Recent research has provided new insights into the pathogenesis of cholesteatoma. Theories of pathogenesis include epithelial cell rests that fail to involute, invagination of squamous epithelium through microscopically injured tympanic membranes, and inclusion or implantation of epithelial cells during pathological middle ear events. Retraction of the tympanic membrane, particularly the pars flaccida, allows the accumulation of keratin debris which can become infected and lead to cholesteatoma formation. Advances in genomics, epigenetics, and immunology research have found alterations in genes and microRNAs involved in proliferation, apoptosis, and inflammation that may contribute to the aggressive growth seen in cholesteatoma.

1. CHOLESTEATOMA-
Recent Advances In
Understanding Its
Pathogenesis
DR. MEGHASHYAM KHYATHI

2. DEFINITION- “SKIN IN THE WRONG PLACE”
• cystic lesion formed from keratinizing
stratified squamous epithelium in the
temporal bone comprising of:
1. Cystic content: desquamated keratin
center
2. Matrix: keratinizing stratified
squamous epithelium
3. Perimatrix: granulation tissue that
secretes multiple proteolytic enzymes
capable of bone destruction
• Synonyms - epidermosis or keratoma

3. LOCATION
Extradural: -middle ear cleft
(most common)
-mastoid
-petrous apex
-EAC
Intradural: Cerebellopontine angle
(most common}

4. HISTORY
• 1683: Joseph Duverney ( french anatomist )
– abscess of bone behind ear – fistula –
shedding – “scales” – “grave accidents”
• 1829: Jean Cruveilhier ( french
anatomist/pathologist) – avascular tumor –
cells of subarachnoid space
• 1838: Johannes Muller ( German
physiologist) - Meant to describe a tumour -
cholesterin + fat - “cholesteatoma”-
MISNOMER

5. CLASSIFICATION
• CONGENITAL
• ACQUIRED : PRIMARY
SECONDARY

6. CONGENITAL CHOLESTEATOMA
Korner’s 1965:
Pearly white mass behind an intact TM in the absence of history of otitis
or otorrhea, TM perforation, or previous otologic procedures
Levenson (1986) criteria:
- Pearly white mass medial to a normal tympanic membrane
- Normal pars flaccida and pars tensa
- No prior history of otorrhea or perforation
- No prior otologic procedure
presence of prior bouts of otitis media does not necessarily exclude the
presence of congenital cholesteatoma

7. CONGENITAL CHOLESTEATOMA
• Origin remains uncertain - theories
• Usually starts from the antero superior
quadrant
• Spreads through the posterior superior
quadrant, attic and finally into the
mastoid cavity
• Mean age of presentation is 4.5 yrs
• M:F ratio is 3:1
• Incidence is 0.12 per 100,000 people
• Anterosuperior quadrant >
Posterosuperior quadrant

8. THEORIES OF PATHOGENESIS OF “CONGENITAL
CHOLESTEATOMA”
• EPITHELIAL CELL REST THEORY
• SQUAMOUS METAPLASIA THEORY
• EPIDERMOID FORMATION THEORY
• INVAGINATION THEORY

9. EPITHELIAL REST THEORY- Failure of involution of
embryonic cell rest :
 Teed (1936)
 Squamous cell rest present in intrauterine life fail to involute
 location - Junction of the first branchial cleft and pouch in the
anterior mesotympanum near geniculate ganglion (ie. Eustachian
tube and middle ear)
 Appears by 10 weeks
 Normally involutes by 33 weeks
 Persistence Congenital cholesteatoma

10. INVAGINATION THEORY (REUDI):
in utero Inflammatory injury to tympanic membrane
Microperforation in the basal layer
Invagination of squamous epithelium (epithelial cones) through
macroscopically intact but microscopically injured TM
Fusion of epithelial cones
Congenital cholesteatoma

11. “ACQUIRED” INCLUSION THEORY (TOS):
 Challenged the “epithelial rest” theory
It was noted that:
Anterosuperior cholesteatoma attached to anterior aspect of the
malleus handle or neck
Posterosuperior cholesteatoma attached to posterior aspect of the
malleus handle and to the incudostapedial joint.
Both are far from the anterior tympanic annulus and the lateral wall
of the Eustachian tube where the epithelial rests are found –
discrepency
STATES that squamous epithelium may be included or implanted into
the tympanic cavity during many pathological events in childhood

