CYNECOLOCX
38, 125-127 (1990)
ONCOLOGY
Lymphocyte Subpopulations in Patients with Cervical lntraepithelial Neoplasia ERIK SOEGAARD
M.D.,*
ANDERSEN,
OLE BJARNE
CHRISTIANSEN,
AND GORM LARSEN, *Department
of Obstetrics
and Gynecology
M.D.,t
CASPER JERSILD,
M.D., D.Sc.,t
M.D.*
and tRegiona1 Centre for Blood Transfusion Section North, Aalborg, Denmark
and Clinical
Immunology,
Aalborg
Hospital,
Received January 9. 1990 Quantitation in
of T-lymphocyte
peripheral
neoplasia formed No
blood and
of
16 patients
15 controls
using
specific
differences
were
with
monoclonal
lymphocytes,
cells.
indicate
immune
results response,
the cervix, the
initiation
observed
are secondary Of Cervical
in
and
cell
patients
killer
ratios,
intraepithelial was
flow
cytometry.
with and
killer
cell-mediated
invasive
cancer
of no significance
0 1990AcademicPIW,
per-
T-sup-
or natural
of the
MATERIALS
cells
cytology
lymphocytes,
disturbances
to the disease Cancer.
cervical
T-helper
Th/T,
natural
cervical
antibodies in
that
and
with
normal
observed
pressor/cytotoxic The
subsets
of in
IIIC.
Initiation and growth of neoplasia are thought to result from a disturbance between the aggressiveness of the neoplasm and the defense mechanisms of the host. Cellmediated immune responses are mediated by T lymphocytes and natural killer (NK) cells, which appear to be important mechanisms in the destruction of neoplastic cells [I]. The use of monoclonal antibodies which specifically bind to various surface antigens of the lymphocytes has made it possible to enumerate the lymphocyte subpopulations. One quantitative measure of cell-mediated immunity is the ratio of T-helper (TJ lymphocytes to T-suppressor/cytotoxic (T,) lymphocytes, the T,/T, ratio. Impaired cellular immunity is often associated with a decreased T,/T, ratio. Evaluation of cell-mediated immune function by measurement of the subpopulations of lymphocytes in patients with invasive and preinvasive lesions of the uterine cervix has been reported only a few times [2-41. It was the aim of the present study to compare peripheral blood lymphocyte subpopulations of patients with cervical intraepithelial neoplasia (CIN) and women with normal cervical cytology.
AND METHODS
Sixteen patients with CIN were included. In 13 cases, the histologic diagnosis was CIN III; in 2 cases, CIN II; and in one case, CIN I. Fifteen controls, all with normal cervical cytology within the year preceding entry into the study, were included. Both patients and controls had no medical history of immunological disease and had not received any medication affecting the immune system. Venous blood was collected from patients and controls, and a complete blood count and differential count were done. A suspension of the peripheral mononuclear cells was made, as described by Oonishi et al. [5], using lymphocyte separation medium (Lymphoprep, Nycorned, 1.077 g/ml). The suspension was adjusted to 5 x lo6 to 2 x 10’ cells/ml, frozen using DMSO as cryoprotectant, and stored in liquid nitrogen. When analyzed, the cell suspension was thawed, washed, and readjusted to 1 x 10’ cells/ml. Lymphocyte viability exceeded 90% for all preparations, using the trypan blue dye exclusion method. Subpopulations of T lymphocytes and NK cells were identified using the monoclonal antibodies CD-3 (Leul), CD-4 (Leu-3), CD-8 (Leu-2), and CD-16 (Leu-11), conjugated with fluorescein isothiocyanate (BectonDickinson, Mountain View, CA). These monoclonal antibodies specifically identify T lymphocytes (T total), Thelper lymphocytes, T-suppressor/cytotoxic lymphocytes, and natural killer cells, respectively. Percentages of lymphocyte subsets were determined by flow cytometry (FACScan, Becton-Dickinson), and their absolute numbers (cells/ml) were calculated based on the total lymphocyte count. The results were evaluated by Student’s t test (using Yates’ correction). 125 oo!30-8258190 $1.50
Copyright 0 1990by Academic Press,Inc. All rights of reproduction in any form reserved.
126
ANDERSEN ET AL.
