit and adjacent structures is
performed, using an endoscopic transnasal or transantral
approach for lesions involving the sinuses. Nowadays, a
transcranial approach is no longer required for orbital biopsy
purposes except in lesions involving the optic canal, superior
orbital fissure, and other intracranial structures.75
Incisional orbital biopsy is the diagnostic method of choice
for lesions that do not require excision for therapy or are too
large for ready excision or to obtain subtyping of a known
malignancy. Such lesions include lymphoproliferative lesions,
IOI, immunoglobulin G4-related disease (IgG4-RD), sarcoid-
osis, autoimmune diseaseerelated changes, and primary
malignant and metastatic tumors. Incisional biopsy is diag-
nostically conclusive in 98% to 99% of orbital cases.41,89
Excisional biopsy is favored in cases where the primary
mode of treatment is removal of the entire lesion. This includes
cystic lesions, cavernous venous malformations, pleomorphic
adenoma of the lacrimal gland, and other circumscribed solid
benign or malignant lesions. To reduce unnecessary lacrimal
gland excision, however, clinicoradiologically suspected
pleomorphic adenoma can be confirmed with intraoperative
fine needle aspiration cytological or frozen section analysis
before excision.47,69
4.2.2. Fine needle aspiration biopsy
Fine needle aspiration biopsy (FNAB) involves sampling of
tumor cells via a hollow needle inserted into the mass. The
aspirated material is stained for cytological examination
under the light microscope but can also be used, after cyto-
spin collection, for immunocytochemical analysis to distin-
guish and characterize malignant lesions.87 With a diagnostic
accuracy (i.e., the cytological diagnosis corresponding with
the histopathological diagnosis) of up to 87%, FNAB can
reliably confirm systemic disorder in known widespread
disease and malignant orbital lesions such as lymphoid tu-
mors and metastasis, or recurrent malignant tumor,
although additional open biopsy should be pursued for
further subtyping of the malignancy.3,94,98 FNAB is valuable
in tumors prone to seeding as in pleomorphic adenoma of
the lacrimal gland, and in coagulopathy and hypervascular
malignant tumors to limit blood loss.30,94 As a primary pro-
cedure with the advantage of a rapid technique, often per-
formed without anesthesia, FNAB can replace open incisional
biopsy in selected cases of palpable lesions. When the lesion
is not palpable, computed tomography or ultrasound guid-
ance can be used.
The nature of the FNAB, composed of disaggregated cells
and without architectural features, however, can pose diffi-
culties in pathological interpretation. With an overall diag-
nostic yield of 74%, cytological diagnosis is less successful in
inflammatory and lymphoproliferative orbital lesions.94
 s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6 745

Fig. 2 e Overview of techniques of orbitotomy without osteotomy for orbital biopsy purposes. A: Axial and B: coronal view of
the orbit showing the areas that can be approached by transcutaneous upper eyelid anterior and lateral orbitotomy. C:
Upper eyelid crease incision for anterior orbitotomy. D: Intraoperative view showing biopsy of a lacrimal gland mass (arrow)
in idiopathic orbital inflammation using transcutaneous anterior orbitotomy. E and F: The lateral areas of the orbit that can
be accessed by lateral canthotomy and retrocanthal orbitotomy. G: Lateral canthotomy (lc) and internal retrocanthal (rc)
incision. H: Intraoperative view showing biopsy of a metastatic lesion of lung carcinoma to the posterior part of the left
lateral rectus muscle (arrow) using lateral canthotomy approach. I and J: The areas of the orbit that can be approached using
a transconjunctival lower anterior orbitotomy. K: Dotted lines showing the internal lower fornix (lf) and subtarsal (st)
conjunctival incision. L: Subtarsal conjunctival incision to biopsy a lesion of the extraperiosteal space. M and N: Spaces of
the orbit accessible through medial orbitotomy. O: Peritomy (p) and retrocaruncular (r) conjunctival incision, and vertical lid
split (vls) for entry to the medial space of the orbit. P: Intraoperative view of the retrocaruncular approach for biopsy of an
extraconal lesion of the deep medial space.

