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Cell-mediated and humoral immune responses to primary oral cancers
J. max.-fac.Surg. 2 (1974)108-113 @ GeorgThieme Verlag,Stuttgart
Cell-Mediated and Humoral Immune Responses to Primary Oral Cancers* Hugh Cannell Department of Oral and Maxillo-Facial Surgery (Head: Prof. Dr. G. R. Seward, M.D., D.M.D.) The London Hospital Medical College, London, U.K. Summary Many human tumours induce an immune response in the host. Correlation and quantitation of immune data with clinical status is described in 9 patients bearing primary oral carcinomata. A microcytotoxicity assay demonstrated that oral carcinoma antigen is tumour specific as judged by the cytotoxic activity or oral tumour patients' effectors against the malignant epithelial cell lines, KB and HEp-2. Serum blocking and enhancement effects were also demonstrated. The results indicated that, this specific assay may prove useful in the prediction of oral malignant tumour recurrence after treatment.
Key-Words: Oral carcinoma; Specific immune responses; Clinical implications.
Introduction Survival (5 year) following i n t r a - o r a l carcinoma varies between 17°/0 and 430/0 depending upon the size (Binnie et al. t972). T h e most important problem during the follow-up period after surgery or r a d i o t h e r a p y remains the detection of small amounts of recurrence or metastasis in scarred and distorted tissues. Clinical examination methods appear unlikely to be able to detect occult tumour masses of less than 1 gram (10" cells). Immune methods have been shown to be capable of responding to as little as 1 mgm (100 ceils) of tumour (Mihulska et al. 1966) and the existence of a specific tumour directed immune response is now well established (Hellstr6m et al. 1968, Bubenik et al. 1970). Oral carcinomata in particular, have also been shown to be antigenic (Cannell 1973). The purpose of this study was to attempt to correlate the presence of an i n t r a - o r a l carcinomatumour burden with a quantitative immune test * Paper will be read at the 2nd Congress for MaxilloFacial Surgery; Zurich 1974.
specific to that tumour burden. In order to carry out the work, use was m a d e of the knowledge that tumours of the same histogenetic origin frequently c a r r y common antigens on the membrane of the m a l i g n a n t cells (Hellstr6m et al. 1971, O'Toole et al. 1972).
Material and Methods Patients: A total of 15 patients with p r i m a r y carcinoma of the oral cavity were studied of whom 9 were investigated in detail after initial experimental problems had been overcome. The stage of the carcinoma at presentation was determined according to the T N M classification and is detailed in T a b l e 1. Diagnosis on each patient was confirmed after incisional biopsy and histopathological report. H e a l t h y n o r m a l controls were matched for ethnic group although their age group was generally younger. Controls of patients bearing m a l i g n a n cies at sites, other than the mouth, were similarly matched including age grouping.
Blood Samples: W e r e taken from a peripheral vein in 20-30 ml amounts from each patient presenting with a p r i m a r y oral carcinoma. F o r every tumour patient sampled at least one normal control was used. Blood samples from each test or control patient were divided and one portion allowed to clot for serum. T h e r e m a i n d e r of the sample was defibrinated by agitation over glass beads in Erichmeyer flasks.
Preparation of lymphoeytes (effector cells): The samples of defibrinated blood were centrifuged down a discontinuous density gradient (Harris and Ukaejiofo 1967) at 3,000 r.p.m, for 15 minutes. T h e buffy layer was then harvested
Cell-Mediated and Humoral Immune Responses to Primary Oral Cancers
109
Table 1 TNM Classification of 9 patients with Primary Intra-Oral Carcinoma. Patient No.
Sex
Age
Site of carcinoma
Histopathology
TNM Classification
1 2 3 4 5 6 7 8 9
9 c~ ~ ~ ~ d ~ ~ c~
,69 46 35 59 65 64 75 63 ,62
Hard palate Tongue-lateral Buccal mucosa Tongue-lateral Alveolar mucosa Palate Multiple-whole mouth Floor of mouth Alveolar mucosa
Acinat cell Ca. Sq. cell Ca. Sq. cell + leuk. Sq. cell Ca. Sq. cell Ca. + leuk. Sq. cell Ca. Sq. cell Ca. Sq. cell Ca. Sq. celt Ca.
T2NoMo T~NoM0 T1NoMo TaNoMo TaNoMo T2NoMo T4N,Mo T~N2M0 T4N1Mo
and washed in medium supplemented with 10°/0 foetal calf serum. After washing the lymphocytes were placed in columns containing lead-free glass wool and incubated for 30 minutes. The lymphocytes were then collected, assessed for purity and viability tested by 0.10/o trypan blue dye exclusion. Purity of mononuelear cell suspensions was always better than 9 5 % lymphocytes with viabilities ranging from 85 °/0 to 98'0/0.
Target cells: Two established cell lines derived from epithelial carcinomata were used as target cells for each of the nine cases finally tested. The cell lines were KB, derived from an oral carcinoma (Eagle 1955) and HEp-2 derived from a laryngeal carcinoma (Moore et al. 1952) H e L a cells derived from a carcinoma of the cervix uteri were also tested but later discarded. Control epithelial target cells were N C T C 2544, derived by Perry et al. (1957) from normal human skin. Each cell line was maintained in culture by continuous passage and che&ed before use for morphology and plating efficiency.
