The enzymatic conversion of cholesterol to the estrogens 17β-estradiol and estrone is a multistep process catalyzed by cytochrome P
450 (CYP), 3β-hydroxysteroid dehydrogenase (HSD3B), and 17β-hydroxysteroid dehydrogenase (HSD17B) family members (), primarily occurring in the granulosa cells of the ovarian follicles in premenopausal women (19
). In contrast, the primary source of estrogen in postmenopausal women is provided by the conversion of androgens into estrogen by aromatase (CYP19) in adipose tissue ().
Fig. 1 Schematic representation of estrogen biosynthesis and metabolism. The biosynthesis (solid boxes) of estrone and estradiol from cholesterol and their subsequent metabolism (dashed boxes) are sequential processes catalyzed by the action of a number of enzymes (more ...)
Estrogen metabolism is a multistep process beginning with CYP1A1-mediated hydroxylation at the 2- or 4-position to produce 2- or 4-hydroxy cathechol estrogens that undergo further oxidation to quinones and semiquinones, respectively (20
). Quinones are reportedly mutagenic, being capable of forming stable DNA adducts or depurinating adducts. The inactivation of estrogens, cathechol estrogens, quinones, and semiquinones is achieved by methylation and subsequent sulfation and conjugation, catalyzed by cathecol-O
-methyl-transferase (COMT), the glutathione S
-transferase (GST) superfamily, and sulfotransferases, respectively (; ref. 19
). Among the enzymes participating in estrogen biosynthesis and metabolism, several have naturally occurring allozymatic variants that, in some instances, exhibit differential catalytic activity (21
). Thus, the genotype of an individual may influence overall tissue estrogen levels. Furthermore, significant interethnic differences in allele frequencies between Caucasian and East Asian populations have been documented for a number of variants (20
To test for an association between polymorphic variants in genes influencing estrogen biosynthesis and metabolism and the prevalence of EGFR-mutant NSCLCs, we genotyped the CYP1A1*2A−6235T/C, CYP1A1*2CIle462Val, CYP17A1−34T/C, CYP19A1
Gly312Ser, COMTVal158Met, GSTM1, and GSTT1 allelic variants within a series of 100 NSCLCs from Japan (). These specific variants were chosen for analysis because they encode allozymes with reported differential functional activity and/or ethnic distributions among Caucasian and East Asian populations (). We focused our analysis on NSCLCs from Japan because the higher frequency of EGFR-mutant lung cancer in East Asia would enhance detection of any bias in the prevalence of these polymorphisms between cases with or without EGFR mutations. NSCLC cases were preselected for equal representation of EGFR-wt and EGFR-mutant genotypes and cases arising in males and females.
Frequency and differential activity of allele variants controlling estrogen biosynthesis and metabolism
Overall, our study cohort seemed to be representative of the Japanese population based on a comparison of the allele frequencies of the variants analyzed here with the reported allele frequencies ().
A comparison of polymorphic genotype distributions between EGFR-wt and EGFR-mutant NSCLC cases revealed a difference for only one estrogen-related variant, CYP1A1*2C (rs1048943; ). We observed an underrepresentation of CYP1A1*2C heterozygotes (Ile/Val) among EGFR-mutant cases as compared with EGFR-wt cases, although this did not reach statistical significance (27% versus 47%; P = 0.08, two-tailed Fisher’s exact test). We also detected a concurrent overrepresentation of CYP1A1*2C Ile/Ile homozygotes among EGFR-mutant cases compared with EGFR-wt cases, although this, too, did not reach statistical significance (69% versus 51%, P = 0.13). The CYP1A1*2C Val genotype (minor G allele) is more prevalent in the East Asian population compared with the Caucasian population () and has been linked to increased production of the 2-hydroxycatecholestrogen metabolite. Thus, whereas interethnic prevalence might favor an association between the CYP1A1*2C Val genotype and EGFR-mutant NSCLC, our analysis of Japanese tumors, in fact, suggests that the CYP1A1*2C Ile genotype was more frequently observed in EGFR-mutant cases. Breakdown of the analysis among male versus female cases did not further support a bias in favor of either CYP1A1*2C variant by sex, with both males and females displaying the trends observed in the combined group (). We also observed a trend toward an increase (35-57%) in the frequency of CYP19A1(TTTA)7 homozygotes in EGFR-mutant female cases compared with wt female cases, although this did not reach statistical significance (). A similar trend for CYP19A1(TTTA)7 was not observed in males. The CYP19A1(TTTA)7 allele occurs at higher frequency in Asian than non-Asian populations (0.69 versus 0.49; ); however, to our knowledge, there is currently no known effect of repeat length of this intronic polymorphism on aromatase activity. No other polymorphism tested, by itself suggested a significant difference in prevalence between Japanese EGFR-mutant and EGFR-wt NSCLC. We note, however, that estrogen levels are modulated by the concerted action of a number of enzymes, and that the limited number of samples prevented analysis of combinatorial associations of all genotypes with respect to EGFR status and sex. In theory, an analysis of 50 wt cases and 50 mutant cases has 80% power to detect differences of ≥28%. Accordingly, larger studies will be required to confirm our observations.
Genotype of NSCLCs by EGFR mutation status and sex
In conclusion, our findings suggest that the selected functionally and/or ethnically distinct variants in the estrogen biosynthesis and metabolism pathways are unlikely to be major genetic determinants of the coincident sex and ethnic bias of EGFR
mutations in NSCLC. Our analysis sets the stage for larger population-based studies aimed at defining genome-wide associations that may underlie the genetic contributions toward the strikingly coincident ethnic and sex bias in the prevalence of EGFR
-mutant lung cancer (10