12. Mechanism of
inclusion of
epithelial cells
A. Adherent and
retracted TM- small
cuff left behind
B. Tangential tear-
remenant cells left
behind
C. Microperforations –
epithelial cones
D. Repeated
inflamations –
epithelial cones

13. CLASSIFICATION OF CONGENITAL CHOLESTEATOMA
Derlacki and Clemis:
1. Petrous pyramid
2. Mastoid
3. Tympanic
Potsic:
Stage 1: Single quadrant with no ossicular or mastoid involvement
Stage 2: Multiple quadrants with no ossicular or mastoid involvement
Stage 3: Ossicular involvement but no mastoid involvement
Stage 4: Mastoid extension

14. CLASSIFICATION OF CONGENITAL CHOLESTEATOMA ( CONTD)
Nelson:
Type 1: Mesotympanum with no incus or stapes erosion
Type 2: Mesotympanum or attic with ossicular erosion but no mastoid
extension
Type 3: Mesotympanum with mastoid extension

15. ACQUIRED CHOLESTEATOMA
PRIMARY ACQUIRED CHOLESTEATOMA
• Cholesteatomas that arise from retraction pockets
• Implies that infection may not have given rise to the
cholesteatoma.
• Tympanic membrane remains intact

16. THEORIES OF PATHOGENESIS OF AQUIRED
CHOLESTEATOMA
• INVAGINATION THEORY ( RETRACTION
POCKET )
• BASAL CELL HYPERPLASIA THEORY
(PAPILLARY INGROWTH)
• EPITHELIAL INVASION/ MIGRATION
THEORY ( IMMIGRATION )
• SQUAMOUS METAPLSIA THEORY
PRIMARY AQUIRED CHOLESTEATOMA
SECONDARY AQUIRED CHOLESTEATOMA
BOTH PRIMARY AND SECONDARY AQUIRED
CHOLESTEATOMA

17. EPITHELIAL INVASION/ MIGRATION THEORY – HABERMANN (1888)
AND BEZOLD (1890)
Inflammation in Prussaks space
causes breaks/ defects in the basal lamina
allowing invasion/ migration of keratinising squamous epithelium
into middle ear
cholesteatoma formation

18. WENDT ( 1873 ) SQUAMOUS METAPLASIA THEORY:
– Low cuboidal and simple squamous epithelium - changed to stratified
squamous epithelium in patients with chronic or recurrent ear
infection -cholesteatoma formation.
– Epithelial cells - pluripotent - can differentiate into other cell types in
presence of inflammation
– Clinically there is little support for this theory - only animal studies

19. INVAGINATION THEORY (WITMAACK 1933): MOST COMMON MECHANISM
Eustachian tube dysfunction
Barometric perturbation of middle ear space
Impaired ventilation
Negative middle ear pressure
Structural weakening of the tympanic membrane and development of retraction
pockets
Pocket deepens and becomes non-self cleaning
Bacterial proliferation and superinfection of the accumulated debris that leads to
chronic infection and epithelial proliferation

21. BASAL CELL HYPERPLASIA OR PAPILLARY INGROWTH: LANGE (1925)
-Initial retraction pocket stage - epithelial migratory pattern is maintained.
-Pocket deepens, drainage pathway narrows, keratin debris accumulates
Accumulated debris becomes infected
Bacterial proliferation
Inflammation
Inflammatory cells influx, production of cytokines, local release of collagenases
Break in the basement membrane
Formation of epithelial cones that grow toward the stroma
-Subepithelial invasion+keratinocyte proliferation (microcholesteatoma)- hallmark of
precholesteatomatous stage of cholesteatoma

22. SUMMARY for theories of aquired cholesteatoma

23. RETRACTION OF TYMPANIC MEMBRANE
• PARS FLACIDA RETRACTION ( MOST COMMON )
• PARS TENSA RETRACTION

24. Classification of Pars flaccida rertraction (Tos)
Stage 1: Pars flaccida is dimpled and more retracted than normal but
not adherent to the malleus
Stage 2: Retraction is adherent to the neck of the malleus and the
full extent of the retraction can be seen
Stage 3: Part of retraction is out of view and there may be partial
erosion of the bony attic wall
Stage 4: Definite erosion of the attic wall with the full extent of the
retraction being uncertain because it is out of view.