RESULTS
The mean age of patients was 34.6 years (range, 2156 years), and that of controls, 33.8 years (range, 21-43 years). The results of the immunological assessment are shown in Table 1. No significant differences were observed, whether absolute values (cells/ml) or percentages were compared. Also, T,/T, ratios were similar in patients and controls. The percentage of NK cells in peripheral blood was reduced approximately 14% in patients, but the difference was not significant. DISCUSSION Defective immune response in patients with invasive cancer of the uterine cervix has previously been reported [3,4,6-131. These studies have demonstrated increasing impairment of the cellular immune response with increasing stage of invasive disease. Few have been able to demonstrate this impairment in early stages of cancer of the cervix [II-131. Caste110et al. [3], demonstrated significantly reduced T,/T, ratios in patients with dysplasia/carcinoma in situ, caused by a significantly increased percentage of T, lymphocytes. Very recently, Ballaram et al. [4] were unable to demonstrate any alterations in the cell-mediated immune response in early stages of cervical cancer. Our results did not show any difference between patients and controls, as far as Th and T, lymphocytes were concerned. NK cells mediate spontaneous, non-HLArestricted cytotoxicity, and can likely be regarded as candidates for mediation of antitumor immunity. We TABLE 1 Results of Immm~ological Assessment in Patients with CIN and Controls”
count per mm’ Lymphocyte count per mm’ T-helper lymphocytes (CD-+
Patients n = 16
Controls n = 15
7700 f 2450
6300 2 1670 NS
2370 2 858
2130 2 404
1091 2 425’ 45.3 ” 7.7’
893 + 216 NS 45.7 2 10.7 NS
Leukocyte
T-suppressor lymphocytes (CD-8) T,/T, cell ratio NK cells (%) NK cells (cells/mm3)
672 29.4 1.63 15.5 370.2
” 2 f ” k
245’ 6.3 0.55 6.98 200
u Means it_ SD compared by Student’s t test. b Cells/mm’. ’ Percent of total lymphocytes.
659 f 248 30.7 + 7.0 1.61 + 0.61 18.0 2 5.70 384.9 f 220
NS
NS NS NS NS NS
were not able to demonstrate any difference in the number of phenotypic NK cells among patients and controls. We are aware that any conclusions as to the function of NK cells are not possible from our results. Ballaram et al. [41 showed reduced NK cell activity against the human erythroleukemia cell line KS62 in all stages of invasive disease, the impairment being more pronounced in higher stages of malignancy. The impaired cellular immunity seen in cases of invasive cancer of the cervix indicate that the immune system contributes to the control of neoplastic cell growth in advanced disease. If a selective immunologic deficiency is of importance for susceptibility to cancer of the cervix, the disturbance should probably be sought in the local defense mechanisms of the cervical epithelium. Recently, a few studies [14-161 have demonstrated a localized immunodeficiency of the cervical epithelium in patients with cervical intraepithelial neoplasia. Further studies of local immune functions in the uterine cervix may be of importance in elucidating the initiation of cancer of the cervix. REFERENCES 1. Burnett, F. M. Immunological aspects of malignant disease, Lancer 1, 1171-1174 (1967). 2. Carson, L. F., Twiggs, L. B., Fukushima, M., Ostrow, R. S., Faras, A. J., and Okagaki, T. Human genital papilloma infections: An evaluation of immunologic competence in the genital neoplasiapapilloma syndrome, Amer. J. Obsret. Gynecol. 155, 784-789 (1986). 3. Castello, G., Esposito, G., Stellato, G., Mora, L. D., Abate, G., and Gfrmano, A. Immunological abnormalities in patients with cervical carcinoma, Gynecol. Oncol. 25, 61-64 (1986). 4. Ballaram, P., Pillai, M. R., Padmanabhan, T. K., Abraham, T., Hareendram, N. K., and Nair, M. K. Immune function in malignant cervical neoplasia: A multiparameter analysis, Gynecol. Oncol. 31, 409-423 (1988). 5. Oonishi, T., Sakashita, K., and Uyesaka, N. Flow cytometric studies of the binding of monoclonal antibodies OKT3, OKT4 and OKT8, J. Immunol. Methods 115, 159-167 (1988). 