Encapsulated tumors that are fibrotic or densely cohesive in
consistency are least likely to yield adequate cellular aspirates
for cytomorphology. Examples include mass lesions with a
significant fibrotic component, such as IOI, IgG4-RD, gran-
ulomatosis with polyangiitis, scirrhous breast carcinoma
metastasis, plexiform neurofibroma, and plasmacytoma.
Furthermore, lesions that are predominantly composed of
fluid or blood, such as cysts and cavernous venous, lymphatic,
and venous-lymphatic lesions, usually yield little diagnostic
cellular aspirate.66
4.2.3. Core needle biopsy
Similar to FNAB, core needle biopsy has the advantage of the
low morbidity inherent with a percutaneous procedure. Using
a large trephine through a stab eyelid skin incision, core
needle biopsy obtains tissue cores with preserved architec-
tural structures, permitting histological and IHC analysis.
There is limited documented experience with core needle bi-
opsy in orbital mass lesions, and with the increased risk of
tumor seeding of the biopsy tract, it should be avoided in
suspected pleomorphic adenoma of the lacrimal gland.97
746 s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6

Table 2 e Overview of the morphological methods and immunophenotyping in the diagnosis of orbital mass lesions
Method Analysis Advantages Disadvantages Use in Indications in Ref
FFPE orbital
tissue tissue
biopsy
18
Frozen sections  Morphological  Rapid  Less reliable diagnosis No  Intraoperative
evaluation of compared to histochemistry control of
cells and tissues from FFPE tissue representative tissue,
 Intraobserver and nature of the lesion,
interobserver variability and surgical margins
Histochemistry  Morphological  Widespread availability  Intraobserver and Yes  All lesions
evaluation of  Low cost interobserver variability
cells and tissues
5,42,65
Immunohisto  Expression of  Widespread availability  Intraobserver and Yes  Lesions with
chemistry specific proteins  Combines morphology interobserver variability abundant
(IHC) in the morphological with protein expression  Sensitivity and specificity nonmalignant
structure differ among cells
different antibodies  Subtyping of
 Tumor heterogeneity and orbital lymphomas
overlapping  Orbital IgG4-RD
morphological features
might obscure
the interpretation
 Lack of clear cutoff values
20
Electron  High  Detailed featuring  Time consuming No  Orbital myopathies
microscopy magnification of cells and organelles  Limited to small
(EM) of cells areas of the specimen
44,82
Flow cytometry  Characterizes the  Quantitative  Difficulties with aggregating Yes  Orbital lymphoma
phenotype of  Expression of multiple cells, for example,  Differentiation
malignant cells molecules on carcinomas and sarcomas between IOI,
 Detects clonality the same cell  Not suitable for orbital IgG4-RD,
 Information from high-throughput settings and EMZL
millions of
cells in a short time
 Rapid
 Low cost
 Widespread availability

FFPE tissue, formalin-fixed and paraffin-embedded tissue; Ref, reference; IgG4-RD, immunoglobulin G4-related disease; IOI, idiopathic orbital
inflammation; EMZL, extranodal marginal zone B-cell lymphoma.

