Comparative ultrastructural study of condylomatous and dysplastic lesions of the human uterine cervix

FACULTY

OF

MEDICINE

Ui

/9XS

C 33S

A THESIS

PRESENTED

TO THE SCHOOL OF GRADUATE STUDIES

OF LAVAL

UNIVERSITY

FOR

THE DEGREE

OF DOCTOR OF PHILOSOPHY (Ph.D.)

BY

MARCOS CASAS-CORDERO

MASTER OF SCIENCE

FROM

THE

UNIVERSITY OF OTTAWA

COMPARATIVE ULTRASTRUCTURAL STUDY OF CQNDYLOMATOUS AND

DYSPLASTIC LESIONS OF THE HUMAN UTERINE CERVIX

RÉSUMÉ

Les condylomes acuminés peuvent se définir comme des lésions intraépithéliales,

causées par une infection virale du papillome humain.

Ces lésions se développpent dans l'épithélium pavimenteux de la région ano-génitale.

Bien qu'ayant toujours été présents au col utérin, ils ont été rarement rapportés.

En 1976, le col utérin a été reconnu comme un endroit propice au développement des

lésions condylomateuses surtout lorsqu'il s'agit de surfaces planes par opposition aux

surfaces papillaires.

L’examen cytologique des lésions condylomateuses fait voir là présence de deux types

de cellules: les koilocytes et les dyskérotocytes.

Les condylomes plans ont des caractéristiques propres:

- parmi les lésions intraépithéliales, ils se trouvent le plus fréquemment au col

utérin;

- le groupe de personnes les plus affectées est relativement jeune (21-25 ans);

- dans le passé, ces lésions étaient diagnostiquées comme des dysplasies;

- et l'examen cytologique démontre que les cellules provenant des lésions planes

ont la même cy to morphologie que le condylome acuminé.

À cause de sa morphologie cellulaire et la structure plane de la surface de l'épi­

thélium, ces lésions ont été appelées condylomes plans pour les distinguer du condy­

lome papillaire classique.

La proposition voulant que les lésions planes soient des condylomes suppose évidem­

ment que l'agent étiologique soit le virus du papillome et que ce dernier pourrait jouer

un rôle important dans le développement du cancer du col utérin.

Au début, une telle affirmation faisait place à une grande incertitude quant à la

terminologie, l'identification histologique et l'étiologie des lésions condylomateuse du

col utérin.

La présente étude se propose comme objectif principal de vérifier si la lésion plane

est d’étiologie virale; d'établir les critères morphologiques qui pourraient aider à faire

un diagnostic qui permettrait de distinguer le condylome de la dysplasie dans le col

utérin.

Des observations au microscope électronique de lésions condylomateuses ont révélé la

présence de particules virales dans 58.88% des condylomes et 3440% des cas où le

condylome était associé avec la dysplasie. La structure des particules virales

correspondait à la morphologie du virus du papillome humain.

La présence d'antigènes du virus du papillome humain détectés à l'aide de la technique

peroxydase-antiperoxydase, a confirmé le type de virus présent dans les lésions

condylomateuses. L'antigène du virus du papillome humain fut trouvé dans 62.63% des

cas de condylome et dans 20.40% des cas de condylome associé à de la dysplasie.

L'interaction virus-cellule épithéliale se manifeste par une altération de la différen­

ciation cellulaire. Cette différenciation anormale est manifestée par la formation de

trois cellules distinctes: les koilocytes qui sont des cellules montrant une zone

perinucléaire claire entourée d'une zone dense composée de tonofilaments; les

dyskérotocytes étant des cellules avec un cytoplasme dense et très kératinisé; et

occasionnellement des cellules "foncées" ayant une apparence fusiforme avec un

cytoplasme dense.

Les altérations morphologiques causées par l'effet cytopathique du virus nous ont

permis d'établir des critères histologiques permettant de différencier le condylome de

la dysplasie.

Le condylome est une infection génératrice de virus du papillome humain dans lequel

on observe l'effet cytopathique des virus (koilocytes), les particules virales et où

l'antigène viral peut être détecté.

La dysplasie ne montre pas de koilocytes. Elle est dépourvue de particule virale et on

note une absence d'antigène viral.

Le condylome associé avec la dysplasie est décrit comme un troisième type de lésion.

Il comprend 34.02% de toutes les lésions condylomateuses étudiées. La morphologie

de ce type de lésions correspond à une association des caractéris tiques du condylome

et de la dysplasie. Il est considéré comme une étape intermédiaire dans la progression

du condylome vers la dysplasie.

L'ensemble de toutes ces observations renforce l'idée que des lésions condylomateuses

du col utérin peuvent être considérées comme une première étape dans la séquence

des altérations qui précèdent le carcinome envahissant du col utérin.

TABLE OF CONTENTS

11

Page

TABLE OF CONTENTS... ii

LIST OF TABLES... vi i

LIST OF FIGURES... ... i x

ACKNOWLEGMENTS... i vx

PREFACE... xvi

CHAPTER I:

INTRODUCTION...

1

1.0 Normal uterine cervix... . . 1

1. 1 General... 1

1.2 Histology... 1

1.2.1 Squamocol umnar junction... ■... 4

1.3 Ultrastructure of normal cervix... 4

1.3.1 Exocervical epithelium... 4 1.3. 1.1 Basal zone. ... ... ... . 5 1.3. 1.2 Intermediate zone... 7 1.3. 1.3 Superficial zone... 8 1.3. 1.4 Clear cells... 9 1.3.2 Endocervical epithelium. ... 10

.1— ■ (Z} Li U H d V 1 U fîl d ... 2.1 History... ... ... 2.2 Cytology... ... .

2

.3 C r i t eria t o i dent i f y c: o n d y 1 om a t o u s .s. ’. .s Histology. ... ... ... 2. 3. 2. 1 F' a p i 11 a r y c o n d y loma... . 2.3.2.2 Fl at condylorna. ... . . 2.3.2.3 Inverted condyloma... 2.3.2.4 Atypica1 condyloma. ... s", n •—1 . FolpOSOOpy. n . . . H n n . n . o n n n . n n n . a 2 „ 3. '3. 1

P

a. pill a r y c o n d y loma... 2.3.3.2 Spi kecl condyl orna. ... 2.3. 3, '3 Flat condyloma... ... . . « 0 II y s g 1 a. s x c:i... ... ...

•0*... (._■ y t o J. o çiy... ... ... 3.3 Histology... ... . . . 3.4 Utrastr uctu.re ... .

4.0 Virus and cancer

4. 1 General.... . .

111

Page

10 10 17 -SU ■-■cr

O

₆

28

28

29

30

30

37

43

4::

IV

Page

5.0 Human papi 11 ornavi rus (HPV)... . 46

5. 2 Structure... ... ... . 49

5.3

Types

o-f HPV... ... ... . 50

6.0 Human papi 11omaviruses and ma1 i g nan t

c on ver

e

i on ... ... ... . 54

6. 1 General... ... . . 54

6.2 Epidermodysplasia verruciformis... 56

6.3 Laryngeal pa.pi 1 lama, ... . 60

6.4 Condyloma acuminatum... ... . 62

7.0 HPV and associ ated f actors in the

squamous carcinoma

of the

uterine

cervix

...,

64

8.0 Virus-host interaction. ... . 67

CHAPTER II: MATERIALS AND METHODS...