Microcytotoxicity tests: The method used was a modification of that first described by Takusugi and Klein (1970). Harvested epithelial target cells from culture were counted, viability tested and then serially diluted so as to be 100-150 cells in 10 btl of tissue culture medium. 10 gl aliquots were then dispensed into wells on microtest plates (Falcon 3034, Falcon Plastics, California, USA). After 6-20 hours incubation at 37 ° C in a humidified 5 % Co2, 95'0/0 air atmosphere, each microtest plate of 60 wells
was examined critically under phase-contrast microscopy. Plates with uneven distribution of target cells were discarded. Each plate used had at least 20 wells per plate counted to ensure accurate knowledge of the number of attached target cells. The medium in the wells was then removed by inversion and the effector cell suspensions in various sera added in 10 }tl aliquots so as to produce effector: target ratios as low as 300:1. At least 12 replicates of each test or control patients' effectors in the various sera (non-heat inactivated) were used. A further 12 replicates of lest or control patients' serum alone were also tested. The wells at the periphery of each plate were always used as internal controls (no effectors, 10~°/o foetal calf serum in growth medimn) of the unhindered normal growth of the target cells and comprised 24 wells on each plate. After addition of effector solutions the plates were further incubated for 40 hours, washed twice with warm medium supplemented with 5 % foetal calf serum, fixed with 100/0 formol saline and stained for counting.
Calculation o[ Cytotoxic El~'ect: The number of cells within the 100 squares of a grid 10 mm square at x 150 magnification was counted in each well. Evidence of cytotoxic activity was taken to be a range from complete destruction of the target cells, to crenation of cell walls with cytoplasmic extrusion or rounding up. Test and control patients' counts (often read blind and decoded later) were evaluated against the growth control counts on the same plate. The percentage cytotoxicity was then expressed according to the formula:
110
H. Cannell
Table 2 Comparison of effects of lymphocytes from other tumour patients on the two malignant cell lines KB and HEp-2. Effectors from patients Malignancy: Rectum Nose Oral Melanoma Skin Melanoma (Fossati et al. 1971)
E.T. ratio
°/o Cytotoxicity KB targets FCS Aut. S.
°/o Cytotoxicity HEp-2 targets FCS Aut. S.
350 350
20 0
0 0
14 0
35 0
500 300
0 0
0 0
0 -
0 -
Table 3 Effects of Lymphocytes on the normal skin cell Targets, NCTC 2544. Malignancy
Oral Ca. Oral Ca. Rectum Nose Normal Controls
E.T. ratio
400 350 350 350 400 350
NCTC 2544 Targets % Cytotoxicity FCS Aut. S.
P.
25 2 11 9 22 10
N.S. N.S. N.S. N.S. -
32 2 38 15 38 19
Means (test replicates) x 100 = % Cells surviving (CS). Mean2 (growth control Then 100- CS = °/o cytotoxicity. replicates) Significance levels were expressed after Student's test and p = 0.05 or less was taken to be significant.
W h e n effectors were suspended in autologous serum, in five cases (Cases 1, 2, 4, 5 and S) significant cytotoxicity was recorded (Fig. 2 b). Effectors from patients bearing an adeno-carcinoma of the rectum with metastases, a basal cell carcinoma of the nose or a m a l i g n a n t melanoma of the p a l a t e did not produce significant cytotoxicity to the KB target cells (Table 2).
Laryngeal Carcinoma Targets (HEp-2): Effectors from oral cancer patients in foetal calf serum, caused significant cytotoxicity in three cases (Cases 1, 3 and 9) (Fig. 2a) and in five cases (Cases 1, 3, 5, 6, and 9) when suspended in autologous serum. Effectors from turnout patients bearing tumours other than oral carcinomata did not produce significant cytotoxicity to the H E p - 2 targets
(Table 2). Normal skin cell targets (NCTC 2544):
Results The percentage cytotoxicities of each test after exposure to effectors and sera from oral cancer bearing patients are on the target cell lines KB and H E p - 2 shown in Figures 1 and 2.
Effectors from patients bearing oral squamous cell carcinoma, basal cell carcinoma of the nose or adeno-carcinoma of the rectum, did not produce significant degrees of cytotoxicity to normal skin cell targets (Table 3).
Normal Controls: Oropharyngeal carcinoma targets (KB): W h e n effectors from oral cancer patients were suspended in foetal calf serum (FCS) and incubated with KB targets, in six cases out of nine significant cytotoxicity r a n g i n g from 95°/0 to 57 % was recorded. In the remaining three cases (patients 7, 8, 9) cytotoxic levels were significantly different from their own test control subjects (Fig. 1 a).
The percentage cytotoxicity, under all conditions, of the normal controls v a r i e d from as little as 5O/o to a more usual 230/0-29'0/0. One normal control showed non-specific cytotoxicity of over 500/0 but this subject on investigation proved to be a h e a v y smoker with concomitant upper respiratory tract illness. Hellstrgm et al. (1971) noted that up to 50/0 of healthy subjects m a y react to both m a l i g n a n t and normal cells. In
Cell-Mediated and Humoral Immune Responses to Primary Oral Cancers
11 ]
ORAL C A N C E R PATIENTS'LYIMPIIOCYTOTOXICITY AGAINST T A R G E T CELLS
FIGURE 1.
