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I I I I I I I I I I I I I I I I A STUDY OF ASSOCIATION OF FEMALE SQUAMOUS CELL CARCINOMA AND ADENOCARCINOMA IN THE LUNG AND HISTORY OF MENSTRUATION Liao Mei-lin, Wang Jian-hwa, Wang Hwei-min, Ou Ai-qin, Wang Xiao jun and Long Wan-qing Shanghai Chest Hospital, Shanghai, China Abstract 181 cases of female squamous cell carcinoma and adenocarcinoma of the lung and 187 normal female controls were age-matched to compare the menstrual history of the two groups. The Epi-infor program, the Chi-square test, or the Bartlett test for homogeneity of variance, were used to evaluate possible statistical significant differences existing between the two groups. Parameters related to menstrual history included: age of menarche, menstrual cycle, number of days of menstrual period, amount of menstrual flow, menstrual pain, breast bloating/tenderness, and total number of inenstrual cycles prior to menopause or diagnosis of lung cancer. The results show that squamous cell carcinoma cases have a higher total number of menstrual cycles than controls, raising the possibility that estrogen may play a role in the induction of squamous cell carcinoma. Since adenocarcinoma cases were found to have shorter menstrual periods than controls, it may be proposed that activity of the corpus luteum is related to the occurrence of adenocarcinoma. Progesterone (PR) and estrogen (ER) receptor levels were also measured in 21 surgical specimens of adenocarcinomas. A positive ER and PR receptor expression was correlated with later menarche and earlier menopause. Biological implications of these findings must be further investigated. Introduction The association of smoking and lung cancer is well known. According to a 1989 World Health Organization report, over 97% of the nations of the world held the view that a history of smoking probably accounted for 80% of the lung cancer cases, especially in the ones involving squamous and small cell carcinomas(1). On the other hand, with regard to the etiology of lung adenocarcinoma, risk factors other than smoking have been proposed and identified, especially in nonsmoking females. A 1988 Shanghai population-based epidemiologic study reported that the development of female lung adenocarcinoma was associated with menstrual and reproductive history, thus raising the possibility that female lung cancer may be related to female sex hormones. The present study compares data on menstrual history using female lung cancer cases in the Shanghai Chest Hospital and controls drawn from healthy current or retired hospital employees. A detailed questionnaire was administered in order to obtain information on menstrual history. The data were analyzed to determine whether an association possibly exists between menstrual history and lung cancer in females. Our results may contribute to a better understanding of the etiology of squamous cell carcinoma and adenocarcinoma of the lung. Materials and Methods N O co ' From January 1993 to June 1994, the files of all the in-patients and part of the out-patients of the j Chest Hospital were randomly screened. Females, between 37-77 years of age, with cytologically and ~ histologically confirmed lung squamous cell carcinoma and adenocarcinoma were identified and used as V ~ tb ,

cases. These included 22 surgical cases in which the surgically-resected specimens were also analyzed for female sex hormone (progesterone (PR) and estrogen (ER)) receptors. Controls, aged 35-78, were drawn from healthy current and retired employees who were determined by physical examination to be free of tumors or other serious disease. Controls were matched for age (± 5 years) with cases. Standardized questionnaires were filled out by trained professionals for both groups. Inquiries were made concerning the following items: age of inenarche; menstrual cycle (in days); length of menstrual periods; whether menstrual flow was heavy (as to affect physical activities), medium (without affecting activities, although the amount was not scanty), or light (basically no awareness that there was a flow); degree of premenstrual breast bloating/tenderness (scored as never, occasional, slight, medium or strong); and menstrual pain (scored as never, occasional, light, medium or strong). Because the ages of menarche and menopause do not completely show the cyclic nature of endocrine functions/changes in females, a formula was used to calculate the menstrual cycle index (MCI) according to the following equation: MCI = (age at which menopause occurs or age at which lung cancer was diagnosed - age of menarche) x 365 + menstrual cycle (in days). The data, from the three groups, i.e., squamous cell carcinoma, adenocarcinoma, and controls, were entered into Fox Base and then analyzed for statistical significance by using the Epi-infor software, or by applying the chi-square (XI) test, or the test for homogeneity of variances according to Bartlett. Data which showed P < 0.05 in the Bartlett test, hence indicative of an abnormal distribution of data points, were further analyzed for statistical significance by the Kruskal-Wallis H value method. These data giving a P of greater than 0.05 in the Bartlett test, and hence indicative of a normal distribution, were evaluated for statistical significance by the ANOVA F method. Results A total of 368 females were analyzed. These included 181 lung cancer cases (19 squamous cell carcinoma and 162 adenocarcinoma) and 187 controls. Comparison of Menstrual History Between Squamous Cell Carcinoma Cases and Controls. The ages of both groups showed a similar normal distribution and there was no statistically significant differences between the two groups (P > 0.05). With regard to menstrual history, no difference was found on age of menarche, length of menstrual period, menstrual cycle, degree of premenstrual breast bloating/tenderness, menstrual flow, menstrual pain, and age of menopause (each with P > 0,05). However, when the total number of menstrual cycles prior to menopause or to diagnosis of lung cancer (MCI) was tested for homogeneity of variance, a mean value of 453.17 was obtained for cases, compared to 413.3 for controls. Since the homogeneity of variances test showed normal distribution of data points, the two groups were evaluated for statistical significance by ANOVA. A P value of less than 0.05 was obtained indicating that squamous cell carcinoma cases have a higher total number of menstrual cycles than the controls. 2. Comparison of Menstrual History Between Adenocarcinoma Cases and Controls. The ages of both groups also showed a normal distribution (P > 0.05). The adenocarcinoma cases, however, had shorter menstrual periods (mean = 4.85 days) than the controls (mean = 5.29 days) with a medium value of 5 days. When these values were tested for homogeneity of variance, a P value of less than 0.005 was obtained indicating high statistical significance. In addition, the frequency/severity -2- I I I I I I , I I I I I I I I I I I I

