- Email: [email protected]
Immunologic evaluation and prognosis in patients with head and neck cancer
Immunologic Evaluation and Prognosis in Patients with Head and Neck Cancer E. Y. Hilal, MD,’ New York, New York H. J. Wanebo, MD,+ New York, New York C. M. Pinsky, MD, New York, New York P. Middleman, MD, New York, New York E. W. Strong, MD, New York, New York H. F. Oettgen, MD, New York, New York
The existence of an immunologic defect in patients with cancer has now been recognized by several investigators [1-B]. This defect has been repeatedly demonstrated in different studies by in vivo testing of delayed hypersensitivity reactions to dinitrochlorobenzene (DNCB) and recall microbial antigens [l-5] and by in vitro testing of lymphocyte responses to mitogens and common antigens [6-8]. This immunologic impairment in cancer patients has been associated with increased recurrence rates [1,9,10] and shortened survival [9,10]. Patients with squamous carcinoma of the head and neck seem to have an unusually high degree of immunosuppression even in the early stages of disease [II--151. The suppression is usually more marked with progression of disease, and may be associated with shortened disease-free. survival after appropriate therapy [11,13,15]. Studies properly evaluating the role of the immune system in head and neck cancer must be performed in carefully staged patients, because it is essential that the patient groups be homogeneous. In the present study we analyzed several tests of immune function in patients with squamous cancer of the head and neck and correlated them with stage of disease and with short-term prognosis to determine their possible usefulness in assessing the risk of recurrence in addition to standard clinical staging. Material and Methods Patients. The study was composed of 183 patients with mucosal squamous cell carcinoma of the head and neck who had been treated at Memorial Hospital between 1970 and 1975. The male to female ratio was approximately 2:l and
the median age was fifty-nine years (range, 23 to 81 years) with a preponderance of patients in the sixth and seventh decades. (Figure 1.) The primary sites were oral cavity (83 patients, 45 per cent), pharynx (52,28.5 per cent), larynx (41,22.5 per cent), and maxillary sinuses (7,4 per cent). Patients were staged according to the TNM system of the American Joint Committee. Approximately half the patients (93) had stage III disease (locally extensive or regionally metastatic disease), and the remaining patients were equally divided between stages I (33 patients), II (32), and IV (25). There were no significant differences in age distribution among the different stages. (Table I.) Immunologic Evaluation. All in vitro tests were performed preoperatively. Peripheral circulating lymphocytes were enumerated in all 183 patients, DNCB skin tests were done in 126 patients, and lymphocyte stimulation studies were done in 112 patients. DNCB skin testing: A sensitizing dose of 2 mg DNCB dissolved in 0.1 ml acetone was applied to either upper arm. Test doses of 100 and 25 pg were applied to the ipsilateral forearm. If during the next two weeks no reaction had developed on the forearm, 25,50, and 100 Mgtest doses were repeated and read 48 hours later. An area of induration of 15 mm in diameter was considered a positive reaction.
60
Medlon age: 59 yrs Ronge. 23-81 yrs * 40E s z 6 30i E 1
20-
IOFrom the Memorial Sloan-Kettering Cancer Center, New York, New York. Presented at tha Twenty-Third Annual Meeting of the Society of Haad and Neck Surgeons, Hilton Head Island. South Carolina, May 4-7, 1977. Present address and reprint requests: Mercy Hospital, 1400 Locust Street, Pittsburgh, Pennsylvania 15219. t Present address: Department of Sugefy, University of Virginia Medical Center. Charlottesville, Virginia 22901. l
Volume 134, October 1977
3 60-69 Age in years
70-79
80-89
Figure 1. Age distribution of 183 patients w/th head and neck cancer.
