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The role of testosterone in laryngeal carcinogenesis
Am J OLolar]~sol 5:344..-349, 19S4
The Role of Testosterone in Laryngeal Carcinogenesis VINKO KAMBI~, M. D., ZORA RAD~EL,1VJ[.D., JANEZ PRE~.ELLM. D., AND MIHA 7,ARGI, M. D. Testosterone levels in the plasma of 25 women and 25 men with carcinoma of the larynx and of 15 women and 15 men with hyperplastic aberrations in the laryngeal mucosa were determined to evaluate the etiologic relation between hyperplastic aberrations in the laryngeal mucosa (including cancer) and the male sex hormones. Patients with laryngeal cancer showed statistically significant increases in plasma testosterone levels compared with the control group. No statistically significant differences were found in plasma testosterone levels or in the testosterone/estradiol index between patients with hyperplastic aberrations of the laryngeal mucosa (including precancerous conditions) and the control subjects. The authors believe that greater attention should be paid to endogenous factors, particularly the male sex hormones, in further studies of the etiology of laryngeal carcinoma.
It is well known that the incidence of laryngeal carcinoma is much higher in men than in women. The ratio of male to female patients is 13'.1 according to Ackerman and Del Regato, ~ 9:1 according to Ogura and Biller, 2 20:1 according to Kambi~, 3 24.6:1 according to Marh et al,, 4 and 16:1 according to the Cancer Registry of Slovenia (for the period from 1970 to 1979). 5 Carcinoma of the larynx, and in particular of the vocal cords, occurs almost exclusively in men, and it occurs at the ages at w h i c h women have an increased i n c i d e n c e of uterine and breast cancer. 6,7 The male larynx also shows a much greater tendency to undergo chronic inflammatory alterations. In a study of 354 rand o m l y selected factory workers from Slovenia (177 men and 177 women), the number of men with chronic hyperplastic aberrations in the laryngeal mucosa was surprisingly higher than that of the women exposed to the same environment (ratio, 7:1). 8 In Yugoslavia there is virtually no difference b e t w e e n the male and female p o p u l a t i o n s in terms of smoking habits and working and living conditions, If exogenous agents only were re-
Received March 27, 1984, from the Clinic of Otorhinolaryngology (Drs. Kambri~, Rad~el, and 7.argi}and the Clinic of Medicine, Department of Endocrinology (Dr. Pre~elj), University Medical Center, Ljubljana, Yugoslavia. Accepted for publication April 25, 1984. Supported by Raziskovalnaskupnost Slovenije, grant rm. C3-176-381/82. Address correspondence and reprint requests to Dr. Kambi~: Clinic of Otorhinolaryngology, University Medical Center Ljubljana, Zalo~ka 2, 61105 LjublJana, Yugoslavia.
sponsible for laryngeal carcinogenesis, the observed male prevalance w o u l d not be so great. The male sex hormones play a decisive role in the physiologic development of the larynx. On the other hand, it has been demonstrated in experimental studies that increased levels of male sex hormones give rise to important changes in the laryngeal mucosa. T e s t o s t e r o n e c o u l d be classed with the substances exhibiting hyperplasia-inducing and cocarcinogenic actions. 7 Following the injection of testosterone, male dogs and ovariectomized female dogs had hyperplasia of the vocal cord epithelium, histologically very similar to carcinoma. 9 The lesions were irreversible. I~ Experiments in mice supported the hypothesis that the larynx represents a target organ for male sex hormones. In mice injected with testosterone, the exogenous agents i n d u c e d p r o n o u n c e d changes in the laryngeal mucosa that could be described as atypical hyperplasia. 11. Hajdutova et al. 12 found elevated testosterone levels in m e n with laryngeal carcinoma, but the values reported were not statistically significant. Saez and Sakai 13 reported the presence of specific androgen receptors in the laryngeal mucosa from subjects of both sexes. They found that the cytosol contains specific protein complexes that bind 5-a-dihydrotestosterone. In their opinion, laryngeal tumors, like certain mammary tumors, could be h o r m o n e - d e p e n d e n t . A u f d e m o r t e et al. 14 demonstrated that the baboon (Papio cynocephalus) larynx is rich in sex steroid receptors. Hussl et el. I~ and Loewit et a]. 18 reported the
344
KAMBICET AL. success of h o r m o n a l therapy in patients with "pachydermia laryngis." These findings support our hypothesis that elevated levels of androgens or their metabolites cause alterations in the laryngeal epithelium, rendering it more susceptible to exogenous carcinogenic agents and thus precipitating the development of precancerous or malignant aberrations in the altered mucosa. The present study was undertaken to determine the concentrations of testosterone in the plasma of male and female patients with cancer or hyperplastic aberrations of the larynx, and to assess the relation between the amount of sex hormones in plasma and the histologic appearance of the laryngeal mucosa. MATERIALS AND METHODS
The subjects were 25 female and 25 male patients with carcinoma of the larynx and 15 female and 15 male patients with hyperplastic aberrations in the laryngeal mucosa, Specimens were obtained from the patients with carcinoma prior to therapy (20 patients) or after treatment (30 patients). Patients with diseases likely to inf l u e n c e plasma testosterone levels (e.g., eun u c h i s m , Cushing's disease, and thyroid dysfunction) were excluded from the study. The control group comprised 80 healthy subjects. For each patient one control subject, m a t c h e d for sex, age, and living and working conditions (workplace, microclimate, residence, cigarette smoking, and alcohol usage), was selected. The patients with carcinoma ranged from 25 to 82 years. The age range of the men and women with hyperplastic aberrations was 30 to 81 years. All of the male patients and 65 per cent of the female patients with laryngeal carcinoma had histories of cigarette smoking. Ninety-five per cent of the male and 60 per cent of the female patients with chronic hyperplastic alterations of the laryngeal mucosa smoked. Excessive use of alcohol was reported by the majority of the male patients with cancer (75 per cent). The histories elicited in the other three groups (i.e., w o m e n with cancer and all patients with hyperplastic aberrations) were not typical of alcoholism; however, for subjective reasons, the data should be interpreted with reservations. The diagnosis was made on the basis of histologic examination of the laryngeal biopsy specimens. The tumors were classified according to the TNM system (UICC, 1978). The hyperplastic changes were classified according to the classification proposed by Kambi6 and Lenart 17 (Table
