Radiol Oncol 1993; 27 : 39-43.
The rate of natural killer cells and their cytotoxic activity in patients with advanced pharyngeal and laryngeal cancer
Lojze Šmid, M Žargi, B Rok, A Župevc, V Kotnik, A Ihan, M Rode
University Department of Otorhinolaryngology and Cervicofacial Surgery, Clinical Center
Ljubljana, Slovenia
The activity of natural killer (NK) cells was assessed In 46 patients with previously untreated advanced squamous cell carcinoma of the head and neck region. The mean NK cell activity of these patients was lower than that observed in a group of 32 age-matched controls (62.5 vs 90), however, the difference was not statistically significant. Patients with regional metastases that grew through the lymph node capsule and those with primary tumor directly invading the surrounding tissue had significantly lower values of NK cell activity than patients with locoregionally controlled tumors (p = 0.02).
Key words: pharyngeal neoplasms; laryngeal neoplasms; killer cells, natura)
Introduction
The role of natural killer (NK) cells in patients with malignomas of the head and neck has not been fully explained yet. Many facts support their possible influence in the destruction of circulating tumor cells,1 and some authors believe that a decreased cytotoxic activity (CA) of these cells attributes to the appearance of distant metastases.2-5 The latter observation seems to be of particular importance and interest in the case of patients with head and neck tumors, as such indicators of the probability of distant metastatic spread would be very helpful for correct treatment selection.
Correspondence to: L. Šmid, MD, University Department of Otorhinolaryngology and Cervicofacial Surgery, Zaloška 2, 61105 Ljubljana, Slovenia
UDC: 616.321-006.6-097:616.22-006.6-097
In our prospective study we were determining the number of NK cells and their CA in the peripheral blood of patients with advanced la-ryngeal and pharyngeal cancer, in order to asses their possible relevance for prognosing the course of disease.
Methods
The study was carried out in 46 patients, 40-81 years of age, with previously untreated and histologically confirmed advanced carcinoma of the pharynx and larynx. The disease was classified as stage 3 or 4 in ali patients.6 In 23 of them the site of origin was the oropharynx, in 15 hypopharynx and in 8 larynx. The prevailing histologic type was poorly differentiated or non-keratinizing planocellular carcinoma (37 patients), whereas well differentiated keratinizing planocellular carcinoma was established in 9
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cases. Twenty-two patients were treated by radiotherapy, 20 were operated on - of these 18 also received postoperative irradiation, and 4 patients were treated by a combined radio-and chemotherapy.
The control group consisted of 32 patients admitted to the University Department of Oto-rhinolaryngology and Cervicofacial Surgery because of some other, non-malignant diseases; the controls were matched to the studied cancer patients by age and sex.
CA of NK cells was determined by means of K 562 tumor cell killing test. These cells were mixed with lymphocytes isolated from the peripheral blood of patients with laryngeal and pharyngeal cancer. After 4-hour incubation, the rate of killed target cells was evaluated, and calculated in lithic units, where their higher value was associated with a higher K 562 tumor cell killing potential of NK cells.
Results and discussion
In patients with advanced cancer NK activity is usually depressed.7"11 Also in our patients, the average CA of NK cells was lower than that observed in the control group (62.5 vs 90), though the difference between both groups was not statistically significant. The average rate of NK cells in patients with laryngeal and pharyngeal cancer was 16.1 % , being lower from that in the control group, though the difference between both groups was not statistically significant.
Comparison of the average rates of NK cell counts and their cytotoxic activity by tumor site and histologic type did not show statistically significant differences, and neither confirmed a correlation between the average rate of these cells and their CA. Similarly, data from single-cell cytotoxicity assays suggest that the defect rests in the activity and not in the number of NK cells.12
Since our investigation was aimed to asses a possible prognostic relevance of NK cells, and since in patients with laryngeal and pharyngeal cancer the best indicator of the probability of
distant dissemination is known to be local and regional tumor spread,13-15 our analysis of the results was centred particularly on these issues. The available literary data on the CA of NK cells in patients with head and neck carcinomas are relatively scarce. There are, however, even less reports on the role and significance of CA of these cells for the appearance of local metastases. Cortesina et al.16 report a decreased CA of NK cells in advanced head and neck cancers. Pross and Baines7 established a decreased CA in patients with metastases in comparison with cases without regional tumor dissemination. Schantz and coworkers,17 however, found an increased CA of NK cells in patients with local or regional tumor invasion of the surrounding tissue.
In our study, the average CA of NK cells in patients with regional metastases in N3 and N2 was found to be significantly lower than that in the control group (p = 0.04 and 0.05 respectively), whereas in patients with NO and Nl no statistically significant difference could be established (Figure 1). The extent of local dissemination (control group - N0-N1-N2-N3 was in correlation with the values of average CA in individual groups: thus, a 5 %o risk was associated with negative correlation (KK = -0.274).
The comparison of NK cell rates and their CA in the group of patients with local or regional spread, and those without evidence of dissemination revealed interesting differences. In the cases when tumor did not show evidence of local or regional invasion into the surrounding tissues the rate of NK cells was on average lower than that in the group with local infiltration (13.5 vs 18.2), though the difference was not statistically significant (p = 0.08). An average CA of NK cells in the group of patients with local tumor invasion (39) was significantly lower than the relevant values in the control group (90) (p = 0.01), and borderline significantly lower than in patients without local infiltration (73) (p = 0.07) (Figure 2). Also, an average CA in the group of patients with regional invasion (44) was significantly lower than that in the control group (90) (p = 0.02) and borderline significantly lower than in the group
NK cells in. patients with advanced pharyngeal and laryngeal cancer
41
125
100
rr:	LTl	WLTl
Figure 2. Mean CA of NK cells according to local tumor infiltration into surrounding tissue (CG = control group; LTI = local tumor infiltration; WLTI = without local tumor infiltration).