469
Hilal et al
Whenever possible, skin tests were completed prior to surgical treatment, but as a rule, this was not possible. The majority of patients were sensitized with DNCB preoperatively and received the challenge postoperatively to avoid delays in definitive treatment. Lymphocyte stimulation studies: In vitro stimulation by mitogens was performed on lymphocytes isolated from heparinized blood on Ficoll-Isopaque@ density gradients. After washing and resuspension in RPM1 1640 supplemented with glutamine, penicillin, streptomycin, and 15 per cent pooled human serum, 0.2 cc of a mononuclear cell suspension containing 1 X lo6 lymphocytes was incubated in microtiter plates. All individual cultures were performed in triplicate and transformation of lymphocytes was measured by incorporation of carbon 14 thymidine. A dose-response curve was derived for each mitogen, using four different concentrations for phytohemagglutinin-P (PHA-P), three different concentrations of concanavalin-A (Con-A), and three different concentrations of pokeweed mitogen (PWM). The maximum response with each mitogen at the optimal concentration was recorded as the mean of the three triplicate values in counts per minute. A value was considered abnormal if it fell below the tenth percentile of the normal control distribution. Healthy volunteer control subjects of both sexes recruited from
TABLE I
Relation of Age and Sex to Stage of Dlsease Stage I
Stage II
Stage Ill
Stage IV
Total
Male Female <80yr >60yr Total
22 11 21 12 33
19 13 22 10 32
62 31 42 51 93
17 8 11 14 25
120 63 96 87 183
TABLE II
Immunologic Reactivity in Head and Neck Squamous Cancer
Parameter
No. of Patients Tested
Per Cent Depressed Responses
DNCB Lymphocyte count PHA Con-A PWM
126 182 112 90 112
46 42 45 28 25
TABLE Ill
Relation of Immune Reactivity to Stage of Disease Per Cent of Patients with Depressed Response Stage I Stage II Stage Ill Stage IV
DNCB Lymphocyte count PHA Con-A PWM
44 21
25 41
60 49
30 45
32 24 9
45 21 23
45 32 33
61 33 23
Significance
l
l l
Significant progressive depression compared with controls (ordered chi-square test). l
470
laboratory and hospital employees underwent in vitro testing simultaneously with all the patients. Statistical Methods. All patients were followed until recurrence or to date, and the time from surgery to recurrence and the disease-free intervals were recorded. Immune parameters in successive stages were compared using the ordered R X C chi-square test. Cochran’s method for combining 2 X 2 tables was used to determine age or sex differences for individual parameters in different stages. Recurrence curves in each stage were drawn with the dif-
ferent parameters using the product limit method. Results
General Obseruations. Significant impairment of the in vivo and in vitro immune parameters was demonstrable in a sizeable proportion of the patients studied. This was more pronounced with lymphocyte counts, DNCB reactivity, and PHA response than with Con-A and PWM responses. (Table II.) Influence of Stage of Disease. In general there was greater depression of all of the immune tests in patients with stages III and IV disease compared with patients with stages I and II disease. There was a significant progressive decrease of the peripheral lymphocyte count and the lymphocyte stimulation responses to PHA and Con-A in comparison with baseline control values. In contrast there was no significant progressive decrease with stage of the DNCB or PWM response. (Table III.) When combinations of parameters were examined in the different stages, no obvious trend was identified, indicating a lack of correlation between the results of the different tests. (Table IV.) Influence of Age and Sex. Our data showed no evidence of decreasing DNCB reactivity with age. (Table III.) The overall proportion of anergic patients below age sixty years was 42 per cent compared with 47 per cent in the group older than sixty years. (Table V.) There was also no significant effect of age on PHA responses in the different stages although the general progressive decline with stage was maintained in the two subdivisions. The overall proportion of low responders was 41 per cent in the patients younger than sixty years and 48 per cent in those older than sixty years. (Table VI.) No consistent differences were found between males and females in either parameter. Influence of Immune Suppression on Prognosis.
The relation of immune suppression to recurrence was examined in patients according to their stage of disease using three tests of immunocompetence: DNCB, lymphocyte count, and PHA. Only a negative DNCB test result in stages I and II correlated significantly with a high recurrence rate. (Figures 2 and 3.) Of eight patients with negative DNCB results in stage I, six developed recurrence by twenty months, The American Journal of Surgery
Immunology in Head and Neck Cancer
whereas none of the ten patients with positive DNCB results had a recurrence after a median follow-up of thirty-five weeks (p
As previously reported by Wanebo et al [13], breakdown by stage revealed a progressive suppression of tests (DNCB, PHA, Con-A) reflecting T cell function with advancing disease. In contrast, decline in B cell function (PWM) with increasing extent of disease was not statistically significant. This suggests that T cell function is depressed by the presence of the cancer. Certain of the tests (PHA) appeared very sensitive to the presence of tumor and showed good correlation with tumor burden. On the other hand, the PHA test results did not correlate well with host-tumor control as reflected by recurrence rates. The DNCB test results did not correlate well with increasing tumor burden but did correlate with recurrence rate in stages I and II disease. These disparate results suggest the need for multiple tests to provide a more complete assessment of immune function. Our own panel of tests is deficient for determining macrophage function per se.
