Gender Differences in p53 Mutational Status in Small Cell Lung Cancer¹

Lung cancer is the leading cause of cancer death in both men and women, with an estimated incidence of 171,600 new cases of lung cancer in the United States in 1999, and an estimated 158,900 deaths (1). The rate of rise in the incidence of lung cancer in women has increased rapidly; in the 30-year period ending in 1985, there was a 396% increase in the incidence of lung cancer in women compared with a 161% increase in men (2). Lung cancer mortality in men decreased at the rate of 1.6% per year between 1990 to 1995 (1), whereas the mortality rate of lung cancer in women increased by 1.7% per year between 1990 and 1994 (3). SCLC³ comprises 15–25% of lung cancer cases in the United States (2, 4). Thompson (5) recently reported that a significantly higher proportion of females with lung cancer present with small cell histology compared with men. This gender difference was especially striking in the younger age group: 34% of females <65 years of age with lung cancer had SCLC compared with 18% of men (5). Although this type of tumor is highly sensitive to cytotoxic chemotherapy, with response rates of 80–90% (6), the duration of responses remains short. Accordingly, 2-year survival rates for patients with extensive disease remain <5%, whereas 5-year survival for patients with limited disease is in the 15–20% range (6).

Mutations in the p53 tumor suppressor gene are one of the most frequently found genetic abnormalities in human cancers. The p53 gene has been found to be mutated in a significantly higher proportion of SCLC compared with NSCLC (70% versus 47%, respectively, in previous series; Refs. 7, 8, 9, 10, 11, 12, 13). However, this conclusion was mainly based on data derived from cell lines or biased gender populations. To determine the mutational frequencies in the p53 tumor suppressor gene and any potential differences between gender, we analyzed primary SCLC tumors from 65 patients (38 males and 27 females) by direct sequencing of the p53 gene between exons 5 and 9. We found that 57% (37 of 65) of the tumors exhibited at least one mutation within the region of the p53 gene analyzed with a total of 42 mutations. Interestingly, there was a trend toward a lower frequency of mutations in the p53 gene in female tumors compared with male tumors.

The study population consisted of 65 patients (37 males and 28 females) with SCLC who were identified on the basis of tissue blocks available from autopsy, biopsy, or surgery at the M. D. Anderson Cancer Center between 1975 and 1995. Corresponding control tissue consisted of kidney or spleens without evidence of tumor infiltration. Analysis of p53 mutational status was performed blinded to clinical outcome of patients. Clinical information was subsequently obtained by retrospective chart review for all patients.

Paraffin-embedded tissue blocks from primary lung tumor samples and control tissues from kidneys or spleens were sectioned using a microtome. A 4-μm section from each block was stained with H&E and reviewed by pathologists to confirm the diagnosis and to locate tumor areas. Tumors were dissected with a scalpel under a stereomicroscope such that tumor cells comprised at least 70% of the cells. Normal control tissues were dissected using the same method. DNA was digested in 200 μl of 50 mm Tris-HCl (pH 8.0) containing 1% SDS-proteinase K and incubated at 42°C for 24 h. DNA was purified using phenol-chloroform extraction, followed by sodium perchloride precipitation.

