Racial Disparity in the Frequency of PTEN Mutations, but not Microsatellite Instability, in Advanced Endometrial Cancers

There is a significant racial disparity in survival among women in the United States with endometrial adenocarcinoma. Five-year survival in the 1989–1994 review by the National Cancer Institute was 86% in whites compared with only 54% in African-Americans (1). Likewise, among 215 patients treated at Duke University Medical Center from 1990 to 1993, survival of African Americans was 20% worse (2). Although survival of African Americans is relatively poor in several types of cancer, the racial disparity in endometrial cancer survival is the greatest seen among all cancers in the Surveillance, Epidemiology, and End Results registry (1).

The etiology of the racial disparity in endometrial cancer incidence and survival has been elucidated to some extent. First, it has been shown that differences in hormone use contribute to the racial disparity in endometrial cancer survival (3). Unopposed estrogen replacement therapy increases the risk of endometrial cancer and is more often prescribed to white women; but because estrogen-induced cancers usually are well differentiated, hormone replacement contributes to more favorable survival in whites. A racial disparity in survival persists after correcting for hormone use,however (2).

The poor outcome in African Americans with endometrial cancer also can be attributed in part to the higher frequency of unfavorable prognostic features (4, 5). Their cancers are more often poorly differentiated, deeply invasive, nonendometrioid, and advanced stage at diagnosis. Although it has been suggested that delayed diagnosis in African Americans might explain their more frequent presentation at an advanced stage, we found that there was no racial disparity in the interval from onset of abnormal uterine bleeding to hysterectomy for endometrial cancer (2). In addition, there was no difference in the intensity of treatment received by African Americans and Caucasians. This suggests that the worse prognosis of African Americans might be attributable to differences in the underlying molecular pathogenesis of the disease.

Recently, some of the genetic alterations involved in the development of endometrial cancer have been elucidated. Overexpression of p53 protein as a result of mutations in the TP53 gene occurs in about 20% of cases and is associated with nonendometrioid histology,advanced stage, and poor outcome (6, 7). We observed p53 overexpression much more frequently in advanced stage cases in which metastatic disease was present (41%) relative to early stage cancers that were confined to the uterus (9%) (8, 9). The incidence of p53 overexpression was strikingly higher in African Americans than whites in both early (10) and advanced stage cases, (11) suggesting that this might contribute to the racial disparity in outcome. Even among advanced stage cases with p53 overexpression, survival of African Americans was worse than that of Caucasians, however (11). This suggests that factors other than p53 also contribute to the racial disparity in outcome.

The PTEN tumor suppressor gene has been found to be mutated in ∼30–40% of endometrial cancers; most frequently in early stage cases (12, 13, 14). In addition, mutations in microsatellite sequences (microsatellite instability) occur in ∼20% of cases (15). There is evidence to suggest that microsatellite instability in endometrial cancers is attributable to methylation of the promoter region of the MLH1 gene (16, 17),rather than to mutations in this or other DNA repair genes (18). Unlike TP53 mutation, which is associated with aggressive nonendometrioid cancers (6, 7, 8, 9), PTEN mutations and microsatellite instability are characteristic of more favorable endometrioid lesions (12, 19). The aim of this study was to determine whether there are racial differences in the incidence of these genetic alterations in advanced endometrial cancers that might contribute to the disparity in outcome.

Snap-frozen tissue samples or paraffin blocks were available from 140 women (78 Caucasian, 62 African American) who underwent hysterectomy for endometrial adenocarcinoma between 1975 and 1997. When paraffin blocks were used, 10 5-μm thick sections of tissue were prepared and dissected to optimize the yield of cancer cells relative to normal cells. Frozen tissue samples were obtained from large tumor masses composed almost entirely of cancer cells.

There were 42 (30%) stage IIIA cases with positive pelvic peritoneal cytology (31) or adnexal metastases (11), 2 (1.4%) stage IIIB cases with vaginal metastases, 47 (33.6%) stage IIIC cases with pelvic and/or aortic lymph node metastases, 2 (1.4%) stage IVA cases with extension of disease to the rectum or bladder, and 47 (33.6%) stage IVB cases with i.p. or other distant spread. Complete clinicopathological information and survival data were abstracted from the hospital records. All of the histological material had been reviewed initially by gynecological pathologists at our institution.