26. Classification of pars tensa retraction (Sade and
Halevy)
Stage 1: Retracted membrane
Stage 2: Retraction onto the incus -not attached
Stage 3: retraction touching the promomtory
Stage 4: retraction attaches to any middle ear structure

27. SECONDARY ACQUIRED CHOLESTEATOMA
 Cholesteatoma secondary to breach in the tympanic membrane with
keratinocyte proliferation in the middle ear at or near the site of a
tympanic membrane perforation which is usually posterosuperior marginal
in location
 Epithelial cells migrate across a denuded surface ‘contact guidance’ and
stop when they encounter another epithelial surface ‘contact inhibition’

28. PATHOGENESIS OF SECONDARY AQUIRED
CHOLESTEATOMA - THEORIES
IMPLANTATION THEORY
Squamous epithelium implanted in the middle ear as a result of
• Foreign body
• Blast injury to TM leaving keratinocyte behind a healed perforation
• At the site of temporal bone fracture
• Iatrogenic introduction (stapedectomy, tympanoplasty, pressure
equalization tube placement, middle ear exploration
SQUAMOUS METAPLASIA THEORY
Chronic otitis media / recurrent otitis media → desquamated
epithelium transformation to keratinized stratified squamous
epithelium

29. EPITHELIAL MIGRATION THEORY
– Squamous epithelium migrates along perforation edge medially
along undersurface of tympanic membrane destroying the columnar
epithelium.
– Secondary to ventilation tube / myringotomy insertion,
tympanoplasty
PAPILLARY INGROWTH / INVASION THEORY
– Inflammatory reaction in Prussack’s space with an intact pars flaccida
causes break in basal membrane → marginal perforation → skin from
EAC wall migrates into the middle ear→ loss of contact inhibition with
the middle ear mucosa (destroyed by infection)
– Posterior superior TM

30. Disturbed homeostasis of keratinocyte growth and programmed cell death:
implicated in cholesteatoma formation
 Elevated level of proliferative marker Ki-67
 Marker of cell death- caspase-3 absent
decreased apoptosis
increased proliferation
TUNEL test- (terminal deoxynucleotide transferase mediated dUTP nick end
labelling technique)
marker of apoptosis

31. BIOFILM THEORY – Garca et al ; 2013, Baysal et al ; 2013
Gram positive and negative bacteria (most commonly pseudomonas aeroginosa found in
extracellular matrix
Aerobic metabolism of P aeroginosa causes imbalance in oxidative processes
Biofilm formation
Induce EGF, upregulate cytokines (IL-6)
Altered epithelial signalling
Hyperkeratotic state
Formation of cholesteatoma matrix and keratin debris

32. • Bacteria in matrix - aggressiveness of the cholesteatoma -
resulting in persistance of the disease.
• The LPS ( lipopolysaccharide ) of bacteria - activate
keratinocyte proliferation.
• Escaped acidic content of bacteria - bone destruction.
• Nguyen et al ; laryngoscope ; 2014 : acid leak through the
cholesteatoma caused by increase in permeability of
epithelium was found to be associated with decrease in
filaggrin protien expression.

33. MUCOSAL TRACTION THEORY – JACKLER et al ;
laryngoscope; may 2015
Sequential adhesion of opposing mucosal surfaces due to bridging mucosal bands
Negative pressure in middle ear
Retraction of pliant portions of TM
Close proximity of of TM and ossicles ( incus )
Conjoined mucosal bilayer absorbed
Trapping of mucosa in middle ear and mastoid
Release of inflamatory cytokines
Proliferation and migration of keratinocytes

34. ADVANCES IN BIOMOLECULAR RESEARCH IN
CHOLESTEATOMA
• 1838: Johannes Muller describes a tumour - cholesterin + fat -
“cholesteatoma”- MISNOMER
• Hayashida and Nomura et al Recently cholesterol found to be an
essential component of cholesteatoma
Desmosterol and delta 7 cholesterol ( lanosterol ) – matrix
• Bloksgaard et al ; 2012 with multiphoton excitation florescence
miccroscopy found all major classes of lipids stored in ODLAND BODIES
in stratum granulosum in cholesteatoma
• Svane knudsen et al found increased increased lipid metabolic activity
in cholesteatoma
No definitive conclusion could be made as cholesterol crystals are a
component of all long standing inflamatory conditions

