Acta path. rnicrobiol. scand. Sect. A, 83: 292-300, 1975
LOSS OF EPITHELIAL BLOOD GROUP ANTIGENS A AND B IN ORAL PREMALIGNANT LESIONS E. DABELSTEEN, B. ROED-PETERSEN and J. J. PINDBORG Departments of Oral Pathology, Royal Dental College, Copenhagen and Aarhus, and Dental Department, University Hospital, Copenhagen, Denmark
Dabelsteen, E., Roed-Petersen, B. & Pindborg, J. J.: Loss of epithelial blood group antigens A and B in oral premalignant lesions. Acta path. microbiol. scand. Sect. A, 83: 292-300, 1975. Tissue from 40 oral premalignant lesions were investigated for the presence of blood group antigens A and B. The material included 18 leukoplakias, 1 erythroplakia, and 3 lichen planus, all with varying degrees of epithelial dysplasia, and 18 leukoplakias without histological evidence of impending malignancy. Thirty-eight benign keratotic oral mucosal lesions were included as a control group. The antigens were demonstrated by a double layer immunofluorescence technique, and the reactivity was compared, by titration, to the reactivity of adjacent normal epithelium from the same patient. All 22 lesions with dysplasia showed decreased reactivity for blood group antigen. Among the 18 leukoplakias without any signs of impending malignancy 4 cases demonstrated loss of antigen reactivity. None of 38 benign control lesions showed any change in antigen reactivity as compared to normal adjacent epithelium. Key words: Blood group antigen A and B; epithelium; oral premalignant lesions.
E. Dabelsteen, Dept. of Oral Pathology, Royal Dental College, Copenhagen, Denmark.
Received 6.ix.74
Accepted 25.xi.74
The term epithelial dysplasia of the oral mucosa is generally used to describe histological changes in the epithelium which are believed to have malignant potential (Krarner 1973, Pindborg et al. 1963). Epithelial dysplasia is most often found in oral leukoplakias and oral erythroplakias (Pindborg et al. 1968, Shear 1972). Recent studies, however, indicate that only some lesions diagnosed as epithelial dysplasia actually develop into cancer, and that cancer may in some cases develop in a leukoplakia without any recognisable interphase of dysplasia (Brocheriou et al. 1973, Mincer et al. 1972, Pindborg & RoedPetersen 1973). Besides the histological changes certain clinical features are known
292
to be precancerous, but even when such characteristics are present we have no way of telling whether cancer will develop and when. Many human tumours are known to have turnour-associated antigens (Hellstrorn & Hellstrom 1971) and furthermore the acquisition of tumour antigen is sometimes acrompanied by loss of normal cytoplasmic antigens (Burtin et al. 1972 a ) . Lappe (1969) and Halpin et al. (1972) have demonstrated that the antigen changes present in murine premalignant lesions can persist unchanged through progression to malignancy. Likewise, preliminary studies on the blood group antigens, which are normally present on the cell membranes of oral epithelial cells, have shown that these disappear
TABLE 1. D a t a
of
Patients Having Oral Lesions w i t h Epithelial Dysplasia
Blood
Clinical
Age
group
diagnosis
B A
0
71 54 54 69 49 60 59 73 71 53 67 73
12
8
66
A
13 15 17 18/a
8 0 0 0
66 40 76 60
A A A A
18/b
-
60
-
19
0
46
B
20 21
8 8
53 62
8
75
Patient/ Lesion
Sex
0
1 2/a 2/b 3 4 5 6 7 8 9 10 11
8 8 8 0 0 8 8 8 0
22
A A A AB A A A A AB
Eryth. Imik. Leuk. Leuk. Lenk. Leuk. Leuk. Leuk. Leuk. Leuk. Leuk. Leuk. Carc.? Leuk. Carc.? Leuk. Leuk. Leuk. Lichen
Localisation
Degree of
Endpoint titre
dYsPlasia
Normal
Buccal Lower lip Tongue Lower lip Commissure Lower lip Buccal mucosa Commissure Commissure Buccal mucosa Buccal mucosa Buccal mucosa
Slight Moderate Moderate Slight Slight Severe Slight Slight Slight Slight Moderate Moderate
1:16 1 :256 1 :256 1 :256 1 :32 1:l I :4 1:16 1:4 1 :32 1 :32 1:16
0/1:16* 0 0 0 0
Floor of the mouth
Carc.-in-situ
1:128
0/1: 16'
Floor of the mouth Floor of the mouth Tongue Tongue
Slight Slight Slight Slight Carc.! Slight Carc.? Severe
1:128 1:128 1:128 1:256
0 0 0 0
1:256
0
1:32
0
Severe Carc.-in-situ + (Carc.) Moderate
1 :64 1:128
0 0
1:32
0
Buccal mucosa Floor of the mouth
A B
Lichen Leuk. Carc.? Leuk. Carc.?
Floor of the mouth Buccal mucosa
A
Leuk.
Buccal mucosa
Lesion
0/1:1* 0/1:4* 0 1 :4 0 0 0
~
Eryth. : Erythroplakia, Leuk. : Leukoplakia, Carc. : Carcinoma. *: endpoint titre in patchy areas.
in most oral carcinomas (Dabelsteen & Pindborg 1973) and in some oral lesions with epithelial dysplasia (Dabelsteen & Fulling 1971). The present investigation was designed to determine further changes in blood group antigen expression in oral premalignant lesions. The hope is that changes in expression of these antigens may prove useful prognostic features in follow-up studies. MATERIAL AND METHODS Leukoplakias were in the present study defined according to the criteria given by Pindborg et al. ( 1963), and erythroplakia according to the criteria given by Shear ( 1972). The material was divided into three groups:
1 ) Lesions w i t h Epithelial Dysplasia Nineteen patients with 22 lesions were investigated. The distribution of sex, age, blood group, clinical diagnosis and localisation is given in Table 1. All patients had a biopsy taken from the lesion. Eleven of the patients had a biopsy taken from clinically normal mucosa-lower lip or contralateral to the lesion-as well. In the remainder of the cases histologically normal mucosa was included in the same biopsy as the dysplastic epithelium. The normal mucosa was obtained to allow comparison between blood group antigen reactivity in dysplastic and normal mucosa of the same patient (Dabelsteen 1972). In nine of the patients follow-up biopsies of the dysplastic lesions (ten lesions) were investigated as well. T h e follow-up period was between 6 months and 10 years (Fig. 3 ) .