DERIVED FROM AN ORO-pHARYNGEAL
CARCINOMA.
(KB CELLS)
100
Fig, 1a
100
Fig. l b
Foetal Call Serum
Autologous Serum
80
8O
6o
¢.
4o
4o
MNC ~2
--MNC
1
T2
TI T3 T3
FIGURE 2.
T4 T4('--CASE NO.
'F2
T1 T3 2'3 T2 T4 T4 T4 B1 B1 B1 - El BI En -
ORAL CANCER PATIENTS'LYMPHOCYTOTOXICITY AGAINST TARGET CELI~ DERIVED FROM A LARYNGEAL CARCINOMA, (HEP-g CELLS) Fig. 2a
108
Fig. 2b
100
Aut ologous Serum
Foetal Calf Serum
80
~
60
~oo
88
x:
~
~
48 .
.
.
.
.
.
40
MNC
~ 2o
~
M
N
C
l# 0
•
KEY:
0
MNC
Mean Percentage
BI
B l o c k i n g of t a r g e t c e l l d e a t h Enhancement
T1-4
Tumouv burdens (TNM
Discussion
Relevance and use of the cell lines: KB cells (Eagle 1955) are epithelial-like cells said to be derived from an oral carcinoma. The original tumour was situated at the back of the mouth and involved the tongue, fauces and soft palate. Growth from well-differentiated intra-oral carcinomata is slow and unlikely to yield large numbers of cells (Cannell 1973). It would appear therefore that KB cells might have been cultured from a highly anaplastic intra-oral carcinoma or even from a naso-pharyngeal carcinoma corn-
3
4
5
6
7
8
9
C y t o t o x i c i t y of n o r m a l c o n t r o l s .
En
our protocol each oral cancer patient had an appropriate healthy control and our results in Figs. 1 and 2 show the mean percentage cytotoxicity rate of those controls (MNC).
2
of t a r g e t c e l l d e a t h classiflcation-See
Table
1. )
pound into the mouth. HEp-2 cells (Moore et al. 1952) derived from a carcinoma of the larynx are also epithelial-like and are easily grown in culture. The use of these two cell lines to test lymphocytotoxicity in oral cancer patients may prove to be of considerable practical importance for methods for the culture and maintenance of the cells are well established. Both cell lines have good epithelial characteristics on histological examination. The plating efficiency of the cells varied little and in our experiments enabled accurate numbers of target cells to be plated.
Specificity of Results: Evidence for the specificity of the cytotoxic activity of oral cancer patients' effectors against oral cancer target cells was provided by (a) Nonsignificant cytotoxic activity of the same effectors
112
H. Cannell: Cell-Mediated and Humoral Immune Responses to Primary Oral Cancers
against normal skin cells. (b) The low cytotoxie activity of effectors from almost all healthy controis, and (c) Non-significant cytotoxic activity of effectors from patients with malignancies other than intra-oral carcinomata. There appeared to be a lower degree of specific cytotoxic ability of effectors from oral cancer patients when exposed to laryngeal carcinoma targets except when suspended in autologous serum (Fig. 2). Our results demonstrated therefore, the presence of oral cancer specific tumour antigens in KB cells and the presence of some different specific antigens in HEp-2 cells. Our results also show that tumour antigens of an intra-oral malignant melanoma appear to be different from the tumour antigens on oral cancer cells. Further but indirect evidence for this latter point is given by the report of Fossati et al. (1971) who were unable to detect a cytotoxic effect of lymphocytes from skin malignant melanoma patients against KB cell targets.
Bloched and enhanced cytotoxic ability: Effectors from patients 7, 8 and 9 showed a poor cytotoxic ability in non-specific serum against oral cancer target cells (Fig. 1A). Each of these patients had a large tumour burden with palpable nodes in the neck. Patient No. 7 was also noncytotoxic to HEp-2 cells. This patient's lack of specific immune response to a range of tumour antigens was in keeping with her clinical status. Originally treated for an intra-oral carcinoma by radiotherapy and surgery some years previously, at the time of the test she had developed leucoplakic areas and four new separate primary intra-oral carcinomata. The blocking of the cytotoxic ability of the effectors when in autologous serum and incubated with the oral cancer cells in three patients (Cases 3, 6 and 7) was comparable with results for other malignant tumours and reported by Hellstr6m et al. (1971). Varying degrees of blocking of cytotoxic effect were also noted in our experiments after autologous serum incubation using HEp-2 targets. In only one patient (Case 2) was the blocking effect demonstrable for the same patient with both cell lines, suggesting that only a few common tumour antigens were involved (Fig. 2 b),
Enhancement of the cytotoxic ability of oral cancer patients effectors against HEp-2 targets (Figs. 2 a and 2 b) was recorded in Cases 5 and 6. Case 8 (Figs. l a and b) showed slightly enhanced activity against KB targets. The increased percentage of cytotoxicity in these three cases was an unexpected finding and may be related to the purity of the effector cell suspensions used in these experiments. The mechanisms of the enhancement of the cytotoxic effect would appear to have several alternative explanations. An antibody in the serum which is effector cell dependent would appear to be likely. On the other hand, cytotoxicity by effectors is known to take place in the absence of added complement, (Perlmann et al. 1974) and complement is a mediator of antibody activity. In our experiments exclusion of thermolabile complement components did not alter the reactivity of the effectors in autologous serum. This would appear to suggest that the non-thermolahile components such as C'3, C'8 or C'9 might be implicated in enhancement of cytotoxic effects. Further evidence for this explanation is given by Perlmamz et al. (1974) who reports that C'8 is associated with lymphocytes from which it cannot be removed by extensive washing procedures. Long-term follow-up and re-testing of the patients would seem to be likely to provide a clue as to whether the presence of enhanced cytotoxic effects in these three patients are related to ultimate prognosis.