TABLE 1. Classification and Distribution of Patients LARYNGEAL CARCINOMA IN M E N
LARYNGEAL CARCINQIvfA IN W O M E N
Extent
Number
Extent
T1NoM0 T2NoMo TaN2M0 T4N~Mo
10 4 9 2
T1NoMo TzNoMo T2N2M0 .T~NzM0
Number 8 7 5
5
I). Atypical hyperplasia ("risky epithelium," precancerous) was found in seven, and abnormal hyperplasia in eight male patients with hyperplastic aberrations, while the female group included four patients with atypical hyperplasia, five with abnormal hyperplasia, and six with simple hyperplasia of the laryngeal mucosa. Concentrations of plasma testosterone and free testosterone in the saliva were determined in all patients and control subjects. Testosterone and serum ~-estradiol determinations were done by the standard radioimmunoassay (RIA) techniques (TESTOK-125, CIS and ESTRIK-125, CIS). The coefficient of variation for both radioimmunoassays in the studied range was less than 10 per cent. Liver function tests (alanine aminotransferase, asparate aminotransferase, lactic dehydrogenase, c~-glutamine transpeptidase) were conducted in patients with carcinoma of the larynx to determine whether liver conditions that could have affected the plasma testosterone levels were present. Patients with pathologic results were excluded. Female patients of childbearing age were tested in the follicular phase of the menstrual cycle. Statistical data evaluation was performed by the Student's t-test for matched pairs. If the data distribution was obviously not normal (salivary testosterone concentrations, testosterone/estradiol index], Wilcoxon's signed ranks test was used. RESULTS
The male patients with laryngeal carcinoma (n = 25) showed significantly increased plasma testosterone levels (17.9 • 7,7 nmol/liter) compared with the control subjects (12.5 + 5.9 nmol/liter) (t = 2.89; P < 0.01) (Table 2). The plasma testosterone concentrations (16.5 + 7.0 nmol/liter) of male patients with hyperplastic aberrations in the laryngeal mucosa (n = 15) did not differ significantly from the values (15.7 - 6.4 nmol/liter) obtained in their control subjects (t = 0.39; P > 0.5) (Table 3).
Volume 5 Number 5 September 1984 345
TESTOSTERONE A N D L A R Y N G E A L
TABLE 2. Plasma Testosterone Levels in Male Patients with Laryngeal Carcinoma and in Matched Control Subjects PLASMu& TESTOSTERONE LEVEL
[NMOL/LITER) PATIENT 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19
20 21 22 23 24 25
AGE(YR) 82 55 52 55 85 69 53 66 54 48 52 60 67 46 66
82 48 60 84 49 60 50 53 71 46
SD
n
American Journal
of Otolaryngology 346
Patient
Control Subject
7.4 13,1 30.3 15.5 14.0 20.3 15.8
3.3 10.5 8.3 27,3 12.5 23.0 8.3 13.1 14.4 15.7 8.7 9.8 16.0 14.6 5.9 11.8 12.5 11.8 5.9 12.5 11.8 13.1 8.3 5.9 26.4 12.5 5.9
27.7 18.1 17.1 23.4 14.0 22.7 35.4 24.7 6.5 21.8 12.3 5.8 8.7 10.4 20.9 15.7
29.2 17.0 17.9
7.7
= 25; t = 2,89; P < 0.01,
The testosterone/estradiol (T/E) index was determined in 11 male patients with hyperplastic laryngeal aberrations. Estradiol values were not available for four patients. The T/E values of these patients (181 _ 67) when compared with those of their control subjects (153 "+ 99) were not significantly elevated (Wilcoxon's signed ranks test, T = 24; P > 0.1) (Table 4). Plasma testosterone levels were 2.8 -+ 1.5 nmol/liter in women with cancer of the larynx (n = 25) and 1.5 _ 0.6 nmol/liter in the control group. The difference was statistically significant (t = 3.70; P < 0.005) (Table 5). Plasma testosterone concentrations in female patients with hyperplastic aberrations in the laryngeal mucosa (n = 15) were not significantly elevated (2.2 _ 1,5 nmol/liter) in comparison with those of ther matched control subjects (1,8 __ 0,7 nmol/liter (t = 1.08; P > 0.2) (Table 6). No significant differences were found in salivary testosterone levels b e t w e e n the w o m e n with hyperplastic aberrations (n = 6) (150 _ 95 pmol/liter) and their control subjects (218 _ 143 pmol/liter (Wilcoxon's signed ranks test, T = 7; P > 0.1) (Table 7]. Similar results were obtained
CANCER
in the group of male patients with hyperplastic aberrations. The T/E index was also calculated for 12 female patients with hyperplastic aberrations in the laryngeal mucosa. Estradiol values were not available for three patients in this group. Although the values obtained in female patients (63 _+_ 73) were higher than those of their control subjects (37 _+ 53), the differences were not statistically significant (Wilcoxon's signed ranks test, T = 24; P > 0.1) (Table 8). DISCUSSION
A p r o g r e s s i v e l y increasing i n c i d e n c e of head and neck cancer, including carcinoma of the larynx, has been reported. ~,19 According to these reports the exogenous factors, particularly cigarette smoking, are responsible for the increased morbidity in both men and women. Our studies in Yugoslavia indicate that the living and working conditions and lifestyles of both sexes are similar. Differences that do exist w o u l d not account for the striking prevalence of laryngeal cancer or chronic h y p e r p l a s t i c aberrations of the laryngeal mucosa in men. These findings led to the hypothesis that an endogenous factor (elevated concentration of male sex hormones) may affect the laryngeal mucosa by increasing its susceptibility to exogenous cocarcinogens. An increasing incidence of laryngeal carcinoma in women has been documented in our female population as well. However, we beTABLE 3. Plasma Testosterone Levels in Male Patients with Hyperplastic Aberrations in the Laryngeal Mucosa and in Matched Control Subjects PLASMA TESTOSTERONE LEVELS (NMOL/LITER) PATmNT
AGE(YR~
Patient
Control Subject 15.1 13.1 12.5 26,6 14.6 26,4 11.8
1 2 3 4 5 5 7
70 50 51 64 46 46 60
20.5 16.1 14.0 13.1 13,1 11.S 18.2
8 9
65
10,5
5.9
44 43 43 riO 32 60 42
11,3 36.2 21.4 11.3 20.9 8.7 21.4 16.5 7.0
22.9 18.5 11.4 11.8 19.7 5,9 18.5 15.7 6.4
10 11 12 13 14 15 SD
n = 15; t = 0.39; P > 0.5,
KAMBIC ET AL.