293
TABLE 2. D a t a of Patients Hauing Oral Premalignant Lesions without A n y Histological Evidence Potential Malignant Development
Sex
Patient No.
8
23 24 25 26
0
28 29 30 31 32 33 34 35 36 37 39 40 41 ~
44 61 63 64 54 59 48 73 73 48 47 82 73 47 67 55 74 69
0 0 8 0
27
0 0
8 0
8 8 8 0 8
8 0
8 ~
~
Localisation of leukoplakia
Blood group
Age
~
~
Endpoint titre Normal Leukoplakia
Commissure Buccal mucosa Birccal mucosa Buccal mucosa Buccal mucosa Buccal mucosa Buccal mucosa Buccal mucosa Buccal mucosa Commissure Buccal mucosa Lower lip Buccal mucosa Buccal mucosa Buccal mucosa Buccal mucosa Buccal mucosa Lower lip ~~
~~
of
1 :32 1 :64 1 :64 1 :32 1:128 1:128 1 :64 1:16 1 :32 1:512 1:64 1:128 1 :32 1 :2 1 :32 1 :32 1:256 1 :32
1 :32 1 :64 1 :64 1 :32 1:128 1:l 1 :64 0 1 :32 1 :256 1 :32 1 :256 1:16 1 :4 1 :32 0 1:32 1 :32
~
'TABLE 3 . Blood T y p i n g of Tissue from Benign Oral Keratotic Lesions. Imniunofluorescence Staining Anti-B test serum
Anti-B test serum absorbed
Phosphate buffered saline
Antihuman IgG/FITC
Antihuman IgG/FITC
Antihuman IgG/FITC
~-
-
-
-
-_
-
-
-
+
-
-
Blood group antisera
Anti-A test serum
Conjugate
Antihuman IgGJFITC
Antihuman IgGJFITC
t
-
+ -
Results of cases typed as A Result of a case with known A reactivity Result of a case with known B reactivity
absorbed
2 ) Leukoplakias without A n y Histological Evidence of Potential Malignant Development Nineteen patients (Nos. 23-41 ) were investigated. The distribution of sex, age, blood group and localisation of leukoplakia is given in Table 2. All patients had a biopsy taken from the leukoplakia and from clinical normal mucosa of the lower lip. The normal mucosa was included for the same reason as mentioned above.
3 ) Benign Keratotic Lesions of the Oral Mucosa This group included 38 fibroepithelial polyps covered with keratinized as well as unkeratinized epithelium. The keratinization of these lesions was
294
believed to have resulted from mechanical trauma. The patients were between 6-78 years with 16 over 42 years. All belonged to blood group A. Blood Grouping of Patients Blood grouping was performed at Statens Seruminstitut, Copenhagen, for the patients in groups 1-2. As no blood groups were available for the 38 control lesions, blood grouping was done by immunofluorescence staining of sections of the formalin-fixed, paraffin-embedded surgical material. T h e readings were made on the unkeratinized buccal epithelium which was included in all of the surgi-
Fig. 1. Borderline between normal and dysplastic epithelium. A. H;iematoxyline and eosin staining. Normal epithelium to the right. Dysplastic epithelium to the left. B. Immunoperoxidase staining of neighbouring section. Black intercellular spaces indicate the presence of blood group antigen ; these are only present in the normal epithelium. x 60.
cal specimens. The sera used in this test are described below and the blood groiiping was performed according to Table 3 .
Handling of Tissue Local infiltration anaesthesia ( 2 per cent lidocaine noradrenaline) was used and care was taken not to inject directly into the site of biopsy. The biopsies were fixed in 10 per cent neutral formalin, paraffin embedded, and sectioned at 5 pm. 'The histology of the tissue was investigated on H & E-stained sections. T h e criteria for epithelial dysplasia have been described previously ( D a belsteen et al. 197 1 ) . The degree of epithelial dysplasia was graded as slight, moderate, severe or carcinoma-in-situ. All the sections were assessed independently by two of the authors (Dabelsteen & P i n d b o r g ) . ?'he defects of such a subjective procedure are recognised, but in the absence of any accurate method for the quantitation of epithelial dysplasia, it was decided to follow the conventional subjective assessment iised in routine histological diagnosis. Detection of Blood Group Antigen , ,. Iissiie from group A and AB persons were examined for blood group antigen A and the tissue from group B persons for blood group B. Blood group antigens in the epithelium were in all cases detected by the imniiinofluorescence ( I F ) staining. When adequate tissue was available, immunoperoxidase ( IP) staining was performed as well ( Weller & Coons 1954, Ai1rarrieas 1969, Dabelsteen & Rygaard 1972). Both methods were used as double-layer techniques-blood group antiserum, conjugate, control reactions and fluorescence microscopy were employed as described previoitsly
(Dabelsteen 1972). T h e amount of antigen in the tissue was estimated by a two-fold titration of the IF staining; dilutions from 1 :1 up to 1:5!2 werc: used. T h e I P stainings wEre performed only with blood group antiserum in !:2 dilutions. Tissues to be compared were always stained simultaneoiisly.
R E S U Id T S In all cases the normal mucosa reacted positively with bright green fluorescence at the cell membrane in the spinous cell layer. Group 1
In Case No. 5 the normal mucosa reacted very weakly. As any decrease in reactivity in this case would thus be difficult to measure because of the uncertainty in the staining reaction, it was excluded from the study. A total loss of blood group antigen in the dysplastic epithelium was seen in 16 cases. In 3 cases the majority of cells reacted negatively but some positive areas were always found (Fig. 1 ) . I n only one case (No. 8) did the dysplastic epithelium demonstrate no change in blood group antigen A reactivity. No correlation was found between the degree of severity of the dysplasia and the blood group antigen changes (Table 1) . The results from the ten cases with followup are summarized in Figs. 2 and 3. I t is seen
295
f i r st
biopsy
first
biopsv
BLOOD
A
HISTOLOGY
P
21 LI)
n
E!
N: as normal mucosa; D: decreased; L : lost.
n
c
ln
Q, c
N : no dysplasia; SI: slight dysplasia; M: moderate dysplasia; Se: severe dysplasia; C: cancer. Fig. 2. Results of follow-up investigation. Each number represents a case number, and the same number is used for the same case in all the figures and tables. A. Histology of first and latest biopsy. B. Antigen reactivity in first and latest biopsy.
