Evaluation of DNA methylation in the human genome: why examine it and what method to use.

PURPOSE: Since its discovery 50 years ago, DNA methylation has been found to be an important part of gene regulation. Newer methods of analysis over the last decade have helped further the understanding of this epigenetic phenomenon. The purpose of this article is to describe current methods of analysis and discuss advantages and disadvantages of each and their possible roles in gynecologic malignancies. RESULTS: The methods for analysis of DNA methylation are divided into two major categories: 1) methods which utilize chemical methods or restriction enzymes to differentially cleave at cytosine versus 5-methylcytosine sites, 2) methods which utilize sodium bisulfite (NaHSO3) to specifically convert unmethylated cytosines to uracil (thymine after PCR). This recently developed method appears to be more sensitive and allows the investigator to specifically delineate the study site(s). CONCLUSION: DNA methylation is important in the human genome. Its role in tumorigenesis is just beginning to be understood. While relying upon newly designed methods of analysis, further understanding of this epigenetic phenomenon and its role in gene expression and tumorigenesis will be forthcoming.

HSP27 in patients with ovarian carcinoma: still an independent prognostic indicator at 60 months follow-up.

OBJECTIVE: Heat shock protein 27 (HSP27) is produced in response to pathophysiologic stress in animal cells. The authors have previously shown that HSP27 is an independent prognostic indicator in patients with ovarian carcinoma. The present study was performed to see whether HSP27 remained an independent prognostic indicator with longer follow-up. METHODS: One hundred and three consecutive patients with epithelial ovarian carcinoma were studied. Slides were prepared from fresh tissue. HPS27 staining was performed as previously described. Patient records were examined for FIGO stage, grade, histology, level of cytoreduction and survival. RESULTS: One hundred and three patients were followed for a mean of 60 months. Twenty patients had FIGO Stage I disease, four Stage II, 59 Stage III, and 20 Stage IV. Immunohistochemical (IHC) staining for HSP27 was not related to histologic grade, level of cytoreduction or histologic subtype. A statistically significant decrease in HSP27 staining was found to correlate with increased FIGO stage (p = 0.008). Using cox-regression analysis, HSP27 staining (p = 0.025), stage (p = 0.0012), and level of cytoreduction (p < 0.0001) were independent predictors of survival in these patients. CONCLUSION: Cox-regression analysis found HSP27 to be an independent indicator of prognosis and survival in patients with ovarian carcinoma who had longer follow-up. Decreased HSP27 staining was related to decreased survival. This study confirms the authors' earlier report on the importance of HSP27 as a prognostic indicator in ovarian carcinoma.

Heat shock protein 27 in the placentas of women with and without severe preeclampsia.

BACKGROUND: Although not fully understood, heat shock proteins (HSP) are well known stress response proteins. The purpose of this analysis was to determine whether staining for HSP27 was different between placentas from pregnancies complicated by severe pre-eclampsia with intrauterine growth restriction (IUGR) as compared to controls. METHODS: Sterile placental tissue was collected from ten women whose pregnancies were complicated by severe preeclampsia with IUGR and from ten women with uncomplicated by severe pre-eclampsia with IUGR and from ten women with uncomplicated term pregnancies. The tissue was then stained for HSP27. RESULTS: The median age of the patients was 27 years (mean 27, range 17-37). The median estimated gestational age at delivery was 38 weeks (mean 37, range 29-41). Overall 12 of 20 placentas stained positively for HSP27 (nuclear and/or cytoplasmic). Eight of ten placentas from women with pre-eclampsia and IUGR stained positively for HSP27 (p = 0.046). CONCLUSION: HSP27 staining of the placenta is twice as common in patients with severe preeclampsia as compared to patients with normal term gestations. These preliminary results warrant the inauguration of a similar but larger study to examine the significance of these findings.

Nuclear and cytoplasmic c-myc staining in endometrial carcinoma and their relationship to survival.