lieve that this finding could be attributed in part to an imbalance of sex h o r m o n e s in these women, as well as to their increased cigarette smoking. The current way of life, characterized by stress and the use of contraceptive ~ud therapeutic h o r m o n e preparations by women, is most likely the cause of the hormonal imbalance and resulting disorders. The results of our study are consistent with the hypothesis of increased testosterone levels in male and female patients with laryngeal carcinoma. Free testosterone levels in the saliva of male and female patients with laryngeal carcinoma in our pilot study were also elevated. However, the results were not taken into consideration because of the small quantity and the changed quality of the saliva, most evident in patients receiving postoperative radiotherapy (ten male and ten female patients). No statistically significant differences were found in the plasma salivary testosterone levels or in the T/E index between patients with hyperplastic aberrations of the laryngeal mucosa and their control subjects. However, the wide spectrum of the histologic features in the cases studied should be taken into consideration: the study included patients with simple, abnormal, and atypical hyperplasia, and, among these lesions, only the last can be classified as precancerous. Atypical hyperplasia is very rare and accounts for only 5.4 per cent of all hyperplastic aberrations involving the laryngeal mucosaJ 7 A study of the testosterone levels in the plasma of patients with atypical hyperplasia during a tenyear period is currently underway. Plasma testosterone values are not a diagnostic tool for the classification of hyperplastic larynTABLE 4. Testosterone/Estradiol Index in Male Patients with Hyperplastic Aberrations in the Laryngeal Mucosa and in Matched Control Subjects TESTOSTERONFJEsTRADIOLINDEX PATIENT 1 2 3 4 5 6 7 8 g 10 11 SD
AGE(YR) 50 51 64 46 46 60 65 44 60 60 42
Patient 146 127 164 109 188 260 131 226 103 302 238 181 • 87
Control 131 114 332 264 27 107 42 203 107 88 264 153 • 99
n = 11; T = 24; P > 0.1 (Wilcoxon's signed ranks test).
TABLE 5. Plasma Testosterone Levels in Female Patients with Laryngeal Carcinoma and in Matched
Control Subjects PLASMA TESTOSTERONELEVELS {NMOL/LITER) PATIENT 1 2 3 4 5 6 7 8 g 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
AGE(YR.]
Patient
Contro 1
52 39 76 81 38 74 33 70 70 25 68 69 54 55 59 61 58 48 68 66 58 46
1,0 4.2 5.1 2,8 4.5 4.7 6,1 4,5 ~.8 3.1 1.4 2,1 1.0 2,1 1.4 2.1 3,6 2.1 1,0 %6 2,4 1,7 0,8 1.0 1.2 2,6 1,5
1,4 1.7 1.7 3.2 1,0 1.4 2.7 i.O 1.0 2,4 1.4 1,7 1.4 1.4 1,0 1,7 1,4 0.9 1.3 1,1 1.4 2.4 0.4 1,4 1,4 1.5 0.6
57 68 58
SD n = 25; t = 3,70; P < 0.005,
TABLE 6. P l a s m a Testosterone Levels in Female Patients with Hyperplastic Aberrations in the Laryngeal Mucosa and in Matched Control Subjects PLASMA TESTOSTERONELEVELS {NMOIJLITER) PATIENT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
AGE(YR1
Patient
Control
30 51 49 38 55 47 51 81 52 40 68 70 68 41 62
1,0 1.6 1,2 0.g 2.7 2,4 4,8 2,7 4.1 4.8 1,2 1.1 0,8 3.6 0.6 2.2 1,5
2.3 2.9 1.3 1.5 2.4 1.0 1.1 1.3 2.9 2.1 2,3 1.3 1.7 2,1 1.0 1.8 0.7
SD n = 15; t = 1.08; P > 0.2.
seal aberrations. Still less can they be used to foretell the outcome of treatment. Their role in laryngeal carcinogenesis, however, should be considered. In addition to the various exogenous
Volume 5 Number 5 September 1984 347
TESTOSTERONE AND LARYNGEAL CANCER
TABLE 7. Salivary Testosterone Levels i n Female Patients with Hyperplastic Aberrations in t h e Laryngeal M u c o s a and in Matched C o n t r o l Subjects
SALIVARYTESTOSTERONELEVELS (P~OL/UTER] PATIENT 1 2 3 4 5 6 SD
AGE(YR)
Patient
Control
30 51 68 51 36 41
86 186 47 262 70 250 150 95
388 308 337 48 131 97 218 143
n = 6; T = 7; P > O.1 (Wilcoxon's signed ranks test].
