British Journal of Obstetrics and Gynaecology May 1916. Vol83. pp 393-394
TUMOUR ASSOCIATED IMMUNITY AND IMMUNOCOMPETENCE IN OVARIAN CANCER BY
L. LEVIN J. E. MCHARDY T. A. POULTON 0. M. CURLING AND
C . N. HUDSON Williurnson Laboratory, Medical College of St Bartholomew’s Hospitul, London, ECIA 7BE Summary Cell-free extracts of autologous ovarian cancer cells evoked a delayed hypersensitivity response (DHR) in skin in four out of six patients in remission from disease and none of seven patients in relapse. These initial results appear to be consistent with in vitro blastogenic assay using the same autologous extracts. To assess whether diminished tumour-associated immunity was due to impaired cellmediated imniunocompetence, blastogenic responses to purified protein derivative of tuberculin (PPD), a secondary recall antigen, and phytohaemagglutinin (PHA) were assessed. Impaired responses to PHA were seen in the relapse group of patients, but responses to PPD did not appear to be impaired in this group.
blastogenic responses which were greater than seen in any of the 10 normal controls. Using a lymphocyte blastogenic assay, evidence for cell-mediated sensitization to extracts of ovarian cancer cells was obtained in 45 ovarian cancer patients (Levin et ul, 19751). The nature of this response appeared to be complex and was seen more frequently in remission than in relapse patients. We have now studied the in vitro cellular immunity in ovarian cancer patients by measuring blastogenic responses to phytohaemagglutinin (PHA) and purified protein derivative of tuberculin (PPD), to assess whether impaired immunocompetence could have been responsible for diminished tumourassociated immunity in relapse patients. During a pilot study of immunotherapy for
A TUMOUR specific cell-mediated immune response has been demonstrated in ovarian cancer patients by in vitro lymphocyte-mediated cytotoxicity of ovarian cancer cells (Hellstrom et cil, 1971; Di Saia et ul, 1971). Furthermore, autologous extracts of ovarian tumour cells have been shown to produce leucocyte migration inhibition (Chen et al, 1973). Chatterjee et ul (1975) found no evidence for a cell-mediated tumour-associated immune response by assessing the ability of lymphocytes from ovarian cancer patients to transform (blastogenesis) when challenged with autologous tumour extracts; they also found no evidence of a delayed skin hypersensitivity response to autologous tumour extracts in their patients. However, 4 out of 12 of their patients had 393
394
LEVIN, MCHARDY, POULTON, CURLING AND HLDSON
ovarian cancer (Levin et al, 1975b), a good correlation was noted in one patient between in vivo delayed hypersensitivity response (DHR) in skin to autologous tumour cell extract and the in vitro response to the same extract. This prompted the present study of the in vivo cell-mediated immune response to autologous tumour extract. METHODS Cell extract Fragments of fresh tumour material from surgical specimens were homogenized in normal saline, frozen rapidly in liquid nitrogen, thawed and the suspension was finally made hypotonic. The tumour homogenate was centrifuged at 400 g for five minutes and the resulting supernatant centrifuged at 3000 g for one hour at 4 "C. The supernatant from the 3000 g centrifugation was estimated for protein content by the FolinLowry technique (Lowry et al, 1952). The extracts were made up to a concentration of 2 mg/ ml in normal saline and stored at -20 "C. All cell extracts were tested for bacterial growth and discarded if bacteria were present.
calf serum in tissue culture medium M199 and, appropriate cultures challenged with 50, 100 or 200 pg of autologous tumour extract. Similar cultures in 12.5 per cent autologous serum or fetal calf serum were challenged with 10 pg PPD or 10 pg PHA. Similar cultures of unstimulated lymphocytes from the same source were set up in parallel. Cultures were kept at 37 "C in 5 per cent COBfor five days and transformation was assessed by incorporating 12"I-5-iodo-deoxyuridine (IUDR) into DNA during the last 6 hours of culture, as described elsewhere (Levin et al, 1975a); the extent of transformation was measured, using a Wilji Gamma Counter, by the difference between the counts per minute (cpm) from stimulated and unstimulated lymphocytes. Where possible, responses were measured in duplicate.