OBJECTIVE: The role of the c-myc proto-oncogene in genomic instability is just becoming more fully understood. However, its role in endometrial cancer is essentially unknown. The objective of this study was to determine the relationship between cytoplasmic and nuclear c-myc staining, DNA index, and survival in patients with endometrial carcinoma. METHODS: One hundred and twenty-one patients with endometrial carcinoma were studied. Image analysis was used to determine DNA index. In addition to cytoplasmic and nuclear c-myc staining and DNA index, histologic type, stage, grade, depth of invasion, lymphvascular space invasion, and peritoneal cytology were evaluated as prognostic indicators. Univariate and multivariate analyses were performed. RESULTS: One hundred and twenty-one patients were followed for over 5 years. c-myc cytoplasmic staining was present in 75.2% of the patients' tumors, and nuclear staining was present in 66.9% (P = 0.99). DNA index was significantly higher in patients with nuclear c-myc staining and no cytoplasmic staining (DNA index 1.38) as compared to those patients whose tumors displayed cytoplasmic c-myc staining but no nuclear c-myc staining (1.18) (P = 0.016). Patients whose tumors stained positively for nuclear c-myc and negatively for cytoplasmic c-myc had significantly worse survival by Kaplan-Meier analysis (P < 0.0001). Seventeen patients died during the follow-up period of this study. By multivariate analysis, positive cytoplasmic c-myc staining with negative nuclear staining (P = 0.0076), negative cytoplasmic c-myc staining with positive nuclear staining (P = 0.011) and FIGO stage (P < 0.0001) were shown to be independent prognostic indicators predictive of survival. CONCLUSION: Nuclear and cytoplasmic c-myc staining, as well as FIGO stage, when assessed by multivariate analysis, were demonstrated to be important factors in predicting survival in the 121 patients in this study. While increasing FIGO stage was prognostic of decreased survival, the specific location of c-myc staining was also associated with prognosis. The expression of the c-myc protein is related to survival in patients with adenocarcinoma of the endometrium.

Nuclear size, shape, and density in endometrial carcinoma: relationship to survival at over 5 years of follow-up. Does analyzing only cells occupying the G0-G1 peak add useful information?

OBJECTIVE: The authors, using image analysis, previously demonstrated nuclear size and summed optical density to be independent prognostic indicators of recurrence in patients with endometrial carcinoma. The same tumors were analyzed by studying the optical features in the G0-G1 peak to see if this changed the values found as well as their importance as prognostic features at greater than 5 years of follow-up. METHODS: Tumors from 74 consecutive patients, surgically treated, with endometrial cancer, were evaluated. Survival, depth of invasion, lymphvascular space invasion, FIGO stage, grade, histology were analyzed. DNA index, progesterone receptor status, as well as nuclear size (NUSZ), shape (NUSH), and summed optical density (NUSD) were evaluated. NUSZ, NUSH, and NUSD were quantified using image analysis. RESULTS: Fifteen patients died from disease during the observation period of the study. Mean follow-up was 82 months with a median of 84 months. Forty-nine patients had stage I cancers, five stage II, 17 stage III, and three stage IV. NUSZ and NUSD were all significantly different between the original (entire cell cycle) and the re-measured (G0G1 only) values (both P < 0.001). Multivariate analysis showed both the original (P = 0.0001) and G0G1-only (P = 0.046) NUSZ and the original (P = 0.0002) and G0G1-only (P = 0.018) NUSD to be independent prognosticators of survival. CONCLUSION: Image analysis is able to quantify cellular and nuclear parameters not otherwise quantifiable. NUSD and NUSZ correlated with traditional prognostic indicators, were demonstrated independent predictors of survival at over 5 years of follow-up. Although the re-measured NUSZ and NUSD from only the G0-G1 peak were significantly different from the original NUSZ and NUSD, they were not as valuable as prognostic factors. Nuclear size and summed optical density measured from the entire cell cycle are independent prognostic indicators of survival at greater than 5 years of follow-up. Measuring nuclear morphometric features in the G0-G1 peak only does not add any new prognostic information.

Methylation: a second hit in the two-hit hypothesis.

This case illustrates that methylation of one BRCA1 allele may serve as the second hit in a patient with a diploid locus and missense mutation on the other allele.

p21 and p53 in ovarian carcinoma: their combined staining is more valuable than either alone.