a I I I I I I I I I I I I I I I I I of breast bloating/tendemess was also significantly different between these two groups (variance = 30.84, degree of freedom = 1, P = 0.000032). Specifically, the adenocarcinoma group had only slight and infrequent "breast bloating/tenderness" and history of menstrual pain. Likewise, statistically significant differences were also found between the two groups with regard to the amount of menstrual flow (P < 0.005). No apparent differences were found in the total number of menstrual cycles, age of menopause and age of menarche between the two groups. (Table 1) 3. Comparison of Menstrual History Between Squamous Cell Carcinoma and Adenocarcinoma Cases. There is no difference between the age of the two groups. The mean menarche ages were 14.79 and 15.488 respectively (P < 0.05), suggesting an earlier menstrual onset for squamous cell carcinoma cases compared to the adenocarcinoma cases. There was no apparent difference in the age of menopause, the length of menstrual period, menstrual cycle, the amount of menstrual flow, and menstrual pain between the two groups. The total number of menstrual cycles was higher for squamous cell carcinoma cases (453.2) than for adenocarcinoma cases (413.3). The difference was statistically significant (P < 0.05). (Table 1) Table 1. Relationship Between History of Menstruation and Female Pulmonary Squamous Cell Carcinoma and Adenocarcinoma Ademcutivmu' C.nmot P. VaLe . F,H P F,H P F,H P Curu 19 181 187 A8e ef inemrche 14.5'/9 15.488 15.3n5 6.085 <0.05 1.858 >0.05 2.197 >0.05 MeemW cycle 27.895 28.895 28.802 1.369 >0.05 0.211 >0.05 1.602 >0.05 Lrngih of 5.368 4.852 5.289 3.211 >0.05 0.053 >0.05 8.703 <0.01 AgeofMrnppwae 50.500 49.273 48.774 1.672 >0.05 2.436 >0.05 1.025 >0.05 Nmnber of IaW 453.176 413.319 413.269 5.014 <0.05 4.818 >0.05 0 >0.05 Amawr m Light 2 26 21 Hary 0 38 52 Mad'~ 17 98 114 Pmnevwal Never 10 104 110 Occas1® 5 35 13 LigM 2 20 42 Medvm 1 1 9 Huvy 1 2 13 Meuswil Pam Nevu Il 108 125 Occasica 5 29 13 L~c I 1a 29 Med'oun 1 3 7 Hary 'Number of life-time meustrual cycles = [(age of menopause age of inenarche) x3651 meastioal cycle (in days) -3- I

