Involved in Epidermodysplasia Verruciformis ...

Characteristics of the Lesions and Risk of Malignant Conversion Associated with the Type of Human Papillomavirus Involved in Epidermodysplasia Verruciformis Gérard Orth, Stefania Jablonska, Maria Jarzabek-Chorzelska, et al. Cancer Res 1979;39:1074-1082.

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[CANCER RESEARCH 39, 1074-1082, March 1979]

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Characteristics of the Lesions and Risk of Malignant Conversion Associated with the Type of Human Papillomavirus Involved in Epidermodysplasia Verruciformis1 Gerard Orth, Stefania Jablonska, Maria Jarzabek-Chorzelska, Slavomir Obalek, Genowefa Rzesa, Michel Favre, and OdIle Croissant Unite de Recherche sur rEtiologie Virale des Cancers Huma!ns, LA 147 CNRS, u 140 INSERM, lnstitut Gustave-Roussy, 94800 villejuIf, France (G. 0., M. F.J; Department of Dermatology, WarsawSchool of Medicine, Warsaw, Poland (S. J., M. J-C., S. 0., G. A.); and unite d'Oncologie virale, Ddpartement de virologie, lnstitut Pasteur, 75015 Paris, France (0. C.J

ABSTRACT

or Bowen's carcinomas), but squamous or basal call carci nomas are also found (9, 11, 15, 25). Many cases occur in Fourteen patients with apidarmodysplasia vermucifommis families, and EV has bean considered as a ganodermatosis, were studied to correlate the type of human papilbomavimus probably transmitted by an autosomal recessive gene (11, (HPV) found in benign lesions to their clinical aspects and 15, 23). Most of the patients thus far investigated showed to the course of the disease; nine patients ware familial abnormal cell-mediated immunity (5, 22). cases, and six had carcinomas. The type of the virus was Flat wart-like EV lesions ware shown to be transmittable determined by the RNA-DNA filter hybridization technique, by auto- or heteroinoculation (10, 11, 14). Typical papibbo using complementary ANA's transcribed from the DNA's of mavirus particles ware regularly found by electron micros the four types of HPV's previously identified, and by immu copy in benign lesions (1, 6, 7, 23, 25, 26, 29). They were nofluorascenca studies, using antisera specific for the four also observed by some authors in in situ carcinomas (3, 25, types of viruses. HPV type 3 was found in seven patients 29) but were never detected in advanced malignant lesions presenting lesions of only the varmuca plana type. These (1 , 3, 8, 9, 25, 27, 29). It was bong thought that the virus cases ware mostly stationary or abortive and showed no found in EV lesions was responsible for all viral skin warts malignant conversion. HPV type 4 was detected in four (19, 24). It is only recently that different types of HPV patients with lesions of different types: vary flat, often showing distinct antiganic properties and only little DNA reddish warts; reddish plaques; and pitymiasis vemsicobor sequence homology were characterized in skin lesions (4, bike pigmented or achmomic plaques. All these patients 17â€”21) and that tha association of EV with particular types showed cancers. Both types of viruses ware found in two of HPV was demonstrated (20, 21). Of the 4 HPV types patients: HPV type 3 in flat wart-like lesions and, for one characterized in our studies, 2 of them, HPV-3 and HPV-4, patient, in large confluent pigmented plaques and HPV type ware found in EV lesions (20). Furthermore, our previous 4 in reddish

or pitymiasis varsicobom-like plaques.

One of

these patients had early malignant lesions (Bowen's dis ease), and the other had multiple carcinomas. For one patient, the type of virus could not be identified

because of

an insufficient amount of material. These results point to the moleof virus in the pathoganasis of epidammodysplasia vammuciformis,together with genetic and immunological factors. They suggest, in addition, dif fement oncogenic potentials for viruses involved in the disease.

INTRODUCTION EV2 (13) is a mare, lifelong

disease

characterized

by

disseminated skin lesions which usually resemble flat warts but may appear also as reddish plaques or pitymiasis vemsi color-bike pigmented or achromic lesions (for review, sea Aafs. 9, 11, 15, and 25). Malignant transformation of some of the lesions has bean observed in 25 to 30% of the cases; cancers are usually of the Bowan's type (Bowan's disease I

Supported

by

Grant

PR6.