We had previously performed mutational analysis of the PTENgene in 66 advanced stage endometrial cancers (19), and for the present study, an additional 74 cases were examined. Eleven primer pairs were used to individually amplify the nine exons and intronic splice sites of the PTEN gene as previously described (13). PCRs consisted of 1–10 ng of genomic DNA;200 μm each of dGTP, dCTP, and dTTP; 20μ m dATP; 10× Taq buffer; 1 unit of Taq DNA polymerase, 0.1 μl of (δ³²P) dATP; and 1.0μ m of each primer in a reaction volume of 10μl. Amplification was then performed using a Perkin-Elmer Cetus 9700 thermocycler (Perkin-Elmer, Sunnyville, CA) using the following protocol: 7 cycles of 95°C for 20 s, 55°C for 20 s,72°C for 30 s; and 30 cycles of 95°C for 20 s, 48°C for 20 s, 72°C for 30 s. Exon 1 was amplified using AmpliTaq Gold with corresponding buffer. PCR products were diluted 1:5 with denaturing loading buffer, heated to 95°C for 5 min, and then cooled on ice. Three μl of each sample were then loaded on nondenaturing 0.5% mutation detection enhancement gels, which underwent electrophoresis at 8 W for ∼16 h. Gels were dried and exposed to Kodak Biomax film for 3–24 h. Abnormally migrating bands were excised from gels after autoradiography and suspended in 100 μl of deionized water, and 1 μl of this solution was used as a template for a subsequent PCR reaction. This product was purified on a Wizard column(Promega, Madison, WI) and sequenced using either the Thermosequenase kit (Amersham, Arlington Heights, IN) or the ABI dye terminator sequencing kit (Perkin-Elmer). Samples were analyzed using the ABI Prism 377 fluorescent DNA sequencer (Perkin-Elmer).

We had previously examined 38 advanced endometrial cancers for microsatellite instability (19), and for the present study, an additional 62 cancer/normal DNA pairs were available for analysis. To determine whether microsatellite instability was present,three polymorphic microsatellite markers [BAT 26,(A_n); D14S65, (CA_n); and D14S297, (GATA_n)] were amplified using the PCR in paired endometrial cancer/blood DNA samples. The PCR products were diluted 1:1 with a loading buffer consisting of 10 mm EDTA(pH 8.0), 98% formamide, 0.02% xylene cyanol B, and 0.02% bromphenol blue and then denatured for 2 min at 95°C. Five μl of this solution were subjected to electrophoresis in 6% polyacrylamide gels containing 8.3 m urea and 25% formamide for 2–3 h at 90 W. The gels were fixed in 10% methanol/10% acetic acid, dried, and exposed to X-ray film at −80°C. Cancer cases were designated as positive when two of the three markers showed alleles of aberrant mobility when compared with corresponding normal tissue.

Proportions among unordered categories were compared using Fisher’s two-tailed exact test, whereas those for ordered categories were compared using the χ² test for trend.

For features with ordered categories, such as age, stage, and parity, aχ ² test for trend was used (20). The logistic regression model was used to analyze the association between PTEN mutation and more than one clinical feature (21). The Cox proportional hazards regression model was used to analyze the relationship between study variables and survival (22). Survival was defined as the interval from diagnosis to death from any cause. Patients still alive were censored in the analysis. Survival curves were constructed using the Kaplan-Meier method, and differences were tested using the log-rank statistic (23, 24). Survival times were truncated at 10 years for the figures, but all statistics were based on the complete data.

Previously observed racial differences in clinical and pathological features of the advanced endometrial cancers (2, 11) were confirmed in this study (Table 1). African American women had cancers with significantly higher stage and grade that were more often nonendometrioid. In addition,overexpression of p53 was more common in cancers of African-American patients. The median survival of African Americans (1.0 years) was significantly less than that of whites (2.5 years; Fig. 1).