Conclusions Although the present series of patients is small, the significant cytotoxic responses, particularly to oral cancer target cells, would appear to form a basis for further follow-up studies. Whilst it is yet too early to state that all oral cancer patients are specifically active immunologically, against the two target cell lines, KB and HEp-2, it is suggested that the present results indicate that these in vitro tests of cell-mediated immunity correlate well with clinical status and tumour burden. Acknowledgement My colleagues in tile Department of Oral and Maxillo-. Facial Surgery and the Department of Radiotherapy, have contributed to these studies by referring their
A. L. Nwoku, H. Koch: The Temporomandibular Joint patients for the immunological tests and by their continuing interest and help. - Grateful a&nowledgement for assistance with laboratory facilities and equipment
113
is made to the London Hospital Cancer Research Committee and the Ernest and Minnie Dawson Cancer Research Trust.
References Binnie, W, H., R. A. Cawson, G. B. Hill: Summary. In: Oral Cancer in England and Wales. HMSO, London 1972
Bubenik, ].,P. Perlmann, K. Helmstein, G. Moberger: Immune response to urinary bladder tumours in man. Int. J. Cancer 5 (1970) 39 Cannell H.: Oral Cancer and Immunity. Brit. J. oral. Surg. 11 (1973) 171 Eagle, H.: Propagation in a fluid medium of a human epidermoid carcinoma strain, KB (21811). Proc. Soc. exp. Biol. Med. 89 (1955) 362
Fossati, G., M, ]. Colnaghi, G. Della Porta, N. Cascinelli, U. Veronesi: Cellular and humoral immunity against human malignant melanoma. Int. J. Cancer 8 (1971) 344 Harris, R., E. D. Uhaejiofo: Rapid preparation of lymphocytes for tissue typing. Lancet 7615 (1969) 327
against its own actively growing primary tumour. J. Nat. Cancer Inst. 36 (1966) 29 Moore, A. E., L. Sabachewcky, H. W. Toolan: Culture Characteristics of 4 permanent lines of human cancer cell. Cancer Res. 15 (1952) 598
O'Toole, C., P. Perlmann, B. Unsquard, G. Moberger, F. Edsmyr: Cellular immunity to human urinary bladder carcinoma. 1. Correlation to clinical stage and radiotherapy. Int. J. Cancer 10 (1972) 17 Perlmann, P., H. Perlmann, P. Lachmann: Lymphocyte-associated complement. Role of C'8 in certain cell-mediated cytic reactions. Scan& J. hmnunol. 3 (1974) 77
Perry, V. P., K. K. Sanford, V. S. Evans, G. W. Hyatt, W. R. Earle: Establishment of clones of epithelial cells from human skin. J. Nat. Cancer Inst. 18
(1957) 709 Takusugi, M., E. Klein: A micro-assay for cell-mediated
Hellstr6m, L, K. E. Hellstr6m, G. E. Pierce, ]. P. S. Young: Cellular and humoral immunity to diffe-
immunity. Transplant. 9 (1970) 219
rent types of human neoplasms. Nature 220 (1968) 1352
Hellstr6m, I., K. E. Hellslr6m, H. O. Sj6gren, G. A. Warner: Demonstration of cell-mediated immunity to human neoplasms of various histological types. hat. J. Cancer 7 (1971) 1 Mikulska, Z. B., C. Smith, P. Alexander: Evidence for an immunological reaction of the host directed
Hugh Cannell, M.Sc., F.D.S.R,C.S., M.R.C.S., L.R.C.P., Department of Oral and Maxillo-Facial Surgery, The London Hospital Medical College, Turner Slreet, London EI 2AD, U.K.
J. max.-fac. Surg. 2 (1974) 113-119 © Georg Thleme Verlag, Stuttgart
The Temporomandibular Joint: A rare Localisation for Bone Tumours* Alagumba L. N. Nwoku, Heribert Koch Clinic for Maxillo-Facial and Plastic Surgery of the Face, Westdeutsche Kieferhlinik (Director: Prof. A. Rehrmann, M.D., D.D.S.), University of Dusseldorf, W. Germany
Summary The literature on tumours of the temporomandibular region has been reviewed, and a classification between true tumours of the condyle and condylar hyperplasia made. Of 3,200 tumours af the head and he& treated in the Clinic for Maxiltofacial and Plastic Surgery of the University of Dfisseldorf from 1. 1. 1955 to * Paper will be read at the 2nd Congress of the European Association for Maxillo-Facial Surgery in Zurich, Switzerland from 16.9.- 21.9. 74.
30, 4. t974, only seven cases of true tumours occurred in the condylar head. One rare and very interesting case, which is not a turnout, has been included to illustrate the complexity and sometimes uncertainty in diagnosis, A possible explanation for the apparent rarity of tumours of the temporomandibular region has been offered. Key-Words: Temporomandibular Joint Tumours; Con-. dylar Hyperplasia; Condylcctomy; Impacted third molar.