CASE NO. YEARS
1
3
7
8
11
13
15
2
1
3
3
2
6
1
17 1
0
18a
18b
1
9 A
i
Fig. 3. Period between first and latest biopsy. The same number is used for the same case in all the figures and tables.
that Case No. 8 which in the first biopsy demonstrated slight dysplasia and no blood group antigen change in the follow-up biopsy showed a loss of blood group antigen and increased at the same time from slight to moderate dysplasia. Three cases developed into carcinoma during the follow-up period. Case No, 13 developed from a lesion with slight dysplasia to carcinoma with adjacent carcinoma-in-situ. I n the first as well as in the follow-up biopsy a total loss of blood group antigen was noted. Two cases (Nos. 17 and 18a) were diagnosed as hyperplastic epithelium with slight dysplasia. One of these cases occurred at the site of a previously excised squamous carcinoma. Both cases demonstrated decrease in blood group reactivity. Follow-up biopsies (Case No. 17 10 years later, Case No. 18 a 1 year later) revealed squamous cell carcinoma in both instances with total loss of blood group 296
reactivity. Case No. 18 b developed from no to moderate dysplasia over a 9-year period and changed at the same time from a ‘‘normal” antigen reactivity to a total loss. The other cases demonstrated no change in histology or blood group reactivity.
Group 2 and 3
A total loss of blood group A was seen in three leukoplakias and in one further case the antigen expression was so weak that it could hardly be detected. The difference in endpoint titre between the leukoplakia and the normal epithelium ranged in these cases between five and eight two-fold titre steps. In one case the leukoplakia reacted positively but with an endpoint titre three two-fold titre steps lower than the normal mucosa from the same patient. In the remainder of the samples only a slight variation between
Fig. 4. Leukoplakia without any histological evidence of malignancy. A. Haematoxyline and eosin. B. Immunofluorescence of neighbouring section. T h e bright intercellular spaces in the spinous cell layer indicates the presence of blood group antigen. x 90.
leukoplakic and normal mucosa could be found (Table 2 ) . I n none of the controls (lesions from group 3 ) was there a variation of blood group antigen reactivity in keratinized and unlieratinized epithelium greater than one endpoint titre (Fig. 4 ) . A previous estimation of the reproducibility of the endpoint titre by triple titration of the same specimen in 2 1 cases (63 stainings) has not given any variation between first, second, and third staining which was greater than two two-fold titration steps. By using this limit and a Fischer’s exact test ( H a l d 1956), a significant difference ( p < 0.01) was found between the leukoplakic material and the fibroepithelial polyps used as control tissue.
DISCUSSION
The present work has investigated the expression of blood group antigens A or B in oral premalignant lesions compared with the antigen expression in uninvolved oral mucosa of the same patients. Previous studies (Dabelsteen 1972, Dabelsteen & Fejerskov 1974a) have shown the existence of interindividual as well as regional variation in reactivity of blood group antigen in normal epithelium of the oral cavity. In group 2 comparisons were made only between blood group antigen reactivity of labial, buccal, and commissural epithelium. The reactivity of normal epithelium from these three regions have been shown to be of the same magnitude (Dabelsteen & Fejerskov 1974 a ) . I n group 1 antigen reactivity of the epithelium of the floor of the mouth was compared with that of the lip in two cases.
297
I t appeared justified because unpublished data indicate that the blood group antigen reactivity of the epithelium from the buccal mucosa and the floor of the mouth are of the same magnitude. I n ten of the cases in group 2 the antigen reactivity in the dysplastic epithelium was compared with the reactivity of adjacent normal epithelium. Previous studies (Dabels t e m & Pindborg 1973, Dabelsteen et al. 1974) have demonstrated that histologically normal epithelium adjacent to carcinomas may, in a few cases, demonstrate blood group antigen changes. The mucosa used as normal control should thus be obtained distant from the lesion. However, in the ten cases in group 2 this was not possible for different practical reasons. As all the oral leukoplakias are keratinizing processes, a benign keratotic lesion of the buccal mucosa was included in the study as a control group to the leukoplakias. By doing so, it was possible to investigate whether antigen changes in leukoplakias were simply the result of the keratinizing process. Premalignant lesions are usually regarded as constituing a spectrum with insignificant epithelial disturbances at one end and late precancerous lesions or early carcinoma a t the other end. Borderlines between the different categories are sometimes difficult to determine. When the sample investigated is divided into lesions with antigen reactivity as in normal epithelium and lesions with loss or decrease in antigen reactivity, it is seen that the cases with decreased or lost reactivity include the dysplastic lesions plus four lesions without any histological signs of malignancy. I t will be of great interest to see whether these five cases are more likely to develop into dysplasia and cancer than the rest of the leukoplakias without severe epithelial changes. I t is of interest to notice that two of the four lesions are from patients who have epithelial dysplasia and loss of blood group antigen in other lesions of the oral cavity. The present findings are especially of interest when one considers the work of Prehn
& Slemmer (1963)) Lappe (1969) and Halpin et al. (1972) who have demonstrated in animal experiments that the antigenic properties of preneoplastic cell populations are carried over into the resulting tumours after the neoplastic transformation has occurred and that antigenicity or lack of it is a property that is already established in the preneoplastic stages of tumour development. That only some of the leukoplakias showed antigen changes corresponds to previous studies which have demonstrated cell-mediated hypersensitivity to some, but not all, leukoplakias (Roed-Petersen et al. 1973). The lesions with antigen changes in the present study have not been investigated for demonstration of cellular hypersensitivity. Although the relationship between epithelial dysplasia in a leukoplakia and malignant transformation of the lesion is debatable, many workers consider that the finding of epithelial dysplasia indicates a likelihood that malignancy will develop. O n this background it should be noted that all the dysplastic lesions in the present study demonstrate loss of blood group antigen. I t may be possible that all the dysplastic lesions investigated have true malignant potential but that the prognosis of the lesions is dependent on extraepithelial factors such as the host irnrnunological responsiveness. Host reactivity against oral leukoplakias with epithelial dysplasia has actually been demonstrated by Roed-Petersen et al. (1973) and by Lehner (1970) although in few patients only. I t is, however, more probable that the antigen changes found in the dysplastic lesions are associated with other factors, such as cell movement and growth rate, rather than malignancy per se. That blood group antigen expression and neoplastic transformation are not necessarily interdependent is demonstrated by the presence of blood group antigens in a few oral carcinomas and by the loss of antigens on epithelial cells taking part in wound healing (Dabelsteen & Fejerskou 1974 b ) . In the six lesions where follow-up biopsies demonstrated a change to a more severe dis-
ease it was interesting to notice that the antigen changes were already present at the lowest stage of morphological change. However, a larger sample, improved criteria for the diagnosis of epithelial dysplasia, and very long follow-up studies are needed before a definite conrlusion ran be made. The present findings are in agreement with Burtin et d ’ s (1972 b ) studies on premalignant lesions of the colon. He investigated undifferentiated non-invasive polyps for three different antigens and roncluded that from an immunological viewpoint polyps behave as cancer. Furthermore, Burtin et al. (1972 a ) found that the loss of normal cytoplasmic antigens in colon tumours were arcompanied by the appearance of new tumour-associated antigen. We have not investigated whether the loss of blood group antigens in the dysplastic lesions is accompanied by the appearance of new tumour associated antigens, against which immunity may exist, but such an investigation would of course be of interest. In conclusion the present study has demonstrated that it is not possible by investigating the distribution of blood group antigens in oral lesions with dysplasia to divide these into different groups. However, by dividing oral precancerous lesions into lesions with antigen reactivity as in normal epithelium and lesions with loss or decrease in antigen reactivity it is seen that the cases with decreased or lost reactkity include the dysplastic lesions plus some lesions without any histological signs of malignancy. These findings may be of prognostic value. The present work was supported by Daell Fonden, Copenhagen, Denmark and Grant DE-1358 from T h e National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland,
USA. The authors want to thank Professor c o h i Smith, Department of Oral Pathology, School of Clinical Dentistry, Sheffield, United Kingdom, and Professor Meruyn Shear, Johannesburg for helpful correction of the manuscript.