'0

1.0 G e n e i a1 2.0

Bi

ops y... . 3. 0 El e c tr on m i c: r o s c o p e t e c h n i q u e. . 3. 1 Fi

xati

on... . 3, 2 Post-fix ati on ... 3.3 Embedding... .

3.4 Block trimmin g and sectioning.

70

78

V

Pape 4.0 Periodic acid schiff (PAS)... ...

5.0 Peroxidase-antiperoxidase reaction (PAP). 5.1 General ... ... 5.2 Immunoperoxidase method. ... 6.0 G a 3.1 o y

i

g 1 u. c:ose technique... ...

CHAPTER III: RESULTS. ...

1.0 General ... ... 2.0 Condyl omatous 1 esi one . ... .

2.1 General ... .

2.2 L i g h t m i c rose o p e obs e r v ations... . 2.2. 1 Koi 1 ocytoti c: one. ... 2.2.2 Deep ep i thel i al zone... . 2.3 E3 ectron microscope observations... 2.3. 1 General ... ... ... . 2.3.2 Pure condyloma. ... 2.3.2.1 Koi1ocytotic zone... ... II I' . .!. . 1 I O i 3 O 1 e ■ n n . . . n « . u e u ■ e . n 2.3.2. 1.2 Dyskeratocytes... . 2.3.2. 1.3 Virus particles... 2.3. 2. 2 Deep epithelial zone... ... •it «■ v* li ■>!. u >it n .1. /TO Z?. .t"> C?. .1 .L “il I 11 Ü. I~1 i?, iinuuuaponnKannaN 2.3.2.2.2 Deep epithelial zone... 2. 3.2.2.3 Per i n u.c 1 ear c 1 ear z one... .

84

84

88

91

94

94

97

97’

99

100

103

103

103

104

1 05

108

1 10

113

113

1 14

1 16

VI

» 0 D ]' 1 3 ~ 1 I4, b n i ■ > K » n i nan ...na a a a a a

3. 1 Li g h t m i c r o s c: o p e

a

b s e r v a t ions. . . . 3.2 E1 sctran micras cope abservations.

3a 2. 1 General. ... Page 1 17 117

118

1 18

3.2.2 U1trestructure of

epithelial

cells... 113

3.2.3 Ultrastructure of basal lamina... 123

4.0 Condyloma associated with dysplasia... 124

4. 1 General ... ... ... . 124

4, 2 Koi 1 ocytot i c: zone. ... ... 125

4.3 Deep ep i thel i al z one.. ... ... . 125

4.3. 1 Dark cells... ... ... . 127

5 „0 P e r i o d i c a c i d s c: h iff ( P A S ) on

epon

thick

sections... ...

129

6.0

Per

o x i d a s e

-an

t i p

63

r

oxidase reaction... ...

1

30

7.0 Gal lot an ic acid technique. . ... . 132

CHAPTER

IV;

DISCUSSION

191

CHAPTER V;

CONCLUSION. ...

CHAPTER VI BIBLIOGRAPHY....

213

215

TABLE 1 TABLE 2 TABLE 3 TABLE 4 TABLE 5

TABLE 6

TABLE

7

TABLE 8

TABLE 9

LIST

OF

TABLES

Clinica1 entitiss associated with human

paxpi 11 ornavi ruses. ... ... . . . Dis t r i b u t ion of c: o n d y 1 o m a t o u s 1 osions

studied according to histologie type... .. Age distrib ution of patien ts according to hi s t o 1 o g i c t y p e o f c o n d y 1 orna t o u s 1 e s i on. . . „ . D e p t h o f k oil o c y t o t i c z o n e

a

c c o r d i n g t o

li i s t o 1 o g i c t y p e o f c o n d y 1 ornat

o

u s 1 e s i o

n....

Frequency of HPV detection by transmission electron mi c: roscop y according to typ e o f c o n d y 1 o m a t ou s lésion... . . Frequency of detection of HPV antigens

by

category of

lesion... ...

Frequen cy of detection o f HPV antigen

s

by

type of candy I amatau.s

lesion. ...

Summar y of u1

trastructural

nuciear

observati ans of candy1omatous

lesions....

S u m m a r y o f u. 11 r a s t r u c t u r a 1 c y t o p 1 a s m i c o b s 91-" v a t i o

n

s o f c o n d y 1 o m a t o u s .1. e s

i

o ns. ... .

VII

'age 101 112

134

VIII

TABLE 10: Summary of ultrastructural nuclear

observations of

dysplasia

... »... 137 T A B L E; 11 :

S

u m m a r y

a

f u 11 r astrue t u r a 1 c y t o p 1 a s m i c

observations of dysplasia... ... 138 TABLE 12: Condyl omatous .lesions : main findings... 195 TABLE 13:

Frequency

of detection of HPVs

by transmission elect non microscop y (TEM)

LIST OF FIGURES

IX Page

FIGURE 1 : Outline at methods followed in the study... 71

FIGURE 2 : Peroxidase-antiperoxidase procedure... 86

FIGURE 3 : Schematic illustration of the depth of the koilocytotic zone... . 102

FIGURE 4 : Schematic illustration of types of condyloma... ... ... 139

FIGURE 5 : Histology of condyloma acuminatum... .140

Figure 6 : Hi gher magnification of Fipure 5... 140

FIGURE 7 : Histology pure condyloma... 141

FIGURE 8 : Higher magnification from Figure 7... 141

F1G U RE 9 : Histology of mi:-: t u. r e o f condylo m

a

t ypes_____ 14 2 FIGURE 10 ; Higher magnification from Figure 9... 142

FIGURE 11 : Higher magnification from Figure 9... 142

FIGURE 12 : Higher magnification from Figure 9... 142

FIG UREE 13 : H i s t ol ogy of n o r mal c o n t r o 1 t issue... 14 3 FIGURE 14 : Histology of normal epithelium and FIGURE 15 : Epon

section

of pure condyloma. ... 144

FIGURE 16 : Higher magnification from Figure 15... . 144

X

FIGURE 18

FIGURE 19

FIGURE 20

FIGURE 21

FIGURE 22

FIGURE 23

FIGURE 24

FIGURE 25

FIGURE 26

FIGURE 27

FIGURE 28

FIGURE 29

FIGURE 30

FIGURE 31

FIGURE 32

FIGURE 33

FIGURE

34

FIGURE

35

FIGURE 36

FIGURE 37

FIGURE 38

Electron micrograph of control tissue... El Bctron micrograph of pure condyloma... Higher magnification of Figure 19... . Electron micrograph of pure condy1oma... Elec t r o n mi c r o g r a p h o f c o n d y.l oma

associated with dysplasia... Electron micrograph of pure condyloma... Elect ron micrograph of pure condy1oma... Elect ron microg raph of vi i- us p artici es... Histology of pure condyloma.. ... .

Electron micrograph of dysksratocytss... Electron micrograph of pure condyloma... Electron micrograph of pure condyloma... Higher magnification from Figure 29... Electr

a

n mic r

a

graph of condy1 orna

associated with dysplasia. ... . .. ... Electron micrograph of control tissue... Electron micrograph of a koilocyte... Electron micrograph of koilocytes.. . . Electron micrograph of condy1oma

associated with dyspl asi a... ... ... E1 e c t r on mi c r o g r a p h o f c o n d y 1 c m a

associated with dysplasia... ... Electron micrograph of a koi1ocyte. ... E1ectron micrograph of contro1 tissus...