Patients Twelve patients with common epithelial (Mullerian) malignant ovarian tumours were
Skin test Three doses of extract were used in each test, these being 0.1 mg, 0.25 mg, and 0 . 5 mg. Injections were given intradermally on the flexor aspect of the forearm. A 0.5 ml volume of normal saline was injected intradermally on the opposite arm as a control. Any response which took 24 hours to appear but disappeared after 72 hours, was indurated and produced a reddened area greater than 3 mm in diameter, was taken to be positive. The skin test was assessed at 6 hours, 24 hours, 48 hours and 72 hours and measured when it was positive. Skin biopsy A 3 mm punch skin biopsy was performed at the site of a positive DHR in three of the patients and the sections examined for histological evidence of DHR. Lymphocyte transformation blastogenic assay Lymphocytes were isolated by centrifuging whole heparinized blood (10 iu preservative-free heparin per ml of blood) at 400 g over a ficoltriosil density gradient. Cultures were set up as 1 x lo6 lymphocytes/ml of 12.5 per cent fetal
FIG.1 Delayed skin hypersensitivity response to 0.25 mg autologous tumour extract.
IMMUNITY IN OVARIAN CANCER
tested with autologous tumour-cell extracts. None of the patients had had chemotherapy or radiotherapy for at least 30 days prior to the test being performed. Six patients were in remission from disease and six were in relapse. All controls were normal females, matched for age with the ovarian cancer patients ; they were mostly patients with prolapse awaiting operation and none were on medication at the time of testing. PHA responses were determined in 16 ovarian cancer patients in remission from disease, 9 in relapse and in 14 normal controls. PPD responses were determined in 10 ovarian cancer patients in remission from disease, 13 in relapse and in 16 normal controls.
RESULTS Delayed hypersensitivity responses ( D H R ) in skin to autologous cell extracts Four of six patients in remission from disease
395
had a positive DHR in skin to autologous tumour extract. The minimum effective dose in three of these patients was 0.25 mg, while in the fourth patient, it was 0 . 5 mg. The diameter of the positive responses was between 4 and 7 mm and in three patients a skin biopsy was taken and examined histologically. Figure 1 shows a typical DHR to tumour extract and Figure 2 shows the typical histological features of this response. One of the remission patients who gave a negative DHR to the extract developed tumour spread to the inguinal lymph nodes four weeks after skin testing. The other patient with a negative DHR was still in remission from disease 18 months after skin testing, when she once again failed to produce a D H R to autologous cell extract. None of the seven patients in relapse developed a DHR to autologous tumour extract.
FIG.2 Punch skin biopsy front a delayed skin hypersensitivity response to autologous tumour extract ( ~ 2 5 0 )The . area was infiltrated almost exclusively with mononuclear cells.
396
LEVIN, MCHARDY, POULTON, CURLING AND HUDSON
In vitro correlation with DHR The blastogenic responses to autologous cell extract for all patients who were skin tested with the same extract are shown in Table I. A positive blastogenic response in this system was taken as one in which the stimulation index was greater than 1.2 and the cpm difference greater than 500 (Levin et al, 1975aJ. All four patients who had a positive DHR to autologous tumour extract, also had a blastogenic response to the same extract. One of the six relapse patients tested had a blastogenic response to autologous tumour extract.
5. I.
AUTOLOGOUS SERUM
FETAL CALF SERUM
. .
0
I
Blastogenic responses to PPD and PHA There was no difference in responses to PPD in the three groups of patients compared (Fig. 3). Blastogenic responses to PPD are shown together with those to tumour extract in Table I, but the data are too limited to make a direct comparison between these two responses. Responses to PHA in ovarian cancer patients and normal controls are shown in Table I1 and Figure 4. PHA responses in ovarian cancer
Controls
Remission
Relapse Controls Remission Relapse
OVARIAN CANCER Xl5.l.) 2.5
2.3
OVARIAN CANCER
2.0
2.9
1.9
2.2
FIG.3 PPD responses in ovarian cancer patients and normal controls. There was no difference in blastogenic responses to PPD in the ovarian cancer patients in remission or relapse, when compared with normal controls.
TABLEI In vivo and in vitro responses to autologous ovarian turnour cell extructs Blastogenic responses
Patient
Remission 1 2 3 4 5*
6t
Relapse 7 8 9 10 11 12 13
Diameter of skin reaction (mm)
Unstimulated
6 7
5 6 0 0
0 0 0 0 0 0 0
Stimulated
Stimulation index
(CPm)
Ovarian cancer cell extract
PPD (cPn4
Ovarian cancer cell extract
2300 1600 1560 2220 2190 1260
3000 4600 3960 3900 2030 1440
2310 2300 4140 2100 3900
1.3 3.0 2.5 1.8 1.0 1.1
1.0 1.4 2.7 1.0
1890 1860 2010 4080
2080 2410 2040 3930 3290 2460
2840 3960 3630 10 300 1260
1.1 1.3 1.0 1.0 1.0 1.1
1.5 2.0
-
3300 2340
* Retested 18 months later (still in remission), and failed to produce DHR.
t Went into relapse with disease spread to inguinal lymph nodes four weeks after testing.