REFERENCES
Aurameas, S.: Coupling of enzymes to proteins with gluteraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry 6: 43-52, 1969. nrocheriou, C., Chomette, P . , Allrial, M . & Helenon, R . : Cancer in situ et micro-invasif de la cayit6 buccale. Etude morphologique et statistique. Path. Microbiol. 39: 214-222, 1973. Burtin, P., Clausell, D . , Bora, J. & uon Kleist, S . : Variations of membrane antigens of human epithelial cells at different stages of differentiation and in malignancy. In: R. Harris, P. Allen and D. Viza (eds.). Cell differentiation, p. 82-85, Munksgaard, Copenhagen (1972 a ) . Burtin, P., Martin, E., Sabine, M . C . & uon Kleist, S . : Immunological study of polyps of the colon. J. National Cancer Inst. 48: 25-29, 1972. Dabelsteen, E.: Quantitative determination of blood group substance A of oral epithelial cells by immunofluorescence and immunoperoxidase methods. Acta path. microbiol. scand. Sect. A, 80: 847-853, 1972. Dabelsteen, E. & Fejerskov, 0.: Distribution of blood group antigen A in human oral epithelium. Scand. J. Dent. Res. 82: 206-21 1, 1974a. Dabelsteen, E . & Fejerskou, 0.: Loss of epithelial blood group antigen A during wound healing in oral mitcoiis membrane. Acta path. microhiol. scand. Sect. A, 82: 431-434, 1974 b. Dabelsteen, E. & Fulling, H.-1.: A preliminary study of blood group substances A and B in oral epithelium exhibiting atypia. Scand. J. Dent. Res. 79: 387-393, 1971. Dabelsteen, E., M y g i n d , N . & Henriksen, B.: Blood group substance A in carcinomas of the larynx. Acta Otolaryngol. 7 7 : 360-367, 1974. Dabelsteen, E. & Pindborg, J. J.: Loss of epithelial blood group substance A in oral carcinomas. Acta path. microbiol. scand. Sect. A, 81: 435 444, 1973. Dabelsteen, E., Roed-Petersen, B., Smith, C . J . & Pindborg, J . 1.: T h e limitations of exfoliative cytology for the detection of epithelial atypia in oral leukoplakias. Br. J. Cancer 25: 21-24, 1971. Dabelsteen, E . & Rygaard, J.: A sensitive immunofluorescence technique for detecting blood group substances A and B. Acta path. microbiol. scand. Sect. A, 80: 433-439, 1972. H a l d , A , : Statistical theory with engineering applications, p. 708. John Wiley & Sons, Inc., New York (1952). Halpin, 7’. Z . , V a a g e , J . & Blair, P . 8 . : Lack of antigenicity of mammary tumors induced by carcinogens in a nonantigenic preneoplastic lesion. Cancer Res. 32: 2197-2200, 1972. Hellstriim, I . & Hellstrom, K . E.: Some effects of
299
the immune defence against cancer. Cancer 28: 1269-1 27 1, 197 1. K r a m e r , I . R . H . : Carcinoma-in-situ of the oral mucosa. Int. Dent. J. 23: 94--99, 1973. Lappd, M . A.: Tumor-specific transplantation antigens: Possible origin in pre-malignant lesions. Nature 223: 82-84, 1969. Lehner, T . : Immunopathology of oral leukoplakia. Br. J. Cancer 24: 442-446, 1970. Mincer, H . H., Coleman, S . A . & Hopkins, K . P . : Observations on the clinical characteristics of oral lesions showing histologic epithelial dysplasia. Oral Surg. 33: 389-399, 1972. Pindborg, J. J., ]elst, O., Renstrup, G . & RoedPeterren, B.: Studies in oral leukoplakia: A preliminary report on the period prevalence of malignant transformation in leukoplakia based on a follow-up study of 248 patients. J. Amer. Dent. Assoc. 76: 767-771, 1968. Pindborg, J . J., Renstrup, G., Poulsen, H . E. & Silverman, S . : Studies in oral leukoplakias. Acta Odont. Scand. 21: 407-414, 1963.
300
Pindborg, J . J . & Roed-Petersen, B.: A follow-up study of 54 patients with oral dysplasia. Annual Meeting of Scandinavian Division for Dental Research. I n : Scand. J. Dent. Res. IADR 1973. Abstract No. 3, p. 17, 1973. Prehn, R . T . & Slemmer, G . L.: The role of immunity as a homeostatic mechanism during oncogenesis. In: Wissler, Dao & Wood, Jr. (eds.) Endogeneous factors influencing hosttumor balance. p. 185-190, 1967. Roed-Petersen, B., Roed-Petersen, J, & Bendixen, G.: Cell-mediated immunity to human oral leukoplakia demonstrated by the leukocyte migration test. Int. J. Cancer 22: 66-72, 1973. Shear, M.: Erythroplakia of the mouth. Int. Dent. J. 22: 460-473, 1972. Weller, T. H . & Coons, A . H . : Fluorescent antibody studies with agents of varicella and herpes zoster propagated in vitro. Proc. SOC.Exp. Biol, (N.Y.) 86: 789-794, 1954.