Mutations in the PTEN tumor suppressor gene were found in 20 of 140 (14%) cancers and were almost exclusively a feature of endometrioid cases (19 of 100; 19%). An example of a cancer in which the PCR products had altered mobility is provided in Fig. 2. DNA sequence analysis of this mutation in codon 234 of exon 7 is shown in Fig. 3. The relationship between clinical and pathological features and PTEN mutation is demonstrated in Table 2.PTEN alterations were detected much more frequently in whites (22%) than in African Americans (5%). In the subset of endometrioid cases, alteration of PTEN also is more frequent in whites than in African Americans (26% versus 8%). Mutation of PTEN also was associated with age <60,microsatellite instability, and normal p53 expression. Multivariate analysis revealed that white race (P = 0.004) and endometrioid histology (P = 0.03) were significantly related to the presence of PTEN mutation after controlling for confounding influences

Microsatellite instability was detected in 15 of 100 (15%) cancers and occurred exclusively in cancers with endometrioid histology (15 of 72;21%). Examples of cancers that exhibited microsatellite instability are shown in Fig. 4. A correlation was not seen between microsatellite instability and other pathological features, such as stage or grade, but instability did correlate with the presence of PTEN mutation and normal p53 expression. (Table 3). There was no racial disparity in the frequency of microsatellite instability. Both microsatellite instability and PTENmutation were associated with favorable survival (Fig. 5). The numbers of cases with PTEN mutation or microsatellite instability were too small to allow for construction of separate survival curves for each race.

The relationship between various prognostic clinical, pathological and molecular prognostic factors, and survival is demonstrated in Table 4. In multivariate analysis, after controlling for stage and age,microsatellite instability (P = 0.03) and PTEN mutation (P = 0.08) remained predictive of survival, whereas race was not (P = 0.26).

African-American women have a lower incidence of endometrial cancer (14.6 per 100,000) than Caucasian women (22.2 per 100,000), but their disease-related mortality is significantly higher (1). Although this disparity in survival is partially attributable to the fact that African-American women more often present with metastatic disease, their survival is significantly worse in both early and advanced stages (25). In a study of patterns of care at our institution, we did not observe a racial disparity in the time from onset of abnormal uterine bleeding to diagnosis of endometrial cancer or in the intensity of treatment (2). Nor were there differences in the intensity of treatment. This suggests that underlying racial differences in molecular pathogenesis might contribute to the disparity in survival.

The TP53 tumor suppressor gene plays a critical role in regulation of cell cycle progression and apoptosis (26, 27, 28). Loss of p53 function is the most frequent molecular alteration observed in human cancers and has been associated with aggressive clinical behavior in many cancer types, including breast, ovarian, and colon cancer (29, 30, 31). Likewise,overexpression of mutant p53 protein in endometrial cancers has been associated with nonendometrioid histology, advanced stage, and poor outcome (8, 9). We have shown that p53 overexpression occurs much more frequently in African Americans relative to whites. Overexpression was three times more frequent in African Americans than in whites in early stage cancers (33% versus 11%; Ref. 10) and two times more frequent in advanced stage cancers(55% versus 25%; Ref. 11). These findings suggest that differences in the frequency of p53 overexpression contribute to the disparity in survival. In advanced stage disease,survival of African-American women remained inferior to that of whites even after controlling for p53 expression, however (11),suggesting that other molecular alterations also may contribute to racial differences in survival.

In the present study, we have extended this line of investigation by examining whether there are racial differences in the incidence of mutations in the PTEN tumor suppressor gene and microsatellite instability in advanced endometrial cancers. Both of these molecular alterations have been associated with endometrioid histology, which is a favorable prognostic factor (19).

The PTEN tumor suppressor gene encodes a phosphatase that inhibits proliferation by opposing the activity of oncogenic kinases,such as those involved in the AKT/PIC-3A pathway (32). In addition, the PTEN gene has a tensin homology domain and may play a role in cellular adhesion (33, 34). Inactivating mutations in the PTEN gene are present in a significant fraction of glioblastomas, melanomas, and prostate cancers (35, 36, 37). In addition, PTEN mutations occur in about one-third of endometrial cancers (13, 14) and are associated with early stage and endometrioid histology (12, 13, 14). In our prior study, mutations were seen in 55%of stage IA cancers compared with only 23% of stage IIIC/IV cases, and PTEN alterations were associated with favorable survival (19). Among endometrioid cases, 5-year survival was about 80% in those with mutations compared with 50% in those lacking mutations (19).