Cell-Mediated and Humoral Immune Responses to Primary Oral Cancers* Hugh Cannell Department of Oral and Maxillo-Facial Surgery (Head: Prof. Dr. G. R. Seward, M.D., D.M.D.) The London Hospital Medical College, London, U.K. Summary Many human tumours induce an immune response in the host. Correlation and quantitation of immune data with clinical status is described in 9 patients bearing primary oral carcinomata. A microcytotoxicity assay demonstrated that oral carcinoma antigen is tumour specific as judged by the cytotoxic activity or oral tumour patients' effectors against the malignant epithelial cell lines, KB and HEp-2. Serum blocking and enhancement effects were also demonstrated. The results indicated that, this specific assay may prove useful in the prediction of oral malignant tumour recurrence after treatment.
Key-Words: Oral carcinoma; Specific immune responses; Clinical implications.
Introduction Survival (5 year) following i n t r a - o r a l carcinoma varies between 17°/0 and 430/0 depending upon the size (Binnie et al. t972). T h e most important problem during the follow-up period after surgery or r a d i o t h e r a p y remains the detection of small amounts of recurrence or metastasis in scarred and distorted tissues. Clinical examination methods appear unlikely to be able to detect occult tumour masses of less than 1 gram (10" cells). Immune methods have been shown to be capable of responding to as little as 1 mgm (100 ceils) of tumour (Mihulska et al. 1966) and the existence of a specific tumour directed immune response is now well established (Hellstr6m et al. 1968, Bubenik et al. 1970). Oral carcinomata in particular, have also been shown to be antigenic (Cannell 1973). The purpose of this study was to attempt to correlate the presence of an i n t r a - o r a l carcinomatumour burden with a quantitative immune test * Paper will be read at the 2nd Congress for MaxilloFacial Surgery; Zurich 1974.
specific to that tumour burden. In order to carry out the work, use was m a d e of the knowledge that tumours of the same histogenetic origin frequently c a r r y common antigens on the membrane of the m a l i g n a n t cells (Hellstr6m et al. 1971, O'Toole et al. 1972).
Material and Methods Patients: A total of 15 patients with p r i m a r y carcinoma of the oral cavity were studied of whom 9 were investigated in detail after initial experimental problems had been overcome. The stage of the carcinoma at presentation was determined according to the T N M classification and is detailed in T a b l e 1. Diagnosis on each patient was confirmed after incisional biopsy and histopathological report. H e a l t h y n o r m a l controls were matched for ethnic group although their age group was generally younger. Controls of patients bearing m a l i g n a n cies at sites, other than the mouth, were similarly matched including age grouping.
Blood Samples: W e r e taken from a peripheral vein in 20-30 ml amounts from each patient presenting with a p r i m a r y oral carcinoma. F o r every tumour patient sampled at least one normal control was used. Blood samples from each test or control patient were divided and one portion allowed to clot for serum. T h e r e m a i n d e r of the sample was defibrinated by agitation over glass beads in Erichmeyer flasks.
Preparation of lymphoeytes (effector cells): The samples of defibrinated blood were centrifuged down a discontinuous density gradient (Harris and Ukaejiofo 1967) at 3,000 r.p.m, for 15 minutes. T h e buffy layer was then harvested
Cell-Mediated and Humoral Immune Responses to Primary Oral Cancers
109
Table 1 TNM Classification of 9 patients with Primary Intra-Oral Carcinoma. Patient No.
Sex
Age
Site of carcinoma
Histopathology
TNM Classification
1 2 3 4 5 6 7 8 9
9 c~ ~ ~ ~ d ~ ~ c~
,69 46 35 59 65 64 75 63 ,62
Hard palate Tongue-lateral Buccal mucosa Tongue-lateral Alveolar mucosa Palate Multiple-whole mouth Floor of mouth Alveolar mucosa
Acinat cell Ca. Sq. cell Ca. Sq. cell + leuk. Sq. cell Ca. Sq. cell Ca. + leuk. Sq. cell Ca. Sq. cell Ca. Sq. cell Ca. Sq. celt Ca.
T2NoMo T~NoM0 T1NoMo TaNoMo TaNoMo T2NoMo T4N,Mo T~N2M0 T4N1Mo
and washed in medium supplemented with 10°/0 foetal calf serum. After washing the lymphocytes were placed in columns containing lead-free glass wool and incubated for 30 minutes. The lymphocytes were then collected, assessed for purity and viability tested by 0.10/o trypan blue dye exclusion. Purity of mononuelear cell suspensions was always better than 9 5 % lymphocytes with viabilities ranging from 85 °/0 to 98'0/0.
Target cells: Two established cell lines derived from epithelial carcinomata were used as target cells for each of the nine cases finally tested. The cell lines were KB, derived from an oral carcinoma (Eagle 1955) and HEp-2 derived from a laryngeal carcinoma (Moore et al. 1952) H e L a cells derived from a carcinoma of the cervix uteri were also tested but later discarded. Control epithelial target cells were N C T C 2544, derived by Perry et al. (1957) from normal human skin. Each cell line was maintained in culture by continuous passage and che&ed before use for morphology and plating efficiency.