fixative, with a sufficient ratio to fixative so that the specimen

5. Analysis and interpretation of orbital
specimens
The different stages of tissue handling after orbital biopsy
include prefixation, fixation, processing, and storing, each of
which are critical to achieve optimal pathological analysis and
interpretation.
5.1. Preanalytical variables
The procedure of specimen processing is complex and vari-
able and, where suboptimal, compromises the morphological,
proteomic, and molecular analytic end points.28 After surgery,
the tissue sample is exposed to cold ischemia that affects the
nucleic acids and proteins and hence influences DNA anal-
ysis.1,6 Delayed fixation of 1 hour compromises the interpre-
tation of fluorescence in-situ hybridization (FISH) analysis.6
On the other hand, analysis with polymerase chain reaction
(PCR) is less sensitive and can afford a fixation delay of up to
4 hours.6 To achieve optimal fixation, the orbital tissue spec-
imen should be promptly immersed in the appropriate
floats freely in the container or else is wrapped in a formalin-
soaked gauze.
Fixation preserves the morphological and cellular details,
destroys the microorganisms, and deactivates the enzymes
responsible for tissue autolysis. Formalin fixation and paraffin
embedding (FFPE) is the preferred method for histological and
IHC staining of most tissue biopsies.1 Formalin, however, can
reduce the quality of RNA and DNA through cross-linking of
proteins, and artifacts such as tissue shrinkage and pre-
cipitations commonly occur. Hence, the impact of formalin
fixation to specimens varies greatly, leading to a wider data
variation compared to fresh frozen samples; however,
methods for optimizing the quality of the macromolecules
allow FFPE blocks with up to 20 years of storage time to be
used for next-generation sequencing (NGS).12,35 In that
respect, pathology archives of FFPE tissue are valuable re-
sources for translational clinical research of orbital tumors, as
long as the use of the samples has been approved by patients
and ethical boards.
Alternative to fixing, the tissue can be snap-frozen in
liquid nitrogen or isopentane. After acquisition, the fresh
 s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6
tissue is wrapped in saline-dampened gauze and promptly
submitted to the laboratory. Fresh-frozen tissue particularly
retains the integrity of the nucleic acids, making it ideal for
biomarker profiling, RNA and DNA analysis, and protein
mass spectrometry; however, as mentioned previously,
many of these tests can nowadays also be performed on
fixed material.2,16,92 Moreover, storage of frozen samples is
costly and space consuming. In the case of suspected in-
fectious agents, a fresh sample is sent to microbiology for
culture and analysis. Other fixatives used in specific cases
include alcohol-based fixatives, HOPE (HEPES-Glutamic Acid
Buffer Mediated Organic Solvent Protection Effect), and ni-
trite pickling salt.23 Fixatives for electron microscopy are
glutaraldehyde and osmium tetroxide, which enhance the
contrast of the cellular structures.
5.2. Pathological methods
Depending on the clinical and differential diagnosis, the
pathological analysis ranges from traditional morphological
assessment to high-throughput molecular assays. An over-
view of the array of morphological and molecular methods is
presented in, respectively Tables 2 and 3.
5.2.1. Morphology and immunophenotyping
5.2.1.1. Frozen sections. Frozen sections are acquired by rapidly
freezing, sectioning, and staining the fresh tissue, usually with
modified hematoxylin and eosin (H&E). Although not a substi-
tute for definite pathological diagnosis, frozen sections allow
prompt intraoperative identification of the nature of the orbital
lesion through gross microscopical evaluation. Artifacts, how-
ever, may limit the interpretation.
Frozen sections guide the course of the orbital proced-
ure by intraoperative assessment of (1) the representativity
of the lesion in an incisional tissue biopsy; (2) the nature of
the lesion that requires excisional biopsy; and (3) the sur-
gical margins in the resection of orbital malignancy, optic
nerve glioma, and intraocular malignancy with optic nerve
invasion.18 Evaluation of frozen sections can be chal-
lenging and requires a close cooperation and consultation
between the surgeon and the pathologist during the
procedure.
5.2.1.2. Histochemistry. Routinely, orbital tissue samples are
histomorphologically examined with H&E-stained sections
after FFPE. Other stains include the following: Giemsa for
mast cells, eosinophils, and plasma cells; the Perls iron stain
to assess the age of hemorrhage; and the periodic acid Schiff
stain for carbohydrate molecules, such as glycogen and
glycoproteins, typically found in fungi. The latter stain is
used in the differential diagnosis of invasive sinu-orbital
adenocarcinoma and parasitic and fungal infections.90
Other stains highlighting microorganisms include the
following: (1) the Gram stain to detect and to distinguish
between gram-positive and gram-negative bacteria; (2) the
Grocott-Gomori methenamine silver stain for fungi; and (3)
the Ziehl-Neelsen stain for acid-fast bacilli, for example,
mycobacterium. Certain organic and inorganic foreign bodies
and substances (e.g., wood, talc, silica, suture, cosmetic filler,
747
amyloid) exhibit birefringency on polarized light microscopy,
which is a useful tool in the context of (giant cell) granulo-
matous inflammation in an orbital lesion.48,62
5.2.1.3. Immunohistochemistry. Using antibodies that specif-
ically bind to antigens, IHC provides data about the expres-
sion of antigens, such as proteins, within the context of the
tissue structure. Based on gain or loss of a specific protein
expression, the grade, intensity, and pattern of nuclear and
cytoplasmic immunostaining grade are evaluated. Comple-
mentary to histochemistry, IHC is widely used in the evalu-
ation of protein expression of orbital mass lesions, especially
in lesions with abundant nonmalignant cells87; however, the
interpretation of antigen expression in IHC can be subjective,
particularly with the use of antibodies of variable sensitivity
and specificity.
Immunophenotyping of orbital tumors has diagnostic,
prognostic, and predictive value. Well-selected immunopa-
nels are typically applied in the diagnosis and subtyping of
orbital lymphoma and in the determination of the primary
organ site of metastasis to the orbit (Figs. 3-5).65 Commonly
used markers include Cluster of Differentiation (CD) 20 anti-
gen in B-cell lymphoma, CD3 antigen in T-cell lymphoma, and
CD68 antigen for macrophages, as well as cytokeratin anti-
gens in the differentiation of epithelial malignancy, and es-
trogen and progesterone receptor and prostate-specific
antigen for metastasis. Immunostaining for IgG4-positive
plasma cells is used in orbital lesions consisting of lympho-
plasmacytic inflammation and fibrosis suggestive of IgG4-
RD.26,95
5.2.1.4. Electron microscopy. With electron microscopy, sub-
cellular elements can be visualized under high magnifica-
tion. Owing to the higher optical resolution of electron
microscopy compared to light microscopy, however, arti-
facts induced by formalin fixation can challenge the inter-
pretation of the specimen. Transmission electron
microscope is used in the diagnosis of orbital myopathy
and in rare cases where IHC has yielded inconclusive
results.20
5.2.1.5. Flow cytometry. The phenotype of malignant cells and
identification of possible clonality can be analyzed by flow
cytometry. Through antibody-antigen complexes on the sur-
face, cytoplasm, and nucleus of the cells, the antigen
expression is characterized in heterogenous cell populations.
Flow cytometry relies on the cellularity of the sample which is
variable between tumor type. Flow cytometers allow for up to
19 different markers of the same cell, although this is not
usually required. The cell concentration should be at least
104 cells/mL in w100 mL of fluid suspension for each sample.
The sample is transported in a medium for optimal cell
viability, for example, RPMI (Roswell Park Memorial Institute)
growth medium, sterile saline, and RNA stabilization agent. A
diagnosis can be provided within 3-4 hours of specimen
receipt. Flow cytometry is commonly used with fresh unfixed
liquid samples such as blood and ocular fluids. The analysis
can also be accomplished on fresh frozen tissue as long as the
cells are frozen down as a cell suspension and intact on
thawing.