PPD
3.1
-
0.9 -
3.3 0.5
IMMUNITY IN OVARIAN CANCER
397
TABLEI1 Blastonenic responses to PHA in ovarian cancer patients rand normal controls Autologous serum Unstimulated Stimulated (CPm) (CPd Ovarian cancer remission: 2880 61440 3330 62040 1940 26970 1680 18600 1950 13830 3510 21205 4210 25630 1740 10000 7200 29700 2480 9000 2660 9363 4800 9600 2400 3940 4480 4480
Fetal calf serum Stimulation Index 21 18.6 14 9.9 7.0 6.0 6.0 5.7 4.2 3.6 3.5 2.0 1-7 1.0 -
Normal controls: 4400 1850 2100 4230 1800 2000 3510 4800 3420 2700 5400 6Ooo
6270 2670 4040 4980 -
49500 26100 12920 5700 2500 11370 2230 1580 -
10400 46620 41700 81360 3 1920 31000 52240 6oooO 3 m 21000 35100 35520 27690 7800 7200 8460 -
-
-
57770 3000 8580
25 1.7 3.9 1.6 1.1 6.0 10.0 3.3 1.8 4.4 31.0 10.0
7.2 6.0 4.3 2.6 1-5 1.5 1.1 0-9 -
9240 3040 1950 1460 3340 1930 1220 1730 5430 1940 4080
-
-
Stimulation Index
-
-
25 25 20 19 18 16.0 15.5 12.5 9.0 7.9 6.5 6.5 4.4 3.0 1.8 1.7 -
Stimulated (cpm)
2280
1740 2150 1920 9240 2850 2380 3730 3600 3020 2960 4140 5430 1590 2120
-
Ovarian cancer relapse : 6840 4700 3000 2190 1710 8080 1850 1780 -
Unstimulated (cpm)
-
8400 1070 1830 2160 4440 5760 3480 4560 -
3060 3260 2280 8220
-
3060 10200 17880 23050 12120 6024 13320 92500 41000 28100 3770 3330
5.2
2.4 1.6
67740 7910 1950 1560 3120 2770 1580 24830 28100 9100 8460
7.0 2.6 1.0 1.1 0.9 1.4 1.3 14.3 5.2 4.7 2.0
38000 17820 1lo00 14880
5.0 17.0 6.0 6.9 3.5 2.4 2.9 -
-
15420 13140 6600 -
-
25320 3420 26640 21660 25020
5.5 1.1 11.2 9.5 3.0
398
LEVIN, MCHARDY, POULTON, CURLING AND HUDSON
s. 1
AUTOLOGOUS SERUM
FETAL CALF SERUM
40
30
.
20
a 0
10
t
0
0
0
0
:
0 0
5 0
. . .
0
0
2
0
5
. Controls Remission Relapse
Controls Remission Relapse
OVARIAN CANCER
x (S.1.)
12
8
3
0
0
OVARIAN CANCER
6
7
4
FIG.4 PHA responses in ovarian cancer patients and normal controls. While there was no differencein PHA responses in remission patients when compared ai t h normal controls, responses in relapse were lower than in the controls in autologous screen (P=O.O3).
relapse patients were lower than PHA responses in normal controls, and this difference was statistically significant (P =O .03) in autologous esrum. Responses to PHA in relapse patients were not different from the normal controls when these were measured in fetal calf serum: there were decreased PHA responses in fetal calf serum in the control group when compared with their responses in autologous serum. DISCUSSION In vivo evidence for a tumour-associated immune response in the 13 ovarian cancer patients tested follows the same pattern as the in vitro response reported by the same authors (Levin et al, 1975a)in that DHR occurred commonly in the remission group of patients. There is prima facie evidence in this series for a correlation
between the in vivo and in vitro test in individual patients, but the number of results is too small to make statistical analysis meaningful. Altogether 33 per cent of all patients tested had a positive DHR to autologous tumour extract, and this compares with positive responses in 25 per cent of patients with solid tumours tested in series conducted by Hughes and Lytton (1964) and Stewart (1969) and 50 per cent of patients who gave positive responses in a series conducted by Oren and Herberman (1971). Although Chatterjee ef al (1975) found that none of their patients with ovarian cancer produced a DHR to autologous tumour extract, they did not define which of their patients had residual disease, or whether they were receiving chemotherapy or radiotherapy at the time of testing. Diminished tumour-associated responsiveness could be due to serum factors (Sjogren et al, 1972; Currie and Basham, 1972; Jose and Seshadri, 1974) or to impaired cell-mediated immunity; it is the latter aspect we examined. Graham and Graham (1962) found impaired DHR to PPD in patients with gynaecological cancers. Khoo and Mackay (1974) confirmed this observation by skin testing patients with a variety of gynaecological cancers with five standard recall antigens, including PPD. We could demonstrate no in vitro evidence for impaired blastogenic responses to PPD in