BACKGROUND: Mutations in p53 are the most common genetic alterations in human malignancies. Expression of its protein product has been linked to decreased survival rate in ovarian carcinoma. Less is known about the importance of p21 expression. The purpose of this study was to determine the value of the combination of p21 and p53 expression in patients with epithelial ovarian malignancies. METHODS: One hundred three consecutive patients with epithelial ovarian carcinoma were studied using snap-frozen tissue specimens. Immunohistochemical staining utilizing the pAb1801 monoclonal antibody to p53 and NCL-WAF-1 monoclonal antibody to p21 was performed. Image analysis was used to determine whether nuclear staining for either antibody was present. In addition to p21 and p53, International Federation of Gynecology and Obstetrics stage, grade, histology, level of cytoreduction, and DNA index were analyzed as prognostic factors. Univariate and multivariate analyses was performed. RESULTS: One hundred three patients were observed for more than 5 years. Immunohistochemical staining for p21 and p53 were significantly inversely related (P = 0.041). Among the patients whose tumors showed p21 staining but no p53 staining, there were no recurrences and all patients were alive at 5-year follow-up. The 5-year survival rate for these patients was significantly better than for the patients with other combinations of p21/p53 staining (P < 0.0001). The DNA index between these 2 groups was not significantly different (P = 0.057). Multivariate analysis shows the combination of p21 and p53 (P = 0.013) staining to be more valuable as a prognostic indicator than either p53 (P = 0.015) or p21 (P = 0.5) alone. CONCLUSIONS: This study confirms the importance of the combination of p21 and p53 nuclear staining in patients with ovarian carcinoma. Cox regression analysis revealed combination of p21 positive and p53 negative to be a better independent indicator of prognosis and survival in patients with ovarian carcinoma than either p21 or p53 alone.

Tumor markers and molecular biological markers in gynecologic malignancies.

Gynecologic oncology is a rapidly growing field due to constant advances in immunohistochemistry and molecular biology. This review serves as an overview of new studies promoting the use of tumor markers and molecular biological prognostic factors in malignancies affecting women. The majority of studies focus on either endometrial or ovarian carcinoma. Other gynecologic malignancies (cervical, vulvar, and vaginal carcinoma) have a much smaller representation in the world literature. Multiple new markers were examined over the last year. We conclude that although some markers show promise as potential new consensus prognostic indicators, more work is needed to confirm results and clarify any existing discrepancies.

Radical hysterectomy in the elderly female: a comparison to patients age 50 or younger.

BACKGROUND: Longevity in the United States is increasing, therefore the knowledge of whether radical surgery can be performed on patients over age 65 with acceptable morbidity and mortality is increasingly important. A retrospective study of elderly patients undergoing radical hysterectomy was performed with a comparison to a younger cohort. METHODS: A retrospective case study of all patients over 65 years of age having undergone a radical hysterectomy for cervical cancer in one practice of gynecologic oncology was performed. Each patient was matched with two patients age 50 or younger having undergone a similar operation during the same year. No patients between July 1, 1965, and March 31, 1998, were knowingly excluded. All patients were analyzed for preexisting medical conditions, length of postoperative stay, morbidity, and postoperative mortality. RESULTS: Sixty-two patients over age 65 having undergone a radical hysterectomy and pelvic lymphadenectomy were found. These patients were matched with 124 patients age 50 or younger having undergone a radical hysterectomy during the same calendar year. No differences in operative morbidity (minor, P > 0.57; major, P > 0.93) or mortality (no deaths in either group) were found. Patients age 65 or older had more comorbidities prior to surgery than the younger cohort (minor, P < 0.001; major, P < 0.001). The mean postoperative hospital stay was significantly longer in the older cohort (P = 0.005). CONCLUSIONS: Radical hysterectomy is a safe surgical procedure in a select population of patients 65 and over. No differences in operative mortality or morbidity were found when compared to a cohort of patients age 50 or younger. However, hospital stay was longer in the older cohort.

Immunohistochemical staining of the mismatch repair gene, hMSH2, and survival in patients with ovarian carcinoma.