Table 3 e Molecular analyses and profiling in the diagnosis of orbital mass lesions
Method Analysis Advantages
Fluorescence in  Detection of  Not influenced by
situ hybridization cytogenetic surrounding cells in
(FISH) abnormalities heterogeneous samples
 Gain or loss of known  Detection of aneuploidies
chromosomal material
and fusion genes
Polymerase  Presence and  Highly sensitive
chain reaction (PCR) quantity of a nucleotide  Useful in the detection
 Fusion gene transcripts of mutations from
circulating-tumor
DNA and specimens with
low content of tumor
tissue
 Possible in specimens
with small amount of, or
highly degraded,
macromolecules
 Rapid determination of
the presence of a nucleo-
tide sequence
Multiplex  Detection of  Rapid
ligation-dependent probe changes in copy  Low cost
amplification (MLPA) number variations,  Technically
point mutations uncomplicated
and DNA methylation  “Semi-high”-throughput:
up to 50 different loca-
tions in one single
reaction
 Identifies the exact
breakpoint of alterations
Sanger sequencing  First-generation  Widespread availability
DNA sequencing method
748
Disadvantages Use in Indications in orbital Ref
FFPE tissue
tissue
17,67
 Cannot detect small DNA Yes  Orbital lymphoma
alterations, such as  Orbital sarcoma
microdeletions,  Optic nerve glioma (NF1)
point mutations  Orbital small-blue-round-
 Labor-intensive cell-tumor
 Not suitable for  Orbital alveolar rhabdo-
high-throughput settings myosarcoma (PAX-3-
 Limited to the availability FOXO1, PAX7-FOXO1,
of probes for different loci PAX3-NCOA1, or PAX3-
 Only performed in spe- AFX)
s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6
cific diagnostic questions  Orbital neuroblastoma
(MYCN)
9
 False-positive results due Yes  Orbital sarcoma
to contamination, even in  Orbital lymphoma
very small fractions
 False negative results
from low-quality
nucleotides
 Hypothesis-based
approach
37
 Not suitable for genome- Yes  Optic nerve pilocytic as-
wide screening trocytoma (BRAF)
 Detects only relative  Orbital malignant mela-
changes in copy number, noma (BRAF)
aberrations might not be  Orbital neuroblastoma
detected in samples (MYCN)
with low tumor cell
content
 Careful choice of refer-
ence genome
 Labor-intensive Yes  Optic nerve pilocytic as-
 Time consuming trocytoma (BRAF)
 Low sensitivity  Orbital malignant mela-
 Requires high tumor cell noma (BRAF)
content (30-50%)
 Only 1 strand at the time
can be sequenced
Microarray  Expression of transcripts  Low cost
or methylation patterns  Low data output
based on  Easy data analysis
complementary compared to sequencing
probe hybridization
DNA  Massive parallel  Germline and somatic
sequencing sequencing of DNA mutations,
interaction between cod-
ing and noncoding re-
gions (WGS)
 Point mutations, small
insertions and deletions,
copy number variations,
 Hypothesis-based, Yes
a priori knowledge of
genes of
interest required
 Lack of reproducibility
 High amount of substrate
compared to sequencing
15,35
 Degradation of DNA/RNA Yes  Orbital lymphoma
due to FFPE is a challenge Fresh/frozen
 Low coverage in tissue is preferable
genome-wide approaches
 High complexity of
workflow
 Advanced bioinformatics,
large amount of data