TABLE IV
Correlation of the Various Parameters
o/22
Stage I
Male Female <60 yr >60 yr
1 ZCB 1 LYfl”ct
stage Ill
7122 (32%) 1120 (5%) 1111 (9%) 1111 (9%)
2118 (11%) l/7 (14%) o/7
1 LYYC$
Our data demonstrate a significant general suppression of immune reactivity in patients with head and neck cancer as measured by all the parameters used. Results of the DNCB test, lymphocyte count, and lymphocyte stimulation with PHA test were more frequently abnormal than the results with the mitogens, Con-A, or PWM, although all are significantly different from the controls. The frequency of DNCB anergy in our series is comparable with that found by others. Our patients, however, had more profoundly decreased lymphocyte reactivity in vitro to PHA than patients studied by Catalona, Sample, and Chretien [S]. It is important that only the DNCB test results correlated with short-term prognosis in patients with stages I and II disease (but not in patients with stages III and IV cancer).
stageII
Stage I
Stage IV
13155 (24%) 20165 (31%) 4126 (15%) 2126 (6%)
2112 (17%) 4120 (20%) 2/11 (16%) l/l0 (10%)
DNCB Note: Lym ct = lymphocyte count.
TABLE V
Relation of DNCB Reactlvlty in Age and Sex Stage I
Stage II
Stage Ill
stage IV
% No. % No. % No. No. % Test- Nega- Test- Nega- Test- Nega- Test- Negative ed tive ed ed tive ed tive Male Fenld0 <60yr >60yr
12 6
50 33
11 9
36 11
43 22
58 64
16 7
31 29
11 6
18 83
15 5
13 60
30 35
63 51
10 13
50 15
Stage II
Stage Ill
Stage IV
No. Tested
Depressed Response
No. Tested
Depressed Response
No. Tested
Depressed Response
No. Tested
Depressed Response
15 7 15 7
40% 14% 33% 29%
14. 8 13 9
50% 38% 38% 55%
37 18 21 34
49% 39 % 43% 47%
8 5 5 8
63 % 60% 60% 63 %
VolumeJ134,oclober1977
471
Hilal et al Stage I
StogeII
-=DNCB @ IO pts. (IO NED) ---=DNCB 0 8 pts. ( 2 NED) V,v=lnd~cates length of followup
--
= (DNCB-I 5pts (2 NED1 = KJNCBtl 15ptr (tONED) 0 = lndlcates length of F”
L__________________
PC.01 t 10 Time
I
I
30
40
to recurrence
(months)
from
surgery
Figure 3. Relation of DNCB reactMy stage II.
07
IO
20
30
50
40
60
I
I
20
Stage
70
I
50
60
to recurrence rate,
IQ
Time from surgery to recurrence (months)
Figure 2. Relation of DNCB reactivity to recurrence rate, stage I. -= (DNCB-I 7pts.llNEDl -- = (DNCB+Il6pts.(5NEDl V.0 = Indxotes ten@h of F”
Stoge IlI
i_E__Y_Y_Y
t IO Time
I
t
t
I
I
20
30
40
50
60
from
surgery
to recurrence
(months)
Figure 5. Relation of DNCB reactivity to recurrence rate, stage IV.
0-
20
IO Time
from
30 surgery
to recurrence
40
50
60
(months1
Figure 4. Relation of DNCB reactivity to recurrence rate, stage Ill.
Our data showed no correlation between DNCB reactivity and lymphocyte counts nor between DNCB and mitogen responses. There was no cumulative suppression of different parameters with increasing stage of disease. A study by Golub et al [16] showed similar results, although these authors did find a correlation between DNCB reactivity and the mixed lymphocyte response to a standardized pool of stimulating lymphocytes. Quantitative and qualitative defects of cellular immunity have been correlated with worsened prognosis in cancer patients in general [1,4,9,10], although not all of the authors confirm this [17]. There have been suggestive though not significant correlations of immune tests with prognosis in head and neck cancer patients [11--131. A major problem with most studies has been the lack of a homogenous
472
population of cancer patients in which to make prognostic correlations with immune function. It is essential to examine properly staged patients to assess any significance of immune impairment, particularly any unique relation of immune response to prognosis beyond what is expect&d from standard clinical staging [IO]. Our study reveals that of all the parameters analyzed, only DNCB reactivity correlated significantly with disease-free survival after surgery and only in stages I and II disease. The lack of correlation between DNCB reactivity and prognosis in patients with advanced head and neck cancer has been previously reported [13]. This observation may have important therapeutic implications. Patients with early disease usually have a favorable prognosis with conventional therapy. Our data suggest that a group of such patients identified by a negative DNCB reaction might benefit from either more aggressive approaches or possibly adjuvant immunotherapy. On the other hand, in patients with advanced disease, the generally unfavorable prognosis seems to be un-
The American
Journal of Surgery
Immunology
TABLE VII
DNCB Lymphocyte count PHA Con-A PWM
Correlation of Depressed Immune Response with Recurrence Rate Stage I
Stage II
+ -
+ -
Note: + = positive correlation: -
Stage Ill
No. of Patients Tested
-
Relation of Immune Suppression to Recurrence
Stage IV
-
= no correlation.