OBJECTIVE: hMSH2 is a mismatch repair gene. The protein of this gene can be demonstrated by immunohistochemical methods. The authors wanted to analyze whether the percent of positive nuclear area staining of the hMSH2 protein correlated with survival in patients with ovarian carcinoma. METHODS: One hundred and two patients with epithelial ovarian carcinoma were studied. Slides were prepared from snap frozen tissue. Quantification of staining and DNA index were performed using image analysis. In addition to hMSH2, FIGO stage, grade, histology, and level of cytoreduction were analyzed as prognostic factors. RESULTS: Mean follow-up was 64 months and median was 59 months. Nineteen patients had stage I cancers, 4 stage II, 59 stage III, and 20 stage IV. Optimal cytoreduction was accomplished in 71% of patients. hMSH2 staining was significantly higher in better differentiated tumors (p=0.006), but there was no difference in staining among the FIGO stages (p=0.43). Cox regression analysis revealed FIGO stage (p=0.0005), level of cytoreduction (p=0.006) and hMSH2 staining (p=0.016) to be independent predictors of survival in patients with ovarian carcinoma. CONCLUSION: The hMSH2 protein can be demonstrated by immunohistochemical methods and quantified by image analysis. hMSH2 staining was shown to be an independent prognostic indicator of survival in patients with ovarian carcinoma.

The great imitator: miliary peritoneal tuberculosis mimicking stage III ovarian carcinoma.

PURPOSE: To report a case of peritoneal tuberculosis initially mistaken at the time of surgery for metastatic ovarian carcinoma. CASE REPORT: A 31-year-old Filipino female was found to have increasing abdominal pain, ascites, early satiety, increasing abdominal girth and bilateral complex masses. Diagnostic laparoscopy revealed the presence of > 4 liters of ascites, a frozen pelvis secondary to what appeared to be bilateral ovarian carcinomas, along with miliary seeding of the entire anterior abdominal wall, omentum, small bowel and small bowel mesentery, right and left diaphragmatic surfaces, and hepatic surface. Frozen section, at the time of laparotomy, revealed necrotizing granulomas most consistent with disseminated tuberculosis that was confirmed at the time of final pathologic review and culture. CONCLUSION: Peritoneal tuberculosis can be mistaken for widely metastatic ovarian carcinoma at time of surgery.

p53 and bcl-2 in epithelial ovarian carcinoma: their value as prognostic indicators at a median follow-up of 60 months.

OBJECTIVE: p53 is the most common tumor suppressor gene involved with human malignancies. Mutations in p53 are present in approximately 50% of human malignancies. bcl-2 is a protooncogene. Expression of its protein product is related to better prognosis in several malignancies. METHODS: One hundred and three patients with epithelial ovarian carcinoma were studied. Immunohistochemical staining using the pAb1801 monoclonal antibody to p53 and the anti-bcl-2 124 monoclonal antibody to bcl-2 was performed. Image analysis was used to measure percentage positive nuclear area staining of mutant p53. In addition to bcl-2 and p53, FIGO stage, grade, histology, and level of cytoreduction were analyzed as prognostic factors. Univariate as well as Cox regression analysis was performed. RESULTS: One hundred and three patients were followed for a mean of 60 months. Twenty patients had FIGO stage I disease, 4 stage II, 59 stage III, and 20 stage IV. Immunohistochemical staining for mutant p53 was not significantly related to DNA index (P = 0.99) but was related to increasing FIGO stage (P < 0.001) and increasing histologic grade (P = 0.039). Using Cox regression analysis, increased mutant p53 staining was an independent predictor of survival in these patients (P = 0.0032), along with stage (P < 0. 0001) and level of cytoreduction (P < 0.0001). Although by itself bcl-2 was not an independent prognostic indicator (P = 0.18), the combination of p53 and bcl-2 was independently predictive of survival (P = 0.038). CONCLUSION: This study confirms the authors' earlier report on the importance of p53 as a prognostic indicator of survival in ovarian carcinoma. Cox regression analysis reveals mutant p53 staining to be a better independent indicator of prognosis and survival in patients with ovarian carcinoma than the combination of bcl-2 and p53.

MIB-1 in endometrial carcinoma: prognostic significance with 5-year follow-up.