Microcytotoxicity tests: The method used was a modification of that first described by Takusugi and Klein (1970). Harvested epithelial target cells from culture were counted, viability tested and then serially diluted so as to be 100-150 cells in 10 btl of tissue culture medium. 10 gl aliquots were then dispensed into wells on microtest plates (Falcon 3034, Falcon Plastics, California, USA). After 6-20 hours incubation at 37 ° C in a humidified 5 % Co2, 95'0/0 air atmosphere, each microtest plate of 60 wells
was examined critically under phase-contrast microscopy. Plates with uneven distribution of target cells were discarded. Each plate used had at least 20 wells per plate counted to ensure accurate knowledge of the number of attached target cells. The medium in the wells was then removed by inversion and the effector cell suspensions in various sera added in 10 }tl aliquots so as to produce effector: target ratios as low as 300:1. At least 12 replicates of each test or control patients' effectors in the various sera (non-heat inactivated) were used. A further 12 replicates of lest or control patients' serum alone were also tested. The wells at the periphery of each plate were always used as internal controls (no effectors, 10~°/o foetal calf serum in growth medimn) of the unhindered normal growth of the target cells and comprised 24 wells on each plate. After addition of effector solutions the plates were further incubated for 40 hours, washed twice with warm medium supplemented with 5 % foetal calf serum, fixed with 100/0 formol saline and stained for counting.
Calculation o[ Cytotoxic El~'ect: The number of cells within the 100 squares of a grid 10 mm square at x 150 magnification was counted in each well. Evidence of cytotoxic activity was taken to be a range from complete destruction of the target cells, to crenation of cell walls with cytoplasmic extrusion or rounding up. Test and control patients' counts (often read blind and decoded later) were evaluated against the growth control counts on the same plate. The percentage cytotoxicity was then expressed according to the formula:
110
H. Cannell
Table 2 Comparison of effects of lymphocytes from other tumour patients on the two malignant cell lines KB and HEp-2. Effectors from patients Malignancy: Rectum Nose Oral Melanoma Skin Melanoma (Fossati et al. 1971)
E.T. ratio
°/o Cytotoxicity KB targets FCS Aut. S.
°/o Cytotoxicity HEp-2 targets FCS Aut. S.
350 350
20 0
0 0
14 0
35 0
500 300
0 0
0 0
0 -
0 -
Table 3 Effects of Lymphocytes on the normal skin cell Targets, NCTC 2544. Malignancy
Oral Ca. Oral Ca. Rectum Nose Normal Controls
E.T. ratio
400 350 350 350 400 350
NCTC 2544 Targets % Cytotoxicity FCS Aut. S.
P.
25 2 11 9 22 10
N.S. N.S. N.S. N.S. -
32 2 38 15 38 19
Means (test replicates) x 100 = % Cells surviving (CS). Mean2 (growth control Then 100- CS = °/o cytotoxicity. replicates) Significance levels were expressed after Student's test and p = 0.05 or less was taken to be significant.
W h e n effectors were suspended in autologous serum, in five cases (Cases 1, 2, 4, 5 and S) significant cytotoxicity was recorded (Fig. 2 b). Effectors from patients bearing an adeno-carcinoma of the rectum with metastases, a basal cell carcinoma of the nose or a m a l i g n a n t melanoma of the p a l a t e did not produce significant cytotoxicity to the KB target cells (Table 2).
Laryngeal Carcinoma Targets (HEp-2): Effectors from oral cancer patients in foetal calf serum, caused significant cytotoxicity in three cases (Cases 1, 3 and 9) (Fig. 2a) and in five cases (Cases 1, 3, 5, 6, and 9) when suspended in autologous serum. Effectors from turnout patients bearing tumours other than oral carcinomata did not produce significant cytotoxicity to the H E p - 2 targets
(Table 2). Normal skin cell targets (NCTC 2544):
Results The percentage cytotoxicities of each test after exposure to effectors and sera from oral cancer bearing patients are on the target cell lines KB and H E p - 2 shown in Figures 1 and 2.
Effectors from patients bearing oral squamous cell carcinoma, basal cell carcinoma of the nose or adeno-carcinoma of the rectum, did not produce significant degrees of cytotoxicity to normal skin cell targets (Table 3).
Normal Controls: Oropharyngeal carcinoma targets (KB): W h e n effectors from oral cancer patients were suspended in foetal calf serum (FCS) and incubated with KB targets, in six cases out of nine significant cytotoxicity r a n g i n g from 95°/0 to 57 % was recorded. In the remaining three cases (patients 7, 8, 9) cytotoxic levels were significantly different from their own test control subjects (Fig. 1 a).
The percentage cytotoxicity, under all conditions, of the normal controls v a r i e d from as little as 5O/o to a more usual 230/0-29'0/0. One normal control showed non-specific cytotoxicity of over 500/0 but this subject on investigation proved to be a h e a v y smoker with concomitant upper respiratory tract illness. Hellstrgm et al. (1971) noted that up to 50/0 of healthy subjects m a y react to both m a l i g n a n t and normal cells. In
Cell-Mediated and Humoral Immune Responses to Primary Oral Cancers
11 ]
ORAL C A N C E R PATIENTS'LYIMPIIOCYTOTOXICITY AGAINST T A R G E T CELLS
FIGURE 1.
DERIVED FROM AN ORO-pHARYNGEAL
CARCINOMA.