26/76

for

Cancer

Research

from

the

Polish

Government and Grants ATP 28.76.60, CRL 76.4.100.1 , ASR 1.005, and 014 from Institut National de Ia Sante et de Ia Recherche MÃ©dicale. 2 The

abbreviations

used

are:

EV,

epidermodysplasia

vemruciformis;

HPV,

human papillomavirus; HPV-3, human papillomavirus type 3; HPV-4, human

papillomavirustype 4; HPV-1, humanpapillomavirustype 1; HPV-2,human papillomavirus type 2; cRNA, complementary RNA. Received July 5, 1978; accepted November 15, 1978.

1074

study involving 11 patients (5 of them with cancers) had suggested that the aspect of EV lesions and the probability of malignant transformation could depend on the virus type (20). The aim of the present

studies

is to extend

these

results to 3 additional patients (one with cancers) and to analyze the correlation between the type of the virus, the clinical aspect of the lesions, and the course of the disease.

MATERIALSAND METHODS Sample Collection. Scrapings and biopsies of lesions were collected from patients with EV who ware attending the Department of Dermatology, Warsaw School of Med i cine (Table 1). For virus purification and viral DNA extrac tion, repeated samples from some patients and, for some of them, from various types of lesions were collected in Eagle's minimum essential medium containing antibiotics and ware stored until use at â€”70Â°. For immunofluorescenca studies, biopsies ware either processed immediately or frozen in liquid nitrogen 12 to 24 hr after collection in culture medium. Virus PurIfication and Viral DNA Preparation. EV HPV's ware purified from the pooled scrapings of each of the

patients, T. G., J. K., E. D., J. D., and S. M., and HPV-1and HPV-2 were purified from deep plantar warts and hand common warts, respectively, as previously described (18, 20). Viral DNA's were obtained

either by extraction

from the

CANCERRESEARCHVOL. 39


Role of Virus in EV vinions (20) or by selective extraction

from the lesions (18),

and DNA concentration was determined by electron micros copy as previously described (18). Molecular Hybridization Experiments. DNA's from HPV 1 , HPV-2, J. D. HPV, and J. K. HPV (taken as prototypical HPV-3 and HPV-4, respectively), as well as from T. G. HPV,

were transcribed in vitro into cANA's, using Escharichia coli ANA polymerasa containing a-factor (a gift from S. Saragosti, Institut Pasteur, Paris, France) (18). cRNA-DNA filter hybridization under paraffin (12) was performed as previously reported (18, 20). Immunofluorescence Studies. Direct and indirect immu nofluomascenca tests were performed mainly as described by Bautnar at al. (2). Specific guinea pig antisera ware raised against full particles of HPV-1 (G 121, G 122), HPV-2 (G 206), HPV-3 [using J. D. HPV (G 280) or E. D. HPV (G 281)], and HPV-4 [using S. M. HPV (G 271)] or against empty

particles of HPV-4 [using J. K. HPV (G 251) or S. M. HPV (G 264)]. These sara had been used in previous studies (18, 20), except G 271 , which was obtained under the conditions

described for other EV HPV's (20). Fluomascain-babeled lgG

fractions with a fbuorasceinto protein molar ratio of 2 or 5 were prepared from anti-HPV-1 antiserum as previously described (16) and ware used in direct immunofluorescanca tests at a concentration of 0.5 mg/mI. Anti-HPV-2, anti-HPV 3, and anti-HPV-4 antisera were used in indirect immunoflu

orescance tests at a dilution of 1/80 to 1/320. Tissues embedded in Tissue-Tek II O.C.T. compound medium (Lab Tek Products) ware cut in a cryostat (SLEE, London, Eng land) at â€”20Â°. Seven-sm-thick sections were set on gelatin coated slides, fixed for 10 mm at â€”2@Y' in acetone, and washed for 20 mm in 0.05 M sodium phosphata/0.1 M NaCI (pH 7.2). For direct immunofluomescence tests, sections

were incubated for 30 mm at 37Â°with fluorescein-labebed anti-HPV-1 bgG. For indirect immunofluorescanca tests, sections ware incubated for 30 mm at 37Â°with diluted

antisera or preimmune sara and then, after washing, with fluorascein-labebad anti-guinea pig IgG rabbit lgG at a concentration of 0.5 mg/mI (fluorescein to protein molar ratio ranging from 1 to 2.5)(16, 20). Sectionsware mounted in buffered glycerol (pH 8.0), examined, and photographed with an American Optical Company microscope or a Zeiss Photomicroscope II. RESULTS

Morphologyof EV Lesionsas Relatedto the VirusType Characteristics of EV Lesions. The 14 patients studied are presented in Tables 1 and 2. Nine were familial cases, and 6 had cancers. All patients had lesions on the domsaof the hands and on the face, and most of them had lesions on the extremities and the trunk. Patients showed various types of lesions: (a) flat wart-bike lesions, somewhat more elevated on the domsa of the hands (Fig. 1) but in some

patients almost at the skin level and often reddish (Fig. 2); (b) reddish plaques, usually on the trunk (Fig. 3); (c) scaling brownish or achmomic lesions resembling pityriasis vemsi color (Fig. 4); (d) large, pigmented, irregularly shaped confluent plaques (Fig. 5); and (a) common warts intemmin glad with vemrucaplana-type lesions (Fig. 6).