RNA  Massive parallel
sequencing sequencing of RNA
 RNA-editing,
novel fusion genes,
noncoding RNAs,
alternative splicing
Epigenome  Bisulfite sequencing
sequencing and ChIP-sequencing
 Can be locus-specific
or genome-wide
s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6
and structural variants  High cost
 High coverage for identi-  Ethical considerations
fication of
low-frequency mutations
(WES, targeted
sequencing)
16,73,92
 Functional information, Yes  Alveolar
differential gene Fresh/frozen rhabdomyosarcoma
expression tissue is preferable
 Low detection limit in
low expressed transcripts
 Low amount of RNA input
required
19,52,71,85
 Methylation status,  Need of a reference Yes  Orbital lymphoma
histone modification, methylome Fresh/frozen  Orbital peripheral nerve
and transcription factor  Requires high tumor tissue is preferable for sheath tumors
binding content due to methyl- comprehensive analysis
 Potential to identify ation heterogeneity
tumors of unknown within a tumor
primary
 Stable modification of
cancer cells,
tissue can be obtained
from body fluids
and fine needle aspirates

IOI, idiopathic orbital inflammation; IgG4-RD, immunoglobulin G4-related disease; EMZL, extranodal marginal zone B-cell lymphoma; WGS, whole-genome sequencing; WES, whole-exome
sequencing; ChIP, chromatin immunoprecipitation.

749
750 s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6

Fig. 3 e Lacrimal gland tumor, first presentation of an extranodal marginal zone B-cell lymphoma. A: A 78-year-old woman
with left upper eyelid ptosis and swelling, and proptosis. Magnetic resonance imaging, B: coronal and C: axial view,
showing homogeneous enlargement of the orbital lobe of the lacrimal gland (asterisk). D: Intraoperative view of the mass
lesion (arrow) using transcutaneous upper eyelid anterior orbitotomy under local anesthesia. E: Histological view of small-
sized lymphoid cells with prominent, round nucleoli and pale cytoplasm (hematoxylin & eosin, original magnification 350,
scale bar [ 500 mm). The molecular genetic studies using polymerase chain reaction demonstrated clonal rearrangements
of the immunoglobulin heavy (IGH) and light (IGK) chain genes.