(KB CELLS)
100
Fig, 1a
100
Fig. l b
Foetal Call Serum
Autologous Serum
80
8O
6o
¢.
4o
4o
MNC ~2
--MNC
1
T2
TI T3 T3
FIGURE 2.
T4 T4('--CASE NO.
'F2
T1 T3 2'3 T2 T4 T4 T4 B1 B1 B1 - El BI En -
ORAL CANCER PATIENTS'LYMPHOCYTOTOXICITY AGAINST TARGET CELI~ DERIVED FROM A LARYNGEAL CARCINOMA, (HEP-g CELLS) Fig. 2a
108
Fig. 2b
100
Aut ologous Serum
Foetal Calf Serum
80
~
60
~oo
88
x:
~
~
48 .
.
.
.
.
.
40
MNC
~ 2o
~
M
N
C
l# 0
•
KEY:
0
MNC
Mean Percentage
BI
B l o c k i n g of t a r g e t c e l l d e a t h Enhancement
T1-4
Tumouv burdens (TNM
Discussion
Relevance and use of the cell lines: KB cells (Eagle 1955) are epithelial-like cells said to be derived from an oral carcinoma. The original tumour was situated at the back of the mouth and involved the tongue, fauces and soft palate. Growth from well-differentiated intra-oral carcinomata is slow and unlikely to yield large numbers of cells (Cannell 1973). It would appear therefore that KB cells might have been cultured from a highly anaplastic intra-oral carcinoma or even from a naso-pharyngeal carcinoma corn-
3
4
5
6
7
8
9
C y t o t o x i c i t y of n o r m a l c o n t r o l s .
En
our protocol each oral cancer patient had an appropriate healthy control and our results in Figs. 1 and 2 show the mean percentage cytotoxicity rate of those controls (MNC).
2
of t a r g e t c e l l d e a t h classiflcation-See
Table
1. )
pound into the mouth. HEp-2 cells (Moore et al. 1952) derived from a carcinoma of the larynx are also epithelial-like and are easily grown in culture. The use of these two cell lines to test lymphocytotoxicity in oral cancer patients may prove to be of considerable practical importance for methods for the culture and maintenance of the cells are well established. Both cell lines have good epithelial characteristics on histological examination. The plating efficiency of the cells varied little and in our experiments enabled accurate numbers of target cells to be plated.
Specificity of Results: Evidence for the specificity of the cytotoxic activity of oral cancer patients' effectors against oral cancer target cells was provided by (a) Nonsignificant cytotoxic activity of the same effectors
112
H. Cannell: Cell-Mediated and Humoral Immune Responses to Primary Oral Cancers
against normal skin cells. (b) The low cytotoxie activity of effectors from almost all healthy controis, and (c) Non-significant cytotoxic activity of effectors from patients with malignancies other than intra-oral carcinomata. There appeared to be a lower degree of specific cytotoxic ability of effectors from oral cancer patients when exposed to laryngeal carcinoma targets except when suspended in autologous serum (Fig. 2). Our results demonstrated therefore, the presence of oral cancer specific tumour antigens in KB cells and the presence of some different specific antigens in HEp-2 cells. Our results also show that tumour antigens of an intra-oral malignant melanoma appear to be different from the tumour antigens on oral cancer cells. Further but indirect evidence for this latter point is given by the report of Fossati et al. (1971) who were unable to detect a cytotoxic effect of lymphocytes from skin malignant melanoma patients against KB cell targets.
Bloched and enhanced cytotoxic ability: Effectors from patients 7, 8 and 9 showed a poor cytotoxic ability in non-specific serum against oral cancer target cells (Fig. 1A). Each of these patients had a large tumour burden with palpable nodes in the neck. Patient No. 7 was also noncytotoxic to HEp-2 cells. This patient's lack of specific immune response to a range of tumour antigens was in keeping with her clinical status. Originally treated for an intra-oral carcinoma by radiotherapy and surgery some years previously, at the time of the test she had developed leucoplakic areas and four new separate primary intra-oral carcinomata. The blocking of the cytotoxic ability of the effectors when in autologous serum and incubated with the oral cancer cells in three patients (Cases 3, 6 and 7) was comparable with results for other malignant tumours and reported by Hellstr6m et al. (1971). Varying degrees of blocking of cytotoxic effect were also noted in our experiments after autologous serum incubation using HEp-2 targets. In only one patient (Case 2) was the blocking effect demonstrable for the same patient with both cell lines, suggesting that only a few common tumour antigens were involved (Fig. 2 b),
Enhancement of the cytotoxic ability of oral cancer patients effectors against HEp-2 targets (Figs. 2 a and 2 b) was recorded in Cases 5 and 6. Case 8 (Figs. l a and b) showed slightly enhanced activity against KB targets. The increased percentage of cytotoxicity in these three cases was an unexpected finding and may be related to the purity of the effector cell suspensions used in these experiments. The mechanisms of the enhancement of the cytotoxic effect would appear to have several alternative explanations. An antibody in the serum which is effector cell dependent would appear to be likely. On the other hand, cytotoxicity by effectors is known to take place in the absence of added complement, (Perlmann et al. 1974) and complement is a mediator of antibody activity. In our experiments exclusion of thermolabile complement components did not alter the reactivity of the effectors in autologous serum. This would appear to suggest that the non-thermolahile components such as C'3, C'8 or C'9 might be implicated in enhancement of cytotoxic effects. Further evidence for this explanation is given by Perlmamz et al. (1974) who reports that C'8 is associated with lymphocytes from which it cannot be removed by extensive washing procedures. Long-term follow-up and re-testing of the patients would seem to be likely to provide a clue as to whether the presence of enhanced cytotoxic effects in these three patients are related to ultimate prognosis.