affected by the immunologic status and may not benefit from an immunopotentiator. To this end, we are currently engaged in a double-blind clinical trial comparing the effects of an immunomodulator, Levamisole with placebo, on local recurrence rates and survival in patients with resectable head and neck cancer after adequate surgery. Other methods of analysis of immunopathology in head and neck cancer have also been pursued. Berlinger and Good [17] found a good correlation between the mixed lymphocyte culture assay and the clinical stage of head and neck cancer patients. Preliminary data also indicate a possible association of an abnormal mixed lymphocyte culture response and poor prognosis in these patients [Ml. Summary
Patients with mucosal squamous cell carcinoma of the head and neck generally have suppressed T cell function, and this suppression tends to be more pronounced with progression of disease. The prognostic relationships of multiple tests of immune function were analyzed in 183 clinically staged patients. Correlation of immune parameters with prognosis was evident only with the DNCB response, which correlated with recurrence in stages I and II disease (but not in stages III and IV). There was no correlation of any of the in vitro tests of immunofunction with recurrence in any stage. There were no correlations between any of the immune parameters and age or sex nor between DNCB reactivity and any of the in vitro responses. References 1. Eilber FR, Morton DL: Impaired immunologic reactivity and recurrence following cancer surgery. Cancer 25: 362, 1970. 2. Solowey AC, Rapaport FT: Immunologic responses in cancer patients. Surg Gynecol Obsfet 121: 756, 1965. 3. Hughes LE, Mackay WD: Suppression of the tuberculin response in malignant disease. Br A&d J 2: 1346, 1965. 4. Pinsky CM, El-Domeiri A, Caron AS, Knapper WH, Oettgen HF: Delayed hypersensltivit$ reactions in patients with cancer. Recent results in cancer research. 47: 37, 1974.
Volume 134. October 1977
TABLE VIII
DNCB Negative Positive PPD Negative Positive Lymphocyte count Low Normal PHA Low Normal Con-A Low Normal PWM Low Normal
in Head and Neck Cancer
Per Cent with No Evidence of Disease
54 67
43 55
53 59
41 51
76 104
46 54
48 63
63 51
24 64
50 56
22 88
36 59
5. Catalona WJ, Chretien PB: Abnormalities of quantitative dinitrochlorobenzene sensitization in cancer patients: correlation with tumor stage and histology. Cancer 31: 353, 1973. 6. Catalona WJ, Sample WF, Chretien PB: Lymphocyte reactivity in cancer patients: correlation with tumor histology and clinical stage. Cancer 31: 65, 1973. 7. Gatti RA. Garrioch DB, Good RA: Depressed PHA responses in patients with non-lymphoid malignancies, p 339. Proceedings of the Fifth Leukocyte Culture Conference (Harris JE, ed). New York, Academic Press, 1970. 8. Sucin-Foca N, Buda J, McManus J: Impaired responsiveness of lymphocytes and serum inhibitory factors in patients with cancer of the head and neck. Am J Surg 128: 534, 1974. 9. Pinsky CM, Wanebo HJ, Mike V, Dettgen HF: Delayed cutaneous hypersensitivity reactions and prognosis in patients with cancer. Ann NY Acad Sci 276: 407,1976. 10. Wanebo l-U, Rao B, Miyazawa N, Martini N, Middleman MP, Oettgen HF, Beattie EJ: Immune reactivity in primary carcinoma of the lung and its relation to prognosis. J Thorac Cardiovasc Surg 72: 339, 1976. 11. Lundy J. Wanebo H, Pinsky C. Strong E, Oettgen H: Delayed hypersensitivity reactions in patients with squamous cell cancer of the head and neck. Am J Surg 128: 530, 1974. 12. Eilber FR, Morton DL. Ketcham AS: Immunologic abnormalities in head and neck cancer. Am J Surg 128: 534, 1974. 13. Wanebo HJ, Jun MY, Strong E, Oettgen HF: T-cell deficiency in patients with squamous cell cancer of the head and neck. Am J Surg 130: 445, 1975. 14. Chretien PB: Unique immunobiological aspects of head and neck squamous carcinoma, p 339. Workshops from the Centennial Conference on Laryngeal Cancer (Alberti PW, Bryce DP, ed). New York, Appleton-Century-Crofts, 1976. 15. Maisel RH, Dgura JH: Dinitrochlorobenzene skin sensitization and peripheral lymphocyte count: predictors of survival in head and neck cancer. Ann Otol85: 517, 1976. 16. Golub SH. O’Connell TX, Morton DL: Cwelation of in vivo and in vitro assays of immunocompetence in cancer patients. Cancer Res 34: 1833, 1974. 17. Berlinger NT, Good RA: Concomitant immunopathology with squamous cell carcinomas of the head and neck regions. Trans Am Acad Opthalmol Otolatyngol62: 588, 1976. 18. Berlinger NT: Immunobiology. Dtolaryngolcgy (Papparella M. Shumrick D, ed). Philadelphia, WB Saunders. (In press.)