Although conventionally used in the diagnostic and
prognostic classification of lymphoma and leukemia, flow
cytometry can also be applied in orbital inflammatory le-
sions. IOI and IgG4-RD, which share overlapping morpho-
logical findings with extranodal marginal zone B-cell
lymphoma, can be distinguished through careful analysis
and integration of the morphological features and
immunoprofiling.44,82
5.2.2. Molecular analyses and profiling
When microscopy and immunoprofiling are inconclusive, or
subclassification of the tumor is required for diagnostic,
prognostic, and predictive purposes, ancillary molecular
testing is performed. Specific genetic alterationsdsuch as
chromosome deletions, gains, balanced or unbalanced trans-
locations, amplifications, and polysomydcan be detected by
cytogenetic and molecular analyses including FISH, PCR, and
multiplex ligation-dependent probe amplification (MLPA).17
Recent advances with deep sequencing array-based plat-
forms and bioinformatic handling allow a comprehensive
analysis of cancer genomes, epigenomes, and transcriptomes
for research and clinical purposes. The rapidly evolving
identification of biomarkers combined with the decreasing
costs of multiplex sequencing has shifted the use from the
research field to the clinical setting; however, ethical consid-
erations and informed consent from the patient are required
to be prepared for coincidental findings, particularly when
examining for germline mutations. The analysis and storage
of the vast amount of data generated are also challenges to be
dealt with.
Molecular analysis is particularly useful in the diagnosis of
poorly differentiated orbital tumors from lymphoproliferative
and mesenchymal origin.67,73 In an expert multicenter study,
the morphological diagnosis of sarcoma was changed after
molecular genetic testing in 14% of cases.40 As it is a priori
unknown which sarcoma will benefit from ancillary testing,
molecular genetic analysis is nowadays standardly performed
on all orbital sarcomas.
5.2.2.1. Polymerase chain reaction. With DNA polymerase and
synthetic oligonucleotides, a known region of a genome can
be amplified in a chain reaction, causing a billion-fold ampli-
fication of a gene product. The advantages of PCR are its high
sensitivity, even from a low input, and rapid determination of
the presence of nucleotide sequences in the biospecimen.
Because of the high sensitivity, however, cross-contamination
can easily occur, leading to false-positive results. Hence,
experience in interpretation is required.
 s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6 751

Fig. 4 e Orbital metastasis of breast adenocarcinoma. A: Left-sided periocular pain, proptosis, and optic neuropathy
developed in an 82-year-old woman, with a history of a primary invasive ductal carcinoma of the breast. Magnetic
resonance imaging, B: coronal and C: axial view showing an ill-defined contrast-enhancing mass in the orbital apex and
inferior rectus muscle (asterisk) with deviation of the optic nerve and thrombus in the superior ophthalmic vein. D: Orbital
tumor tissue, procured through lower anterior orbitotomy, showing epithelial cells clustered in sheets and nests with
variable amount of ductal differentiation admixed with lymphocytes. The tumor cells are pleomorphic with prominent
nucleoli (hematoxylin & eosin, scale bar [ 200 mm). E and F: Immunohistochemistry showed high expression of E: CK7
antigen and F: estrogen receptor, confirming the mammary origin of the lesion (scale bar [ 500 mm).

PCR is routinely used as an adjunct to morphological
analyses in the screening for translocations associated with
orbital sarcoma and lymphoma. An example is the hallmark
reciprocal translocation t(14;18) (q32;q21), which is present
in up to 90% of follicular lymphoma and is used to monitor
treatment response.9 PCR is routinely used to detect clonality
of a B or T cell population within an orbital lymphoprolifer-
ative lesion.22 Clonal rearrangement of the immunoglobulin
heavy chain genes on PCR (IgH-PCR), however, can confirm
the diagnosis of a Benon-Hodgkin lymphoma (B-NHL) only
with associated morphological and immunohistological
features.22 Similarly, with orbital T-cell proliferations, a
clonal product on PCR directed against the T-cell receptor
would speak for a T-NHL should the corresponding
morphology and immunohistology also match these find-
ings. On the other hand, a polyclonal amplification product
would be suggestive of a reactive orbital inflammatory
process.5
reaction is dependent on the ligation of the two MLPA oligo-
nucleotides that serve as a template for PCR reaction. The
highly specific ligase enzyme facilitates only the ligation of
probes with a 100% match of the two MLPA oligonucleotides
with the target sequence. In cases with low tumor cell con-
tent, however, aberrations may not be detectable through
MLPA.
Mutation-specific MLPA probes should only be used in
cases where the finding of the mutation has diagnostic,
prognostic, or predictive value, such as, for example, the BRAF
mutation.37 Evidence of BRAF mutations can be found in
pilocytic astrocytomaetype low-grade optic nerve glioma and
in histiocytic lesions of the orbit.53 The finding of BRAF mu-
tation in orbital melanoma contributes to identify the cuta-
neous (metastatic) versus uveal (local) origin.63 In orbital
neuroblastoma, MLPA analysis may detect the oncogene
MYCN, which is indicative of aggressive behavior and high
metastatic potential of the tumor.37

5.2.2.2. Multiplex ligation-dependent probe amplification. 5.2.2.3. Fluorescence in situ hybridization. Based on the ability
Multiplex ligation-dependent probe amplification is a PCR- to hybridize complementary nucleic acids, FISH detects spe-
based technique that enables a sensitive multiplex amplifi- cific sequences and thereby studies the presence and fre-
cation of up to 50 different genomic locations at once.37 The quency of cytogenetic abnormalities and rearrangements.
752 s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6