Page

145

146

146

147

147

148

149

149

150

150

151

152

152

153

154

155

155

156

157

159

FIGURE FIGURE FIGURE FIBURE FIGURE

FIGURE

FIGURE FIGURE FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE FIGURE FIGURE FIGURE

XI

Page 39 Electron micrograph of _{p u. r e c o n d y 1 o m a . " - " » "} 160 40 _{: Hig her magn1fication f rom Figure 39... - " " " -} 160 41 : E1ectron micrograph o f _{c o n t r o I t i s s u e. ° “ " “ “} 161 42 E1 e c t r o n m i c r o g r a p h o f _{p u r e c o n d y 1 o m a. - " " ■ "} 162 43 _: Eî 1 e c t r o n m i c r o g r a p h o f p u. r e c o n d v 1 n m a. 163 44

•

_{H i g h e r m a g n i f i c a t i o n f rom Figure 43... " * " " "} 163 45 _■ E1ectron micrograph o f _{contro1 tiesue. " “ 1 • "} 164 4 6 _: E1 e c t r o n m i c r o g r a p h o f _{control tissue. - - " a "} 165 47 _: E-! 1 e c t r a n m i c r o g r a p h o f c o n t r o 1 t i s s u e. _{- ■ -} 166

C

O

N

: EE 1 e c t r o n m i crog r a p h o f pure condyloma. - - ” " " 167 49 _: E1 e c t r o n m i. c r o g r a p h o f candy1 orna

associated with dyspla _{- - " " “} 167 50 ; Elect i" o n mi c r o g r a p h o f _{p u. r e c o n d y 1 o m a. ° “ « - ■} 168 51 “ E1 e c: t r o n m i c r o g r a p h o f p u r e c o n d y 1 orna. _{" a "} 168 52 _: E1 e c t r o n m i c r o g r a p h o f _{k oi1 ocytosis... ■ ■ " ■ ■} 169 : S c h e m a t i c il 1 u. s t r a t ion of k o i 1 o c y t o t i. c p r o cess. .

170

54 _: H i s t o 1 o g y o f c o n cl y 1 o m a associated w i t

h

d y s p 1 a s i a... . . 171 55': Higher magnification from Figure 54... .. 171 Higher ma.gni f i c at i on f r am Fi gure 54... 171

57 : Electron micrograph of dar k

58 :

Electron micrograph of

_dark

173 59 : Electron micrograph of dar k C- S 1 J. S" un»un«<t>i»Ei 174

FIGURE FIGURE FIGURE FIGURE FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE FIGURE

FIGURE

FIGURE Page

61 : Histology

of normal control

tissue.... 176

6 2

:

H i stolo g

y

o f

n

a

r rn a 1 a n d d y s p 1 a s t i c

tiss

u e. .

17 6 63 : Epon section of dysplasi a... 177 64 : Higher magnification from Figure 63.... 177 65 s Higher magnification from Figure 63... 177 66 : Epon

section

of normal

tissue and dysplasia

178

XII

67 : Higher magnification from Fi g ure 66 6 8 : H i g hi e r m a

gnific

a t. i o n f r o m F i g u r e o 6 6? : Hiet

ology

of condyloma

associated

with

dysplasia...

70 i Higher magnification from Figure 69 71 : E pon s e c t i o n o f p u. r e c o n d y 1

oma. . . . .

72 : Higher mag nificati

a

n f rom Figure 71

73

: Epon

section

of

dysplasia. ...

74 H i g h e r m a g n i f i c a t i

a

n from Figur e 7

75

;

El e c t r o n m i c r o g r

a

p h

of dysplasi a. »

76 _" Electron micr

ograp

h

of

dysplasi ân t

;

E .1 e c t r o n m i c r o g r a p h

_{of dysplasi}

a. «

78

_■

H i g he r m a g n i -f i c a 't i o n from Figur e 7'

79

: E1 e c t r o n micro g r a p hi of

dysplasi

a. a a

80

_*

H i g h e r m a g n i f i c a t i o n from Figur e 7( 81

_-

E1eatron m

icrogr

aph of dysplasi a. ■ »

82

: Electr

a

n micrograph

of dysplasi

a, a

83

_•

Electron micrograph of

dysplasi

a ■ u

84

H i g h e r m a g n i f i c a t i

a

n from Figur s- y..

178

178

1 79

180

180

180

180

181

182

183

183

184

.1. 84

185

186

187

187

XIII

FIGURE FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

Page

85 : Epon section normal tissue with PAS. ... 188

86 :

Epon section pure condyloma with PAS.... 188

87 : Hig her magnific ation from Figure 86... 188

88

:

Higher magnification from Fi gure 86. ... . 188

89 : Purs condyloma with peroxidase-antiperoxidase. ... 189

90 : Higher magnification from Figure 89.... . 189

91 : Pure condyloma with immunoperoxidase. ... 189

92 : Higher magnification from Figure 91... 189

93 : Condy1oma associated with dysp1 asia, immunoperoxidase... 190

94 : Higher magnification from Figure 93... . . 190

95 : Condy1oma associat ed with dysplasia, immunoperoxidase. ... 190

96 : Higher magnification from Figure 95. ... 190

IVX

ACKNOWLEDGMENTS

I sincerely thank Dr. Alexander Meisels, Professor and Head of the Department of Laboratory Medicine, for providing the

opportunity for me to undertake this study, and for his

encouragement, guidance and support throughout the progress of this work.

I gratefully acknowledge Carol Morin for his constructive critisism and generous advice in reviewing the manuscript.

Special thanks to Doctors Michel Roy and Michel Fortier for suppling the biopsies used in this study,

For their competent and generous technical assistance I am much obliged to Mme

Claire

For.land, Miss Linda Chevalier, Mme

Suzamne Martel and Mme Lisset Bertran.

I greatly appreciated the excellent assistence of Aristides F'ouster 1 a with the electron microscope technique.

The

material

and financial support of the National Cancer I netitute of Canada is thankful1 y ac know!edged.

X

I

am

very

grateful to Ms Mary Hilton for her efficient and ess e ntia 1 w or k in t h e? c: o r recti o n a nd revi s ion of t h e

manuseript.

My deepest gratitude to rny wife Susan, without whose hard work and

moral

support this thesis could not have been finished.

Special thanks to my

sons

Marc and

Rene,

for all the

interruptions in my work to remind me that life is not only in books, and that there are also other

parts

of the the body

as

PREFACE

Since the mid-1970s when the characteristic cellular pattern of condy1omatous lesions of the human cervix was first described by Meisels and Fortin

(1976)

.in observations of Papanicolaou smears, the squamous epithelium of the cervix has been

recognized as the main tissue

target for

the development of condy1omatous lesions. The cytologic pattern of condylomatous lesions as reported

by the

authors was characterized by the presence of two distinct types

of

cells: koi1ocytes and dyskeratocyt.es.

A year after that study appeared, two

research

groups, one from Canada (Meisels et al.,

1977)

and one from Finland (F'urola and Savia,

1977),

reported the existence of flat epithelial lesions in the human cervix. These lesions were observed to have

distinctive characteristics: they occurred more frequently than any other type of intraepithelial lesion

of

the

cervix;

they occurred in a relatively young age-group (21-25

years); they

had previously been diagnosed

as dysplasia;

and the cytological examination showed that the cells from

these

new flat lesions had the same cytomorphology as the cells from condylomata a cuminata. Bec a u s e o f t h e s i m i 1 a r c e 1 1 m o r p h o 1 o g y ai n d their

XVII

•flat surface, these lesions were called flat condylomata to distinguish them from papillary and classical condyloma

acuminatum, which for many years have been considered

as

rare pathologic entities in the human cervix. The assumption that flat lesions are also condylomata suggests that a condyloma virus could be the étiologie agent and that the virus could be involved in the development of cancer of the cervix.