Molecular HybridizationData. In our previousstudies (20), characterization

of the virus

present

in lesions

of

patients with EV was performed by molecular hybridization without taking into account the clinical types of the lesions. However, results indicated the occurrence of HPV-3 in

Table 1

Patientswith EVâ€• Age at1 Stmalig nantcon var

Cell-me

at onset of PatientsSexAge munity@'Remarks1.

im (ym)Camcinomass'diated

(ym)Age disease (yr)sion

D.2. T.G.M52543++â€”BmothemofJ.

K.andH.

J.K.F38517+++â€”3.

H.D.F4611â€”MothemofE.D.,D.D.,andW.D.4.E.D.F227â€”5.D.D.F21715+â€”6. W. D.

disappeared after biopsies

7. R. P.M P.8.A.P.F2510+9. M9

475.5 Early childhood+

NDdWarts

E. I.F184â€”Fathem

Father of A. with warts on

hands10.

J. D.M43Early case11

childhood37++

long-standing,

flat

+â€”Nonfamilial

. J. G.M247â€”Nonfamiliab case12. M. G.M217â€” case13. 5. M.M22820+ case14.

to

Â±Nonfamilial

+â€”Nonfamilial

R.M.M36235+Â±Nonfamilialcase Cl Characteristics

b

of

@,premalignant

carcinomas; C Depressed

benign

lesions

lesions

of

ama

described

Bowen's

type

+ + + , invasive carcinomas, (â€” ),

lowered

(Â± ), om

preserved

in

Table

(carcinoma

2.

in situ)

and

single

carcinoma;

++,

multiple

Bowen's

at various sites. (+

) nonspecific

cell-mediated

immunity,

as

checked

by

in

vitro

methods

and cutaneoustests (5).3 d ND, not done.

MARCH 1979


1075

G. Orthatal.

EVPatientsExtensionaMorphology

Table 2 of the benign lesions of patients

Characteristics

and distribution

with

of skin lesions@'Activityâ€•HPV

wart type typedFlat Elevated

warts1.

Reddish plaques

Very flat

T.G.++Hands,face thighs+++42.

J. K.++++Hands,

feer

Face, ex-

tmemities++++43. H.D.++Hands,feat,

Pitymiasis vemsi- Pigmented color type plaquesCommon

Trunk

Tmunk,amms,

Whole skin

Trunk, arms

Neck,tmunk, arms,

Tmunk,thighs

Trunk

Trunk,amms,

Faca,ex tmemities

forearms,

legsâ€”34. ED.++AsH.D. AsH.D.+35,

D.D.+++AsH.D.

AsH.D.

thighs++++3,46.

W. D.+Face, handsâ€”37. P.+Handsâ€”Inconclusive8. R. A. P.+Hands Faceâ€”39.

E. I.+Hands Faceâ€”310. J.D.++++Face,exthighs++++3,411. J.G.+Hands

Legs

tmemities FaceHands,

leg, foot+3,212. Face,

MG.++Hands

thighs, legs++313.

S.M.+++Face,extremities+++414. R.M.+++Face,ex-

tmemities @

a

hands and face; ++,

b Flat

wart-type

Tmunk,ex-

Trunk

Tmunk,extremities++4

Trunk

tmemities

lesions

hands, face, and some at other locations;

are

either

somewhat

elevated

or vary

flat,

+++,

almost

widespread; at the

skin

++++,

level.

generalized.

Reddish

plaques

are

flat

maculam

lesions

(up

to 2 cm). Pitymiasis vemsicobom-typelesions are brownish scaling or achmomic plaques. Pigmented plaques are dark brown, large, confluent, and immegulamly shaped lesions. Common warts are elevated hypemkematoticpapules. C. _ , no

new

lesions

d As determined e The

typical

for

many

years;

in a previous elevated

flat

lesions are clearly distinct

+

study

wart-like

D.,

in

confluent

+ + + + , from

and

lesions

in this

pigmented

a few

new

while

at first

gmadually

HPV-4

was

found in patients showing reddish plaques and/or pityniasis varsicobor-lika lesions together with somewhat flatter and reddish vamrucaplana-typa lesions (Table 2). Molecular

hybridization

data reported

in Table 3 ware

obtained on 3 new patients and on different types of lesions of several patients, using cANA's specific for the 4 types of HPV's, with J. D. and J. K. HPV's as prototypical HPV-3 and HPV-4,

respectively.