OBJECTIVE: MIB-1, a monoclonal antibody to the Ki-67 antigen, has presumptively been shown to be predictive of recurrent disease in patients with endometrial cancer. In order to more conclusively establish whether MIB-1 staining can be used as a prognostic indicator of recurrent disease or survival, a larger group of patients with a minimum follow-up of 5 years was analyzed. METHODS: The tumors from 147 consecutive patients receiving primary surgical therapy for endometrial carcinoma were evaluated with the MIB-1 monoclonal antibody. Proliferation index was quantified by image analysis. Patients were followed for a minimum of 60 months. In addition to MIB-1 staining, histologic type, stage, grade, depth of invasion, lymphovascular space invasion, and peritoneal cytology were evaluated as prognostic indicators. RESULTS: Twenty-five of 147 patients died during the study period. MIB-1 staining was not significantly elevated in advanced (stage II, III, and IV) as opposed to early (stage I) carcinomas (P = 0.38). In patients whose tumor MIB-1 staining was less than 33.0%, no deaths occurred. By multivariate analysis, only MIB-1 staining (P < 0.001), FIGO stage (P = 0.005), and LVI (P = 0.005) were shown to be independent prognostic indicators predictive of survival. CONCLUSION: In this series of 147 consecutive patients with endometrial carcinoma, the monoclonal antibody MIB-1 was shown to be an independent prognostic indicator of 5-year survival. This follow-up further validates the previous work regarding the significance and potential usefulness of MIB-1 as a prognostic indicator.

What staging surgery should be performed on patients with uterine papillary serous carcinoma?

OBJECTIVE: While uterine papillary serous carcinoma (UPSC) is an aggressive histologic subtype, it fortunately is not as common as some other histologic subtypes. Overall, patients with UPSC have a poor survival rate. Since the optimal surgical procedure to perform on patients with this tumor is unknown, the authors wanted to determine what the optimal surgical management of patients with UPSC should be. METHODS: All patients with the preoperative or frozen section intraoperative diagnosis of UPSC were treated with a staging or cytoreductive procedure analogous to patients with serous carcinoma of the ovary. Patients analyzed underwent surgery from March 1983 to September 1995. RESULTS: Sixty-five patients with UPSC were found. Twenty patients had FIGO stage I tumors, 6 stage II tumors, 8 stage III tumors, and 31 stage IV tumors. Twenty-nine patients had upper abdominal disease (17 gross disease and 12 microscopic disease only). Forty-eight patients underwent pelvic and paraaortic lymphadenectomy, with 6 of 48 having positive lymph nodes. All 14 patients with lymphovascular space invasion had stage IV disease. Thirty-one of sixty-five patients had positive cytology at the time of surgery. CONCLUSION: Based on the clinical experience of these investigators, patients with UPSC should undergo a staging laparotomy similar to the procedure undertaken for patients with ovarian carcinoma. The surgery should include at least partial omentectomy, total abdominal hysterectomy and bilateral salpingo-oophorectomy, peritoneal washings, peritoneal biopsies, and pelvic and paraaortic lymphadenectomy similar to an ovarian cancer staging procedure if no gross disease > or =2 cm is found at time of surgery. If disease > or =2 cm is found, cytoreduction should be undertaken when feasible.

p53 expression as a prognostic indicator of 5-year survival in endometrial cancer.

BACKGROUND: One of the most common genetic alterations to occur in human cancers is an alteration of the p53 tumor suppressor gene. The purpose of this article was to build upon the authors' previous work with p53 and determine whether p53 was a prognostic indicator of 5-year survival. METHODS: One hundred thirty-seven consecutively surgically treated patients with endometrial cancer had their p53 expression studied by immunoperoxidase staining and quantified by image analysis. All patients were evaluable for 5-year survival. RESULTS: One hundred three patients had endometrioid adenocarcinoma; 6, adenosquamous carcinoma; 14, papillary serous carcinoma; 10, clear cell carcinoma; and 4, undifferentiated carcinoma. p53 expression ranged from 0.0 to 58.2% positive nuclear area with a mean of 11.5% (median 2.6%) for the cohort. For the patients with endometrioid carcinoma, the mean p53 expression was 7.1% while for the nonendometrioid tumors it was 24.6% (P<0.001). Fifty-nine of the 103 endometrioid tumors (57.3%) stained positive for p53 while 32 of the 34 nonendometrioid (94.1%) tumors stained positive (P<0.001). Increasing histologic grade correlated with an increasing p53 expression (P = 0.003). The percentage of tumors expressing p53 was found to be higher in FIGO stage II, III, and IV than in FIGO stage I cancer (P = 0.003). However, mean p53 expression did not differ between early (stage I) and advanced (stage II, III, and IV) cancers (P = 0.088). Utilizing 5-year survival as the endpoint for multivariate analysis, FIGO stage (P = 0.0028) and p53 expression (P<0.001) were the only independent prognostic indicators found. CONCLUSION: p53 expression is more commonly found in nonendometrioid than in endometrioid adenocarcinoma of the endometrium. It, along with FIGO stage, is an independent prognostic indicator of 5-year survival.