At the time the present study began, there

was

still much uncertainty concerning the proper histological identification and the true etiology of flat condylomatous lesions of the

cervix. These uncertainties gave rise to misinterpretations and the use of inconsistent terminology, which of course made it difficult to settle on the appropriate treatment. Thus,

research to clarify the nature of the flat lesion appeared to be justified.

I therefore decided to undertake the present study.

My

purpose was to search for the true viral etiology

of

flat lesions, and establish morphological criteria that would assist in making a differential diagnosis between condylomata and dysplasia of the uterine cervix.

Since it was assumed that flat condylomata were lesions of a viral origin, it followed that virus particles and/or viral antigens would be seen or identified in the tissue from such

XVIII

lesions. To confirm these assumptions, two basic techniques were used for the purposes of this study: the electron

microscope and the peroxidase-immunoperoxidase technique. The electron microscope was used to look for human papillomavirus particles in ultrathin tissue sections and to describe the ultrastructural features of flat condylomatous lesions as compared with dysplasia. The peroxidase-immunoperoxidase

technique was used to assist in the identification of the human papillomavirus antigens in paraffin sections.

While this study was in progress, several research groups published similar studies. At the same time, partial results from this thesis have been reported in a number of

CHAPTER I

INTRODUCTION

1.0 NORMAL UTERINE CERVIX

1.1 General

The cervix uteri is a tubular structure located

at

the narrow, lower end of the uterus. It

measures

approximately 4 cm in length and 3 c:m in diameter. The cervix

consists

of two s

e

g m

e

n t s, t h e e ndoc e r v i a n d t h e e x o c e r v i x . E m b r yo.logically, the endocervix

derives

from the paramesonephric area, and the exocer vix f rom t he urogen it a1 sinus (Forsherg, 1973; Jor dan and Singer,

1976).

1.2 Histology

The histology

of

the

cervix

corresponds to the general plan

for

hoi 1ow organs described in histol

ogy

textbooks (Rhodin, 1981). The structure of the

cervix resembles

the remainder of the

uterus. It consists of a mucosa composed of an epithelium and a 1 a mina prop ria. The

se

gment of mucosa t hat

s

how s

branc

hinq

glandular folds on its surface is the endocervix. It is lined by a single layer of mucus-producing columnar epithelium, composed of tall clear cells. Occasional cells

are

ciliated. The exocervix is the portion of the mucosa that projects into the vagina; it is lined by a stratified squamous epithelium similar to that of the vagina.

The lamina propria is a framework of reticular fibers and

stroma with more fibers than cells. The squamous epithelium is joined with the lamina propria by a basement membrane; this membrane has been demonstrated by transmission electron microscope (Dougherty and Low, 1958; Ashworth et al., 1961; Younes et al., 1965). The mucosa rests upon the myometrium, which is composed chiefly of bundles of smooth muscle with intervening strands of connective tissue. An outer, more longitudinal layer of muscle continues

into

the vagina.

According to observations made in the light microscope by Dierk (1927), Papanicolaou, Traut and Marchetti (1943), Papanicolaou (1954), Fluhmann (1961), and Davies and Woolf (1963), the

normal cervical squamous epithelium is composed of four cellular zones:

1. Basal zone or stratum germinativum

This

layer consists

of a single

row

of columnar cells that

rests

on the cervical stroma. The cells have prominent nuclei and small amounts of cytoplasm. The epithelium margin bordering

the stroma and the basement membrane fold into the epithelium forming structures into which the stroma extends deeply

as

"pegs" or papillae.

2.

Parabasal zone

or

stratum spinosum profundum

This zone is made up of several rows of polyhedral cells with numerous intercellular bridges. The intercellular spaces become more clearly marked toward the superficial layer. The cytoplasm of some cells stains delicately with the Periodic Acid Schiff

(PAS) method.

3. Intermediate zone or stratum spinosum superficiale

This zone is composed of five to twelve rows of cells. They show progressive changes in shape, becoming flattened on the surface. Heavy deposits of

glycogen

are found in the cells of this zone. Some of the cells contain two nuclei.

4. Superficial zone or stratum superficiale

This zone consists of several rows of flattened cells lying parallel to the surface. Here the nuclei are pyknotic and

remain in situ until the cells are desquamated, in contrast to the epidermis, which has a superficial stratum of non-nuc1eated keratinized cells called squame (s).

4

1.2.1 The squamocolumnar junction

The area of the cervical epithelium where the squamous epithelium joins the columnar epithelium is called the

squamocolumnar junction. During

certain

periods of life this area shows

dynamic cellular

changes known as

metaplasia,

in which, by a

physiological or pathological process,

the columnar epithelium is

partially replaced by

squamous epithelium.

Several authors

(Reagan

and Patten,

1962;

Johnson et al., 1973; Koss, 197?)

clearly

state

that metaplastic

changes

make the

squamoco1umnar

junction

an area with ma

rked ne

op1 astic

potenti al

Singer and Jordan

₍₁₉₇₆

} believe 1::hat met apiastic

changes "

introduce

an

el ement

_of

i

nstabill

ty

to

the junctional

interface

between the two

epi

thel i

_{al types".}

1,3 U ]. t r a s t r u c t u r e of an o r m

a

1 c e r v i x 1.3.1

Exocervical

epithelium I n s

_tudyir

_'9 t h e

me

>rp that _{t h e c er vice 1 ep} harm ones,

p tr~' C I ci i iy

■f

or

ex amp 1 e, i nd n umb

_{er of}

_ce

1 1 ]. ■er t h i c i: n ess

o

f

the epi

hology of

the cervix

it must be remembered

ithelium responds to the stimulation of

of estrogen

and

progesterone. Estrogen,

cell differentiation,

which increases the

s, at

the same

time

increasing

the

thelium.

For this

reason

it is to

be

n -3 c I" cj rl •{•• _.t

_the

_number

_an

_{ickness of}

_{1 1 a}

_{ire of}

the epithelium may

vary

somewhat.

Because changes resulting from cell differentiation are

gradual, it is impossible to identify all the layers described under the optic microscope. However, observation of the

epithelium in an electron microscope (Dierks et al.,

1927;

Ashworth et. a!^1960; Hackemann et al., 1968; make it possible to distinguish only a clear division between three zones. The zones described are: the basal zone or stratum germinativum, the intermediate zone or stratum spinosum, and the superficial zone or stratum functionalis.

1.3.1.1 Basal zone or stratum germinativum

The cells in the basal zone are elongated, with the long axis perpendicular to the basal membrane. Desmosomes are

observed

on the upper border connecting the basal cells with the parabasal cells. On the lateral border, the desmosomes are relatively scanty compared with the cells of the intermediate zone. The structure of desmosomes is the classic type described in the squamous epithelium (Fawcett, 1966). The desmosome consists of two dense plaques in the plasma membrane of

the

adjacent

cells;

each plaque is joined to the inner leaflet of the cell

membrane. These plaques

are

attachment sites for converging tonofilaments. The intercellular

spaces

are varied in width

and

6

The basal cells are joined to the basal lamina by a large

number of hemidesmosomes (half- desmosomes), which form several electron-opaque pyramid-shaped areas. The bases of the pyramids are contiguous. Cytoplasmic tonof i 1 aments converge above the pyramids. Pinocytoti c: vesicles in various stages of development are usually observed between the hemi desmosomes (Younes et al.,

1965).