LOSS OF EPITHELIAL BLOOD GROUP ANTIGENS A AND B IN ORAL PREMALIGNANT LESIONS E. DABELSTEEN, B. ROED-PETERSEN and J. J. PINDBORG Departments of Oral Pathology, Royal Dental College, Copenhagen and Aarhus, and Dental Department, University Hospital, Copenhagen, Denmark
Dabelsteen, E., Roed-Petersen, B. & Pindborg, J. J.: Loss of epithelial blood group antigens A and B in oral premalignant lesions. Acta path. microbiol. scand. Sect. A, 83: 292-300, 1975. Tissue from 40 oral premalignant lesions were investigated for the presence of blood group antigens A and B. The material included 18 leukoplakias, 1 erythroplakia, and 3 lichen planus, all with varying degrees of epithelial dysplasia, and 18 leukoplakias without histological evidence of impending malignancy. Thirty-eight benign keratotic oral mucosal lesions were included as a control group. The antigens were demonstrated by a double layer immunofluorescence technique, and the reactivity was compared, by titration, to the reactivity of adjacent normal epithelium from the same patient. All 22 lesions with dysplasia showed decreased reactivity for blood group antigen. Among the 18 leukoplakias without any signs of impending malignancy 4 cases demonstrated loss of antigen reactivity. None of 38 benign control lesions showed any change in antigen reactivity as compared to normal adjacent epithelium. Key words: Blood group antigen A and B; epithelium; oral premalignant lesions.
E. Dabelsteen, Dept. of Oral Pathology, Royal Dental College, Copenhagen, Denmark.
Received 6.ix.74
Accepted 25.xi.74
The term epithelial dysplasia of the oral mucosa is generally used to describe histological changes in the epithelium which are believed to have malignant potential (Krarner 1973, Pindborg et al. 1963). Epithelial dysplasia is most often found in oral leukoplakias and oral erythroplakias (Pindborg et al. 1968, Shear 1972). Recent studies, however, indicate that only some lesions diagnosed as epithelial dysplasia actually develop into cancer, and that cancer may in some cases develop in a leukoplakia without any recognisable interphase of dysplasia (Brocheriou et al. 1973, Mincer et al. 1972, Pindborg & RoedPetersen 1973). Besides the histological changes certain clinical features are known
292
to be precancerous, but even when such characteristics are present we have no way of telling whether cancer will develop and when. Many human tumours are known to have turnour-associated antigens (Hellstrorn & Hellstrom 1971) and furthermore the acquisition of tumour antigen is sometimes acrompanied by loss of normal cytoplasmic antigens (Burtin et al. 1972 a ) . Lappe (1969) and Halpin et al. (1972) have demonstrated that the antigen changes present in murine premalignant lesions can persist unchanged through progression to malignancy. Likewise, preliminary studies on the blood group antigens, which are normally present on the cell membranes of oral epithelial cells, have shown that these disappear
TABLE 1. D a t a
of
Patients Having Oral Lesions w i t h Epithelial Dysplasia
Blood
Clinical
Age
group
diagnosis
B A
0
71 54 54 69 49 60 59 73 71 53 67 73
12
8
66
A
13 15 17 18/a
8 0 0 0
66 40 76 60
A A A A
18/b
-
60
-
19
0
46
B
20 21
8 8
53 62
8
75
Patient/ Lesion
Sex
0
1 2/a 2/b 3 4 5 6 7 8 9 10 11
8 8 8 0 0 8 8 8 0
22
A A A AB A A A A AB
Eryth. Imik. Leuk. Leuk. Lenk. Leuk. Leuk. Leuk. Leuk. Leuk. Leuk. Leuk. Carc.? Leuk. Carc.? Leuk. Leuk. Leuk. Lichen
Localisation
Degree of
Endpoint titre
dYsPlasia
Normal
Buccal Lower lip Tongue Lower lip Commissure Lower lip Buccal mucosa Commissure Commissure Buccal mucosa Buccal mucosa Buccal mucosa
Slight Moderate Moderate Slight Slight Severe Slight Slight Slight Slight Moderate Moderate
1:16 1 :256 1 :256 1 :256 1 :32 1:l I :4 1:16 1:4 1 :32 1 :32 1:16
0/1:16* 0 0 0 0
Floor of the mouth
Carc.-in-situ
1:128
0/1: 16'
Floor of the mouth Floor of the mouth Tongue Tongue
Slight Slight Slight Slight Carc.! Slight Carc.? Severe
1:128 1:128 1:128 1:256
0 0 0 0
1:256
0
1:32
0
Severe Carc.-in-situ + (Carc.) Moderate
1 :64 1:128
0 0
1:32
0
Buccal mucosa Floor of the mouth
A B
Lichen Leuk. Carc.? Leuk. Carc.?
Floor of the mouth Buccal mucosa
A
Leuk.
Buccal mucosa
Lesion
0/1:1* 0/1:4* 0 1 :4 0 0 0
~
Eryth. : Erythroplakia, Leuk. : Leukoplakia, Carc. : Carcinoma. *: endpoint titre in patchy areas.
in most oral carcinomas (Dabelsteen & Pindborg 1973) and in some oral lesions with epithelial dysplasia (Dabelsteen & Fulling 1971). The present investigation was designed to determine further changes in blood group antigen expression in oral premalignant lesions. The hope is that changes in expression of these antigens may prove useful prognostic features in follow-up studies. MATERIAL AND METHODS Leukoplakias were in the present study defined according to the criteria given by Pindborg et al. ( 1963), and erythroplakia according to the criteria given by Shear ( 1972). The material was divided into three groups:
1 ) Lesions w i t h Epithelial Dysplasia Nineteen patients with 22 lesions were investigated. The distribution of sex, age, blood group, clinical diagnosis and localisation is given in Table 1. All patients had a biopsy taken from the lesion. Eleven of the patients had a biopsy taken from clinically normal mucosa-lower lip or contralateral to the lesion-as well. In the remainder of the cases histologically normal mucosa was included in the same biopsy as the dysplastic epithelium. The normal mucosa was obtained to allow comparison between blood group antigen reactivity in dysplastic and normal mucosa of the same patient (Dabelsteen 1972). In nine of the patients follow-up biopsies of the dysplastic lesions (ten lesions) were investigated as well. T h e follow-up period was between 6 months and 10 years (Fig. 3 ) .
293
TABLE 2. D a t a of Patients Hauing Oral Premalignant Lesions without A n y Histological Evidence Potential Malignant Development
Sex
Patient No.