remission or relapse patients. Oppenheim (1968) found that blastogenic responses to PPD in guinea pigs inoculated with BCG was a more sensitive test of the cell-mediated response than DHR, often occurring before in vivo evidence of sensitization. However, there are certain aspects of cell-mediated immunity which blastogenesis does not measure, such as effective secretion of lymphokines, and the patient’s ability to produce an effective inflammatory response, both of which would be necessary for a DHR to develop. Reduced PHA responses in relapse patients is consistent with the findings of Wolff and De Oliveira (1979, who also found impaired lymphocyte responses to PHA in ovarian cancer relapse patients, although responses in remission patients were normal. PHA is a powerful mitogen and lymphocytes require no in vivo pre-sensitization to respond to it-the mechanism of action therefore differs
IMMUNITY IN OVARIAN CANCER
fundamentally from the blastogenesis induced by secondary recall antigens. Changes in the lymphocyte cytoplasmic matrix in cancer patients with progressive disease may result in decreased PHA responses (Cerzek et al, 1974). If this is the case, it does not necessarily follow that these changes impede lymphocyte responsiveness to secondary recall antigens ; since PHA and PPD induce blastogenesis predominantly in T-lymphocytes (Kreeftenberg et al, 1975), our data would support such a hypothesis. A tumour-associated response would require in vivo pre-sensitization to be demonstrable by blastogenesis and this would parallel the response to PPD more closely than to PHA. Since the response to PPD is not impaired, diminished tumour-associated immunity in relapse cannot be attributed to impaired cell-mediated immunocompetence to recall antigens. ACKNOWLEDGEMENTS This work was supported by the Cancer Research Campaign, and the research programme into ovarian cancer is under the auspices of the Association of Obstetricians and Gynaecologists and Regional Histopathologists Group of the N.E. Thames Region, supported by the Regional Health Authority. We are grateful to all colleagues who have kindly supplied fresh tumour material and allowed us access to patients. We thank Mr Peter Crocker for the microphotograph. REFERENCES Cerzek, L., Cerzek, B., and Franklin, C. I. V. (1974): British Journal of Cancer, 29, 345.
399
Chatterjee, M., Barlow, J. J., Allen, H. J., Chung, W. S., and Piver, M. S. (1975): Cancer, 36, 956. Chen, S., Koffler, D., and Cohen, C. J. (1973): American Journal of Obstetrics and Gynecology, 115, 467. Currie, G. A., and Basham, C. (1972): British Journal of Cancer, 26, 427. Di Saia, P. J., Rutledge, F. N., Smith, J. P., and Sinkovics, J. G. (1971): Cancer, 28, 1129. Graham, J. B., and Graham, R. M. (1962): Surgery, Gynecology and Obstetrics, 114, 1. Hellstrom, I., Hellstrom, K. E., Sjogren, H. O., and Warner, G. A. (1971): International Journal of Cancer, 7 , 1 . Hughes, L. E., and Lytton, B. (1964): British Medical Journal, 1, 209. Jose, D. G., and Seshadri, R. (1974): International Journal of Cancer, 13, 824. Khoo, S. K., and Mackay, E. V. (1974): Journal of Obsteirics and Gynaecology of the British Commonweafrh, 81, 229. Kreeftenberg, J. G., Leerling, M. F., and Loggen, H. G . (1975) : Clinical and Experimental Immunology, 22, 121. Levin, L., McHardy, J. E., Curling, 0. M., and Hudson, C. N. (1975a): British Journal of Cancer, 32, 152. Levin, L., McHardy, J. E., Curling, 0. M., and Hudson, C. N. (19756): Diagnosis and Treatment of Ovarian Neoplastic Alterations. Edited by de Watteville. Excerpta Medica/American Elsevier, p. 21 1. Lowry, 0. H., Roseborough, N. J., and Farr, A. L. (1952) : Journal of Biological Chemistry, 193, 265. Oppenheim, J. J. (1968) : Federation Proceedings, 27, 21. Oren, M. E., and Herberman, R. B. (1971): Clinical and Experimental Immunology, 9, 45. Sjogren, H. O., Hellstrom, I., Bansal, S. C., Warner, G. A., and Hellstrom, K. E. (1972): International Journal of Cancer, 9, 274. Stewart, T. H. M. (1969): Cancer, 23, 1368. Wolff, J. P., and De Oliveira, C. F. (1975): Obstetrics and Gynecology, 45, 656.