I 4. Analysis of Estrogen and Progesterone Receptors and History of Menstruation in the 22 Surgical Cases of Female Squamous Cell Carcinoma and Adenocarcinoma. Of the 22 surgical cases analyzed, 21 were adenocarcinomas and only one had squamous cell carcinoma. Positive estrogen receptors (ER) were found in 16 cases (72.7% of total cases), 15 of which were adenocarcinoma and 1 was squamous cell carcinoma. Fourteen cases (representing 63.6% of total cases and 66.6% of adenocarcinoma cases), all adenocarcinoma were also found to be positive for progesterone receptors (PR). The relationship between sex hormones and the history of menstruation is illustrated in Table 2. Table 2. A Comparison of Menstrual Histories of 21 Female Adenocarcinoma Cases with Positive and Negative of Hormone Receptors +. No. EM01- ~ S' C. 15 71.4 6 2A.6 14 66.6 7 33.4 Meruche (Age) 15.1 13.8 <0.05 14.2 <0.05 Numbv af D.y, of Memuv.l Pulod 5 5 >0.05 5 5 >0.05 MemuW CSCIe(Days) 28.0 28.6 <0.05 28.1 2&4 <0.05 Menywtt (Aye) 45.7 48.4 <0.05 47.3 49.8 <0.05 Adenocarcinoma cases with positive ER and PR had later menarche than those with negative ER and PR, P < 0.05. The age at which menopause occurred was also earlier for the adenocarcinoma cases with positive ER and PR than those with negative ER and PR, P < 0.05. There was no difference in the number of days of the menstrual period or menstrual cycle, and no difference in breast bloating/tenderness, menstrual pain and amount of menstrual flow. Discussion In attempting to understand the etiology of lung cancer in females, an effort has been made to study risk factors other than smoking. For example, deficiency of vitamin A being associated with the occurrence of lung cancer has been supported both by epidemiological research and in animal experiments. Many studies in China have explored nonsmoking risk factors, such as cooking fumes(3), history of tuberculosis(4), burning of coal(5), and menstrual history(2). This study is a statistical analysis of the menstrual history of 181 female lung squamous cell carcinoma and adenocarcinoma cases and 187 age-matched normal female controls. The purpose was to find out whether similarities or differences existed in the menstrual history between the squamous cell carcinoma and adenocarcinoma cases and whether the occurrence of female lung squamous cell carcinoma/adenocarcinoma is related to the status of sex hormones. The results of our study show that the total number of menstrual cycles prior to menopause or before lung cancer was diagnosed were -4- I I I I I , I I I I I I I I

I I I I I I I I I I I I I I I I I I significantly higher for squamous cell carcinoma cases (453) than for controls (417). Other parameters related to menstruation showed no appreciable differences between the two groups. A greater number of menstrual cycles could imply a greater frequency of elevated estrogen levels. Since estrogens are known to stimulate growth of cells, especially the epithelial cells, they may be a candidate for induction of squamous cell carcinoma. On the other hand, the total number of life-time menstrual cycles for the adenocarcinoma cases were not found to be higher than the controls. In fact, adenocarcinoma cases showing positive ERs and PRs actually had a later menarche and an earlier menopause. These observations suggest that sex hormone, as well as levels and functions of receptors may be involved in controlling the growth of lung cells. Our study found that the 162 adenocarcinoma cases had shorter menstrual periods than controls. The length of the menstrual period is often related to the regularity of the follicular cycles which in turn depends on the functions of the corpus luteum. A hyper-active corpus luteum function, for example, would shorten menstrual periods. Accordingly, it may be hypothesized that adenocarcinoma patients have a more active corpus luteum, compared to the controls. In conclusion, studies of female lung cancer risk factors must incorporate measurement of sex hormone levels, and assay of progesterone/estrogen receptor expression. Moreover, considerations must be given to the interrelationship of the hypothalamus-pituitary-ovary axis. Further case-control studies integrating assessment of endocrinological parameters and epidemiologic approaches are needed in order to understand the significance of sex hormone levels and receptor functions on female lung cancer. -5- ,

References 1. Stjernsward, J.; Stanley, K.; "Etiology, Epidemiology and Prevention," Lung Cancer 4 (Supp.): 11-14, 1988. 2. Zheng, W.; Gao, Y.T.; Sun, L.; "A Study of the Association of Menstrual History and Lung Cancer," Tumor 8(3): 150-152, 1988. 3. Gao, Yu-Tong; Blot, W.J.; Zheng, W., et al.; "Lung Cancer Among Chinese Women," Int_J. Cancer 40: 604-609, 1987. 4. Zheng, W.; Blot, W.J.; Liao, M.L., et al.; "Lung Cancer and Prior Tuberculosis Infection in Shanghai," Br. J. Cancer 56(4): 501-504, 1987. 5. Du, Y.X.; "Progress in Lung Cancer Prevention Research in Guangzhou," First Seminar of Guangzhou Research Center for Lung Cancer, Guangzhou, China, May 10, 1985. -6- I I I I I I I I I I I I I I I 1 I I I