Individual exons (5, 6, 7, 8, 9) of the p53 gene were amplified separately. The primers used for amplifying exons were as follows: 5′ fragment of exon 5 (181 bp); sense primer 5′-CTTCCAGTTGCTTTATCTG-3′; antisense primer 5′-TCATGTGCTGTGACTGCTTG-3′; 3′ fragment of exon 5 (237 bp): sense primer 5′-CAACAAGATGTTTTGCCAAC-3′; antisense primer 5′-TGAGGAATCAGAGGCCTGG-3′; exon 6 (265 bp), sense primer 5′-CTGCTCAGATAGCGATGG-3′; antisense primer 5′ -CACTGACAACCACCCTTAAC-3′; exon 7 (215 bp): sense primer 5′ -CCAAGGCGCACTGGCCTC-3′; antisense primer 5′ -GAGGCAAGCAGAGGCTGG-3′; exon 8 (257 bp): sense primer 5′ -ACCTGATTTCCTTACTGCC-3′; antisense primer 5′ -GGTGATAAAAGTGAATCTGAG-3′; exon 9 (176 bp): sense primer 5′ -CAAGGGTGCAGTTATGCC-3′; antisense primer 5′ -CTGGAAACTTTCCACTTGAT-3′. Sequencing primers were as follows: 5′ fragment of exon 5: 5′-TTCACTTGTGCCCTGACTT-3′; 3′ fragment: 5′-AACCAGCCCTGTCGTCTC-3′; exon 6: 5′ -GAGACGACAGGGCTGGTT-3′; exon 7: 5′-CACAGCAGGCCAGTGTGC-3′; exon 8: 5′-TGAATCTGAGGCATAACTGC-3′; exon 9: 5′ -CTGGAAACTTTCCACTTGAT-3′.

Each PCR reaction contained 10% DMSO, 6.7 mm MgCl₂, 1.5 mm dNTP, 6 ng/μl of each primer, 0.05 units of Taq polymerase (Life Technologies, Grand Island, NY), and ∼0.8 ng/μl template DNA in a final volume of 50 μl. Thermocycling was performed in a Hybaid thermocycler; amplified DNA was then subjected to cycle sequencing in the presence of [γ-³³P] ATP-labeled primer using the Amplicycle sequencing kit (Perkin-Elmer, Branchburg, NJ) according to the manufacturer’s protocol. PCR products were run on a 6% Long-Range sequencing gel (FMC BioProducts, Rockland, ME) and exposed to film.

Survival curves were estimated by the Kaplan-Meier method, and the resulting curves were compared using the log-rank test. P < 0.05 was considered statistically significant. Statistical analysis of the association between two categorical variables was performed using Fisher’s exact test for numbers 5 and the χ² test for numbers 6. Statistical analysis was performed using the SAS statistical package (SAS Institute, Cary, NC).

Clinical features of the study population are detailed in Table 1. Of the 65 patients with SCLC included in this series, 28 were female and 37 were male. The mean age of this study population was 58.9 years, with a range of 37–80 years. There was no significant difference between males and females in this series in mean age, race, or response to chemotherapy. There was a trend toward greater tobacco exposure in males compared with females (72 pack-years versus 59 pack-years, respectively); however, this difference was not statistically significant (P = 0.33). Similarly, there was a nonsignificant trend toward better performance status at diagnosis in females compared with males (P = 0.16). The only clinical variable in this series that was significantly associated with gender was stage of disease, with a higher proportion of females presenting with limited stage disease at the time of diagnosis (P = 0.04). The association between alcohol consumption and gender was of borderline statistical significance, with a higher proportion of males reported to have heavy alcohol consumption than females (37% versus 13%, respectively; P = 0.06).

A total of 65 primary tumors were analyzed for p53 mutations by direct sequencing of exons 5–9. Thirty-seven (57%) of the 65 tumors were found to have one or more mutations, with a total of 42 mutations (Table 2). Four tumors had more than one mutation, including one with three mutations. Among 28 tumors from females, 13 (46%) contained p53 mutations, compared with 24 (65%) of 37 tumors from males (P = 0.14). Furthermore, tumors from 4 (17%) of the 24 males had more than one mutation in the p53 gene, compared with none of the tumors from females.

Mutations were demonstrated throughout all exons analyzed; however, the majority of mutations (34 of 42; 81%) were within exons 5–7. The location of mutations by exon were as follows: 11 of 42 (26%) of the total mutations were located in exon 5; 12 of 42 (29%) were in exon 6; 11 of 42 (26%) were in exon 7; 5 of 42 (12%) were in exon 8; and 1 of 42 (2%) was within exon 9.