6. Hagen, C., Froland, A., and Weberg, E. Lymphocyte transformation in cancer patients, Lancet 1, 1340 (1972). 7. Twomey, P. L., Rogentine, G. N., and Cliretin, P. B. Lymphocyte function and HLA antigen frequency in gynecologic squamous cancer, ht. Surg. 59, 468-470 (1974). 8. Khoo, S. K., and MacKay, E. V. Relation of cell mediated immunity in women with genital tract cancer to origin, clinical stage and subsequent behaviour of neoplasm, J. Obstet. Gynecol. 81, 229-235 (1974). 9. Levy, S., Kopersztych, S., Musatti, C. C., Souen, J. S., Salvatore, C. A., and Mendes, N. F. Cellular immunity in squamous cell carcinoma of the uterine cervix, Amer. J. Obstet. Gynecol. 130, 160-164 (1978). 10. Alsabti, E. A. K. The immunostatus of untreated cervical carcinoma, Gynecol. Oncol. 9, 6-11 (1980). 11. Ishiguro, T., Sugitachi, and Katoli, K. T and B lymphocytes in
LYMPHOCYTE
SUBPOPULATIONS
patients with squamous cell carcinoma of the uterine cervix, Gynecol. Oncol. 9, 80-85 (1980). 12. Kietlinska, Z. T and B lymphocyte counts and blast transformation in patients with stage I cervical cancer, Gynecol. Oncol. 18, 247256 (1984). 13. Saxena, V. K., Chander, S., Chandra, M., Sachdeva, S., Ghosh, D., and Saxena, H. M. K. A study of T lymphocyte population in cancer of the cervix. Effects of pelvis irradiation, Int. J. Cancer 22, 173-177 (1985).
IN CIN
127
14. Morris, H. H. B., Gatter, K. C., Sykes, G., Casemore, V., and Mason, D. Y. Langerhans’ cells in human cervical epithelium: Effects of wart virus infection and intraepithelial neoplasia, Brit. .I. Obstet. Gynaecol. 90, 412-420 (1983). 15. Edwards, J. N. T., and Morris, H. H. B. Langerhans’ cells and lymphocyte subsets in the female genital tract, Brit. J. Obstet. Gynaecol. 92, 974-982 (1985). 16. Tay, S. K., Jenkins, D., Maddox, P., Campion, M., and Singer, A. Subpopulations of Langerhans’ cells in cervical neoplasia, Brit. J. Obstet. Gynaecol. 94, lo-15 (1987).
38, 125-127 (1990)
ONCOLOGY
Lymphocyte Subpopulations in Patients with Cervical lntraepithelial Neoplasia ERIK SOEGAARD
M.D.,*
ANDERSEN,
OLE BJARNE
CHRISTIANSEN,
AND GORM LARSEN, *Department
of Obstetrics
and Gynecology
M.D.,t
CASPER JERSILD,
M.D., D.Sc.,t
M.D.*
and tRegiona1 Centre for Blood Transfusion Section North, Aalborg, Denmark
and Clinical
Immunology,
Aalborg
Hospital,
Received January 9. 1990 Quantitation in
of T-lymphocyte
peripheral
neoplasia formed No
blood and
of
16 patients
15 controls
using
specific
differences
were
with
monoclonal
lymphocytes,
cells.
indicate
immune
results response,
the cervix, the
initiation
observed
are secondary Of Cervical
in
and
cell
patients
killer
ratios,
intraepithelial was
flow
cytometry.
with and
killer
cell-mediated
invasive
cancer
of no significance
0 1990AcademicPIW,
per-
T-sup-
or natural
of the
MATERIALS
cells
cytology
lymphocytes,
disturbances
to the disease Cancer.
cervical
T-helper
Th/T,
natural
cervical
antibodies in
that
and
with
normal
observed
pressor/cytotoxic The
subsets
of in
IIIC.
Initiation and growth of neoplasia are thought to result from a disturbance between the aggressiveness of the neoplasm and the defense mechanisms of the host. Cellmediated immune responses are mediated by T lymphocytes and natural killer (NK) cells, which appear to be important mechanisms in the destruction of neoplastic cells [I]. The use of monoclonal antibodies which specifically bind to various surface antigens of the lymphocytes has made it possible to enumerate the lymphocyte subpopulations. One quantitative measure of cell-mediated immunity is the ratio of T-helper (TJ lymphocytes to T-suppressor/cytotoxic (T,) lymphocytes, the T,/T, ratio. Impaired cellular immunity is often associated with a decreased T,/T, ratio. Evaluation of cell-mediated immune function by measurement of the subpopulations of lymphocytes in patients with invasive and preinvasive lesions of the uterine cervix has been reported only a few times [2-41. It was the aim of the present study to compare peripheral blood lymphocyte subpopulations of patients with cervical intraepithelial neoplasia (CIN) and women with normal cervical cytology.