TUMOUR ASSOCIATED IMMUNITY AND IMMUNOCOMPETENCE IN OVARIAN CANCER BY
L. LEVIN J. E. MCHARDY T. A. POULTON 0. M. CURLING AND
C . N. HUDSON Williurnson Laboratory, Medical College of St Bartholomew’s Hospitul, London, ECIA 7BE Summary Cell-free extracts of autologous ovarian cancer cells evoked a delayed hypersensitivity response (DHR) in skin in four out of six patients in remission from disease and none of seven patients in relapse. These initial results appear to be consistent with in vitro blastogenic assay using the same autologous extracts. To assess whether diminished tumour-associated immunity was due to impaired cellmediated imniunocompetence, blastogenic responses to purified protein derivative of tuberculin (PPD), a secondary recall antigen, and phytohaemagglutinin (PHA) were assessed. Impaired responses to PHA were seen in the relapse group of patients, but responses to PPD did not appear to be impaired in this group.
blastogenic responses which were greater than seen in any of the 10 normal controls. Using a lymphocyte blastogenic assay, evidence for cell-mediated sensitization to extracts of ovarian cancer cells was obtained in 45 ovarian cancer patients (Levin et ul, 19751). The nature of this response appeared to be complex and was seen more frequently in remission than in relapse patients. We have now studied the in vitro cellular immunity in ovarian cancer patients by measuring blastogenic responses to phytohaemagglutinin (PHA) and purified protein derivative of tuberculin (PPD), to assess whether impaired immunocompetence could have been responsible for diminished tumourassociated immunity in relapse patients. During a pilot study of immunotherapy for
A TUMOUR specific cell-mediated immune response has been demonstrated in ovarian cancer patients by in vitro lymphocyte-mediated cytotoxicity of ovarian cancer cells (Hellstrom et cil, 1971; Di Saia et ul, 1971). Furthermore, autologous extracts of ovarian tumour cells have been shown to produce leucocyte migration inhibition (Chen et al, 1973). Chatterjee et ul (1975) found no evidence for a cell-mediated tumour-associated immune response by assessing the ability of lymphocytes from ovarian cancer patients to transform (blastogenesis) when challenged with autologous tumour extracts; they also found no evidence of a delayed skin hypersensitivity response to autologous tumour extracts in their patients. However, 4 out of 12 of their patients had 393
394
LEVIN, MCHARDY, POULTON, CURLING AND HLDSON
ovarian cancer (Levin et al, 1975b), a good correlation was noted in one patient between in vivo delayed hypersensitivity response (DHR) in skin to autologous tumour cell extract and the in vitro response to the same extract. This prompted the present study of the in vivo cell-mediated immune response to autologous tumour extract. METHODS Cell extract Fragments of fresh tumour material from surgical specimens were homogenized in normal saline, frozen rapidly in liquid nitrogen, thawed and the suspension was finally made hypotonic. The tumour homogenate was centrifuged at 400 g for five minutes and the resulting supernatant centrifuged at 3000 g for one hour at 4 "C. The supernatant from the 3000 g centrifugation was estimated for protein content by the FolinLowry technique (Lowry et al, 1952). The extracts were made up to a concentration of 2 mg/ ml in normal saline and stored at -20 "C. All cell extracts were tested for bacterial growth and discarded if bacteria were present.
calf serum in tissue culture medium M199 and, appropriate cultures challenged with 50, 100 or 200 pg of autologous tumour extract. Similar cultures in 12.5 per cent autologous serum or fetal calf serum were challenged with 10 pg PPD or 10 pg PHA. Similar cultures of unstimulated lymphocytes from the same source were set up in parallel. Cultures were kept at 37 "C in 5 per cent COBfor five days and transformation was assessed by incorporating 12"I-5-iodo-deoxyuridine (IUDR) into DNA during the last 6 hours of culture, as described elsewhere (Levin et al, 1975a); the extent of transformation was measured, using a Wilji Gamma Counter, by the difference between the counts per minute (cpm) from stimulated and unstimulated lymphocytes. Where possible, responses were measured in duplicate.