473
The existence of an immunologic defect in patients with cancer has now been recognized by several investigators [1-B]. This defect has been repeatedly demonstrated in different studies by in vivo testing of delayed hypersensitivity reactions to dinitrochlorobenzene (DNCB) and recall microbial antigens [l-5] and by in vitro testing of lymphocyte responses to mitogens and common antigens [6-8]. This immunologic impairment in cancer patients has been associated with increased recurrence rates [1,9,10] and shortened survival [9,10]. Patients with squamous carcinoma of the head and neck seem to have an unusually high degree of immunosuppression even in the early stages of disease [II--151. The suppression is usually more marked with progression of disease, and may be associated with shortened disease-free. survival after appropriate therapy [11,13,15]. Studies properly evaluating the role of the immune system in head and neck cancer must be performed in carefully staged patients, because it is essential that the patient groups be homogeneous. In the present study we analyzed several tests of immune function in patients with squamous cancer of the head and neck and correlated them with stage of disease and with short-term prognosis to determine their possible usefulness in assessing the risk of recurrence in addition to standard clinical staging. Material and Methods Patients. The study was composed of 183 patients with mucosal squamous cell carcinoma of the head and neck who had been treated at Memorial Hospital between 1970 and 1975. The male to female ratio was approximately 2:l and
the median age was fifty-nine years (range, 23 to 81 years) with a preponderance of patients in the sixth and seventh decades. (Figure 1.) The primary sites were oral cavity (83 patients, 45 per cent), pharynx (52,28.5 per cent), larynx (41,22.5 per cent), and maxillary sinuses (7,4 per cent). Patients were staged according to the TNM system of the American Joint Committee. Approximately half the patients (93) had stage III disease (locally extensive or regionally metastatic disease), and the remaining patients were equally divided between stages I (33 patients), II (32), and IV (25). There were no significant differences in age distribution among the different stages. (Table I.) Immunologic Evaluation. All in vitro tests were performed preoperatively. Peripheral circulating lymphocytes were enumerated in all 183 patients, DNCB skin tests were done in 126 patients, and lymphocyte stimulation studies were done in 112 patients. DNCB skin testing: A sensitizing dose of 2 mg DNCB dissolved in 0.1 ml acetone was applied to either upper arm. Test doses of 100 and 25 pg were applied to the ipsilateral forearm. If during the next two weeks no reaction had developed on the forearm, 25,50, and 100 Mgtest doses were repeated and read 48 hours later. An area of induration of 15 mm in diameter was considered a positive reaction.
60
Medlon age: 59 yrs Ronge. 23-81 yrs * 40E s z 6 30i E 1
20-
IOFrom the Memorial Sloan-Kettering Cancer Center, New York, New York. Presented at tha Twenty-Third Annual Meeting of the Society of Haad and Neck Surgeons, Hilton Head Island. South Carolina, May 4-7, 1977. Present address and reprint requests: Mercy Hospital, 1400 Locust Street, Pittsburgh, Pennsylvania 15219. t Present address: Department of Sugefy, University of Virginia Medical Center. Charlottesville, Virginia 22901. l
Volume 134, October 1977
3 60-69 Age in years
70-79
80-89
Figure 1. Age distribution of 183 patients w/th head and neck cancer.