AND METHODS
Sixteen patients with CIN were included. In 13 cases, the histologic diagnosis was CIN III; in 2 cases, CIN II; and in one case, CIN I. Fifteen controls, all with normal cervical cytology within the year preceding entry into the study, were included. Both patients and controls had no medical history of immunological disease and had not received any medication affecting the immune system. Venous blood was collected from patients and controls, and a complete blood count and differential count were done. A suspension of the peripheral mononuclear cells was made, as described by Oonishi et al. [5], using lymphocyte separation medium (Lymphoprep, Nycorned, 1.077 g/ml). The suspension was adjusted to 5 x lo6 to 2 x 10’ cells/ml, frozen using DMSO as cryoprotectant, and stored in liquid nitrogen. When analyzed, the cell suspension was thawed, washed, and readjusted to 1 x 10’ cells/ml. Lymphocyte viability exceeded 90% for all preparations, using the trypan blue dye exclusion method. Subpopulations of T lymphocytes and NK cells were identified using the monoclonal antibodies CD-3 (Leul), CD-4 (Leu-3), CD-8 (Leu-2), and CD-16 (Leu-11), conjugated with fluorescein isothiocyanate (BectonDickinson, Mountain View, CA). These monoclonal antibodies specifically identify T lymphocytes (T total), Thelper lymphocytes, T-suppressor/cytotoxic lymphocytes, and natural killer cells, respectively. Percentages of lymphocyte subsets were determined by flow cytometry (FACScan, Becton-Dickinson), and their absolute numbers (cells/ml) were calculated based on the total lymphocyte count. The results were evaluated by Student’s t test (using Yates’ correction). 125 oo!30-8258190 $1.50
Copyright 0 1990by Academic Press,Inc. All rights of reproduction in any form reserved.
126
ANDERSEN ET AL.
RESULTS
The mean age of patients was 34.6 years (range, 2156 years), and that of controls, 33.8 years (range, 21-43 years). The results of the immunological assessment are shown in Table 1. No significant differences were observed, whether absolute values (cells/ml) or percentages were compared. Also, T,/T, ratios were similar in patients and controls. The percentage of NK cells in peripheral blood was reduced approximately 14% in patients, but the difference was not significant. DISCUSSION Defective immune response in patients with invasive cancer of the uterine cervix has previously been reported [3,4,6-131. These studies have demonstrated increasing impairment of the cellular immune response with increasing stage of invasive disease. Few have been able to demonstrate this impairment in early stages of cancer of the cervix [II-131. Caste110et al. [3], demonstrated significantly reduced T,/T, ratios in patients with dysplasia/carcinoma in situ, caused by a significantly increased percentage of T, lymphocytes. Very recently, Ballaram et al. [4] were unable to demonstrate any alterations in the cell-mediated immune response in early stages of cervical cancer. Our results did not show any difference between patients and controls, as far as Th and T, lymphocytes were concerned. NK cells mediate spontaneous, non-HLArestricted cytotoxicity, and can likely be regarded as candidates for mediation of antitumor immunity. We TABLE 1 Results of Immm~ological Assessment in Patients with CIN and Controls”
count per mm’ Lymphocyte count per mm’ T-helper lymphocytes (CD-+
Patients n = 16
Controls n = 15
7700 f 2450
6300 2 1670 NS
2370 2 858
2130 2 404
1091 2 425’ 45.3 ” 7.7’
893 + 216 NS 45.7 2 10.7 NS
Leukocyte
T-suppressor lymphocytes (CD-8) T,/T, cell ratio NK cells (%) NK cells (cells/mm3)
672 29.4 1.63 15.5 370.2
” 2 f ” k
245’ 6.3 0.55 6.98 200
u Means it_ SD compared by Student’s t test. b Cells/mm’. ’ Percent of total lymphocytes.