8
23 24 25 26
0
28 29 30 31 32 33 34 35 36 37 39 40 41 ~
44 61 63 64 54 59 48 73 73 48 47 82 73 47 67 55 74 69
0 0 8 0
27
0 0
8 0
8 8 8 0 8
8 0
8 ~
~
Localisation of leukoplakia
Blood group
Age
~
~
Endpoint titre Normal Leukoplakia
Commissure Buccal mucosa Birccal mucosa Buccal mucosa Buccal mucosa Buccal mucosa Buccal mucosa Buccal mucosa Buccal mucosa Commissure Buccal mucosa Lower lip Buccal mucosa Buccal mucosa Buccal mucosa Buccal mucosa Buccal mucosa Lower lip ~~
~~
of
1 :32 1 :64 1 :64 1 :32 1:128 1:128 1 :64 1:16 1 :32 1:512 1:64 1:128 1 :32 1 :2 1 :32 1 :32 1:256 1 :32
1 :32 1 :64 1 :64 1 :32 1:128 1:l 1 :64 0 1 :32 1 :256 1 :32 1 :256 1:16 1 :4 1 :32 0 1:32 1 :32
~
'TABLE 3 . Blood T y p i n g of Tissue from Benign Oral Keratotic Lesions. Imniunofluorescence Staining Anti-B test serum
Anti-B test serum absorbed
Phosphate buffered saline
Antihuman IgG/FITC
Antihuman IgG/FITC
Antihuman IgG/FITC
~-
-
-
-
-_
-
-
-
+
-
-
Blood group antisera
Anti-A test serum
Conjugate
Antihuman IgGJFITC
Antihuman IgGJFITC
t
-
+ -
Results of cases typed as A Result of a case with known A reactivity Result of a case with known B reactivity
absorbed
2 ) Leukoplakias without A n y Histological Evidence of Potential Malignant Development Nineteen patients (Nos. 23-41 ) were investigated. The distribution of sex, age, blood group and localisation of leukoplakia is given in Table 2. All patients had a biopsy taken from the leukoplakia and from clinical normal mucosa of the lower lip. The normal mucosa was included for the same reason as mentioned above.
3 ) Benign Keratotic Lesions of the Oral Mucosa This group included 38 fibroepithelial polyps covered with keratinized as well as unkeratinized epithelium. The keratinization of these lesions was
294
believed to have resulted from mechanical trauma. The patients were between 6-78 years with 16 over 42 years. All belonged to blood group A. Blood Grouping of Patients Blood grouping was performed at Statens Seruminstitut, Copenhagen, for the patients in groups 1-2. As no blood groups were available for the 38 control lesions, blood grouping was done by immunofluorescence staining of sections of the formalin-fixed, paraffin-embedded surgical material. T h e readings were made on the unkeratinized buccal epithelium which was included in all of the surgi-
Fig. 1. Borderline between normal and dysplastic epithelium. A. H;iematoxyline and eosin staining. Normal epithelium to the right. Dysplastic epithelium to the left. B. Immunoperoxidase staining of neighbouring section. Black intercellular spaces indicate the presence of blood group antigen ; these are only present in the normal epithelium. x 60.
cal specimens. The sera used in this test are described below and the blood groiiping was performed according to Table 3 .
Handling of Tissue Local infiltration anaesthesia ( 2 per cent lidocaine noradrenaline) was used and care was taken not to inject directly into the site of biopsy. The biopsies were fixed in 10 per cent neutral formalin, paraffin embedded, and sectioned at 5 pm. 'The histology of the tissue was investigated on H & E-stained sections. T h e criteria for epithelial dysplasia have been described previously ( D a belsteen et al. 197 1 ) . The degree of epithelial dysplasia was graded as slight, moderate, severe or carcinoma-in-situ. All the sections were assessed independently by two of the authors (Dabelsteen & P i n d b o r g ) . ?'he defects of such a subjective procedure are recognised, but in the absence of any accurate method for the quantitation of epithelial dysplasia, it was decided to follow the conventional subjective assessment iised in routine histological diagnosis. Detection of Blood Group Antigen , ,. Iissiie from group A and AB persons were examined for blood group antigen A and the tissue from group B persons for blood group B. Blood group antigens in the epithelium were in all cases detected by the imniiinofluorescence ( I F ) staining. When adequate tissue was available, immunoperoxidase ( IP) staining was performed as well ( Weller & Coons 1954, Ai1rarrieas 1969, Dabelsteen & Rygaard 1972). Both methods were used as double-layer techniques-blood group antiserum, conjugate, control reactions and fluorescence microscopy were employed as described previoitsly
(Dabelsteen 1972). T h e amount of antigen in the tissue was estimated by a two-fold titration of the IF staining; dilutions from 1 :1 up to 1:5!2 werc: used. T h e I P stainings wEre performed only with blood group antiserum in !:2 dilutions. Tissues to be compared were always stained simultaneoiisly.
R E S U Id T S In all cases the normal mucosa reacted positively with bright green fluorescence at the cell membrane in the spinous cell layer. Group 1
In Case No. 5 the normal mucosa reacted very weakly. As any decrease in reactivity in this case would thus be difficult to measure because of the uncertainty in the staining reaction, it was excluded from the study. A total loss of blood group antigen in the dysplastic epithelium was seen in 16 cases. In 3 cases the majority of cells reacted negatively but some positive areas were always found (Fig. 1 ) . I n only one case (No. 8) did the dysplastic epithelium demonstrate no change in blood group antigen A reactivity. No correlation was found between the degree of severity of the dysplasia and the blood group antigen changes (Table 1) . The results from the ten cases with followup are summarized in Figs. 2 and 3. I t is seen
295
f i r st
biopsy
first
biopsv
BLOOD
A
HISTOLOGY
P
21 LI)
n
E!
N: as normal mucosa; D: decreased; L : lost.
n
c
ln
Q, c
N : no dysplasia; SI: slight dysplasia; M: moderate dysplasia; Se: severe dysplasia; C: cancer. Fig. 2. Results of follow-up investigation. Each number represents a case number, and the same number is used for the same case in all the figures and tables. A. Histology of first and latest biopsy. B. Antigen reactivity in first and latest biopsy.