469
Hilal et al
Whenever possible, skin tests were completed prior to surgical treatment, but as a rule, this was not possible. The majority of patients were sensitized with DNCB preoperatively and received the challenge postoperatively to avoid delays in definitive treatment. Lymphocyte stimulation studies: In vitro stimulation by mitogens was performed on lymphocytes isolated from heparinized blood on Ficoll-Isopaque@ density gradients. After washing and resuspension in RPM1 1640 supplemented with glutamine, penicillin, streptomycin, and 15 per cent pooled human serum, 0.2 cc of a mononuclear cell suspension containing 1 X lo6 lymphocytes was incubated in microtiter plates. All individual cultures were performed in triplicate and transformation of lymphocytes was measured by incorporation of carbon 14 thymidine. A dose-response curve was derived for each mitogen, using four different concentrations for phytohemagglutinin-P (PHA-P), three different concentrations of concanavalin-A (Con-A), and three different concentrations of pokeweed mitogen (PWM). The maximum response with each mitogen at the optimal concentration was recorded as the mean of the three triplicate values in counts per minute. A value was considered abnormal if it fell below the tenth percentile of the normal control distribution. Healthy volunteer control subjects of both sexes recruited from
TABLE I
Relation of Age and Sex to Stage of Dlsease Stage I
Stage II
Stage Ill
Stage IV
Total
Male Female <80yr >60yr Total
22 11 21 12 33
19 13 22 10 32
62 31 42 51 93
17 8 11 14 25
120 63 96 87 183
TABLE II
Immunologic Reactivity in Head and Neck Squamous Cancer
Parameter
No. of Patients Tested
Per Cent Depressed Responses
DNCB Lymphocyte count PHA Con-A PWM
126 182 112 90 112
46 42 45 28 25
TABLE Ill
Relation of Immune Reactivity to Stage of Disease Per Cent of Patients with Depressed Response Stage I Stage II Stage Ill Stage IV
DNCB Lymphocyte count PHA Con-A PWM
44 21
25 41
60 49
30 45
32 24 9
45 21 23
45 32 33
61 33 23
Significance
l
l l
Significant progressive depression compared with controls (ordered chi-square test). l
470
laboratory and hospital employees underwent in vitro testing simultaneously with all the patients. Statistical Methods. All patients were followed until recurrence or to date, and the time from surgery to recurrence and the disease-free intervals were recorded. Immune parameters in successive stages were compared using the ordered R X C chi-square test. Cochran’s method for combining 2 X 2 tables was used to determine age or sex differences for individual parameters in different stages. Recurrence curves in each stage were drawn with the dif-
ferent parameters using the product limit method. Results
General Obseruations. Significant impairment of the in vivo and in vitro immune parameters was demonstrable in a sizeable proportion of the patients studied. This was more pronounced with lymphocyte counts, DNCB reactivity, and PHA response than with Con-A and PWM responses. (Table II.) Influence of Stage of Disease. In general there was greater depression of all of the immune tests in patients with stages III and IV disease compared with patients with stages I and II disease. There was a significant progressive decrease of the peripheral lymphocyte count and the lymphocyte stimulation responses to PHA and Con-A in comparison with baseline control values. In contrast there was no significant progressive decrease with stage of the DNCB or PWM response. (Table III.) When combinations of parameters were examined in the different stages, no obvious trend was identified, indicating a lack of correlation between the results of the different tests. (Table IV.) Influence of Age and Sex. Our data showed no evidence of decreasing DNCB reactivity with age. (Table III.) The overall proportion of anergic patients below age sixty years was 42 per cent compared with 47 per cent in the group older than sixty years. (Table V.) There was also no significant effect of age on PHA responses in the different stages although the general progressive decline with stage was maintained in the two subdivisions. The overall proportion of low responders was 41 per cent in the patients younger than sixty years and 48 per cent in those older than sixty years. (Table VI.) No consistent differences were found between males and females in either parameter. Influence of Immune Suppression on Prognosis.
The relation of immune suppression to recurrence was examined in patients according to their stage of disease using three tests of immunocompetence: DNCB, lymphocyte count, and PHA. Only a negative DNCB test result in stages I and II correlated significantly with a high recurrence rate. (Figures 2 and 3.) Of eight patients with negative DNCB results in stage I, six developed recurrence by twenty months, The American Journal of Surgery
Immunology in Head and Neck Cancer
whereas none of the ten patients with positive DNCB results had a recurrence after a median follow-up of thirty-five weeks (p
As previously reported by Wanebo et al [13], breakdown by stage revealed a progressive suppression of tests (DNCB, PHA, Con-A) reflecting T cell function with advancing disease. In contrast, decline in B cell function (PWM) with increasing extent of disease was not statistically significant. This suggests that T cell function is depressed by the presence of the cancer. Certain of the tests (PHA) appeared very sensitive to the presence of tumor and showed good correlation with tumor burden. On the other hand, the PHA test results did not correlate well with host-tumor control as reflected by recurrence rates. The DNCB test results did not correlate well with increasing tumor burden but did correlate with recurrence rate in stages I and II disease. These disparate results suggest the need for multiple tests to provide a more complete assessment of immune function. Our own panel of tests is deficient for determining macrophage function per se.
TABLE IV
Correlation of the Various Parameters
o/22
Stage I
Male Female <60 yr >60 yr
1 ZCB 1 LYfl”ct
stage Ill
7122 (32%) 1120 (5%) 1111 (9%) 1111 (9%)
2118 (11%) l/7 (14%) o/7
1 LYYC$
Our data demonstrate a significant general suppression of immune reactivity in patients with head and neck cancer as measured by all the parameters used. Results of the DNCB test, lymphocyte count, and lymphocyte stimulation with PHA test were more frequently abnormal than the results with the mitogens, Con-A, or PWM, although all are significantly different from the controls. The frequency of DNCB anergy in our series is comparable with that found by others. Our patients, however, had more profoundly decreased lymphocyte reactivity in vitro to PHA than patients studied by Catalona, Sample, and Chretien [S]. It is important that only the DNCB test results correlated with short-term prognosis in patients with stages I and II disease (but not in patients with stages III and IV cancer).
stageII
Stage I
Stage IV
13155 (24%) 20165 (31%) 4126 (15%) 2126 (6%)
2112 (17%) 4120 (20%) 2/11 (16%) l/l0 (10%)
DNCB Note: Lym ct = lymphocyte count.