Patients Twelve patients with common epithelial (Mullerian) malignant ovarian tumours were
Skin test Three doses of extract were used in each test, these being 0.1 mg, 0.25 mg, and 0 . 5 mg. Injections were given intradermally on the flexor aspect of the forearm. A 0.5 ml volume of normal saline was injected intradermally on the opposite arm as a control. Any response which took 24 hours to appear but disappeared after 72 hours, was indurated and produced a reddened area greater than 3 mm in diameter, was taken to be positive. The skin test was assessed at 6 hours, 24 hours, 48 hours and 72 hours and measured when it was positive. Skin biopsy A 3 mm punch skin biopsy was performed at the site of a positive DHR in three of the patients and the sections examined for histological evidence of DHR. Lymphocyte transformation blastogenic assay Lymphocytes were isolated by centrifuging whole heparinized blood (10 iu preservative-free heparin per ml of blood) at 400 g over a ficoltriosil density gradient. Cultures were set up as 1 x lo6 lymphocytes/ml of 12.5 per cent fetal
FIG.1 Delayed skin hypersensitivity response to 0.25 mg autologous tumour extract.
IMMUNITY IN OVARIAN CANCER
tested with autologous tumour-cell extracts. None of the patients had had chemotherapy or radiotherapy for at least 30 days prior to the test being performed. Six patients were in remission from disease and six were in relapse. All controls were normal females, matched for age with the ovarian cancer patients ; they were mostly patients with prolapse awaiting operation and none were on medication at the time of testing. PHA responses were determined in 16 ovarian cancer patients in remission from disease, 9 in relapse and in 14 normal controls. PPD responses were determined in 10 ovarian cancer patients in remission from disease, 13 in relapse and in 16 normal controls.
RESULTS Delayed hypersensitivity responses ( D H R ) in skin to autologous cell extracts Four of six patients in remission from disease
395
had a positive DHR in skin to autologous tumour extract. The minimum effective dose in three of these patients was 0.25 mg, while in the fourth patient, it was 0 . 5 mg. The diameter of the positive responses was between 4 and 7 mm and in three patients a skin biopsy was taken and examined histologically. Figure 1 shows a typical DHR to tumour extract and Figure 2 shows the typical histological features of this response. One of the remission patients who gave a negative DHR to the extract developed tumour spread to the inguinal lymph nodes four weeks after skin testing. The other patient with a negative DHR was still in remission from disease 18 months after skin testing, when she once again failed to produce a D H R to autologous cell extract. None of the seven patients in relapse developed a DHR to autologous tumour extract.
FIG.2 Punch skin biopsy front a delayed skin hypersensitivity response to autologous tumour extract ( ~ 2 5 0 )The . area was infiltrated almost exclusively with mononuclear cells.
396
LEVIN, MCHARDY, POULTON, CURLING AND HUDSON
In vitro correlation with DHR The blastogenic responses to autologous cell extract for all patients who were skin tested with the same extract are shown in Table I. A positive blastogenic response in this system was taken as one in which the stimulation index was greater than 1.2 and the cpm difference greater than 500 (Levin et al, 1975aJ. All four patients who had a positive DHR to autologous tumour extract, also had a blastogenic response to the same extract. One of the six relapse patients tested had a blastogenic response to autologous tumour extract.
5. I.
AUTOLOGOUS SERUM
FETAL CALF SERUM
. .
0
I
Blastogenic responses to PPD and PHA There was no difference in responses to PPD in the three groups of patients compared (Fig. 3). Blastogenic responses to PPD are shown together with those to tumour extract in Table I, but the data are too limited to make a direct comparison between these two responses. Responses to PHA in ovarian cancer patients and normal controls are shown in Table I1 and Figure 4. PHA responses in ovarian cancer
Controls
Remission
Relapse Controls Remission Relapse
OVARIAN CANCER Xl5.l.) 2.5
2.3
OVARIAN CANCER
2.0
2.9
1.9
2.2
FIG.3 PPD responses in ovarian cancer patients and normal controls. There was no difference in blastogenic responses to PPD in the ovarian cancer patients in remission or relapse, when compared with normal controls.
TABLEI In vivo and in vitro responses to autologous ovarian turnour cell extructs Blastogenic responses
Patient
Remission 1 2 3 4 5*
6t
Relapse 7 8 9 10 11 12 13
Diameter of skin reaction (mm)
Unstimulated
6 7
5 6 0 0
0 0 0 0 0 0 0
Stimulated
Stimulation index
(CPm)
Ovarian cancer cell extract
PPD (cPn4
Ovarian cancer cell extract
2300 1600 1560 2220 2190 1260
3000 4600 3960 3900 2030 1440
2310 2300 4140 2100 3900
1.3 3.0 2.5 1.8 1.0 1.1
1.0 1.4 2.7 1.0
1890 1860 2010 4080
2080 2410 2040 3930 3290 2460
2840 3960 3630 10 300 1260
1.1 1.3 1.0 1.0 1.0 1.1
1.5 2.0
-
3300 2340
* Retested 18 months later (still in remission), and failed to produce DHR.
t Went into relapse with disease spread to inguinal lymph nodes four weeks after testing.