TABLE 8. Testosterone/Estradiol Index in F e m a l e Patients with Hyperplastic Aberrations in the Laryngeal M u c o s a and in Matched Control S u b j e c t s TESTOSTERONFJESTRADIOL INDEX PATIENT
ACE(YR)
Patient
1 2
30 51
1 160
CONCLUSION
Americon Journal
of Otolaryngology 348
Our investigations demonstrated testosterone levels in patients of both sexes with laryngeal cancer that were significantly higher than those in their healthy counterparts of the same age, habits, and living and working conditions. This is in agreement with the hypothesis that the development of laryngeal carcinoma is related to the amount of circulating testosterone. The action of male sex hormones may increase the susceptibility of the laryngeal mucosa to exogenous factors, which, in connection with a yet obscure (possibly immunobiologic) response of the organism, leads to malignancy after a latency period. The larynx--a target organ for male sex h o r m o n e s - - s e e m s to be d e p e n d e n t on the amount of testosterone, not only in its physio-
33 15
3
49
4
3
4
36
90
10
5 6
41
5
69 22 242
4 2
7 8
55 47 51
9
40
7
4
10 11 12
68 40 70
40 69 42 63 73
130
SD
factors, such as smoking, alcohol consumption, and poor microclimatic living and working conditions, the role of male sex hormones should also be suspected in the pathogenesis of the disease and should receive more attention in furthor research. The possibility of treating patients with laryngeal carcinoma by hormones was discussed by Saez and Sakai. 13 Hussl et al} ~ and Loewit et al. le reported on successful antiandrogenic treatment with a testosterone antagonist in patients with hyperplastic aberrations of the laryngeal mucosa. As our study failed to demonstrate a statistically significant increase in testosterone levels in male and female patients with laryngeal hyperplasia, the theoretic basis of the success of hormonal therapy remains to be clarified.
Control
3 110
130 37 53
n = 12; T = 24; P > 0.1 (Wilcoxon's signed ranks test].
logic development but also in the pathogenesis of disease, including laryngeal carcinoma. Further investigations should be undertaken to identify the role of testosterone in the development of epithelium at risk for cancer (atypical hyperplasia) in the laryngeal mucosa. These studies would also clarify the roles of hormones in the etiology of laryngeal carcinoma.
References 1. Ackerman LV, Del Regato ]A: Cancer--diagnosis, treatment and prognosis. St. Louis, CV Mosby Co, 1970, pp 3 0 8 - 3 2 9 2. Ogura JH, Biller HF: Cysts and tumors of the larynx, in Paparella MM, Shumrick DA (eds): Otolaryngology, vol 3 (Head and Neckl. Phfladilphia, WB Sounders Co, 1973, pp 6 5 8 - 6 6 5 3. Kambi~ V: Otorinolaringologija. Ljubljana, Mladinska knjiga, 1975, p 170 4. M a h r HR, F i k e n t s c h e r R, B r u c h m f i l l e r W: Besonderheiten des w e i b l i c h e n Larynxkarzinoms. Laryingol Rhinol 56:998-1003, 1977 5. Cancer incidence in Slovenia (1970-1979). Cancer registry of S l o v e n i a - - I n s t i t u t e of Oncology, Ljubljana, 1973-1983 6. Kambi~ V: Quelques idles sur los ~tats pr~cancereux du larynx. JFORL 14:827-837, 1965 7. Kambi~ V, Lenart I: Erosio laryngis falsa. Symp Otolaryngol Iug 5:97-101, 1970 8. Kambl~ V: Profesionalna obolenja v otorinolaringologiji. Zbornik Radova VIII. Kongresa Otorinolaringologa ]ugoslavije, SkopJe, 1969, pp 1 7 3 - 1 7 6 9. Kambi~ V, Lenart I'. Untersuchungen fiber die Wirkung y o n T e s t o s t e r o n auf die K e h l k o p f s c h l e i m h a u t des Hundes. HNO 16:327-330, 1968 10. Karnbi~ V, Lenart I, Lenart V, et ah Beitrag zur Frage der Rsversibilit/it bzw: Irreversibilit~lt v a n Gewebsver~ind e r u n g e n , die bei H u n d e n n a c h der i.m. Verabreichung v a n Testosteran an dar laryngealen Schlelmhaut auftreten. HNO 21:300-303, 1973 11. Kambi~ V, Rad.~el Z, ~argi M: V e r ~ n d e r u n g e n an der
KAMBI~ ET AL.
12. 13. 14.
15.
laryngealen M~luseschleimhaut als Folge endogener und exogener Faktoren. HNO 25:249-252, 1977 Hajdutova R, Dimitrova S, Melamed M: Izsledvane na testosterona v krvnija serum na bolni ot rak no larinksa. Onkologia (Sofia) 16:22-26, 1979 Saez S, Sakai F: Androgen receptors of human pharyngolaryngeal mucosa and pharyngo-laryngeal epithelioma. J Steroid Biochem 7:919-921, 1976 Aufdemorte TB, Sheridan PJ, Holt GR: Autoradiographic evidence of sex steroid receptors in laryngeal tissues of the baboon (Papio cynocephalus). Laryngoscope 93:1607-1611, 1983 Hussl B, Loewit K, Richter E, et el: First clinical expe-
16.