125
100 -
CG	RTI	WRTI
Figure 3. Mean CA of NK cells according to regional tumor infiltration into surrounding tissue (CG = control group; RTI= regional tumor infiltration; WRTI = without regional tumor infiltration).
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125
100
[/J
75
a

50
25
p-0.004	p = 0.02
CG
LRTI
WLRTI
Figure 4. Mean CA of NK cells according to local and regional tumor infiltration into surrounding tissue (CG = control group; LRTI = local and/or regional tumor infiltration into surrounding tissue; WLRTI =± without local and/or regional tumor infiltration into surrounding tissue).
without regional invasion (75) (p = 0.07) (Figure 3). The difference is apparent also in the group of operated patients in whom the removed lymph nodes were histologically examined.
When the average CA in the group without locoregional invasion into the surrounding tissues was compared with that found in the group with locally and regionally infiltrating tumor the difference was statistically significant (p = 0.02) (Figure 4). These findings can be presented from yet another aspect: in only one of 18 patients without evidence of locoregional invasion CA was lower than 30 lithic units (LU) (5.5 %). On the other hand, in the group of 28 patients with tumor invasion into the surrounding tissues CA was below 30 LE in as many as 17 patients (61 %). Therefore CA lower than 30 LE could be considered as unfavourable prognostic sign.
Conclusion
The findings of our investigation indirectly support the hypothesis that in patients with laryn-geal and pharyngeal cancer the course of disease in reflected in the CA level of NK cells.
Nevertheless, it should be kept in mind that our results are only preliminary, and therefore their final evaluation would require further follow up of the patients. Only in this way the hypothetical correlation between the CA of NK cells and prognosis of the disease could be confirmed.
References
1.	Gorelik E, Fogel M, Segal M, Feldman S. Differences in resistance of metastatic tumor cells and cells from local tumor growth to cytotoxicity of natural killer cells. J Natl Cancer Inst 1979; 63: 1397-404.
2.	Gorelik E, Wiltrout RH, Okumura K, Habu S, Herberman RB. Role of NK cells in the control of metastatic spread and growth of tumor cells in mice. Int J Cancer 1982; 30: 107-12.
3.	Hanna N. Expression of metastatic potential of tumor cells in young nude mice is correlated with low levels of natural killer cell-mediated cytotoxicity. Int J Cancer 1980; 26: 675-80.
4.	Hanna N, Fidler I. Role of natural killer cells in the destruction of circulating tumor emboli. J Natl Cancer Jnst 1980; 65: 800-12.
5.	Talmadge J, Meyers K, Prieur D, Starkey J. Role of NK cells in tumor growth and metastasis in beige mice. Natuce (Lond) 1980; 284: 622-24.
6.	TNM classification of malignant tumors. International Union Against Cancer, Geneve 1987.
7.	Pross HF, Baines MG. Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. The effect of malignant disease. Int J Cancer 1976; 18: 593-8.
8.	Takasugi M, Ramseyer A, Takasugi J. Decline of natural nonselective cell-mediated cytotoxicity in patients with tumor progression. Cancer Res 1977; 37: 413-9.
9.	Cunningham-Rundles S, Filippa DA, Braun DW, Antonelli P, Ashikari H. Natural cytotoxicity of peripheral blood lymphocytes and regional lymph node cells in breast cancer in women. J Natl Cancer Inst 1981; 67: 585-92.
10.	Kadish AS, Doyle AT, Teinhauer EH, Ghossein NA. Natural cytotoxicity and interferon production in human cancer: deficient natural killer activity and normal interferon production in patients with advanced disease. J Immunol 1981; 127: 1817-22.
11.	Pandolfi T, Semenzato G, De Rossi G, Strong DM, Quinti L, Pezzutto A, Mandelli F, Aiuti F. Heterogeneity of T-CLL defined by monoclonal antibodies in nine patients. Clin Immunol Immu-nopathol 1982; 24: 330-5.
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12.	Steinhauer EH, Doyle AT, Reed J, Kadish AS. Defective natural cytotoxicity in patients with cancer: normal number of effector cells but decreased recycling capacity in patients with advanced disease. J Immunol 1982; 129: 2255-32.
13.	Merino OR, Lindberg RD, Fletcher GH. An analysis of distant metastases from squamous cell carcinoma of the upper respiratory and digestive tracts. Cancer 1977; 40: 145-51.
14.	Johnson JT, Barmes EL, Myers EN. The extracapsular spread of tumors in cervical node metastases. Arch Otolaryngol Head Neck Surg 1981; 107: 725-9.
15.	Vikram B, Strong EW, Shah JP. Failure at distant sites following multimodality treatment for advanced head and neck cancer. Head Neck Surg 1984; 6: 730-3.
16.	Cortesina G, Sartoris A, Di Fortunato V. Natural killer-mediated cytotoxicity in patients with laryngeal carcinoma. Ann Otol Rhinol Laryngol 1984; 3: 189-91.
17.	Schantz SP, Poisson L. Natural killer cell response to regional lymph node metastases. Arch Otolaryngol Head Neck Surg 1986; 112: 45-51.