TABLE V
Relation of DNCB Reactlvlty in Age and Sex Stage I
Stage II
Stage Ill
stage IV
% No. % No. % No. No. % Test- Nega- Test- Nega- Test- Nega- Test- Negative ed tive ed ed tive ed tive Male Fenld0 <60yr >60yr
12 6
50 33
11 9
36 11
43 22
58 64
16 7
31 29
11 6
18 83
15 5
13 60
30 35
63 51
10 13
50 15
Stage II
Stage Ill
Stage IV
No. Tested
Depressed Response
No. Tested
Depressed Response
No. Tested
Depressed Response
No. Tested
Depressed Response
15 7 15 7
40% 14% 33% 29%
14. 8 13 9
50% 38% 38% 55%
37 18 21 34
49% 39 % 43% 47%
8 5 5 8
63 % 60% 60% 63 %
VolumeJ134,oclober1977
471
Hilal et al Stage I
StogeII
-=DNCB @ IO pts. (IO NED) ---=DNCB 0 8 pts. ( 2 NED) V,v=lnd~cates length of followup
--
= (DNCB-I 5pts (2 NED1 = KJNCBtl 15ptr (tONED) 0 = lndlcates length of F”
L__________________
PC.01 t 10 Time
I
I
30
40
to recurrence
(months)
from
surgery
Figure 3. Relation of DNCB reactMy stage II.
07
IO
20
30
50
40
60
I
I
20
Stage
70
I
50
60
to recurrence rate,
IQ
Time from surgery to recurrence (months)
Figure 2. Relation of DNCB reactivity to recurrence rate, stage I. -= (DNCB-I 7pts.llNEDl -- = (DNCB+Il6pts.(5NEDl V.0 = Indxotes ten@h of F”
Stoge IlI
i_E__Y_Y_Y
t IO Time
I
t
t
I
I
20
30
40
50
60
from
surgery
to recurrence
(months)
Figure 5. Relation of DNCB reactivity to recurrence rate, stage IV.
0-
20
IO Time
from
30 surgery
to recurrence
40
50
60
(months1
Figure 4. Relation of DNCB reactivity to recurrence rate, stage Ill.
Our data showed no correlation between DNCB reactivity and lymphocyte counts nor between DNCB and mitogen responses. There was no cumulative suppression of different parameters with increasing stage of disease. A study by Golub et al [16] showed similar results, although these authors did find a correlation between DNCB reactivity and the mixed lymphocyte response to a standardized pool of stimulating lymphocytes. Quantitative and qualitative defects of cellular immunity have been correlated with worsened prognosis in cancer patients in general [1,4,9,10], although not all of the authors confirm this [17]. There have been suggestive though not significant correlations of immune tests with prognosis in head and neck cancer patients [11--131. A major problem with most studies has been the lack of a homogenous
472
population of cancer patients in which to make prognostic correlations with immune function. It is essential to examine properly staged patients to assess any significance of immune impairment, particularly any unique relation of immune response to prognosis beyond what is expect&d from standard clinical staging [IO]. Our study reveals that of all the parameters analyzed, only DNCB reactivity correlated significantly with disease-free survival after surgery and only in stages I and II disease. The lack of correlation between DNCB reactivity and prognosis in patients with advanced head and neck cancer has been previously reported [13]. This observation may have important therapeutic implications. Patients with early disease usually have a favorable prognosis with conventional therapy. Our data suggest that a group of such patients identified by a negative DNCB reaction might benefit from either more aggressive approaches or possibly adjuvant immunotherapy. On the other hand, in patients with advanced disease, the generally unfavorable prognosis seems to be un-
The American
Journal of Surgery
Immunology
TABLE VII
DNCB Lymphocyte count PHA Con-A PWM
Correlation of Depressed Immune Response with Recurrence Rate Stage I
Stage II
+ -
+ -
Note: + = positive correlation: -
Stage Ill
No. of Patients Tested
-
Relation of Immune Suppression to Recurrence
Stage IV
-
= no correlation.