With

the

conditions

used,

50

to

60%

of

the radioactivity binds to filters when homologous DNA's and cANA's are hybridized, while almost no annealing is observed between hatemobogous prototypical cRNA's and DNA's. Of the 3 new patients, Patient T. G., showing reddish plaques and pityniasis varsicolor-lika lesions, was found to be infected by HPV-4, while Patient M. G., showing only wart-bike lesions, was infected by HPV-3. The limited amount of material from Patient A. P. did not allow conclu sions. A lesser extant of annealing was observed when T. G. and J. K. HPV cANA's ware hybridized

with J. K. and

T. G. DNA's, respectively, as compared to homologous hybridizations, confirming the genetic heterogeneity al ready reported for viruses belonging to type 4 (20). Data further

1076

reported

in Table

3 show

that

viral

to

extensive

spreading

at

the

time

of

studies.

flattened

throughout

the

years.

Although

still

slightly

raised,

the

relatives (Cases 3 to 6).

lesions and, for Patient

plaques,

lesions

study.

observed

from those of the patient's

patients showing only flat wart-type J.

to

(20)

DNA

obtained

from

the

reddish

and

pityniasis

versicobor-like

brownish

plaquespresenton theback of Patient J.D. forsome years (which have been spreading lately) annealed only with HPV 4 cANA. These lesions had not been tasted previously. Two viral DNA preparations obtained from lesions collected from

the lags of this patient (Fig. 5) at a 1-year interval ware both of HPV-3 type, but the most macant showed also some annealing

with HPV-4 cANA, coinciding

with the eruption

of a few reddish and brownish plaques at this location. Clear evidence for infection by HPV-3 and HPV-4 was also found for Patient D. D. , whose wart-like lesions [analogous tothoseof hermother,H. D.,and sister, E. D. (Fig.1)]ama clearly due to HPV-3 and whose reddish plaques [similar to those of her aunt, J. K. (Fig. 3), and uncle, T. G.] are due to HPV-4.

HPV-2

was

detected

in the

typical

common

warts

of

Patient J. G. (Fig. 6), while the viral DNA found in his flat wart-like lesions hybridized only with HPV-3 cRNA. The lesser extant of hybridization observed, as compared to HPV-3

prototypical

DNA,

confirms

previous

results

obtained

for this patient, which showed the genetic heterogeneity of viruses belonging to type 3 (20). However, HPV-4 was found both in flat vamruca plana-typa lesions and in reddish and pityniasis vemsicobor-bike plaques (Fig. 4) of Patient A. M., as

previously found for the different lesions of Patients J. K. CANCER RESEARCH VOL. 39


Role of Virus in EV Table 3 of HPV present in EV lesions by cRNA-DNA hybridization for17 Filter hybridization under paraffin (12) was performed at 37Â° withabout hr in 0.3 M NaCI/0.03 M sodium citmata/50% fommamide of[3H]cRNA 50 ng of viral DNA (except A. P. DNA) and 3000 cpm Identification

per filter (specific 20).% activity, 3 to 4 x 10@ cpm/@g)(18, of radioactivity bound on filtemsafter hy bmidization with [3H]RNA's complementary

to

with

anti-HPV-3

antiserum

(Fig.

7, a and b), while

her

reddish plaques gave a positive reaction only with anti-HPV 4 antiserum (Fig. 7, C and d). In addition, results show that, for most patients, no antigen could be detected with the 4

types of antisera in some of the samples tested. Widely variable amounts of viral antigens were also observed in a previous study of numerous common warts of a patient infected with HPV-2 (18).

different DNA'sbHPV-3

MalignantConversionas Relatedto the VIrusType Found

HPV-4(J.D.

T.G.DNA

(J.K.