Conclusions Although the present series of patients is small, the significant cytotoxic responses, particularly to oral cancer target cells, would appear to form a basis for further follow-up studies. Whilst it is yet too early to state that all oral cancer patients are specifically active immunologically, against the two target cell lines, KB and HEp-2, it is suggested that the present results indicate that these in vitro tests of cell-mediated immunity correlate well with clinical status and tumour burden. Acknowledgement My colleagues in tile Department of Oral and Maxillo-. Facial Surgery and the Department of Radiotherapy, have contributed to these studies by referring their
A. L. Nwoku, H. Koch: The Temporomandibular Joint patients for the immunological tests and by their continuing interest and help. - Grateful a&nowledgement for assistance with laboratory facilities and equipment
113
is made to the London Hospital Cancer Research Committee and the Ernest and Minnie Dawson Cancer Research Trust.
References Binnie, W, H., R. A. Cawson, G. B. Hill: Summary. In: Oral Cancer in England and Wales. HMSO, London 1972
Bubenik, ].,P. Perlmann, K. Helmstein, G. Moberger: Immune response to urinary bladder tumours in man. Int. J. Cancer 5 (1970) 39 Cannell H.: Oral Cancer and Immunity. Brit. J. oral. Surg. 11 (1973) 171 Eagle, H.: Propagation in a fluid medium of a human epidermoid carcinoma strain, KB (21811). Proc. Soc. exp. Biol. Med. 89 (1955) 362
Fossati, G., M, ]. Colnaghi, G. Della Porta, N. Cascinelli, U. Veronesi: Cellular and humoral immunity against human malignant melanoma. Int. J. Cancer 8 (1971) 344 Harris, R., E. D. Uhaejiofo: Rapid preparation of lymphocytes for tissue typing. Lancet 7615 (1969) 327
against its own actively growing primary tumour. J. Nat. Cancer Inst. 36 (1966) 29 Moore, A. E., L. Sabachewcky, H. W. Toolan: Culture Characteristics of 4 permanent lines of human cancer cell. Cancer Res. 15 (1952) 598
O'Toole, C., P. Perlmann, B. Unsquard, G. Moberger, F. Edsmyr: Cellular immunity to human urinary bladder carcinoma. 1. Correlation to clinical stage and radiotherapy. Int. J. Cancer 10 (1972) 17 Perlmann, P., H. Perlmann, P. Lachmann: Lymphocyte-associated complement. Role of C'8 in certain cell-mediated cytic reactions. Scan& J. hmnunol. 3 (1974) 77
Perry, V. P., K. K. Sanford, V. S. Evans, G. W. Hyatt, W. R. Earle: Establishment of clones of epithelial cells from human skin. J. Nat. Cancer Inst. 18
(1957) 709 Takusugi, M., E. Klein: A micro-assay for cell-mediated
Hellstr6m, L, K. E. Hellstr6m, G. E. Pierce, ]. P. S. Young: Cellular and humoral immunity to diffe-
immunity. Transplant. 9 (1970) 219
rent types of human neoplasms. Nature 220 (1968) 1352
Hellstr6m, I., K. E. Hellslr6m, H. O. Sj6gren, G. A. Warner: Demonstration of cell-mediated immunity to human neoplasms of various histological types. hat. J. Cancer 7 (1971) 1 Mikulska, Z. B., C. Smith, P. Alexander: Evidence for an immunological reaction of the host directed
Hugh Cannell, M.Sc., F.D.S.R,C.S., M.R.C.S., L.R.C.P., Department of Oral and Maxillo-Facial Surgery, The London Hospital Medical College, Turner Slreet, London EI 2AD, U.K.
J. max.-fac. Surg. 2 (1974) 113-119 © Georg Thleme Verlag, Stuttgart
The Temporomandibular Joint: A rare Localisation for Bone Tumours* Alagumba L. N. Nwoku, Heribert Koch Clinic for Maxillo-Facial and Plastic Surgery of the Face, Westdeutsche Kieferhlinik (Director: Prof. A. Rehrmann, M.D., D.D.S.), University of Dusseldorf, W. Germany
Summary The literature on tumours of the temporomandibular region has been reviewed, and a classification between true tumours of the condyle and condylar hyperplasia made. Of 3,200 tumours af the head and he& treated in the Clinic for Maxiltofacial and Plastic Surgery of the University of Dfisseldorf from 1. 1. 1955 to * Paper will be read at the 2nd Congress of the European Association for Maxillo-Facial Surgery in Zurich, Switzerland from 16.9.- 21.9. 74.
30, 4. t974, only seven cases of true tumours occurred in the condylar head. One rare and very interesting case, which is not a turnout, has been included to illustrate the complexity and sometimes uncertainty in diagnosis, A possible explanation for the apparent rarity of tumours of the temporomandibular region has been offered. Key-Words: Temporomandibular Joint Tumours; Con-. dylar Hyperplasia; Condylcctomy; Impacted third molar.