CASE NO. YEARS
1
3
7
8
11
13
15
2
1
3
3
2
6
1
17 1
0
18a
18b
1
9 A
i
Fig. 3. Period between first and latest biopsy. The same number is used for the same case in all the figures and tables.
that Case No. 8 which in the first biopsy demonstrated slight dysplasia and no blood group antigen change in the follow-up biopsy showed a loss of blood group antigen and increased at the same time from slight to moderate dysplasia. Three cases developed into carcinoma during the follow-up period. Case No, 13 developed from a lesion with slight dysplasia to carcinoma with adjacent carcinoma-in-situ. I n the first as well as in the follow-up biopsy a total loss of blood group antigen was noted. Two cases (Nos. 17 and 18a) were diagnosed as hyperplastic epithelium with slight dysplasia. One of these cases occurred at the site of a previously excised squamous carcinoma. Both cases demonstrated decrease in blood group reactivity. Follow-up biopsies (Case No. 17 10 years later, Case No. 18 a 1 year later) revealed squamous cell carcinoma in both instances with total loss of blood group 296
reactivity. Case No. 18 b developed from no to moderate dysplasia over a 9-year period and changed at the same time from a ‘‘normal” antigen reactivity to a total loss. The other cases demonstrated no change in histology or blood group reactivity.
Group 2 and 3
A total loss of blood group A was seen in three leukoplakias and in one further case the antigen expression was so weak that it could hardly be detected. The difference in endpoint titre between the leukoplakia and the normal epithelium ranged in these cases between five and eight two-fold titre steps. In one case the leukoplakia reacted positively but with an endpoint titre three two-fold titre steps lower than the normal mucosa from the same patient. In the remainder of the samples only a slight variation between
Fig. 4. Leukoplakia without any histological evidence of malignancy. A. Haematoxyline and eosin. B. Immunofluorescence of neighbouring section. T h e bright intercellular spaces in the spinous cell layer indicates the presence of blood group antigen. x 90.
leukoplakic and normal mucosa could be found (Table 2 ) . I n none of the controls (lesions from group 3 ) was there a variation of blood group antigen reactivity in keratinized and unlieratinized epithelium greater than one endpoint titre (Fig. 4 ) . A previous estimation of the reproducibility of the endpoint titre by triple titration of the same specimen in 2 1 cases (63 stainings) has not given any variation between first, second, and third staining which was greater than two two-fold titration steps. By using this limit and a Fischer’s exact test ( H a l d 1956), a significant difference ( p < 0.01) was found between the leukoplakic material and the fibroepithelial polyps used as control tissue.
DISCUSSION
The present work has investigated the expression of blood group antigens A or B in oral premalignant lesions compared with the antigen expression in uninvolved oral mucosa of the same patients. Previous studies (Dabelsteen 1972, Dabelsteen & Fejerskov 1974a) have shown the existence of interindividual as well as regional variation in reactivity of blood group antigen in normal epithelium of the oral cavity. In group 2 comparisons were made only between blood group antigen reactivity of labial, buccal, and commissural epithelium. The reactivity of normal epithelium from these three regions have been shown to be of the same magnitude (Dabelsteen & Fejerskov 1974 a ) . I n group 1 antigen reactivity of the epithelium of the floor of the mouth was compared with that of the lip in two cases.
297
I t appeared justified because unpublished data indicate that the blood group antigen reactivity of the epithelium from the buccal mucosa and the floor of the mouth are of the same magnitude. I n ten of the cases in group 2 the antigen reactivity in the dysplastic epithelium was compared with the reactivity of adjacent normal epithelium. Previous studies (Dabels t e m & Pindborg 1973, Dabelsteen et al. 1974) have demonstrated that histologically normal epithelium adjacent to carcinomas may, in a few cases, demonstrate blood group antigen changes. The mucosa used as normal control should thus be obtained distant from the lesion. However, in the ten cases in group 2 this was not possible for different practical reasons. As all the oral leukoplakias are keratinizing processes, a benign keratotic lesion of the buccal mucosa was included in the study as a control group to the leukoplakias. By doing so, it was possible to investigate whether antigen changes in leukoplakias were simply the result of the keratinizing process. Premalignant lesions are usually regarded as constituing a spectrum with insignificant epithelial disturbances at one end and late precancerous lesions or early carcinoma a t the other end. Borderlines between the different categories are sometimes difficult to determine. When the sample investigated is divided into lesions with antigen reactivity as in normal epithelium and lesions with loss or decrease in antigen reactivity, it is seen that the cases with decreased or lost reactivity include the dysplastic lesions plus four lesions without any histological signs of malignancy. I t will be of great interest to see whether these five cases are more likely to develop into dysplasia and cancer than the rest of the leukoplakias without severe epithelial changes. I t is of interest to notice that two of the four lesions are from patients who have epithelial dysplasia and loss of blood group antigen in other lesions of the oral cavity. The present findings are especially of interest when one considers the work of Prehn
& Slemmer (1963)) Lappe (1969) and Halpin et al. (1972) who have demonstrated in animal experiments that the antigenic properties of preneoplastic cell populations are carried over into the resulting tumours after the neoplastic transformation has occurred and that antigenicity or lack of it is a property that is already established in the preneoplastic stages of tumour development. That only some of the leukoplakias showed antigen changes corresponds to previous studies which have demonstrated cell-mediated hypersensitivity to some, but not all, leukoplakias (Roed-Petersen et al. 1973). The lesions with antigen changes in the present study have not been investigated for demonstration of cellular hypersensitivity. Although the relationship between epithelial dysplasia in a leukoplakia and malignant transformation of the lesion is debatable, many workers consider that the finding of epithelial dysplasia indicates a likelihood that malignancy will develop. O n this background it should be noted that all the dysplastic lesions in the present study demonstrate loss of blood group antigen. I t may be possible that all the dysplastic lesions investigated have true malignant potential but that the prognosis of the lesions is dependent on extraepithelial factors such as the host irnrnunological responsiveness. Host reactivity against oral leukoplakias with epithelial dysplasia has actually been demonstrated by Roed-Petersen et al. (1973) and by Lehner (1970) although in few patients only. I t is, however, more probable that the antigen changes found in the dysplastic lesions are associated with other factors, such as cell movement and growth rate, rather than malignancy per se. That blood group antigen expression and neoplastic transformation are not necessarily interdependent is demonstrated by the presence of blood group antigens in a few oral carcinomas and by the loss of antigens on epithelial cells taking part in wound healing (Dabelsteen & Fejerskou 1974 b ) . In the six lesions where follow-up biopsies demonstrated a change to a more severe dis-
ease it was interesting to notice that the antigen changes were already present at the lowest stage of morphological change. However, a larger sample, improved criteria for the diagnosis of epithelial dysplasia, and very long follow-up studies are needed before a definite conrlusion ran be made. The present findings are in agreement with Burtin et d ’ s (1972 b ) studies on premalignant lesions of the colon. He investigated undifferentiated non-invasive polyps for three different antigens and roncluded that from an immunological viewpoint polyps behave as cancer. Furthermore, Burtin et al. (1972 a ) found that the loss of normal cytoplasmic antigens in colon tumours were arcompanied by the appearance of new tumour-associated antigen. We have not investigated whether the loss of blood group antigens in the dysplastic lesions is accompanied by the appearance of new tumour associated antigens, against which immunity may exist, but such an investigation would of course be of interest. In conclusion the present study has demonstrated that it is not possible by investigating the distribution of blood group antigens in oral lesions with dysplasia to divide these into different groups. However, by dividing oral precancerous lesions into lesions with antigen reactivity as in normal epithelium and lesions with loss or decrease in antigen reactivity it is seen that the cases with decreased or lost reactkity include the dysplastic lesions plus some lesions without any histological signs of malignancy. These findings may be of prognostic value. The present work was supported by Daell Fonden, Copenhagen, Denmark and Grant DE-1358 from T h e National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland,
USA. The authors want to thank Professor c o h i Smith, Department of Oral Pathology, School of Clinical Dentistry, Sheffield, United Kingdom, and Professor Meruyn Shear, Johannesburg for helpful correction of the manuscript.