affected by the immunologic status and may not benefit from an immunopotentiator. To this end, we are currently engaged in a double-blind clinical trial comparing the effects of an immunomodulator, Levamisole with placebo, on local recurrence rates and survival in patients with resectable head and neck cancer after adequate surgery. Other methods of analysis of immunopathology in head and neck cancer have also been pursued. Berlinger and Good [17] found a good correlation between the mixed lymphocyte culture assay and the clinical stage of head and neck cancer patients. Preliminary data also indicate a possible association of an abnormal mixed lymphocyte culture response and poor prognosis in these patients [Ml. Summary
Patients with mucosal squamous cell carcinoma of the head and neck generally have suppressed T cell function, and this suppression tends to be more pronounced with progression of disease. The prognostic relationships of multiple tests of immune function were analyzed in 183 clinically staged patients. Correlation of immune parameters with prognosis was evident only with the DNCB response, which correlated with recurrence in stages I and II disease (but not in stages III and IV). There was no correlation of any of the in vitro tests of immunofunction with recurrence in any stage. There were no correlations between any of the immune parameters and age or sex nor between DNCB reactivity and any of the in vitro responses. References 1. Eilber FR, Morton DL: Impaired immunologic reactivity and recurrence following cancer surgery. Cancer 25: 362, 1970. 2. Solowey AC, Rapaport FT: Immunologic responses in cancer patients. Surg Gynecol Obsfet 121: 756, 1965. 3. Hughes LE, Mackay WD: Suppression of the tuberculin response in malignant disease. Br A&d J 2: 1346, 1965. 4. Pinsky CM, El-Domeiri A, Caron AS, Knapper WH, Oettgen HF: Delayed hypersensltivit$ reactions in patients with cancer. Recent results in cancer research. 47: 37, 1974.
Volume 134. October 1977
TABLE VIII
DNCB Negative Positive PPD Negative Positive Lymphocyte count Low Normal PHA Low Normal Con-A Low Normal PWM Low Normal
in Head and Neck Cancer
Per Cent with No Evidence of Disease
54 67
43 55
53 59
41 51
76 104
46 54
48 63
63 51
24 64
50 56
22 88
36 59
5. Catalona WJ, Chretien PB: Abnormalities of quantitative dinitrochlorobenzene sensitization in cancer patients: correlation with tumor stage and histology. Cancer 31: 353, 1973. 6. Catalona WJ, Sample WF, Chretien PB: Lymphocyte reactivity in cancer patients: correlation with tumor histology and clinical stage. Cancer 31: 65, 1973. 7. Gatti RA. Garrioch DB, Good RA: Depressed PHA responses in patients with non-lymphoid malignancies, p 339. Proceedings of the Fifth Leukocyte Culture Conference (Harris JE, ed). New York, Academic Press, 1970. 8. Sucin-Foca N, Buda J, McManus J: Impaired responsiveness of lymphocytes and serum inhibitory factors in patients with cancer of the head and neck. Am J Surg 128: 534, 1974. 9. Pinsky CM, Wanebo HJ, Mike V, Dettgen HF: Delayed cutaneous hypersensitivity reactions and prognosis in patients with cancer. Ann NY Acad Sci 276: 407,1976. 10. Wanebo l-U, Rao B, Miyazawa N, Martini N, Middleman MP, Oettgen HF, Beattie EJ: Immune reactivity in primary carcinoma of the lung and its relation to prognosis. J Thorac Cardiovasc Surg 72: 339, 1976. 11. Lundy J. Wanebo H, Pinsky C. Strong E, Oettgen H: Delayed hypersensitivity reactions in patients with squamous cell cancer of the head and neck. Am J Surg 128: 530, 1974. 12. Eilber FR, Morton DL. Ketcham AS: Immunologic abnormalities in head and neck cancer. Am J Surg 128: 534, 1974. 13. Wanebo HJ, Jun MY, Strong E, Oettgen HF: T-cell deficiency in patients with squamous cell cancer of the head and neck. Am J Surg 130: 445, 1975. 14. Chretien PB: Unique immunobiological aspects of head and neck squamous carcinoma, p 339. Workshops from the Centennial Conference on Laryngeal Cancer (Alberti PW, Bryce DP, ed). New York, Appleton-Century-Crofts, 1976. 15. Maisel RH, Dgura JH: Dinitrochlorobenzene skin sensitization and peripheral lymphocyte count: predictors of survival in head and neck cancer. Ann Otol85: 517, 1976. 16. Golub SH. O’Connell TX, Morton DL: Cwelation of in vivo and in vitro assays of immunocompetence in cancer patients. Cancer Res 34: 1833, 1974. 17. Berlinger NT, Good RA: Concomitant immunopathology with squamous cell carcinomas of the head and neck regions. Trans Am Acad Opthalmol Otolatyngol62: 588, 1976. 18. Berlinger NT: Immunobiology. Dtolaryngolcgy (Papparella M. Shumrick D, ed). Philadelphia, WB Saunders. (In press.)
473