17. 18. 19.
riences with hormone-therapy of pachydarmia laryngis. Arch Otolaryngol 221:221-225, 1978 Loewit K, Hussl B, Richter E, et ah Anti-androgen therapy in pachydermia of the female larynx: a new therapeutic possibility. Arch Otolaryngol 215:75-79, 1977 Kambi~ V: Difficulties in management of vocal cord precancerous lesions. J Laryngol Otol 92:305-315, 1978 McGuirt WF: Head and neck cancer in women: a changing profile. Laryngoscope 93:106-107, 1983 Wynder EL, Covey LS, Mabuchi K, et el: Environmental factors in cancer of the larynx. Cancer 38:1591-1601, 1976
Volume 5 Number 5 September 1984 349
The Role of Testosterone in Laryngeal Carcinogenesis VINKO KAMBI~, M. D., ZORA RAD~EL,1VJ[.D., JANEZ PRE~.ELLM. D., AND MIHA 7,ARGI, M. D. Testosterone levels in the plasma of 25 women and 25 men with carcinoma of the larynx and of 15 women and 15 men with hyperplastic aberrations in the laryngeal mucosa were determined to evaluate the etiologic relation between hyperplastic aberrations in the laryngeal mucosa (including cancer) and the male sex hormones. Patients with laryngeal cancer showed statistically significant increases in plasma testosterone levels compared with the control group. No statistically significant differences were found in plasma testosterone levels or in the testosterone/estradiol index between patients with hyperplastic aberrations of the laryngeal mucosa (including precancerous conditions) and the control subjects. The authors believe that greater attention should be paid to endogenous factors, particularly the male sex hormones, in further studies of the etiology of laryngeal carcinoma.
It is well known that the incidence of laryngeal carcinoma is much higher in men than in women. The ratio of male to female patients is 13'.1 according to Ackerman and Del Regato, ~ 9:1 according to Ogura and Biller, 2 20:1 according to Kambi~, 3 24.6:1 according to Marh et al,, 4 and 16:1 according to the Cancer Registry of Slovenia (for the period from 1970 to 1979). 5 Carcinoma of the larynx, and in particular of the vocal cords, occurs almost exclusively in men, and it occurs at the ages at w h i c h women have an increased i n c i d e n c e of uterine and breast cancer. 6,7 The male larynx also shows a much greater tendency to undergo chronic inflammatory alterations. In a study of 354 rand o m l y selected factory workers from Slovenia (177 men and 177 women), the number of men with chronic hyperplastic aberrations in the laryngeal mucosa was surprisingly higher than that of the women exposed to the same environment (ratio, 7:1). 8 In Yugoslavia there is virtually no difference b e t w e e n the male and female p o p u l a t i o n s in terms of smoking habits and working and living conditions, If exogenous agents only were re-
Received March 27, 1984, from the Clinic of Otorhinolaryngology (Drs. Kambri~, Rad~el, and 7.argi}and the Clinic of Medicine, Department of Endocrinology (Dr. Pre~elj), University Medical Center, Ljubljana, Yugoslavia. Accepted for publication April 25, 1984. Supported by Raziskovalnaskupnost Slovenije, grant rm. C3-176-381/82. Address correspondence and reprint requests to Dr. Kambi~: Clinic of Otorhinolaryngology, University Medical Center Ljubljana, Zalo~ka 2, 61105 LjublJana, Yugoslavia.
sponsible for laryngeal carcinogenesis, the observed male prevalance w o u l d not be so great. The male sex hormones play a decisive role in the physiologic development of the larynx. On the other hand, it has been demonstrated in experimental studies that increased levels of male sex hormones give rise to important changes in the laryngeal mucosa. T e s t o s t e r o n e c o u l d be classed with the substances exhibiting hyperplasia-inducing and cocarcinogenic actions. 7 Following the injection of testosterone, male dogs and ovariectomized female dogs had hyperplasia of the vocal cord epithelium, histologically very similar to carcinoma. 9 The lesions were irreversible. I~ Experiments in mice supported the hypothesis that the larynx represents a target organ for male sex hormones. In mice injected with testosterone, the exogenous agents i n d u c e d p r o n o u n c e d changes in the laryngeal mucosa that could be described as atypical hyperplasia. 11. Hajdutova et al. 12 found elevated testosterone levels in m e n with laryngeal carcinoma, but the values reported were not statistically significant. Saez and Sakai 13 reported the presence of specific androgen receptors in the laryngeal mucosa from subjects of both sexes. They found that the cytosol contains specific protein complexes that bind 5-a-dihydrotestosterone. In their opinion, laryngeal tumors, like certain mammary tumors, could be h o r m o n e - d e p e n d e n t . A u f d e m o r t e et al. 14 demonstrated that the baboon (Papio cynocephalus) larynx is rich in sex steroid receptors. Hussl et el. I~ and Loewit et a]. 18 reported the
344
KAMBICET AL. success of h o r m o n a l therapy in patients with "pachydermia laryngis." These findings support our hypothesis that elevated levels of androgens or their metabolites cause alterations in the laryngeal epithelium, rendering it more susceptible to exogenous carcinogenic agents and thus precipitating the development of precancerous or malignant aberrations in the altered mucosa. The present study was undertaken to determine the concentrations of testosterone in the plasma of male and female patients with cancer or hyperplastic aberrations of the larynx, and to assess the relation between the amount of sex hormones in plasma and the histologic appearance of the laryngeal mucosa. MATERIALS AND METHODS
The subjects were 25 female and 25 male patients with carcinoma of the larynx and 15 female and 15 male patients with hyperplastic aberrations in the laryngeal mucosa, Specimens were obtained from the patients with carcinoma prior to therapy (20 patients) or after treatment (30 patients). Patients with diseases likely to inf l u e n c e plasma testosterone levels (e.g., eun u c h i s m , Cushing's disease, and thyroid dysfunction) were excluded from the study. The control group comprised 80 healthy subjects. For each patient one control subject, m a t c h e d for sex, age, and living and working conditions (workplace, microclimate, residence, cigarette smoking, and alcohol usage), was selected. The patients with carcinoma ranged from 25 to 82 years. The age range of the men and women with hyperplastic aberrations was 30 to 81 years. All of the male patients and 65 per cent of the female patients with laryngeal carcinoma had histories of cigarette smoking. Ninety-five per cent of the male and 60 per cent of the female patients with chronic hyperplastic alterations of the laryngeal mucosa smoked. Excessive use of alcohol was reported by the majority of the male patients with cancer (75 per cent). The histories elicited in the other three groups (i.e., w o m e n with cancer and all patients with hyperplastic aberrations) were not typical of alcoholism; however, for subjective reasons, the data should be interpreted with reservations. The diagnosis was made on the basis of histologic examination of the laryngeal biopsy specimens. The tumors were classified according to the TNM system (UICC, 1978). The hyperplastic changes were classified according to the classification proposed by Kambi6 and Lenart 17 (Table