The nucleus occupies about a third of the cell. The nuclear sap (nuclear matrix or karyoplasma) contains a homogeneous

background of fine granules of chromatin (euchromatin) and some small, patchy areas of coarsely granular material

(heterochromatin). The nucleolus is clearly visible and usually eccentrically placed in the nucleus and sharply demarcated from the surrounding nuclear

sap.

The cytoplasm contains multiple organelles. The Golgi complex is well developed; it is usually found in the superior pole

of

the nucleus. There

are

many mitochondria concentrated close to the inferior pole of the nucleus. Some of them contain

irregular dense bodies, (600 to 800 A) in diameter. Small segments of endoplasmic reticulum are

observed

between the ribosome granules. Lysosomes are present mainly in the

cytoplasmic

zone

close

to the basal lamina. Tonofilaments are

scattered

throughout the cytoplasm in a random arrangement. The 1 argest coneentrati

a

n usua11 y converges on the desmoso

mes.

The stratum germinativum is joined with the stroma of

connective tissue by the basement membrane, which is made up of the lamina lucida (300 to 350

A)

and the lamina densa (300 to 400 A). Underneath the lamina densa there is a zone of

connective tissue made up of collagen fibers. Some of the fibers are thick (600 to 700 A in diameter), others are fine

(250 to 300 A in diameter), and still others are packages of fibrils (150 to 200 A in diameter).

1.3.1.2 Intermediate zone or stratum spinosum

The cells in the intermediate zone are larger than those in the basal layers. Their shape is polygonal but they become

progressively flatter and more elongated, with the long axis parallel to the surface.

The intercellular spaces are often wider here than in the basal layer, but they may be similar or sometimes narrower. In

general, they narrow toward the surface. Desmosomes are present in large numbers. Another type of junction, called the nexus or gap junction, has also been described in large numbers in this zone (Scott et al., 1971). The cells of the lower part of this zone show numerous long, prominent, fine cytoplasmic processes.

8

The cytoplasm shows the same organelles observed in the cells of the stratum germinativum, but they are arranged in a random manner. The cytoplasmic tonofilaments appear to be thicker and densely stained. Mitochondria are spares in the cytoplasm

instead of concentrated at a specific point in the cytoplasm.

The most prominent feature in the cytoplasm is the glycogen. It is generally displayed in small granular concentrations in the deep layers, and as large areas (lake zones) in the superficial zones, where the abundant glycogen granules occupy nearly

two-thirds of the cell.

The nuclei of cells in the intermediate zone are round to oval with smooth borders; they become slightly smaller closer to the

surface. The nuclear-cytoplasmic ratio is reduced. The nuclear sap contains fine granules of chromatin homogeneously

dispersed. The nucleoli are prominent.

1.3.1.3 Superficial zone or stratum functionalis

The cytoplasm of the nuclei of this zone is considerably •flattened. The cell sap is usually devoid of organelles.

Degenerating 1ysosome-like bodies and mitochondria often fill the cytoplasm. There is an increased quantity of tonofilaments, which are coarser and more densely stained. Sometimes small granules of keratohyalin with high electron density are

9

embedded in the tonofibrils. Desmosomes appear distorted, poorly differentiated, with a marked electron density. The nuclei are small and pyknotic.

1.3. 1.4 Clear cells

A few clear cells are observed throughout the epithelium.

Usually they are observed isolated in the stratum germinativum and the stratum spinosum profundum. This type of cell,

described in 1968 by Hackemann et al., is characterized

by

a relatively clear cytoplasm. The cell sap contains the classic organelles, such as endoplasmic reticulum, aggregates of ribosomes, and a large number of smooth-walled vesicles. The Golgi complex is well developed. The most distinct feature of these cells is the presence of numerous, striated vesicles with rounded ends. Some of these vesicles are expanded at one end in the shape of a tennis racket. Often the expansion is central or subterminal. Generally the vesicles are located in the

endoplasmic zone, and occasionally they appear attached to the cytoplasmic membrane. The nuclei have irregular outlines. Clear cells are considered to correspond to the Langerhans cells

depicted by Birbeck et al.

(1961)

in the squamous epithelium of the skin.

1.3.2 Endocervical epithelium

The epithelium of the endocervical segment is lined by a single layer of columnar cells. It is for the most part a

pseudostratified layer, in which the nuclei lie at varying levels of the epithelium. The bases of the columnar cells are attached to the basal lamina by hemidesmosomes. Adjacent cells are connected with junctional comp 1 ex (Fawcett, 1966).

The cytoplasmic sap shows abundant ribonucleic-like granules, mitochondria and small vacuoles of the endoplasmic reticulum. The apical part of the cytoplasm is packed with large droplets of mucus, which appear flocculent. Usually present in this apical area are cytoplasmic microvi11ae.

2.0

CONDYLOMA

According to the definition and description given in

standard

pathology books (Anderson, 1971), condylomata are wart-type lesions characterized by papillary-like proliferations

occurring on the skin and mucosa of the urogenital tract and sometimes involving the anal area.

2.1 Hi story

11

something like "round tumor". This general term appears to have been used by the ancient Romans and Greeks to identify the

proliferative exophytic lesions that develop mainly in the anogenital area (Bafverstedt, 1967).

At that time condy1omatous lesions were the focus of attention of medical and classical writers. In Latin, the words "ficus" and "thymus" were used to describe the macroscopic morphology of this disease. "Ficus" comes from the Greek term "sykon" which means "fig", and "thymus" comes from the Greek, "thymos, thymi ons".

At the beginning of the first century A.D., Celsus described in a medical writing the disease called thymi on:

But that which is named thymi on projects above

the surface like a little wart, narrow near the skin, wider above, hardish and at the top very rough.

The top in colour is like flowers of thyme, whence its name, and there it is readily split and made to bleed;,..Sometimes one is alone, generally several grow together,

either on the palms or soles of the feet.

The worst, howewer, are situated upon the genitals, and there they bleed the most.

that have long been considered as characteristic of condylomatous lesions.

The term "fig" used in those times was considered obscene because a person who had "fig" was believed to be a pederast Probably for this reason, Celsus avoided using the term. As usual, classical literature was a reflection of

society,

as see in the Martial Satires and Epigrams (Bafverstedt, 1967) which one of the writers makes special refererence to the Familia ficosa (fig family):

Father and mother, daughter and son-in-law and grandchild all have the fig; not even the steward or the bailiff and the serfs in the field are spared the shameful complaint. When each and every one has the fig, is it not strange that not a fig grows in a single field !

This epigram strongly suggests that sexual (and incestuous) relationships allow the transmission of condyloma.

The classical

view

of condylomata indicated the warty nature and suggested the venereal transmission

of

this pathologic entity.

we i n

1

respectable diagnosis, began to disappear from medical and classical literature. However, "ficus" or fig, which was kept outside of refined language, survived and was still in use until the late 19th century.

Later studies show that condylomata acuminatum was included in the pathologic term called papilloma. Since 1921 several

papillary lesions have been reported, but few were located in the cervix. Wharton (1921) described three cases of exophytic lesion, one of which was a typical example of condyloma

acuminatum. Wharton commented on the rarity of the lesion. The same year, Meyer (1921), reviewing the German literature, found four cases of condyloma acuminatum; one case showed an invasive carcinoma at its base. In the cases observed in Meyer's own laboratory, two of the lesions were found in pregnant women, and one papilloma had transformed to a low-grade malignancy. Meyer stated that endocervical polyps with squamous metaplasia were not to be considered papillomata. He also said that the location of the base of the growth would help to differentiate the two lesions.