659 f 248 30.7 + 7.0 1.61 + 0.61 18.0 2 5.70 384.9 f 220
NS
NS NS NS NS NS
were not able to demonstrate any difference in the number of phenotypic NK cells among patients and controls. We are aware that any conclusions as to the function of NK cells are not possible from our results. Ballaram et al. [41 showed reduced NK cell activity against the human erythroleukemia cell line KS62 in all stages of invasive disease, the impairment being more pronounced in higher stages of malignancy. The impaired cellular immunity seen in cases of invasive cancer of the cervix indicate that the immune system contributes to the control of neoplastic cell growth in advanced disease. If a selective immunologic deficiency is of importance for susceptibility to cancer of the cervix, the disturbance should probably be sought in the local defense mechanisms of the cervical epithelium. Recently, a few studies [14-161 have demonstrated a localized immunodeficiency of the cervical epithelium in patients with cervical intraepithelial neoplasia. Further studies of local immune functions in the uterine cervix may be of importance in elucidating the initiation of cancer of the cervix. REFERENCES 1. Burnett, F. M. Immunological aspects of malignant disease, Lancer 1, 1171-1174 (1967). 2. Carson, L. F., Twiggs, L. B., Fukushima, M., Ostrow, R. S., Faras, A. J., and Okagaki, T. Human genital papilloma infections: An evaluation of immunologic competence in the genital neoplasiapapilloma syndrome, Amer. J. Obsret. Gynecol. 155, 784-789 (1986). 3. Castello, G., Esposito, G., Stellato, G., Mora, L. D., Abate, G., and Gfrmano, A. Immunological abnormalities in patients with cervical carcinoma, Gynecol. Oncol. 25, 61-64 (1986). 4. Ballaram, P., Pillai, M. R., Padmanabhan, T. K., Abraham, T., Hareendram, N. K., and Nair, M. K. Immune function in malignant cervical neoplasia: A multiparameter analysis, Gynecol. Oncol. 31, 409-423 (1988). 5. Oonishi, T., Sakashita, K., and Uyesaka, N. Flow cytometric studies of the binding of monoclonal antibodies OKT3, OKT4 and OKT8, J. Immunol. Methods 115, 159-167 (1988). 6. Hagen, C., Froland, A., and Weberg, E. Lymphocyte transformation in cancer patients, Lancet 1, 1340 (1972). 7. Twomey, P. L., Rogentine, G. N., and Cliretin, P. B. Lymphocyte function and HLA antigen frequency in gynecologic squamous cancer, ht. Surg. 59, 468-470 (1974). 8. Khoo, S. K., and MacKay, E. V. Relation of cell mediated immunity in women with genital tract cancer to origin, clinical stage and subsequent behaviour of neoplasm, J. Obstet. Gynecol. 81, 229-235 (1974). 9. Levy, S., Kopersztych, S., Musatti, C. C., Souen, J. S., Salvatore, C. A., and Mendes, N. F. Cellular immunity in squamous cell carcinoma of the uterine cervix, Amer. J. Obstet. Gynecol. 130, 160-164 (1978). 10. Alsabti, E. A. K. The immunostatus of untreated cervical carcinoma, Gynecol. Oncol. 9, 6-11 (1980). 11. Ishiguro, T., Sugitachi, and Katoli, K. T and B lymphocytes in
LYMPHOCYTE
SUBPOPULATIONS
patients with squamous cell carcinoma of the uterine cervix, Gynecol. Oncol. 9, 80-85 (1980). 12. Kietlinska, Z. T and B lymphocyte counts and blast transformation in patients with stage I cervical cancer, Gynecol. Oncol. 18, 247256 (1984). 13. Saxena, V. K., Chander, S., Chandra, M., Sachdeva, S., Ghosh, D., and Saxena, H. M. K. A study of T lymphocyte population in cancer of the cervix. Effects of pelvis irradiation, Int. J. Cancer 22, 173-177 (1985).
IN CIN
127
14. Morris, H. H. B., Gatter, K. C., Sykes, G., Casemore, V., and Mason, D. Y. Langerhans’ cells in human cervical epithelium: Effects of wart virus infection and intraepithelial neoplasia, Brit. .I. Obstet. Gynaecol. 90, 412-420 (1983). 15. Edwards, J. N. T., and Morris, H. H. B. Langerhans’ cells and lymphocyte subsets in the female genital tract, Brit. J. Obstet. Gynaecol. 92, 974-982 (1985). 16. Tay, S. K., Jenkins, D., Maddox, P., Campion, M., and Singer, A. Subpopulations of Langerhans’ cells in cervical neoplasia, Brit. J. Obstet. Gynaecol. 94, lo-15 (1987).