PPD
3.1
-
0.9 -
3.3 0.5
IMMUNITY IN OVARIAN CANCER
397
TABLEI1 Blastonenic responses to PHA in ovarian cancer patients rand normal controls Autologous serum Unstimulated Stimulated (CPm) (CPd Ovarian cancer remission: 2880 61440 3330 62040 1940 26970 1680 18600 1950 13830 3510 21205 4210 25630 1740 10000 7200 29700 2480 9000 2660 9363 4800 9600 2400 3940 4480 4480
Fetal calf serum Stimulation Index 21 18.6 14 9.9 7.0 6.0 6.0 5.7 4.2 3.6 3.5 2.0 1-7 1.0 -
Normal controls: 4400 1850 2100 4230 1800 2000 3510 4800 3420 2700 5400 6Ooo
6270 2670 4040 4980 -
49500 26100 12920 5700 2500 11370 2230 1580 -
10400 46620 41700 81360 3 1920 31000 52240 6oooO 3 m 21000 35100 35520 27690 7800 7200 8460 -
-
-
57770 3000 8580
25 1.7 3.9 1.6 1.1 6.0 10.0 3.3 1.8 4.4 31.0 10.0
7.2 6.0 4.3 2.6 1-5 1.5 1.1 0-9 -
9240 3040 1950 1460 3340 1930 1220 1730 5430 1940 4080
-
-
Stimulation Index
-
-
25 25 20 19 18 16.0 15.5 12.5 9.0 7.9 6.5 6.5 4.4 3.0 1.8 1.7 -
Stimulated (cpm)
2280
1740 2150 1920 9240 2850 2380 3730 3600 3020 2960 4140 5430 1590 2120
-
Ovarian cancer relapse : 6840 4700 3000 2190 1710 8080 1850 1780 -
Unstimulated (cpm)
-
8400 1070 1830 2160 4440 5760 3480 4560 -
3060 3260 2280 8220
-
3060 10200 17880 23050 12120 6024 13320 92500 41000 28100 3770 3330
5.2
2.4 1.6
67740 7910 1950 1560 3120 2770 1580 24830 28100 9100 8460
7.0 2.6 1.0 1.1 0.9 1.4 1.3 14.3 5.2 4.7 2.0
38000 17820 1lo00 14880
5.0 17.0 6.0 6.9 3.5 2.4 2.9 -
-
15420 13140 6600 -
-
25320 3420 26640 21660 25020
5.5 1.1 11.2 9.5 3.0
398
LEVIN, MCHARDY, POULTON, CURLING AND HUDSON
s. 1
AUTOLOGOUS SERUM
FETAL CALF SERUM
40
30
.
20
a 0
10
t
0
0
0
0
:
0 0
5 0
. . .
0
0
2
0
5
. Controls Remission Relapse
Controls Remission Relapse
OVARIAN CANCER
x (S.1.)
12
8
3
0
0
OVARIAN CANCER
6
7
4
FIG.4 PHA responses in ovarian cancer patients and normal controls. While there was no differencein PHA responses in remission patients when compared ai t h normal controls, responses in relapse were lower than in the controls in autologous screen (P=O.O3).
relapse patients were lower than PHA responses in normal controls, and this difference was statistically significant (P =O .03) in autologous esrum. Responses to PHA in relapse patients were not different from the normal controls when these were measured in fetal calf serum: there were decreased PHA responses in fetal calf serum in the control group when compared with their responses in autologous serum. DISCUSSION In vivo evidence for a tumour-associated immune response in the 13 ovarian cancer patients tested follows the same pattern as the in vitro response reported by the same authors (Levin et al, 1975a)in that DHR occurred commonly in the remission group of patients. There is prima facie evidence in this series for a correlation
between the in vivo and in vitro test in individual patients, but the number of results is too small to make statistical analysis meaningful. Altogether 33 per cent of all patients tested had a positive DHR to autologous tumour extract, and this compares with positive responses in 25 per cent of patients with solid tumours tested in series conducted by Hughes and Lytton (1964) and Stewart (1969) and 50 per cent of patients who gave positive responses in a series conducted by Oren and Herberman (1971). Although Chatterjee ef al (1975) found that none of their patients with ovarian cancer produced a DHR to autologous tumour extract, they did not define which of their patients had residual disease, or whether they were receiving chemotherapy or radiotherapy at the time of testing. Diminished tumour-associated responsiveness could be due to serum factors (Sjogren et al, 1972; Currie and Basham, 1972; Jose and Seshadri, 1974) or to impaired cell-mediated immunity; it is the latter aspect we examined. Graham and Graham (1962) found impaired DHR to PPD in patients with gynaecological cancers. Khoo and Mackay (1974) confirmed this observation by skin testing patients with a variety of gynaecological cancers with five standard recall antigens, including PPD. We could demonstrate no in vitro evidence for impaired blastogenic responses to PPD in remission or relapse