REFERENCES
Aurameas, S.: Coupling of enzymes to proteins with gluteraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry 6: 43-52, 1969. nrocheriou, C., Chomette, P . , Allrial, M . & Helenon, R . : Cancer in situ et micro-invasif de la cayit6 buccale. Etude morphologique et statistique. Path. Microbiol. 39: 214-222, 1973. Burtin, P., Clausell, D . , Bora, J. & uon Kleist, S . : Variations of membrane antigens of human epithelial cells at different stages of differentiation and in malignancy. In: R. Harris, P. Allen and D. Viza (eds.). Cell differentiation, p. 82-85, Munksgaard, Copenhagen (1972 a ) . Burtin, P., Martin, E., Sabine, M . C . & uon Kleist, S . : Immunological study of polyps of the colon. J. National Cancer Inst. 48: 25-29, 1972. Dabelsteen, E.: Quantitative determination of blood group substance A of oral epithelial cells by immunofluorescence and immunoperoxidase methods. Acta path. microbiol. scand. Sect. A, 80: 847-853, 1972. Dabelsteen, E. & Fejerskov, 0.: Distribution of blood group antigen A in human oral epithelium. Scand. J. Dent. Res. 82: 206-21 1, 1974a. Dabelsteen, E . & Fejerskou, 0.: Loss of epithelial blood group antigen A during wound healing in oral mitcoiis membrane. Acta path. microhiol. scand. Sect. A, 82: 431-434, 1974 b. Dabelsteen, E. & Fulling, H.-1.: A preliminary study of blood group substances A and B in oral epithelium exhibiting atypia. Scand. J. Dent. Res. 79: 387-393, 1971. Dabelsteen, E., M y g i n d , N . & Henriksen, B.: Blood group substance A in carcinomas of the larynx. Acta Otolaryngol. 7 7 : 360-367, 1974. Dabelsteen, E. & Pindborg, J. J.: Loss of epithelial blood group substance A in oral carcinomas. Acta path. microbiol. scand. Sect. A, 81: 435 444, 1973. Dabelsteen, E., Roed-Petersen, B., Smith, C . J . & Pindborg, J . 1.: T h e limitations of exfoliative cytology for the detection of epithelial atypia in oral leukoplakias. Br. J. Cancer 25: 21-24, 1971. Dabelsteen, E . & Rygaard, J.: A sensitive immunofluorescence technique for detecting blood group substances A and B. Acta path. microbiol. scand. Sect. A, 80: 433-439, 1972. H a l d , A , : Statistical theory with engineering applications, p. 708. John Wiley & Sons, Inc., New York (1952). Halpin, 7’. Z . , V a a g e , J . & Blair, P . 8 . : Lack of antigenicity of mammary tumors induced by carcinogens in a nonantigenic preneoplastic lesion. Cancer Res. 32: 2197-2200, 1972. Hellstriim, I . & Hellstrom, K . E.: Some effects of
299
the immune defence against cancer. Cancer 28: 1269-1 27 1, 197 1. K r a m e r , I . R . H . : Carcinoma-in-situ of the oral mucosa. Int. Dent. J. 23: 94--99, 1973. Lappd, M . A.: Tumor-specific transplantation antigens: Possible origin in pre-malignant lesions. Nature 223: 82-84, 1969. Lehner, T . : Immunopathology of oral leukoplakia. Br. J. Cancer 24: 442-446, 1970. Mincer, H . H., Coleman, S . A . & Hopkins, K . P . : Observations on the clinical characteristics of oral lesions showing histologic epithelial dysplasia. Oral Surg. 33: 389-399, 1972. Pindborg, J. J., ]elst, O., Renstrup, G . & RoedPeterren, B.: Studies in oral leukoplakia: A preliminary report on the period prevalence of malignant transformation in leukoplakia based on a follow-up study of 248 patients. J. Amer. Dent. Assoc. 76: 767-771, 1968. Pindborg, J . J., Renstrup, G., Poulsen, H . E. & Silverman, S . : Studies in oral leukoplakias. Acta Odont. Scand. 21: 407-414, 1963.
300
Pindborg, J . J . & Roed-Petersen, B.: A follow-up study of 54 patients with oral dysplasia. Annual Meeting of Scandinavian Division for Dental Research. I n : Scand. J. Dent. Res. IADR 1973. Abstract No. 3, p. 17, 1973. Prehn, R . T . & Slemmer, G . L.: The role of immunity as a homeostatic mechanism during oncogenesis. In: Wissler, Dao & Wood, Jr. (eds.) Endogeneous factors influencing hosttumor balance. p. 185-190, 1967. Roed-Petersen, B., Roed-Petersen, J, & Bendixen, G.: Cell-mediated immunity to human oral leukoplakia demonstrated by the leukocyte migration test. Int. J. Cancer 22: 66-72, 1973. Shear, M.: Erythroplakia of the mouth. Int. Dent. J. 22: 460-473, 1972. Weller, T. H . & Coons, A . H . : Fluorescent antibody studies with agents of varicella and herpes zoster propagated in vitro. Proc. SOC.Exp. Biol, (N.Y.) 86: 789-794, 1954.