In 1952, Marsh reported a compilation of cases of cervical

papillomatous lesion from literature published between 1922 and 1950. Of 31 cases, ten were diagnosed as condyloma acuminatum, six of which were found in pregnant women ; only three of the ten were considered to be malignant. During this period, the patho1ogy textbooks had al

read

y a

11

empte

d

to define papilloma;

14

Ewing stated in 1940 that papilloma was a benign tumor of pavement epithelium with supporting tissue in normal

arrangement. Marsh in 1952 emphasized that the definition of papilloma had excluded the benign tumor of the columnar

epithelium, as well as those composed of both columnar and stratified squamous epithelium, and epithelial growth such as hypertrophy and hyperplasia. Marsh revived the controversy about the papillomatous lesion and the condylomatous lesion. Finally, he suggested that papilloma be defined as a "benign epithelial neoplasm usually composed of pavement epithelium with supporting connective tissue and frequently papillary in structure"; then he added, "in regard to the cervix, the term papilloma should be limited to benign epithelial neoplasm of stratified squamous epithelium with supporting connective

tissue, frequently but not necessarily papillary in structure". Marsh also recognized that the true nature of condyloma

acuminatum was still obscure, although earlier Sutton and

Sutton (1949) and Ormsby and Montgomery (1948) had suggested a viral etiology for lesions in association with pregnancy and some histologic atypi a.

Up to that time, the literature indicated great confusion concerning the description and terminology of papilloma and

condyloma

lesions. However, a\ few years later,

Raftery and

Payne (1954) used data compiled from the literature to define condyloma acuminatum as a cauliflower growth characterized by:

(a) thick papillary stratified squamous epithelium with : -acanthosis

-parakeratosis

-spongiosis with cytoplasmic vacuolization and intranuclear inclusion bodies

(b) fine dermal fronds that are: -few in number

-diffusely and focal 1 y infiltrated by chronic inflammatory cells.

Using the above criteria, Raffery and Payne reviewed 587

biopsies of the uterine cervix. The conclusion of their study was that condyloma acuminatum was a relatively common lesion

(3% of all cervical biopsies); it was found that the age range was

18 to

32

years

old; it was also pointed out that clinically and histologically condyloma may be

confused

with squamous cell carci noma.

Around the

same

time, Goforth (1952) and Kister and Hertig (1952)

suggested

that condyloma acuminatum could be

distinguished from squamous papilloma because it was a

disease

transmissible

from

patient to patient, probably

of

viral etiology, and with no causal relationship to neoplastic

disease.

16

morphology of squamous papillary lesions, there was still confusion in defining condyloma and papilloma of the cervix.

It was not until 1958 that Woodruff and Peterson put end to the disagreement by establishing criteria to diagnose condyloma acuminatum. These authors reported that the most important histological characteristics of cervical condyloma acuminatum were "elongation and thickening of the rate pegs, marked

acanthosis, parakeratosis, papillomatosis, mild, and

cytoplasmic vacuolization". Although there is a degree of overlapping of certain features, these criteria validate diagnosis. The same authors also stressed that although malignant changes were rare in these lesions, such changes could make it difficult to arrive at a differential diagnosis.

For several decades it was believed that condylomata acuminata or genital wart lesions had a viral origin (Ciuffo, 1907). This idea

was

never proved, although for long a time several authors shared the opinion that the common wart, juvenile wart, and genital wart were caused by the same virus (Waelsch and

Habermann, 1924; Young, 1964; Rowson and Mahy, .1967; Lever and Schaumburg- Lever, 1975).

The presence of intranuclear virus particles in condyloma acuminatum was confirmed by Dunn and Ogilvie (1968) and Oriel and Almeida (1970) with the transmission electron microscope. These virus particles were morphologically similar to the human

17

papillomavirus (HF'V) observed in wart lesions in the squamous epithelium of the skin.

The HF'V virus attracted attention as a possible new virus to be related to the development of cancer of the external genitalia. Nevertheless it was already known that condyloma acuminatum was rare in the human cervix, compared with the external genitalia, where it was very common pathology.

2.2 Cytology

It is widely accepted in the field of cytology that

Papanicolaou smears provide a highly effective method and an indispensable tool for the detection of cervical lesions

(Quizibash, 1974; Meisels and Morin, 1984

In the study of cervical condylomata the observation of cells in smears was important in establishing criteria for

differentiating the lesions.

Using cytologic

smears

from the uterine cervix, George Papanicolaou (1960) was the first to describe cells

originating from condylomata acuminata. He found that the cells showed nuclear enlargements and that hyperchromasi a was a

18

cells were well differentiated with an abundant cytoplasmic supply. Papanicolaou

also

warned that these cells could be confused with cells from malignant squamous lesions, and he noticed that some cells had a perinuclear halo.

Cells with a perinuclear halo, however, have been studied since the late 1940. Ernest Ayre (1949) was the first to point out the? morphology of certain cells observed in the Papanicolaou smears. These cells were characterized by the presence of a perinuclear clear zone, which Ayre plainly illustrated in his publication. The morphology shown in Ayre's illustration leaves no doubt that those cells today would quickly be identified as typical of the condyloma.

A few years later in another publication about cancer cytology of the uterus, Ayre (1951) again presented excellent pictures of cells with a\ cytoplasmic clearing which he named "halo cells". In addition he illustrated a few sections of tissue from a flat zone of condyloma, similar to those that would be described two decades 1 ateh

as

flat condylomata (Mei sel s et al., 1977; Purola and Savia, 1977). Ayre thought that the

morphologic changes such as "halos" should be considered to be the precursor of preinvasive cancer. Thus he called these

lesions

"nearocarcinoma" (Greek "nearo" = earliest stage).

George Papanicolaou (1954) also focused attention on a type of cell that

showed

features similar to those of the cells

19

described

previously by Ayre. He stated that one of the patients whose smears showed these particular cells had a biopsy showing epidermoid carcinoma in situ, thus suggesting that the

cells

with a perinuclear clear zone may have some kind

of

relationship with genital cancer. In the same publication Papanicolaou used the term "perinuclear halo" to describe a perinuclear clear zone that was usually observed in smears from cases of vaginal trichomoniasis.

The fact that "peri nuclear halo" (Papanicolaou) and "halo cell" (Ayre) were similar in appearance brought confusion and a

variety of interpretations among clinicians. Two years later, Koss and Durfee (1956) defined the perinuclear cytoplasmic clearing as koilocytotic atypi a, described as an "unusual pattern of the squamous epithelium of the uterine cervix",

which histologically was characterized by the presence of large cells with small, irregular and hyperchromâtic nuclei.

Surrounding the nuclei was a clear cytoplasmic zone, which gave the impression that the nucleus was suspended in an empty

space. By means of this description, Koss and Durfee could help clinicians to avoid confusing koilocytotic atypi a with other cervical lesions. Furthermore they concluded that although the role of koilocytotic atypi a in the earlier stages

of

the

development of cervical cancer was not clear, the possibility that some of these lesions could progress to cancer could not be excluded.

The illustrations of cells and tissue published by Ayre (1949 and 1951) strong1 y show that the "halo cell" corresponds to epithelial cells with koilocytotic atypi a reported by Koss and Durtee (1956).