Fig. 5 e Orbital embryonal rhabdomyosarcoma. A: A 14-year-old boy without previous medical history presented with
swelling and redness of the right upper eyelid, a palpable solid mass of the upper orbit, and decreased motility of the eye. B:
Computed tomography scan, sagittal view, showing a diffuse mass lesion in the upper orbit (asterisk). C: Intraoperative
view of the biopsy of the mass using transcutaneous upper eyelid anterior orbitotomy. D and E: The histopathological
examination revealed small-round-blue-cells with a high mitotic activity (Hematoxylin & Eosin, scale bar [ 80 mm
 s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6
Approximately 30% of all sarcomas harbor relatively simple
cytogenetic aberrations, such as specific translocations,
which are well suited for FISH analysis.17
FISH analysis is widely used in orbital lymphoproliferative
and soft tissue malignancies, including the rare small-blue-
round-cell tumors of the orbit.73 In childhood orbital malig-
nancy, detecting translocations is relevant in the diagnosis,
prognosis, and prediction of targeted therapeutic response.
These include PAX3-FOXO1, PAX7-FOXO1, PAX3-NCOA1, and
PAX3-AFX fusions in alveolar rhabdomyosarcoma and MYCN
in neuroblastoma (Fig. 5).24,39
5.2.2.4. High-throughput techniques for molecular profiling.
5.2.2.4.1. Genome profiling. Based on the knowledge of the
human genome, multiple DNA molecules can be simulta-
neously sequenced. Whole-genome sequencing involves the
process of the complete coding and noncoding DNA sequence.
One tumor can harbor tens of thousands of somatic mutations
due to the genomic instability of the tumor cells. Many mu-
tations in noncoding regions, however, have uncertain impact
on the carcinogenesis and are, to date, mostly considered
without any clinical value. On the other hand, whole-exome
sequencing determines the coding DNA sequence only.
Compared to whole-genome sequencing, whole-exome
sequencing is less comprehensive and a more affordable
strategy. DNA extracted from FFPE tissue can be used, making
whole-exome sequencing suitable for clinical and research
purposes.35
With an a priori knowledge of candidate genes, small re-
gions of cancer genes characterized as mutational hotspots
can be sequenced, achieving a high depth of sequencing
(termed “next generation sequencing,” NGS). Compared to
PCR, the throughput of targeted sequencing is higher and,
dependent on the size of the gene panel, less time-consuming
and cost-consuming. This is relevant in the common setting
of small-sized and heterogeneous diagnostic samples from
the orbit.
Nowadays, targeted sequencing is becoming increasingly
integrated into the routine diagnosis of orbital tumors. Diffuse
large B-cell and marginal zone lymphoma of the orbit display
different profiles and rates of somatic mutations as compared
to nonorbital lymphomas, suggesting the involvement of
other mechanisms during the lymphomagenesis in this re-
gion.15 This finding emphasizes the necessity of including the
primary site of origin in the interpretation of genomic
analyses.
5.2.2.4.2. Methylome profiling. Epigenetic modifications
such as DNA methylation, histone modification, and non-
coding RNAs are heritable alterations not due to DNA changes.
They play a profound role in the promotion and evolution of
cancer. Although healthy cells display a finely tuned balance
between methylated and demethylated sites, cancer cells
=and 20 mm, respectively). The differential diagnosis included rhabdomyosarcoma, Ewing sarcoma, neuroblastoma, and
lymphoma. F: The proliferation marker Ki-67 was positive in 40e50% of the tumor cells. G: Immunohistochemical
examination showed diffuse strongly positive staining for the muscle-specific myogenic factor 4 (scale bar [ 20 mm).
Ancillary molecular testing was negative in fusion protein PAX-3/FOXO1 and PAX-7/FOXO1, and EWSR1 (Ewing sarcoma) by
FISH analysis, confirming the diagnosis of embryonal rhabdomyosarcoma.
753
exhibit aberrant methylation patterns. An epigenetic hall-
mark of cancer appears to be general DNA hypomethylation
and locus-specific hypermethylation.52 To evaluate the
methylation profile, techniques such as global methylation
quantification, locus-specific DNA methylation, and genome-
wide DNA methylation are used. To evaluate the latter,
microarray techniques and bespoke NGSs are increasingly
applied in various tumors, using DNA extracted from FFPE
samples.
The use of methylation profiling can help to separate and
subclassify different types of peripheral nerve sheath tu-
mors of the orbit, such as neurofibroma, schwannoma, and