Ovarian carcinoma metastatic to bilateral axillary lymph nodes. A case report.

BACKGROUND: Ovarian cancer is a major cause of cancer deaths in women and usually presents with diffuse abdominal disease. Lymph node metastases are common, but axillary lymph nodes are rarely involved. CASE: A 63-year-old woman, initially presenting with abdominal symptoms and massive ascites, underwent optimal cytoreduction followed by intravenous chemotherapy with paclitaxel and carboplatin. The patient subsequently underwent a second-look surgery revealing only microscopically positive disease and then received intraperitoneal chemotherapy with cisplatin. At a follow-up visit, she was found to have bilateral axillary lymph node enlargement. Mammography revealed no lesions in either breast. Fine needle aspiration dissection confirmed the diagnosis of metastatic, recurrent ovarian adenocarcinoma and subsequently axillary lymph node dissection was undertaken. CONCLUSION: Ovarian carcinoma, which usually follows typical patterns of metastatic involvement, can appear in unusual areas.

A study of heat shock protein 27 in endometrial carcinoma.

OBJECTIVE: Heat shock protein 27 (HSP27) is a relatively small protein produced in response to pathophysiologic stress. The purpose of this study was to determine prospectively whether HSP27 was associated with known prognostic factors in patients with endometrial carcinoma. METHODS: One hundred fifty-three consecutive patients with endometrial carcinoma were studied. Slides were prepared from fresh tissue. HSP27 was analyzed using a semiquantitative measurement. Patient records were examined for FIGO stage, grade, depth of myometrial invasion, histology, lymphovascular space invasion, time to recurrence, and survival. RESULTS: The mean follow-up was 53 months (median 56 months, range 30-68 months). Endometrioid tumors showed significantly higher HSP27 staining than nonendometrioid tumors (P = 0.005). Patients alive at the conclusion of this study had significantly higher mean HSP27 staining than patients who were deceased (P < 0.001). Logistic regression revealed HSP27 staining (P = 0.02), FIGO stage (P = 0. 014), and lymphovascular space invasion (P = 0.046) to be independently predictive of survival. CONCLUSION: HSP27 staining is significantly higher in endometrioid than nonendometrioid tumors. HSP27 staining is an independent prognostic indicator in patients with endometrial carcinoma, the most common gynecologic malignancy in the United States.

Markov optical texture parameters as prognostic indicators in ovarian carcinoma.

Texture is a descriptive property of a surface describing the morphometric heterogeneity of complex structures. Computer aided image analysis allows optical texture measurement and analysis of gray-scale images. The authors, utilizing image analysis, prospectively studied Markov nuclear texture features to determine their relevance as prognostic indicators of survival in patients with epithelial ovarian carcinoma. Ninety-nine consecutive patients with ovarian cancer, treated initially with surgery were evaluated for their length of survival, level of cytoreduction, FIGO stage, grade, histology, and DNA index, as well as 20 Markov texture features. Markov nuclear texture features were quantified using image analysis. Mean follow-up for the study population was 64 months (median 59) with a range from 51 to 89 months. Five optical texture features showed significant correlation with length of survival. Difference entropy (P = 0.033) and information measure A (P = 0.041) were both indirectly correlated with survival while information measure B (P = 0.030), correlation coefficient (P = 0.045), and the maximum correlation coefficient (P = 0.041) were directly correlated. Only sum entropy (P = 0.035), FIGO stage (P = 0.0031), and level of cytoreduction (P < 0.0001) were independent predictors of survival in this population. Optical texture can be quantified by image analysis. Utilizing multivariate analysis, the Markov texture feature, sum entropy, was demonstrated to be an independent prognostic indicator of survival in patients with epithelial ovarian cancer. FIGO stage and optimal cytoreduction also were independent prognostic indicators of survival.