Several causes of koi1ocytosis have been suggested in the past. Sagiroglu (1959) speculated that the peri nuclear cavity could

be a mechanically produced artifact, caused by scraping the epithelium of the cervix with a wooden spatula used to collect the exfoliative cells. Similar cytoplasmic alterations were reported as a result of topical applications of podophyl1in on the cervix (Saphir

et

al., 1959). Electron microscope studies made by Okagaki et al. in 1973 have depicted koi1ocytosis as an abnormal karyopyknosis, possibly caused by abnormal

physicochemical dynamics of the cells. De Giroi ami, Ayre and Claudatus (1959) considered koi1ocytosis a "nuclear disease" in which the nuclear membrane was the outside border of the

cavity. At that time no one had even pointed out a possible manifestation of viral disease, until Ayre (I960) in an exceptional paper described the "halo cell" as a cell

characterized by a perinuclear translucent area surrounding the nucleus, mainly in cells

of

superficial or intermediate origin. The-? perinuclear area is well defined by a sharp, dark, and

intense border. What was very interesting in light of today's knowledge was Ayre's early idea, which deserves special

21

The thought is, then, that halo cells represent the earliest manifestation of malignancy in human cells, that they are caused by some nucleic

acrid-viral infectious activity in an estrogenic environment which produces derangement of cell metabolism and orderly replication of cells, and that they are probably a mutant deviation towards malignant cell growth.

A year later Naib and Masukawa (1961) published a paper in which they depicted cells from a classical condyloma

acuminatum. The cells reported were large intermediate squamous cells that appeared swollen and edematous with a thick, clear, basophilic, poorly keratinized cytoplasm and ill-defined

borders. The chromatin pattern was bland; the nucleolus was usually prominent. Intranuclear basophilic inclusion bodies varying in number, shape, and size were sometimes seen. Cytoplasmic degenerative vacuoles, perinuclear halo,

bi nuci eation, karyorrhexis and nuclear pyknosis were considered as secondary characteristics. The authors did not find any

morphological criteria to indicate malignant changes. Their description making reference to intranuclear basophilic inclusion bodies appears more likely to be derived from the lesion known to be produced by herpesvirus II.

In 1963, in studying perinuclear alterations, Sagiroglu

clear area of 5 u large at the most surrounding the nucleus (Koss and Wolinska, 1959; De Giro! ami, .1.967), and commonly seen in inflammation by trichomonad infestation, from the

"paranuclear halo", corresponding to

the

clear zone but usually ■formed

at one

side

of

the nucleus. At that time

this

alteration was still considered

by

Sagiroqlu to be a mechanically produced artifact.

A few years later, on the basis

of

accurate observations of cells with perinuclear clearings. De Girolami (1967) attempted

to

clarify the agents that produced the perinuclear halo. He concluded that this halo was associated with

acute

and chronic cervi citis, especi a11 y due to trichomonas vaginalis, and that koi1 ocytot ic atypi a was not associated with such infections, but was a manifestation

of

marked dysplasia,

possibly

of viral origin.

Several

studies have been done since Papanicolaou (1960) made the

first observations

of cell

samples

showing condyloma

a c

u

minatum. However , a

diagnostic

c: y tologic p a t tern o f t h i s lesion was not correctly identified until recently when Me i sels and Fortin (1976) described the characteristic cytomorphology o f c o n d y 1 o m a t o u

s

.1.

esio

n s o f t h e c e r v

i x

. In a sec o n d p a p e r , Meisels

et

al.

(1977)

made a correlation study by

means

of c: y t o

logy,

his t o 1 o g y a n d c o 1 p oscopy.

The

a u t h o r s d e s c r i b eel t h e

"flat" condylomatous lesion, which had never before been recognized in the

cervix.

The same

year

two Finnish authors,

F'urol a and

Savia (1977),

reported almost identical conclusions.

These flat condylomatous lesions were observed to have

distinctive characteristics: they ocurred in

a

relatively young age-group (21-25

years);

they ocurred more frequently than any other type of intraepithelial lesion of the cervix; the

cytological examination showed that the cell showed the same cytomorphology as the cell from condyloma acuminatum; and they had previously been diagnosed as dysplasia.

Since this discovery, research focused on this "new" pathologic entity has steadily been increasing (Syrjanen, 1979; 1980; Reid et al., 1980; Walker et al., 1983; Crum et al., 1983; Mei sels et al., 1983; Zuna, 1984; Coleman and Ri chman, 1985.

2.3 Criteria to identify condylomatous lesions in the cervix

The criteria to identify condylomatous lesions of the uterine cervix will be described here under three main headings:

Cytology,

Histology and Colposcopy.

2.3.1

Cytology

The cellular pattern of condylomatous lesions

of

the cervix described by Meisels and Fortin (1976) and F'urol a and Savia

24

have been proved to occur more frequently than the classical exophytic type. According to Meisels, "The smear was usually clean, with few or no inflammatory cells. The cell population consisted of intermediate and superficial cells. " Two types of cells were observed to be peculiar to condylomata — the

dyskeratocyte and the koilocyte.

Dyskeratocytes

These are isolated cells, or more often small aggregates of cells showing dyskeratotic changes with a dense yellowish or orangiophi1ic cytoplasm and small, slightly irregular, dense nuclei of the size normally found in intermediate cells.

Koi1 ocytes

The "balloon cell"

(Meieel's

term) is an intermediate cell, often quite enlarged, containing one or two and occasionally several hyperchromât.ic, slightly irregular nuclei. The

chromatin is dense and may be coarsely granular. Nucleoli are either absent or inconspicuous. The nucleus is surrounded by an irregular clear area, which may vary in size from a small halo to a large empty space, occupying most of the cytoplasmic area. Peripheral to the nuclear clearing, the cytoplasm is quite

dense, almost

glassy

in appearance and shows a characteristic amphophilic staining.

It is important to point out that the "balloon cells"

correspond to the cells that were previously described by Koss and Durfee (1956) as "koi1ocytotic atypia" and as early cancer cells by Ayre (1960).

2.3.2 Histology

According to recent histopathological descriptions (Meisels and Fortin, 1977; F'urola and Savia, 1977; Meisels et al.,1981),

•four different histologic types of lesion are diagnosed as

condyloma in the cervix: papillary or exophytic, flat, inverted or endophytic, and atypical.

2.3.2.1 The papillary condyloma (Figures 4, 5)

This exophytic lesion corresponds to the classical type previously described in the literature respecting the vulva

(Gardner and Kaufman 1965) and the cervix (Woodruff and

Peterson, 1958). Woodruff and Peterson described the histologic features of the papillary cervical condyloma:

- a marked degree of acanthosis, considered to be the most prominent feature of the papillary condyloma. The prickle-cell layer is hypertrophic, but the epithelium usually appears with an orderly arrangement and, as a rule, neoplasia can be readily dismissed.

26

- cytoplasmic vacuolization in the acanthotic zone of the epithelium is frequently seen, but is considered of rather secondary importance for the diagnosis.

- parakeratosis is consistently noted to a varying degree. However, hyperkeratosis rarely occurs.

- elongation and thickening of the rete pegs. They are

interconnected and may thus present a confusing picture to the inexperienced observer. This is especially true when tangential sections of the tissue are made.

- papillomatosis (exophytic papillary proliferation) produces the typical papillary or tree-like architecture. The papillae are narrow and often edematous with a chronic inflammatory infi1trate.