35. RECENT ADVANCES IN GENOMICS OF CHOLESTEATOMA
• CONNEXIN 26 (gap junction B-2) protien coded by the – GJB2
gene - transmembrane protien- cochlea/skin- mutaions cause –
non syndromic sensorineural hearing loss & hyperkeratotic skin
disorders
• Choung et al 2006 – found upregulation of connexin 26 in
middle ear cholesteatoma.
• Klenke et al 2012 – found higher expression of GJB2 gene in
middle ear cholesteatoma than the EAC skin
Found positive correlation between aggressiveness of the
lesion with the levels of GJB2 gene levels

36. GENOMIC INSTABILITY IN CHOLESTEATOMA
• Recent studies- Alterations in proto-oncogenes – c-myc and c-jun-
implicated in multifactorial pathogenesis
• Downregulation of tumor supressor genes in cholesteatoma – p53,
p27, CDH18,19, ID4, PAX3, LAMC2, TRAF2B
• Albino et al - Higher expression of p53 in cholesteatoma
• Vassar et al -Upregulation and activation of EGFR ( epidermal
growth factor receptor) – keratinocyte proliferation in basal layer of
epidermis in cholesteatoma
Ergun et al - over expression of TGF-alfa- stimulation of EGFR and
stimulator of cell growth

37. Chung et al ; 2015 jun- Cellular FLICE ( FADD- like IL-B converting
enzyme) and c-FLIP ( inhibitory protien ) – antiapoptotic
regulator associated with human malignancies found in
cholesteatoma.
They also found increased ki-67 – marker of hyperproliferation
in benign hyperplastic epithelial disease .
GENOMIC INSTABILITY IN CHOLESTEATOMA (contd)

38. Lee and Chung et al ;2015 april : reduced expression of E –
cadherin & beta catenin in cholesteatoma.
• Reduced expression - increases invasiveness of SCC in
malignancies which is also seen in cholesteatoma.
• Acquired cholesteatoma > congenital cholesteatoma
• Explains increased agressiveness of acquired cholesteatoma.
Thus the existence of a link between genomic alterations and
pathogenesis of cholesteatoma can be made.
And also an underlying association between cholesteatoma and
neoplasm can be asserted.

39. EPIGENETIC REGULATION IN CHOLESTEATOMA –
role of micro RNA in pathogenesis
Kuo CL ; laryngoscope ;2015: MicroRNA are small non coding
molecules which regulate expression of post transcriptional
messenger RNA - dysregulation of which have now been
implicated in pathogenesis of cholesteatoma
Friedland et al ; 2009 - upregulation of micro RNA 21
supression of PTEN & PDCD4( tumor supressor genes)
Increased migration , growth and invasion in cholesteatoma.

40. Chen and Qin; 2011 – found increased levels of micro RNA 21
and pronounced reduction of PTEN and PDCD4 in pediatric
patients.
They also found upregulation of microRNA-let-7a and concurrent
down regulation of HMGA2 (an oncogene seen in neoplasms)
reduction in proliferation of cholesteatoma cells and increased
keratinocyte apoptosis.

41. RECENT ADVANCES IN IMMUNOLOGY IN
PATHOGENESIS OF CHOLESTEATOMA
recent studies have Implicated the role of innate immunity in the
pathogenesis of cholesteatoma
Szczepanski et al; 2006 – strong expression of TLR ( Toll Like
Receptors ) TLR-2, TLR-3, TLR-4 in acquired cholesteatoma
Leichtle et al ; 2015 : TLR and NOD ( nucleotide binding
oligomerization domain ) induction and thus activation of
innate immunity in cholesteatoma.
relationship between NOD2 mRNA levels and development of
acquired cholesteatoma

42. ADVANCES IN ANGIOGENESIS IN CHOLESTEOTOMA
• Olszewska et al; 2004: Angiogenesis in cholesteotoma – role in its
aggressiveness.
VEGF, IL8, COX2 – most potent angiogenic factors
• Fukodome s et al; 2013: found that these three factors are
regulated by transcription factor inhibitor of DNA binding ( Id1).
Thus Id1 can be a potential target for regulation of
cholesteatoma progression and its aggressiveness