patients. Oppenheim (1968) found that blastogenic responses to PPD in guinea pigs inoculated with BCG was a more sensitive test of the cell-mediated response than DHR, often occurring before in vivo evidence of sensitization. However, there are certain aspects of cell-mediated immunity which blastogenesis does not measure, such as effective secretion of lymphokines, and the patient’s ability to produce an effective inflammatory response, both of which would be necessary for a DHR to develop. Reduced PHA responses in relapse patients is consistent with the findings of Wolff and De Oliveira (1979, who also found impaired lymphocyte responses to PHA in ovarian cancer relapse patients, although responses in remission patients were normal. PHA is a powerful mitogen and lymphocytes require no in vivo pre-sensitization to respond to it-the mechanism of action therefore differs
IMMUNITY IN OVARIAN CANCER
fundamentally from the blastogenesis induced by secondary recall antigens. Changes in the lymphocyte cytoplasmic matrix in cancer patients with progressive disease may result in decreased PHA responses (Cerzek et al, 1974). If this is the case, it does not necessarily follow that these changes impede lymphocyte responsiveness to secondary recall antigens ; since PHA and PPD induce blastogenesis predominantly in T-lymphocytes (Kreeftenberg et al, 1975), our data would support such a hypothesis. A tumour-associated response would require in vivo pre-sensitization to be demonstrable by blastogenesis and this would parallel the response to PPD more closely than to PHA. Since the response to PPD is not impaired, diminished tumour-associated immunity in relapse cannot be attributed to impaired cell-mediated immunocompetence to recall antigens. ACKNOWLEDGEMENTS This work was supported by the Cancer Research Campaign, and the research programme into ovarian cancer is under the auspices of the Association of Obstetricians and Gynaecologists and Regional Histopathologists Group of the N.E. Thames Region, supported by the Regional Health Authority. We are grateful to all colleagues who have kindly supplied fresh tumour material and allowed us access to patients. We thank Mr Peter Crocker for the microphotograph. REFERENCES Cerzek, L., Cerzek, B., and Franklin, C. I. V. (1974): British Journal of Cancer, 29, 345.
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Chatterjee, M., Barlow, J. J., Allen, H. J., Chung, W. S., and Piver, M. S. (1975): Cancer, 36, 956. Chen, S., Koffler, D., and Cohen, C. J. (1973): American Journal of Obstetrics and Gynecology, 115, 467. Currie, G. A., and Basham, C. (1972): British Journal of Cancer, 26, 427. Di Saia, P. J., Rutledge, F. N., Smith, J. P., and Sinkovics, J. G. (1971): Cancer, 28, 1129. Graham, J. B., and Graham, R. M. (1962): Surgery, Gynecology and Obstetrics, 114, 1. Hellstrom, I., Hellstrom, K. E., Sjogren, H. O., and Warner, G. A. (1971): International Journal of Cancer, 7 , 1 . Hughes, L. E., and Lytton, B. (1964): British Medical Journal, 1, 209. Jose, D. G., and Seshadri, R. (1974): International Journal of Cancer, 13, 824. Khoo, S. K., and Mackay, E. V. (1974): Journal of Obsteirics and Gynaecology of the British Commonweafrh, 81, 229. Kreeftenberg, J. G., Leerling, M. F., and Loggen, H. G . (1975) : Clinical and Experimental Immunology, 22, 121. Levin, L., McHardy, J. E., Curling, 0. M., and Hudson, C. N. (1975a): British Journal of Cancer, 32, 152. Levin, L., McHardy, J. E., Curling, 0. M., and Hudson, C. N. (19756): Diagnosis and Treatment of Ovarian Neoplastic Alterations. Edited by de Watteville. Excerpta Medica/American Elsevier, p. 21 1. Lowry, 0. H., Roseborough, N. J., and Farr, A. L. (1952) : Journal of Biological Chemistry, 193, 265. Oppenheim, J. J. (1968) : Federation Proceedings, 27, 21. Oren, M. E., and Herberman, R. B. (1971): Clinical and Experimental Immunology, 9, 45. Sjogren, H. O., Hellstrom, I., Bansal, S. C., Warner, G. A., and Hellstrom, K. E. (1972): International Journal of Cancer, 9, 274. Stewart, T. H. M. (1969): Cancer, 23, 1368. Wolff, J. P., and De Oliveira, C. F. (1975): Obstetrics and Gynecology, 45, 656.
