Model for the molecular genetic diagnosis of endometrial cancer using K-ras mutation analysis.

Endometrial cancer is the most common female genital tract cancer in Western societies (1). The prognosis for endometrial cancer is closely associated with tumor stage and histology (2). Currently, almost all cases are diagnosed following presentation with postmenopausal bleeding. Although the value of screening women in the general population for endometrial cancer is questionable, there are arguments for screening selected populations, such as women who belong to cancer-susceptible families [e.g., those with the Lynch II cancer family syndrome (3)] and women who are taking tamoxifen (4). New technologies such as the polymerase chain reaction (PCR) offer exciting opportunities for the molecular detection of cancer. For example, K-ras and p53 gene mutations have been identified in the stools of patients with colorectal cancer (5–8) and pancreatic cancer (9), the sputum of patients with lung cancer (10), and the urine of patients with bladder cancer (11), in some cases up to 1 year prior to clinical diagnosis (8,10). The aim of this study was to develop a model for the application of molecular diagnostic techniques to a gynecological cancer. We have chosen endometrial cancer because the endometrium is easily accessible for biopsy sampling, detection of poor-prognosis endometrial cancers at an early stage could conceivably result in an improvement in causespecific mortality, and K-ras (KirstenRAS) mutations are known to occur in endometrial cancer (12–14). Although K-ras mutations are relatively infrequent in this type of cancer, they are a suitable target for study because the mutations are mostly confined to codons 12 or 13 of the gene (15–18) and they occur relatively early in the process of endometrial carcinogenesis (13,18–20). In this study, we tested the hypothesis that Kras mutations could be detected in DNA samples prepared from cells in endometrial aspirates and cervical smear specimens taken from patients with endometrial cancer. Tissue specimens were collected at the time of surgery from 42 endometrial cancer patients aged 42 years through 93 years. These samples included a cervical smear (taken with a cotton swab), an endometrial aspirate biopsy specimen (taken with a pipelle suction curette), and a small portion of the primary tumor. All samples were placed in sterile Eppendorf tubes, snap-frozen in liquid nitrogen, and stored at −80 °C. The 42 tumors examined by histologic methods were classified by tumor type, stage, and grade as follows: 30 were adenocarcinomas, seven were endometrioid carcinomas, two were carcinosarcomas, two were clear-cell adenocarcinomas, and one was a mullerian mixed tumor; 33 were International Federation of Gynecology and Obstetrics (FIGO) stage I, three were FIGO stage II, two were FIGO stage III, two were FIGO stage IV, and two were not staged (21); 16 were grade 1, 15 were grade 2, eight were grade 3, and the two clear cell carcinomas and the one mullerian mixed tumor were not graded (21). Cell lines with known K-ras mutations (SW480 human colon carcinoma cell line and Hec1-A human endometrial carcinoma cell line) and without K-ras mutations (HEK293 normal human embryonic kidney cell line) were used as positive and negative controls, respectively. DNA was extracted from all tissue specimens and cultured cell samples by use of standard techniques. Tumor samples were minced by use of a sterile scalpel and then digested overnight in 5–10 mL of lysis buffer (150 mM NaCl, 10 mM Tris–HCl, 10 mM EDTA, 0.5% sodium dodecyl sulfate, and 250 mg/mL proteinase K) at 37 °C. The blood sample, which was collected in an EDTA-containing tube, was first treated with distilled water to lyse the red blood cells and then centrifuged at 2500 rpm at room temperature for 10 minutes to concentrate the lymphocytes. The supernatant was discarded, and the pellet was resuspended in lysis buffer. DNA was prepared from the cells by phenol– chloroform–isoamyl alcohol extraction (25 : 24 : 1) and ethanol precipitation. The DNA precipitate was dissolved in sterile water. DNA was prepared from cells in endometrial aspirate specimens in the same way as for the tumor specimens but with the use of a smaller volume (2–3 mL) of lysis buffer. We extracted DNA from the cells in the cervical smear specimens by first washing the cotton swab in 0.5 mL of lysis solution before continuing with the rest of the extraction procedure as described above. Confluent cultured cells were first washed in phosphate-buffered saline and then placed in lysis buffer before the rest of the extraction procedure was continued as described above. So that we could minimize the risk of contamination, extractions of DNA from cells in the cervical smears, endometrial aspirates, and in vitro cell cultures were performed in a separate laboratory where no DNA or PCR work had previously been performed. All reagents, tools, and equipment used for DNA extraction were handled with a strict protocol to eliminate the possibility of contamination of PCR reactions. Samples were processed separately with the use of a fresh change of gloves, a bench cover, and a polystyrene tube rack for each preparation. Sterile disposable, plastic Pasteur pipettes were used for all steps. Primary tumors were initially screened for mutations in codons 12 and 13 of the K-ras gene by use of the