HPVHPV-1 on fiItems@' HPV-1 0.8HPV-2

HPV-2

HPV)

HPV)

0.1

0.3

58.0

0.5

0.1

0.3 â€”0.2

61 .2 â€”0.1

0.6

0.4

0.3

0.1 0.1 ND

0.9 0.8 ND

44.2 45.3 0.3

0.6 â€”0.1 â€”0.1

â€”0.1

0.1

â€”0.1

15.4

0.2 0.1

0.1 0.2

42.6 56.4

1.0 8.3

0.1 â€”0.1

â€”0.1 0.2

46.0 47.5

0.4 2.4

0.1

â€”0.2

2.2

28.7

0.1

â€”0.1

55.9

0.3

1.0HPV-3

0.2

â€”0.5HPV-4 (J. D. HPV) 28.3T.G.(J. K. HPV)

0.1 0.5

57.5M. G.Face;A' NDâ€•Hands; â€”A. A â€”J. P. (hands;A)

0.2 49.0

13.4

D.Back; â€”Lags;D B, C

â€”Legs; â€”D. 0 0.Hands;A â€”Legs; â€”Tmunk;B,C A

â€”J. G.Hands; â€”Leg;EA

â€”Palm;E â€”A.

9.6

â€”0.2

â€”0.3 53.4 â€”0.2 33.4

0.8 1.0

â€”0.2 1.0

â€”0.3

0.1

13.5

M.Hands;

in EV Lesions Malignant conversion of lesions was observed only in the 6 patients infected with HPV-4, 2 of them being infected also with HPV-3 (Tables 1, 2, and 5). In Patient 0. 0., infected with both viruses, lesions were of early Bowen's disease type (carcinoma in situ) (Fig. 8) and ware controlled by topical 5-fluorouracil. In other patients, malignant be sions were of Bowen's carcinoma type; they were usually multiple,

mostly

0.3

parts of the body, and

DISCUSSION Our studies indicate that the clinical aspect of the lesions, the course of the disease, and the malignant conversion in EV are rebated to the type of HPV responsible for the infection. Patients infected with HPV-3 alone show flat wart like lesions, a more protracted course of the disease, and no malignant

â€”Legs;A' A'

on light-exposed

sometimes invasive (Fig. 9); metastases were never ob served.

conversion

(Tables 1 and 2). This includes

abortive and/or stationary familial cases; in one of them, â€”Back; â€”0.2 0.2 1.8 20.5 the lesions did not reappear after removal. HPV-4 infection -Tmunk;B,C B, C ND ND 3.4 23.1 results in flatter vemmucaplana-lika lesions, often reddish, â€”Human ND ND 0.6 11.9 only slightly elevated on the dorsa of the hands, associated 0.5Humanthymus 0.2 0.2 0.6 0.5 liver 0.1 0.1 0.1 1 .5 ND with reddish plaques and pityriasis versicobor-like lesions mostly on the trunk. It was always associated with carcino a Viral DNA's were obtained from vinions extracted from a single plantam wart (HPV-1), pooled common warts of a single patient mas. (HPV-2), limb lesions of patient J. 0. (HPV-3), and whole-body Two patients were found to be infected by both viruses. lesions of Patients J. K. (HPV-4) and T. G. Other viral DNA's were One of them showed all the different types of lesions and selectively extracted from lesions of different sites and momphol ogy: flat wart-like lesions, elevated (A) or very flat (A'); reddish had multiple premalignant or early malignant lesions of plaques (B); pitymiasis vemsicobom-like plaques (C); pigmented Bowan's disease type. It is worth stressing that her mother, plaques (0); and common warts (E). A. P. DNA corresponded to sister, and brother were infected with HPV-3 and showed DNA selectively extracted from very few lesions and showing no no malignant lesions, whereas hemaunt and uncle were viral DNA molecules when checked by electron microscopy. infected with HPV-4 and had multiple cancers. The second b [3H]cRNA's were obtained by in vitro tmanscmiption with E. coli RNA polymerase of pumified Form 1 HPV-1 or HPV-2 DNA molecules and of J. 0., J. K., and T. G. HPV DNA molecules extracted from vimions(18, 20). C ND,

not

done.

and S.M. (20). Immunofluorescence

Studies. The association of distinct

types of HPV with particular cutaneous lesions has been studied by the immunofluorescenca technique, using spa

cific antisera against each of the 4 HPV types. Previous studies have shown that viral capsid antigens are detected in the nuclei of the upper keratinizing cells and in kamatin ized cells of the lesions (18, 20). Aesults reported in Table 4 confirm the data obtained by molecular hybridization axpem imants. This is best illustrated for Patient D. 0. , whose typical flat wart-like lesions gave a positive reaction only

patient

showed,

in addition,

large,

pigmented,

confluent

plaques on the begsdue to HPV-3 and had cancers, includ ing an invasive carcinoma of the forehead. Ha was first considered to be infected only by HPV-3 (20) on the basis of molecular hybridization data obtained with 6 DNA prepama tions from limb lesions including large pigmented plaques until

the

plaques

on

his

back

were

tasted.