Thirty-one of 42 mutations (74%) were missense mutations resulting in the substitution of one amino acid for another. Two of 42 mutations were silent mutations. A double mutation was found in one tumor consisting of nucleotide substitutions for the second and third bases in the same codon. The substitution of the third base alone would have resulted in a silent mutation; thus it was the substitution of the second bp that resulted in a functionally important missense mutation. Two of 42 (5%) mutations were located at intron/exon boundaries, which might have affected RNA splicing. Two of 42 mutations (5%) were nonsense mutations resulting in the substitution of a termination codon. Four of 42 mutations (10%) were small deletions or insertions causing frameshifts in coding for the p53 protein. One of 41 (2%) was a deletion that could cause a five-amino acid deletion and a splicing abnormality. All of these mutations were confirmed to be present in tumor tissue only and not in the corresponding constitutional DNA.

The pattern of nucleotide substitutions in this series of SCLC tumors is shown in Fig. 1. The most common type of nucleotide substitution in both males and females was the A:T→G:C transversion, comprising 28% of all tumors, 23% of primary tumors in females, and 35% of tumors in males. The second most frequent nucleotide substitution overall was the G:C→T:A transversion, comprising 17% of all tumors and 20% of tumors in males but only 8% of tumors in females. In females, G:C→A:T transitions and A:T→T:A transversions were both more common than G:C→T:A transversions, each comprising 15% of the total. Other types of nucleotide substitutions were present in <10% of tumors. These differences in the patterns of nucleotide substitutions in males and females did not reach statistical significance.

The association between p53 mutational status and a number of clinical features are shown in Table 3. There was no significant association between p53 mutational status and survival, age, race, cumulative tobacco exposure, and response to chemotherapy. There was a nonsignificant trend toward a higher frequency of p53 mutations in patients with extensive disease stage (P = 0.20), heavy alcohol consumption (P = 0.45), and worse performance status (P = 0.07). There was no significant difference in overall survival between patients with p53 mutations and those without p53 mutations (P = 0.23). After adjusting for disease stage, there was no significant association between gender and frequency of p53 mutation by the Mantel-Haenszel test.

To our knowledge, this is the largest study to date analyzing p53 mutations in SCLC primary tumors that has included a significant number of female patients with SCLC. The mutational frequency (57%) in our series was slightly lower than that reported in other studies of primary SCLC, which range from 60 to 80% (7, 8,11, 13, 14). Possible explanations for the difference between our results and those of previous studies in SCLC include smaller sample sizes of previous studies; these differences included a smaller sample size (≤28 SCLC primary tumors), biased gender populations in previous studies, analysis of codons outside of exons 5–9 in some studies, and inclusion of tumor cell lines in some previous studies (7, 8, 11, 13, 14).

The spectrum of nucleotide substitutions in SCLC primary tumors has been demonstrated to be more diverse than that in NSCLC primary tumors (12). In our study, A:T→G:C transitions were the most common mutation (28% of all mutations), followed by G:C→T:A transversions (17%) and G:C→A:T transitions (12%). In a review of p53 mutations in 92 SCLC cell lines and primary tumors, Greenblatt et al. (12) found that G:C→T:A transversions comprised 46% of the mutations, G:C→A:T transitions comprised 21% of the mutations, and A:T→G:C comprised 9% of the mutations. In another report, however, A:T→G:C substitutions were the second most commonly identified abnormality, occurring in 33% of the tumors analyzed (7), a frequency close to that in our study. Although mutational “hotspots” in the p53 gene have been identified in NSCLC (7, 12, 15), we and others (13, 14) did not identify specific mutational hotspots of p53 mutations in SCLC tumors. It is well established that the type of mutations reflects the type of mutagens involved (7). G:C→T:A transversions are associated with benzo[a]pyrene (16, 17, 18, 19), whereas A:T→G:C transitions may be induced by N-ethyl-N-nitrosourea (20, 21, 22). Interestingly, a high rate of A:T→G:C transitions in SCLC previously has been demonstrated only in a Japanese population. Multiple explanations have been suggested for the distinct nucleotide substitution patterns seen in the Japanese SCLC population compared with United States and United Kingdom populations (7). It has been postulated that Japanese patients may have a higher level of exposure to certain carcinogens that can cause A:T→G:C transitions, or that Japanese may be more sensitive to certain carcinogens than their American or British counterparts (7). Our study is the first to demonstrate that A:T→G:C transitions in the p53 gene occur frequently in an American population with SCLC. Thus, it may be that patients in our study have been exposed to or are susceptible to carcinogens, such as N-ethyl-N-nitrosourea, similar to those of Japanese patients included in the study by Takahashi et al. (7).