TABLE 1. Classification and Distribution of Patients LARYNGEAL CARCINOMA IN M E N
LARYNGEAL CARCINQIvfA IN W O M E N
Extent
Number
Extent
T1NoM0 T2NoMo TaN2M0 T4N~Mo
10 4 9 2
T1NoMo TzNoMo T2N2M0 .T~NzM0
Number 8 7 5
5
I). Atypical hyperplasia ("risky epithelium," precancerous) was found in seven, and abnormal hyperplasia in eight male patients with hyperplastic aberrations, while the female group included four patients with atypical hyperplasia, five with abnormal hyperplasia, and six with simple hyperplasia of the laryngeal mucosa. Concentrations of plasma testosterone and free testosterone in the saliva were determined in all patients and control subjects. Testosterone and serum ~-estradiol determinations were done by the standard radioimmunoassay (RIA) techniques (TESTOK-125, CIS and ESTRIK-125, CIS). The coefficient of variation for both radioimmunoassays in the studied range was less than 10 per cent. Liver function tests (alanine aminotransferase, asparate aminotransferase, lactic dehydrogenase, c~-glutamine transpeptidase) were conducted in patients with carcinoma of the larynx to determine whether liver conditions that could have affected the plasma testosterone levels were present. Patients with pathologic results were excluded. Female patients of childbearing age were tested in the follicular phase of the menstrual cycle. Statistical data evaluation was performed by the Student's t-test for matched pairs. If the data distribution was obviously not normal (salivary testosterone concentrations, testosterone/estradiol index], Wilcoxon's signed ranks test was used. RESULTS
The male patients with laryngeal carcinoma (n = 25) showed significantly increased plasma testosterone levels (17.9 • 7,7 nmol/liter) compared with the control subjects (12.5 + 5.9 nmol/liter) (t = 2.89; P < 0.01) (Table 2). The plasma testosterone concentrations (16.5 + 7.0 nmol/liter) of male patients with hyperplastic aberrations in the laryngeal mucosa (n = 15) did not differ significantly from the values (15.7 - 6.4 nmol/liter) obtained in their control subjects (t = 0.39; P > 0.5) (Table 3).
Volume 5 Number 5 September 1984 345
TESTOSTERONE A N D L A R Y N G E A L
TABLE 2. Plasma Testosterone Levels in Male Patients with Laryngeal Carcinoma and in Matched Control Subjects PLASMu& TESTOSTERONE LEVEL
[NMOL/LITER) PATIENT 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19
20 21 22 23 24 25
AGE(YR) 82 55 52 55 85 69 53 66 54 48 52 60 67 46 66
82 48 60 84 49 60 50 53 71 46
SD
n
American Journal
of Otolaryngology 346
Patient
Control Subject
7.4 13,1 30.3 15.5 14.0 20.3 15.8
3.3 10.5 8.3 27,3 12.5 23.0 8.3 13.1 14.4 15.7 8.7 9.8 16.0 14.6 5.9 11.8 12.5 11.8 5.9 12.5 11.8 13.1 8.3 5.9 26.4 12.5 5.9
27.7 18.1 17.1 23.4 14.0 22.7 35.4 24.7 6.5 21.8 12.3 5.8 8.7 10.4 20.9 15.7
29.2 17.0 17.9
7.7
= 25; t = 2,89; P < 0.01,
The testosterone/estradiol (T/E) index was determined in 11 male patients with hyperplastic laryngeal aberrations. Estradiol values were not available for four patients. The T/E values of these patients (181 _ 67) when compared with those of their control subjects (153 "+ 99) were not significantly elevated (Wilcoxon's signed ranks test, T = 24; P > 0.1) (Table 4). Plasma testosterone levels were 2.8 -+ 1.5 nmol/liter in women with cancer of the larynx (n = 25) and 1.5 _ 0.6 nmol/liter in the control group. The difference was statistically significant (t = 3.70; P < 0.005) (Table 5). Plasma testosterone concentrations in female patients with hyperplastic aberrations in the laryngeal mucosa (n = 15) were not significantly elevated (2.2 _ 1,5 nmol/liter) in comparison with those of ther matched control subjects (1,8 __ 0,7 nmol/liter (t = 1.08; P > 0.2) (Table 6). No significant differences were found in salivary testosterone levels b e t w e e n the w o m e n with hyperplastic aberrations (n = 6) (150 _ 95 pmol/liter) and their control subjects (218 _ 143 pmol/liter (Wilcoxon's signed ranks test, T = 7; P > 0.1) (Table 7]. Similar results were obtained
CANCER
in the group of male patients with hyperplastic aberrations. The T/E index was also calculated for 12 female patients with hyperplastic aberrations in the laryngeal mucosa. Estradiol values were not available for three patients in this group. Although the values obtained in female patients (63 _+_ 73) were higher than those of their control subjects (37 _+ 53), the differences were not statistically significant (Wilcoxon's signed ranks test, T = 24; P > 0.1) (Table 8). DISCUSSION
A p r o g r e s s i v e l y increasing i n c i d e n c e of head and neck cancer, including carcinoma of the larynx, has been reported. ~,19 According to these reports the exogenous factors, particularly cigarette smoking, are responsible for the increased morbidity in both men and women. Our studies in Yugoslavia indicate that the living and working conditions and lifestyles of both sexes are similar. Differences that do exist w o u l d not account for the striking prevalence of laryngeal cancer or chronic h y p e r p l a s t i c aberrations of the laryngeal mucosa in men. These findings led to the hypothesis that an endogenous factor (elevated concentration of male sex hormones) may affect the laryngeal mucosa by increasing its susceptibility to exogenous cocarcinogens. An increasing incidence of laryngeal carcinoma in women has been documented in our female population as well. However, we beTABLE 3. Plasma Testosterone Levels in Male Patients with Hyperplastic Aberrations in the Laryngeal Mucosa and in Matched Control Subjects PLASMA TESTOSTERONE LEVELS (NMOL/LITER) PATmNT
AGE(YR~
Patient
Control Subject 15.1 13.1 12.5 26,6 14.6 26,4 11.8
1 2 3 4 5 5 7
70 50 51 64 46 46 60
20.5 16.1 14.0 13.1 13,1 11.S 18.2
8 9
65
10,5
5.9
44 43 43 riO 32 60 42
11,3 36.2 21.4 11.3 20.9 8.7 21.4 16.5 7.0
22.9 18.5 11.4 11.8 19.7 5,9 18.5 15.7 6.4
10 11 12 13 14 15 SD
n = 15; t = 0.39; P > 0.5,
KAMBIC ET AL.