Finally,

HPV-2

was detected in typical common warts intermingled with elevated vammucaplana-lika lesions due to HPV-3 in one patient. This virus has bean shown to be preferentially associated with common warts (18, 19), so its possible role in the few cases described as EV and showing hypertrophic typical common warts (15, 28) should be considered. In conclusion, this study shows that the morphological aspects of lesions and the type of HPV associated with them may be of prognostic significance. It further reinforces our

MARCH 1979


1077

G. Orthetal. Table 4 of the virus present in different EV lesions by immunofluorescence

techniquesViral Characterization

capsid antigens were detected in frozen 7-j.@m-thick sections of lesions by a direct immunofluomescence technique with fluomescein-labeled anti-HPV-1 guinea pig lgG or by an indirect immunofluorescence technique with anti-HPV-2, anti-HPV-3, and anti-HPV-4 guinea pig antisera and fluomescein-labebed anti-guinea pig IgG rabbit lgG

under conditions described in â€œMaterials and Methods.â€•HPV in:Flat

type

wart-type tested1 Patientsâ€•

Elevated

Very flat

. T. G.

2. J. K. 3. 4. 5. 6. 8. 9. 10.

lesions

H.D. E. D. D. D. W.D. A.P. El. J. D.

Reddish plaques

Pitymiasis ver sicolom-type lesions

HPV-4 (1/5)

HPV-4 (1/2)'@

HPV-4 (3/5)

HPV-4(1/4)

HPV-4(2/3)

HPV-4(4/5)

HPV-3(2/2) HPV-3 (4/5) HPV-3 (7/10) (0/2) HPV-3(1/2) HPV-3(1/3)

HPV-3(1/2) HPV-3 (1/1) HPV-3 (2/3)

HPV-4 (5/6)

HPV-3 (6/16)

HPV-4 (5/5)

11 . J. G.

HPV-3 (7/15)

12. MG.

HPV-3(5/6)

Pigmented plaques: HPV-3 (2/4) Common

13. S. M.

HPV-4 (4/4)

HPV-4 (1/1)

14. R. M.

HPV-4 (2/2)

HPV-4 (1/3)

a No sample available for Patient R. P. (Case 7). b Numbers in parentheses, numbem of positive lesions

Table5 Carcinomasin patients withV as related to the virus typeVirus

HPV-42/2 for Patient

warts: HPV-2 (3/4)

HPV-4 (2/3)

versus

number

of lesions

tested.

ously or through interaction with environmental factors, such as actinic radiations. Nevertheless, although mobecu lamhybridization and immunofluomascanca evidence for the persistence and the expression of the viral genoma in EV carcinomas must be obtained, our data strongly suggest that HPV-4 is associated somehow with malignant progmas

conversionHPV-30/7HPV-44/4HPV-3, typeMalignant

a HPV type was not determined

HPV-4 (1/1)

Other types of lesions

A. P. His lesions weme

analogous to those of his daughter, A. P., who was infected by HPV-3.

sion and, thus, that one HPV, at least, has an oncoganic potential. Note Added in Proof

previous conclusions on the importance of the virus in the pathoganasis of EV, together with other factors (20). Ga netic

factors

are known

to play a major

role in this disease,

and compilation of literature data on the genetics of EV supports transmission by an autosomal recessive gene (15). Nine of the 14 patients

of this study ware familial

3 S.

Obalek,

W.

in preparation.

1078

Glinski,

M.

Haftek,

and

S.

Jablonska.

Comparative

proposed. While the designation of HPV-1, HPv-2, and HPv-3 remains unmodified, HPv-4 should now be designated HPv-5 (Coggin, J. H., Jr., and

zur Hausen,H. Meetingreport: workshopon papillomavirusesand cancer. Cancer Res. 39: 545-546, 1979).

cases.

Three of them belonged to a family of 6 siblings with no evidence of EV in the parents (Patients 1 to 3) (Table 1) (5). Three families among the 4 studied showed EV in 2 succas sive generations, without evidence of consanguinity (Pa tiants 3 to 9). These rates are unexpected in the hypothesis of a rare autosomal recessive disease. Immunological fac tons appear to be important as wall (5, 22); cell-mediated immunity was abnormal in all the patients of this study except those showing abortive or regressive evolution of the disease (Table 1) (5).3 Our data, especially those on familial cases, indicate that the type of infecting HPV determines the clinical aspects of the lesions and bead to a consideration of the robe of the virus in their malignant conversion. It cannot be excluded, however, that a particular genetic constitution may have 2 distinct consequences: a higher susceptibility to HPV-4 infection, resulting in lifelong, widespread, benign skin lesions, and a higher risk for skin cancer, either spontana on cell-mediated

Since this manuscript was submitted, a classification of human papillo

mavirusesbased on the chronological order of their identification was

studies

immunity in patients with various types of warts, manuscript

ACKNOWLEDGMENTS We thank Peter Sheldrick and FranÃ§,oiseBreitburd for fruitful discussion and critical reading of the manuscript; Nicole Jibard and Dominique Fortin for careful assistance; and Daniel Cany and AyszamdWierczak for preparation of the illustrations.