43. HOW DOES CHOLESTEATOMA ERODE BONE?
1. LIPOPOLYSACCHARIDE (component of bacterial cell wall)
(higher concentration in cholesteatoma with bony erosion)
Preosteoclastic cells having receptor activator NF-kB (RANKL)
Release of cytokines (EGF,TNF-a, IL-1a, IL-1b, IL-6,INF-b, PTHrP)
Conversion of preosteoclastic cells to osteoclastic cells
Bone erosion

44. Manuia et al; 2014 : Matrix-metalloprotinases ( MMP ) –
proteolytic enzymes shown to promote aggressiveness of
cholesteatoma by destruction of bony tissue
upregulated MMP ( MMP 1, MMP9, MMP10, MMP 12 )and
downregulation of tissue inhibitor of metalloprotienases is
associated with the matrix degradation
Pediatric cholesteatomas are more aggressive than adult
cholesteatomas due to greater number of metalloprotienases
in children

45. 2. NITRIC OXIDE TYPE 2:
Cytokines (TNF-a, IL-1b and IFN-g)
Formation of nitric oxide type 2
Enhanced osteoclastic activity
Bone erosion
A recent meta-analysis by Chen AP et al ; acta otolaryngol ; 2015 : shows a
significant corelation between the RANKL/OPG/RANK system with middle
ear cholesteatoma .
positive correlation exists between RANKL expression and cholesteatoma
OPG expression showed an inverse association with cholesteatoma

46. Cholesteatoma expansion theory:
Accumulation of debris inside the cholesteatoma
Expansion of its size escape of proteolytic enzymes in perimatrix
Pressure over surrounding tissue marked granulomatous reaction
Pressure necrosis of surrounding bone bone destruction
Hyperemic decalcification theory:
release of cytokines from the perimatrix
Vascular dialatation and angiogenesis
Increased blood flow to the cholesteatoma
Increased transport of calcium away from the site
More release of calcium through osteoclast stimulation

47. PATHWAYS OF SPREAD
Most common sites of origin in
order of frequency
 Posterior epitympanum
 Posterior mesotympanum
 Anterior epitympanum

48. Posterior epitympanic Cholesteatoma:
most common
extends posteriorly from Prussack’s space
Superior incudal space(lateral to incus body)
Aditus ad antrum
Antrum

49. ALTERNATIVE ROUTE
Posterior epitympanic cholesteatoma
Descends floor of prussack’s space
Posterior space of Von troeltsch
Middle ear (posterior mesotympanum)
RARELY
Anterior spread from the prussack’s space
Anterior to the head of malleus
Anterior epitympanum
Downward into the anterior mesotympanum via the anterior
pouch of von troeltsch

50. Posterior mesotympanic cholesteatoma
• Posterior portion of pars tensa
retracts to form cholesteatomatous
sac
• Sinus tympani and facial recess
commonly involved
• Extension to mastoid occurs via
posterior tympanic isthmus and
inferior incudal space (medial to
incus and malleus)

51. Anterior epitympanic cholesteatoma
 Retractions form anterior to the
malleus head
 Reach the middle ear through the
anterior pouch of Von troeltsch
 Floor of anterior epitympanum
related to the horizontal portion of
the facial nerve and geniculate
ganglion- facial nerve dysfunction
may occur

52. THANK YOU

53. The Prussack’s Space
Boundaries:
• Superior Limit: Lateral Malleolar Fold.
• Anterior Limit: Thin, Membranous Fold Among The Tympanic
Membrane And The Anterior Malleolar Ligament Fold.
• Medial And Inferior Limit: Neck And Short Process Of The Malleus
Respectively.
• Lateral Limit: Shrapnell’s membrane
• Posteriorly: Represented By A Large Posterior Pocket Of Von Troltsch

54. • Posterior pouch of Von troeltsch:
between the tympanic membrane and the posterior
mallear fold
 Anterior pouch of Von troeltsch:
between the tympanic membrane and the anterior
mallear fold.

59. Epitympanic Diaphragm
 3 Malleolar Ligament Folds (Anterior, Lateral, And Posterior)
 Posterior Incudal Fold
 2 Duplicated Membranous Folds (Tensor Fold And The Lateral Incudomalleolar Fold)
Associated With The Incus And The Malleus.