lieve that this finding could be attributed in part to an imbalance of sex h o r m o n e s in these women, as well as to their increased cigarette smoking. The current way of life, characterized by stress and the use of contraceptive ~ud therapeutic h o r m o n e preparations by women, is most likely the cause of the hormonal imbalance and resulting disorders. The results of our study are consistent with the hypothesis of increased testosterone levels in male and female patients with laryngeal carcinoma. Free testosterone levels in the saliva of male and female patients with laryngeal carcinoma in our pilot study were also elevated. However, the results were not taken into consideration because of the small quantity and the changed quality of the saliva, most evident in patients receiving postoperative radiotherapy (ten male and ten female patients). No statistically significant differences were found in the plasma salivary testosterone levels or in the T/E index between patients with hyperplastic aberrations of the laryngeal mucosa and their control subjects. However, the wide spectrum of the histologic features in the cases studied should be taken into consideration: the study included patients with simple, abnormal, and atypical hyperplasia, and, among these lesions, only the last can be classified as precancerous. Atypical hyperplasia is very rare and accounts for only 5.4 per cent of all hyperplastic aberrations involving the laryngeal mucosaJ 7 A study of the testosterone levels in the plasma of patients with atypical hyperplasia during a tenyear period is currently underway. Plasma testosterone values are not a diagnostic tool for the classification of hyperplastic larynTABLE 4. Testosterone/Estradiol Index in Male Patients with Hyperplastic Aberrations in the Laryngeal Mucosa and in Matched Control Subjects TESTOSTERONFJEsTRADIOLINDEX PATIENT 1 2 3 4 5 6 7 8 g 10 11 SD
AGE(YR) 50 51 64 46 46 60 65 44 60 60 42
Patient 146 127 164 109 188 260 131 226 103 302 238 181 • 87
Control 131 114 332 264 27 107 42 203 107 88 264 153 • 99
n = 11; T = 24; P > 0.1 (Wilcoxon's signed ranks test).
TABLE 5. Plasma Testosterone Levels in Female Patients with Laryngeal Carcinoma and in Matched
Control Subjects PLASMA TESTOSTERONELEVELS {NMOL/LITER) PATIENT 1 2 3 4 5 6 7 8 g 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
AGE(YR.]
Patient
Contro 1
52 39 76 81 38 74 33 70 70 25 68 69 54 55 59 61 58 48 68 66 58 46
1,0 4.2 5.1 2,8 4.5 4.7 6,1 4,5 ~.8 3.1 1.4 2,1 1.0 2,1 1.4 2.1 3,6 2.1 1,0 %6 2,4 1,7 0,8 1.0 1.2 2,6 1,5
1,4 1.7 1.7 3.2 1,0 1.4 2.7 i.O 1.0 2,4 1.4 1,7 1.4 1.4 1,0 1,7 1,4 0.9 1.3 1,1 1.4 2.4 0.4 1,4 1,4 1.5 0.6
57 68 58
SD n = 25; t = 3,70; P < 0.005,
TABLE 6. P l a s m a Testosterone Levels in Female Patients with Hyperplastic Aberrations in the Laryngeal Mucosa and in Matched Control Subjects PLASMA TESTOSTERONELEVELS {NMOIJLITER) PATIENT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
AGE(YR1
Patient
Control
30 51 49 38 55 47 51 81 52 40 68 70 68 41 62
1,0 1.6 1,2 0.g 2.7 2,4 4,8 2,7 4.1 4.8 1,2 1.1 0,8 3.6 0.6 2.2 1,5
2.3 2.9 1.3 1.5 2.4 1.0 1.1 1.3 2.9 2.1 2,3 1.3 1.7 2,1 1.0 1.8 0.7
SD n = 15; t = 1.08; P > 0.2.
seal aberrations. Still less can they be used to foretell the outcome of treatment. Their role in laryngeal carcinogenesis, however, should be considered. In addition to the various exogenous
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TESTOSTERONE AND LARYNGEAL CANCER
TABLE 7. Salivary Testosterone Levels i n Female Patients with Hyperplastic Aberrations in t h e Laryngeal M u c o s a and in Matched C o n t r o l Subjects
SALIVARYTESTOSTERONELEVELS (P~OL/UTER] PATIENT 1 2 3 4 5 6 SD
AGE(YR)
Patient
Control
30 51 68 51 36 41
86 186 47 262 70 250 150 95
388 308 337 48 131 97 218 143
n = 6; T = 7; P > O.1 (Wilcoxon's signed ranks test].