REFERENCES 1. Aaronson, C. M., and Lutzner, M. A. Epidermodysplasia verruciformis

and epidermoidcarcinoma.Electronmicroscopicobservations.J. Am. Med.Assoc.,201:775-777,1967. 2. Beutner, E. H., Chorzelski, T. P., Bean, S. F., and Jordon, A. E. Immunopathology of the skin: labeled antibody studies. pp. 147-197. Stroudsburg: Dowden, Hutchinson, and Ross Inc., 1973.

3, Delescluse,C., PruniÃªras, M., Regnier,M., Moreno,G., andArouete.J. Epidermodysplasia verruciformis. I. Electron microscope autoradiogra phy and tissue culture studies. Arch. Dermatol. Forsch. , 242: 202-215, 1972. 4. Gissmann, L. , Pfister, H., and Zur Hausen, H. Human papilloma viruses (HPv). Characterization of four different isolates. Virology, 76: 569â€”580, 1977.

5, Glinski, W., Jablonska,S., Langner,A., Obalek, S., Haftek, M., and Proniewska,M. Cell-mediatedimmunity in epidermodysplasiaverruci formis. Dermatologica, 153: 218-227, 1976.

6. Grupper,C., PruniCras,M., Delescluse,C., Arouete,J., and Garelly,E. Epidermodysplasie

verruciforme:

etude ultrastructurale

et autoradi

CANCERRESEARCHVOL. 39


Role of Virus in EV ographique. Ann. Dermatol. Syphiligr., 98: 33â€”47, 1971. 7. Jablonska, S., Biczysko, W., Jakubowicz, K., and Dabrowski, J. On the viral etiology of epidermodysplasla verruciformis Lewandowsky-Lutz. Electron microscope studies. Dermatologica, 137: 113â€”125, 1968. 8. Jablonska, S., Biczysko, W., Jakubowicz, K., and Dabrowski, J. The ultrastructure of transitional states to Bowen's disease and invasive Bowen's carcinoma in epidermodysplasia verruciformis. Dermatologica, 140: 186-194, 1970. 9. Jablonska, S., Dabrowski, J., and Jakubowicz, K. Epidermodysplasia vermuciformis as a model in studies on the role of papovaviruses in oncogenesis. Cancer Res., 32: 583-589, 1972. 10. Jablonska, S., Fabjanska, L., and Fommas,I. On the viral aetiology of epidermodysplasia verruciformis. Demmatologica, 132: 369-385, 1966. 11. Jablonska, S., Maciejewski, W., Dabrowski, J., and Langner, A. Epider modysplasia vermuciformis. In: M. PruniÃ©mas (ed), Biomedical Aspects of Human Wart Virus Infection. pp. 113-135. Lyon: Fondation MÃ©rieux, 1976. 12. Kourilsky, P.. Mercereau, 0., Gros, D., and Tremblay, G. Hybridization on filters with competitor DNA in the liquid phase in a standard and a microassay. Biochimie (Paris), 56: 1215â€”1221 , 1974.

human papillomavirus that causes skin warts. J. Virol., 24: 108â€”120,

1977. 19. Orth, G.. Jablonska, S., Breitburd, F., Favre. N., and Croissant, 0. The human papillomaviruses. Bull. Cancer (Paris), 65: 151-164, 1978. 20. Orth, G., Jablonska, S., Favre, M., Croissant, 0., Jarzabek-Chorzelska. M. , and Rzesa, G. Characterization of two types of human papillomavi ruses in lesions of epidermodysplasia

verrucifommis. Proc. Natl. Aced.