2.3.2.2 The flat condyloma (Figures 6, 7)

This lesion was described by Meisels and Fortin (1977) and F'urola and Savia (1977). It is characterized by ax virtually unaltered architecture and lacks the classical exophytic

morphology of the condyloma acuminatum. The squamous epithelium may be of normal thickness, often increased thickness or

occasionally very thin. The papillae of the connective tissue and small vessels are elongated and widened. Usually there is a flat focus of acanthotic epithelium with

27

mildly accentuated rete pegs. The cells of the basal layers are dense, with abundant cytoplasm, in contrast to the superficial cells. The basal cells maintain normal polarization. The upper

layer of the epithelium displays marked perinuclear cytoplasmic clearing (koi1ocytosis) that gives to the superficial zone of the lesion its characteristic appearance. The surface layers often show keratinization or dyskeratotic changes. The nuclei of the deeper cell layers are relatively large and display finely dispersed chromatin, whereas the nuclei of the upper layers are retracted, hyperchromatic, rather large and

sometimes pyknotic or karyorrhectic. There is bi- and

multi nuci eation. Nucleoli are generally very visible. Sometimes the flat lesions show small

spikes

on the surface of the

epithelium, which becomes irregular. This variation of the flat condyloma is called the spiked condyloma.

2.3.2.3 The inverted condyloma (Figures 8, 9, ID)

The inverted condyloma was also described by Meisels and Fortin in 1977. It was depicted

as

a flat lesion

that

resembles the inverted papilloma of the nose. The main feature is a papi liar- proliferation into the glandular neck, obliterating and taking the

place

of the columnar epithelium. The histologic

2.3.2.4 Atypical condyloma

In 1981, Meisels et al. reported that some condylomatous

lesions (atypical condyloma) showed marked nuclear atypia. Such lesions have the same general morphology as the flat condyloma. The authors state that around 9 % of atypical condyloma show indisputable evidence of progression to dysplasia and carcinoma in situ. This capacity to progress to a malignant lesion gave the authors the idea that atypical condyloma represents a further step in the evolution toward cervical cancer. The

authors also warn that these lesions can easily

be

misdiagnosed as dysplasia or even carcinoma in situ.

2.3.3 Colposcopy

Condyloma acuminatum is a lesion that most clinicians agree is easy to identify with the naked

eye.

However, the morphology of the flat condyloma described by Meisels et al. (1977) is

similar to that of the dysplastic

lesion.

For this

reason,

both these lesions are difficult to distinguish using a col poseope.

Three types of lesion (papillary, spiked and flat condylomata) can be identified with the colposcope (Meisels et al.,

1977,

1979; Roy et al., 1981, 1983).

1 The papillary or exophytic condyloma

It is observed as a raised, dense, thick, white lesion with finger-like projections resulting in an irregular surface

contour. Capillary loops are usually seen in the stroma of each projection. Hyperkeratosis is a common finding that sometimes masks the capillary vessels.

2.3.3.2 The spiked condyloma

It is a flat, white

area

characterized by the presence of small projections that look like spikes. Usually there is no

capillary loop visible in the spikes. This type of lesion was originally called "early condyloma" (Meisels et al., 1977) but

in a subsequent publication, Roy et. al. (1981) stated that it was more reasonable to

call

it "spiked condyloma" because there was no proof yet that it would eventual 1 y develop into a florid 1esion.

2.3.3.3 The flat condyloma

The surface is flat and frequently shows a

mosaic

pattern. It appears as a white lesion with a somewhat granular surface. Usually no capillary vessels are seen in the flat condyloma. Often the surface reveals some dotted areas. The flat condyloma observed with the colposcope cannot be distinguished from

0 DYSPLASIA

3.1 History

When the early studies on the origin of cervical cancer

appeared, several investigators thought that a surface lesion with the histologic characteristics of malignancy could be? “early" carcinoma (Schawenstein, 1909; Schot1 sender, 1908; Rubin 1910, and Schott1aender and Kermanner, 1912), Later, Schiller (1927), and Meyer (1941) stated that cancer of the? cervix developed in two stages: a long latent period or noninvasive stage, and a later period, the?

“invasive

stage". addition, these investigators thought that tumor malignancy appeared as a fully developed cancer with no intermediate stage, and that it extended along the epithelial surface by transforming the neighbouring normal cells (Schiller, 1927) oi adjacent tissue (Meyer, 1941).

Subsequent studies by Younger and Hertig

(1949),

Fund

et

al. (194-8), Stoddar (1952), Reagan

(1953),

Mackay et al.

(1959),

and Fluhmann (1961) indicated that malignant

cells

gradually

evolved

from

areas

in which the cells

showed obvious

atypi a. These authors also said that the

type of

atypi a differed markedly from the one observed in a fully developed cervical carcinoma.

31

Further studies based an data from several sources (Reagan, 1964) stated that certain morphologic differences could be discerned among preneoplastic lesions: those in which the epithelium showed slight alterations, and those characterized by the presence of a uniform population of small cells showing anaplastic changes. The second group was named "carcinoma in situ", and lesions with fewer abnormalities were called

dysplasia. Today cancer in situ is a widely accepted term that was first suggested by Schott 1aender and Kermauner in 1912.

The word dysplasia comes from the Greek language (dys = bad, plasia - moulding). The use of this name in cytology was

suggested by Papanicolaou (1949), although it was Reagan et al, (1953) who introduced it into the terminology used in

gynecologic pathology. The concept of dysplasia was rapidly adopted by clinicians to characterize some abnormalities of the squamous epithelium that were believed to be earlier neoplastic changes than the ones described as carcinoma in situ. Perhaps because the preneoplastic changes progressed through stages of advancing severity until the invasion took place, a multitude of terms have been used to describe these changes:

leucoparakeratosis or hyperkeratosis (Schiller 1938) , atypi a (Hoffman, 1949), atypical (Hinselmann, 1949; Bechtold and Reicher, 1952) , restless, (Held, 1952), unquiet (Bajardi,

1961), basal cell hyperactivity (Galvin et al., 1949),

and Te Linde, 1949; Novak and Galvin, 1951), atypical

hyperplasia (Figge et al., 1962; Mckay, 1959; Reagan et al., 1955) , anaplasia (Mckay et al., 1959; Younge and Hertig, 1949), dissociated intraepithelial anaplasia (Fund and Lacy, 1951), precancerous metaplasia (Carson and Go'll, 1954), koilocytotic atypi a (Koss and Dur-fee, 1956), atypical condyloma (Meisels et al., 1981), atypical immature metaplasia (Crum et al., 1983).

Reagan and Hamonic (1956) subclassified dysplasia into three groups: mild, moderate, and severe. In 1969, Patten introduced an additional subdivision of dysplasia — keratinizing,

non-keratinizing and "metaplastic. Thus, the above terms could be made even longer and more complex thanks to a choice of

six

further additional adjectives.

The differences in terminology are due mainly to the broad

histological spectrum seen in tissue that is gradually changing as the lesion evolves, and to the lack of clear morphologic criteria on which to base diagnoses. The use of varying

terminology has resulted in confusion in interpreting cervical lesions. Indeed, a group of prominent pathologists and

clinicians came together in an effort to obtain much-needed uniformity of internationally accepted terminology for

lesions

of the uterine cervix (International Agreement on Histological Terminology for Lesions of the Uterine Cervix, 1962).

The members of this group agreed to define dysplasia in the following terms :