TABLE 8. Testosterone/Estradiol Index in F e m a l e Patients with Hyperplastic Aberrations in the Laryngeal M u c o s a and in Matched Control S u b j e c t s TESTOSTERONFJESTRADIOL INDEX PATIENT
ACE(YR)
Patient
1 2
30 51
1 160
CONCLUSION
Americon Journal
of Otolaryngology 348
Our investigations demonstrated testosterone levels in patients of both sexes with laryngeal cancer that were significantly higher than those in their healthy counterparts of the same age, habits, and living and working conditions. This is in agreement with the hypothesis that the development of laryngeal carcinoma is related to the amount of circulating testosterone. The action of male sex hormones may increase the susceptibility of the laryngeal mucosa to exogenous factors, which, in connection with a yet obscure (possibly immunobiologic) response of the organism, leads to malignancy after a latency period. The larynx--a target organ for male sex h o r m o n e s - - s e e m s to be d e p e n d e n t on the amount of testosterone, not only in its physio-
33 15
3
49
4
3
4
36
90
10
5 6
41
5
69 22 242
4 2
7 8
55 47 51
9
40
7
4
10 11 12
68 40 70
40 69 42 63 73
130
SD
factors, such as smoking, alcohol consumption, and poor microclimatic living and working conditions, the role of male sex hormones should also be suspected in the pathogenesis of the disease and should receive more attention in furthor research. The possibility of treating patients with laryngeal carcinoma by hormones was discussed by Saez and Sakai. 13 Hussl et al} ~ and Loewit et al. le reported on successful antiandrogenic treatment with a testosterone antagonist in patients with hyperplastic aberrations of the laryngeal mucosa. As our study failed to demonstrate a statistically significant increase in testosterone levels in male and female patients with laryngeal hyperplasia, the theoretic basis of the success of hormonal therapy remains to be clarified.
Control
3 110
130 37 53
n = 12; T = 24; P > 0.1 (Wilcoxon's signed ranks test].
logic development but also in the pathogenesis of disease, including laryngeal carcinoma. Further investigations should be undertaken to identify the role of testosterone in the development of epithelium at risk for cancer (atypical hyperplasia) in the laryngeal mucosa. These studies would also clarify the roles of hormones in the etiology of laryngeal carcinoma.
References 1. Ackerman LV, Del Regato ]A: Cancer--diagnosis, treatment and prognosis. St. Louis, CV Mosby Co, 1970, pp 3 0 8 - 3 2 9 2. Ogura JH, Biller HF: Cysts and tumors of the larynx, in Paparella MM, Shumrick DA (eds): Otolaryngology, vol 3 (Head and Neckl. Phfladilphia, WB Sounders Co, 1973, pp 6 5 8 - 6 6 5 3. Kambi~ V: Otorinolaringologija. Ljubljana, Mladinska knjiga, 1975, p 170 4. M a h r HR, F i k e n t s c h e r R, B r u c h m f i l l e r W: Besonderheiten des w e i b l i c h e n Larynxkarzinoms. Laryingol Rhinol 56:998-1003, 1977 5. Cancer incidence in Slovenia (1970-1979). Cancer registry of S l o v e n i a - - I n s t i t u t e of Oncology, Ljubljana, 1973-1983 6. Kambi~ V: Quelques idles sur los ~tats pr~cancereux du larynx. JFORL 14:827-837, 1965 7. Kambi~ V, Lenart I: Erosio laryngis falsa. Symp Otolaryngol Iug 5:97-101, 1970 8. Kambl~ V: Profesionalna obolenja v otorinolaringologiji. Zbornik Radova VIII. Kongresa Otorinolaringologa ]ugoslavije, SkopJe, 1969, pp 1 7 3 - 1 7 6 9. Kambi~ V, Lenart I'. Untersuchungen fiber die Wirkung y o n T e s t o s t e r o n auf die K e h l k o p f s c h l e i m h a u t des Hundes. HNO 16:327-330, 1968 10. Karnbi~ V, Lenart I, Lenart V, et ah Beitrag zur Frage der Rsversibilit/it bzw: Irreversibilit~lt v a n Gewebsver~ind e r u n g e n , die bei H u n d e n n a c h der i.m. Verabreichung v a n Testosteran an dar laryngealen Schlelmhaut auftreten. HNO 21:300-303, 1973 11. Kambi~ V, Rad.~el Z, ~argi M: V e r ~ n d e r u n g e n an der
KAMBI~ ET AL.
12. 13. 14.
15.
laryngealen M~luseschleimhaut als Folge endogener und exogener Faktoren. HNO 25:249-252, 1977 Hajdutova R, Dimitrova S, Melamed M: Izsledvane na testosterona v krvnija serum na bolni ot rak no larinksa. Onkologia (Sofia) 16:22-26, 1979 Saez S, Sakai F: Androgen receptors of human pharyngolaryngeal mucosa and pharyngo-laryngeal epithelioma. J Steroid Biochem 7:919-921, 1976 Aufdemorte TB, Sheridan PJ, Holt GR: Autoradiographic evidence of sex steroid receptors in laryngeal tissues of the baboon (Papio cynocephalus). Laryngoscope 93:1607-1611, 1983 Hussl B, Loewit K, Richter E, et el: First clinical expe-
16.
17. 18. 19.
riences with hormone-therapy of pachydarmia laryngis. Arch Otolaryngol 221:221-225, 1978 Loewit K, Hussl B, Richter E, et ah Anti-androgen therapy in pachydermia of the female larynx: a new therapeutic possibility. Arch Otolaryngol 215:75-79, 1977 Kambi~ V: Difficulties in management of vocal cord precancerous lesions. J Laryngol Otol 92:305-315, 1978 McGuirt WF: Head and neck cancer in women: a changing profile. Laryngoscope 93:106-107, 1983 Wynder EL, Covey LS, Mabuchi K, et el: Environmental factors in cancer of the larynx. Cancer 38:1591-1601, 1976
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