We have demonstrated a trend toward a lower frequency of p53 mutations in primary SCLC tumors of females compared with males, suggesting that males with SCLC may be more susceptible to certain carcinogens in tobacco smoke that preferentially induce mutations in p53. This difference is intriguing because it may partially explain biological differences in tumors of males and females with SCLC. A similar trend has been demonstrated in patients with NSCLC: Kure et al. (23) previously found a trend toward a higher frequency of p53 mutations in males with NSCLC compared with females (53% versus 36%, respectively).

Women with lung cancer are more likely to present with small cell histology compared with men; however, women with SCLC have been well documented to have improved survival compared with men with SCLC (2, 24, 25, 26). The biological reasons for these differences between males and females with SCLC have not been well elucidated. Thompson (5) in a recent analysis of 1044 patients with lung cancer reported that a significantly higher proportion of females with lung cancer present had small cell histology compared with men. This gender difference was more striking in younger patients: 34% of females <65 years of age with lung cancer had SCLC compared with 18% of men (5). Furthermore, Ferguson et al. (2) found that women with SCLC were significantly younger than men at the time of diagnosis, with a mean age of 57.4 years compared with 60.2 years for men. Additionally, cumulative cigarette consumption was significantly lower in women with SCLC than in men with SCLC (2).

It has long been postulated that women are more susceptible to the effects of carcinogens in tobacco than are men (27). Zang and Wynder (28) demonstrated a higher risk of lung cancer in females when controlled for cumulative tobacco exposure. In a case-control study, Zang and Wynder (28) found that the dose-response odds ratio for the development of lung cancer was ∼1.5-fold higher in women than in men for SCLC, squamous cell, and adenocarcinoma subtypes. Potential biological mechanisms for this gender difference include differences in nicotine metabolism, gender differences in cytochrome P-450 enzymes, and hormonal influences on tumor development (28).

The association between alcohol consumption and lung cancer risk is controversial. Numerous case-control studies and prospective studies have demonstrated an association between alcohol intake and lung cancer risk, whereas other studies have not found an association (29, 30, 31, 32, 34, 35, 36). In our study, a higher proportion of males were reported to have heavy alcohol consumption than females (37% versus 13%, respectively; P = 0.06). Furthermore, the proportion of patients whose tumors had p53 mutations was slightly higher in patients with a history of heavy alcohol consumption compared with those without a history of heavy alcohol consumption (64% versus 53%), but this difference was not statistically significant (P = 0.45)

Paradoxically, although women appear to be at higher risk for the development of SCLC when controlled for tobacco exposure and other prognostic variables, multiple studies have demonstrated that women with SCLC had superior survival compared with men with SCLC (2, 24, 25, 26, 37). Moreover, among patients with limited disease, females have a significantly higher rate of complete response than males (26). Given the increased risk for the development of SCLC in females, the improved survival of females with SCLC is somewhat surprising. The lower frequency of p53 mutations in SCLC tumors from females may be one possible mechanism for this improved survival. However, our study was unable to demonstrate such an association between p53 mutation status and survival.

In summary, we have demonstrated a trend toward lower frequency of mutations in the p53 gene in primary SCLC tumors from females compared with tumors from males, suggesting the presence of distinct biological mechanisms of development of female SCLC compared with male SCLC. The distinct mutational spectrum in SCLC in this series suggests a complex influence of various carcinogens in tobacco smoke.