In the present study, the incidence of PTEN mutations (14%)was relatively low, as would be predicted, because these were all advanced stage cancers and a high fraction were nonendometrioid. Similar to prior studies of the full spectrum of endometrial cancer (19), we found that PTEN mutation was associated with favorable prognostic factors, such as endometrioid histology and lower grade and stage. In addition, PTENmutation was associated with favorable survival as in a prior study (19), but the statistical significance was borderline because of the small number of cases with mutations. The incidence of mutations was much lower in African Americans (5%) than whites (22%),suggesting that this contributes to the relatively poor survival of African Americans.

It could be argued that the lower frequency of PTENmutations in African Americans is simply a reflection of their higher incidence of nonendometrioid and stage IV cancers. On the other hand,our present understanding of the pathogenesis of cancer suggests that clinical features such as grade, histological type, and stage are the end result of the underlying genetic mutations that caused the cancer. There is strong evidence that mutation of the PTEN gene is an early event in endometrial carcinogenesis that occurs before loss of normal differentiation or histology. In this regard, we have shown that PTEN mutations are found in ∼20% of premalignant endometrial hyperplasias (38). Thus, we would suggest that African Americans have more nonendometrioid, advanced stage endometrial cancers because p53 is a more frequent target for mutations than PTEN in their endometrium. Conversely, in Caucasians, the PTEN gene is the more frequent target and endometrioid; early stage cancers predominate.

Microsatellites are short repeat DNA sequences (e.g. AAAA or CACACA) that occur throughout the genome, both within coding regions of genes and intervening noncoding regions. Because of their repetitive sequences, microsatellites have an increased susceptibility to mutation during DNA replication. Most of these mutations normally are recognized and corrected by a family of DNA mismatch repair enzymes that act in concert (39). About 20% of endometrial cancers have mutations in multiple microsatellite sequences attributable to inactivation of the MLH1 DNA repair gene (16, 40). Although the MLH1 gene may be inactivated by mutations in hereditary nonpolyposis colon cancer syndrome,inactivation of MLH1 in sporadic endometrial cancers appears to be attributable to methylation of the promoter region of the gene,resulting in inhibition of transcription (16, 40). Microsatellite instability is a feature of more favorable diploid endometrioid endometrial cancers, (15, 41) but it does not correlate with grade or stage (42, 43, 44).

In the present study, microsatellite instability was seen in 15%of all cases and was exclusively a feature of endometrioid cancers,occurring in 21% of these cases. As in prior studies, microsatellite instability did not correlate with stage or grade (19, 42, 43, 44), but it was predictive of better survival. The favorable prognostic significance of microsatellite instability in this study was greater than that of PTEN mutation. On the other hand, there was no racial disparity in the frequency of microsatellite instability in the entire group of patients (whites 16%, blacks 13%) or in the subset with endometrioid histology (whites 20%, blacks 22%). Thus,although microsatellite instability is a favorable prognostic factor,it does not appear to contribute to the racial disparity in survival.

Survival of African-American women with advanced endometrial cancer in this study and others is consistently worse than that of whites. A better understanding of these differences is essential if we hope to decrease the high mortality rate of African-American women with endometrial cancer. The reasons for this disparity are no doubt complex and include factors such as differences in the frequency of hormone replacement therapy, which is more common in whites (2, 3). We have shown that two of the genetic alterations that underlie the development of some endometrial cancers appear to contribute to the racial disparity in survival. Overexpression of mutant p53 correlates with more virulent behavior and is more common in African Americans (10, 11). Conversely, mutations of the PTEN tumor suppressor gene are associated with favorable outcome and are more common in whites.

Although alterations in several genes have been described in endometrial cancers, none of these changes occurs in a majority of cases. This suggests that there are several pathways that can lead to the development of endometrial cancer. More detailed molecular epidemiological studies that consider behavioral and environmental risk factors, inherited susceptibility, and acquired molecular alterations are needed to elucidate the etiology of what likely is a heterogeneous disease in both African Americans and Caucasians.