Sci.U.S.A.,75:1537-1541.1978. 21. Pass, F., Reissig, M., Shah, K. V.. Eisinger, M., and Orth, G. Identifica tion of an immunologically distinct papillomavirus from lesions of epidermodysplasia verruciformis. J. NatI. Cancer Inst., 59: 1107-1112,

1977. 22. Prawer, 5. E., Pass, F., Vance, J. C., Greenberg, L. J., Yunis. E. J.. and Zelickson, A. S. Depressed immune function in epidemmodysplasia verrucifommis. Arch. Demmatol., 113: 495â€”499, 1977. 23. Rajagopalan, K., Bahru, J., Loo, D. S. C., Tay, C. H., Chin, K. N., and Tan. K. K. Familial epidemmodysplasiavemruciformisof Lewandowsky and Lutz. Arch. Dermatol., 105: 73-78. 1972. 24. Rowson, K. E. K., and Mahy, B. W. J. Human papova (wart) virus.

Bacteriol.Rev.,31: 110-131,1967. 13. Lewandowsky,F.,and Lutz.W. EmFalleinerbishernicht beschriebenen 25. Aueda, L. A., and Rodriguez, G. Verrugas humanas per virus papova. Hauterkrankung (Epidermodysplasia vermucifommis).Arch. Dermatol. Sy

philol., 141:193-203,1922. 14. Lutz, W. A propos de l'epidermodysplasie verrucifomme. Dermatologica,

92: 30-42,1946.

15. Lutzner, M. A. Epidermodysplasia verrucifommis: an autosomal recessive disease characterized by viral warts and skin cancer. A model for viral oncogenesis. Bull. Cancer (Paris), 65: 169-182, 1978. 16. Orth, G., Breitburd, F., and Favre, M. Evidence for antigenic determi nants shamed by the structural polypeptides of (Shops) rabbit papillo

mavirus and human papillomavirus type 1. Virology, in press, 1978.

17. Orth, G., Breitburd, F., Favre,M., and Croissant,0. Papillomaviruses: possible role in human cancer. In: H. H. Hiatt, J. D. Watson, and J. A. Winsten (ads.), Origins of Human Cancer, pp. 1043-1068. Cold Spring Harbor: Cold Spring Harbor Laboratory, 1977. 18. Orth, G., Favre, M., and Croissant, 0. Characterization of a new type of

Correlacion clinica, histologica y ultraestructural. Med. Cutanea Ibero LatinoAmericana,2: 113-136, 1976. 26. Ruiter, M., and Van Mullem, P. J. Demonstration by electron microscopy

of an lntmanuclearvirus In epidermodysplaslaverrucifommis.J. Invest. Dermatol., 47: 247-252, 1966. 27. Ruiter, M., and Van Mullem, P. J. Behavior of virus in malignant degeneration of skin lesion in epidemmodysplasia verrucifommis. J. In

vest.Dermatol., 54:324-331 , 1970.

28. Van Der Meer, J. B. Epidermodysplasiavemruclformls.Report of a case

from Utmecht.In: M. PrunlÃ©ras (ed), BiomedicalAspectsof HumanWart Virus Infection, pp. 137-143. Lyon: Fondation MÃ©rleux,1976. 29. Yabe, Y., and Sadakane, H. The virus of epidermodysplasla verruclfor mis: electron microscopic and fluorescent antibody studies. J. Invest. Dermatol., 65: 324-330, 1975.

Fig. 1. Typical flat wart-like lesions. Dorsum of the hand of Patient E. D. Fig. 2. Very flat, reddish, wart-type lesions. Dorsum of the hand of Patient S. M. Fig. 3. ReddIsh plaques with some brown and achromic lesions resembling pityriasisversicolor. Chest of Patient J. K. Fig. 4. Achromic plaques resembling pltyriasls versicolor lesions together with a few reddish and brown-reddish small plaques. Back of Patient R. M. Fig. 5. Large, pigmented (dark brown). Irregularly shaped, confluent plaques. Leg of Patient J. D. Fig. 6. Common warts (arrows) together with elevated flat wart-like lesions. Hand of Patient J. G. Fig. 7. DetectIon of viral antigens In the superficial layers of different lesions of Patient D. D. by the indirect immunofluorescence test. Adjacent frozen sections of a flat wart-like lesion (a and b) and of a reddish plw@ue(c and d) were incubated with antl-HPV-3 antiserum (G 290) at a 1/160 dIlution (a and c) or with antl-HPV-4 antiserum (G 251) at a 1/160 dilutIon (b and d) and, after washing, with fluorescein-labeled anti-guinea pig lgG rabbit globulins. x 260. Fig. 8. Multiple lesions of Bowen's disease type (carcinomain situ) intermingled with flat wart-like lesions. Forehead of Patient D. D. Fig. 9. InvasIve carcinoma. Forehead of Patient J. K.

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