malignant peripheral nerve sheath tumors.85 Some of these
tumors have a similar genetic profile but may differ on an
epigenomic level. Furthermore, loss of H3K27 trimethyla-
tion has shown predictive value in malignant peripheral
nerve sheath tumors and is associated with a poor
prognosis.19
5.2.2.4.3. Transcriptome profiling. With the use of high-
throughput sequencing technology, RNA sequencing
comprehensively and accurately assesses the gene expression
profile by in-detecting tumor-specific alternative splicing, long
noncoding RNAs, and interpreting the significance of DNA
mutational status. Both the presence and frequency of an RNA
sequence can be determined, allowing quantitative evaluation
of the gene expression.
Many cancers, especially those deriving from mesodermal
tissue, feature a high incidence of gene fusions and sub-
groups.93 In some cases, as in alveolar orbital rhabdomyo-
sarcoma, the presence or absence of a fusion gene has
important prognostic value.73 Traditionally investigated on a
hypothesis-driven approach with low-throughput techniques
such as FISH or reverse-transcription PCR, the use of RNA
sequencing will augment subclassifications in various orbital
tumors.
6. Conclusion
Orbital mass lesions pose a diagnostic challenge because of
their diversity of causes. Despite distinct clinical and imaging
features in some lesions, a tissue biopsy is frequently required
to reach at a conclusive diagnosis essential for appropriate
management. An orbital biopsy, however, is only as good as
the combined skills of the surgeon and the experience of the
laboratory, including the technicians and the pathologist. An
appreciation of the limitations of a diagnostic orbital biopsy is
as important as an understanding of its many useful appli-
cations. Despite FNAB and core needle biopsy being less
invasive alternatives, open biopsy via small incision orbitot-
omy is the preferred approach because it confers the highest
diagnostic value. Close collaboration between the surgeon and
754 s u r v e y o f o p h t h a l m o l o g y 6 4 ( 2 0 1 9 ) 7 4 1 e7 5 6
the pathologist is key in the sharing of important clinical data
and the construction of differential diagnoses. Peroperative
variables, that is, the size, site, and quality of the specimen, as
well as preanalytic variables, that is, fixation, processing, and
ancillary testing, all determine a successful pathological
outcome.
Recent advances in genomic, transcriptomic, and epi-
genomic testing of tissue biopsies constitute a new diag-
nostic paradigm, with a shift from low- to high-throughput
assays, and thereby understanding the molecular profile in
a more comprehensive perspective. Traditional diagnosis
from tissue of the primary tumor and treatment with
cohort-based protocols is gradually evolving into patient-
tailored management. By correlating clinical data with the
histological and molecular profile, tumor progression can be
assessed and targeted therapy selected.71 Some centers
have already implemented high-throughput techniques,
such as comprehensive targeted NGS panels, as an inte-
grated part in the diagnostics of orbital tumors. By exploit-
ing pathology resources of orbital tissue biopsies, detailed
correlation between molecular features and clinical
behavior of orbital tumors can be studied. Through inter-
national collaborations such as the International Cancer
Genome Consortium and Cancer Genome Atlas, novel sub-
classes and new taxonomies of tumors have been identi-
fied.14,21,74 Comparatively, to increase data collection of rare
diseases, cases with orbital mass lesions should be sub-
jected to multicenter studies. These efforts will generate
new and robust insights in the phenotypic-genotypic profile
of orbital mass lesions, ultimately to offer precision targeted
therapy to the patient.
6.1. Methods of literature search
The data were identified by searches of MEDLINE (via
PubMed), Current Contents, and references from relevant ar-
ticles, using the following search terms “orbit” AND “diag-
nosis”, “imaging”, “biopsy”, “fine needle aspiration”, “core
needle biopsy”, and the specific diseases and methods dis-
cussed in this review. As additional source and cross-check
for the reliability of the search strategy, we reviewed
comprehensive textbooks.29,36
7. Disclosures
None of the authors have proprietary or commercial interest
that represents a conflict of interest with the material in this
study.
Acknowledgments
Thanks to Rita Van Ginderdeuren, MD (University Hospitals
Leuven) for contributing the pathological images used in
Figures 3 and 4.
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