Cervical cancer: prevention, diagnosis, and therapeutics.

Cervical cancer is a leading cause of cancer deaths in women worldwide. Because of its association with human papilloma virus infection, as well as the ability to screen for premalignant stages of the disease, it is now largely a preventable disease. This article describes the molecular basis for cervical cancer, and presents a clinical overview of current treatment approaches and technological advances, emphasizing the unique aspects of this viral disease as it relates to the immune system and vaccination or other immunotherapeutic strategies.

Generation of tumor-specific cytotoxic T lymphocytes by stimulation with HPV type 16 E7 peptide-pulsed dendritic cells: an approach to immunotherapy of cervical cancer.

OBJECTIVE: The aim of this study was to generate HPV-16 E7 peptide-specific cytotoxic T lymphocytes (CTLs) in vitro for future adoptive immunotherapy of cervical cancer. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from HLA-A2+ healthy donors. The PBMCs were incubated with HPV-16 E7(11-20) peptide and varying cytokines in the primary culture. Restimulation was performed weekly with peptide-pulsed, irradiated autologous PBMCs. Alternatively, the PBMCs were depleted of abundant CD4+ cells and stimulated with HPV-16 E7(11-20) peptide-pulsed dendritic cells. Cytolytic activity was determined by a standard 4-h (51)Cr-release assay. RESULTS: After 6 weeks in culture, we were able to establish peptide-specific CTL lines in one of seven donors by incubating PBMCs with HPV-16 E7(11-20) peptide. When we employed autologous peptide-pulsed dendritic cells to stimulate CD8+ cell-enriched PBMCs, we obtained CTL lines in four of seven donors. The primed CTLs were able to lyse the HLA-A2+ and HPV-16+ cervical cancer cell line Caski. SiHa, an HLA-A2-, but HPV 16+, cervical cancer cell line could be lysed only after transfection with HLA-A2. In addition, a high cytotoxicity (>80%) was obtained against peptide-pulsed, but not unpulsed, targets such as autologous Ebstein-Barr virus-immortalized B cells or allogeneic lipopolysaccaride-stimulated PBMCs. DCs were clearly the most potent of all tested antigen presenting cells to stimulate a CTL response in a proliferation assay. CONCLUSION: HPV-16 E7 peptide-specific CTLs could be generated in vitro. A practical protocol to expand the CTLs to a sufficient number for an application in a clinical trial is in progress.

Epidemiology and biology of cervical cancer.

Worldwide, cancer of the cervix is the second leading cause of cancer death in women: each year, an estimated 500,000 cases are newly diagnosed. Among populations, there are large differences in incidence rates of invasive cervical cancer: these reflect the influence of environmental factors, screening Papanicolaou (Pap) tests, and treatment of pre-invasive lesions. The high-risk human papillomavirus (HPV) subtypes 16, 18, 31, 33, and 51 have been recovered from more than 95% of cervical cancers. We have made great strides in understanding the molecular mechanism of oncogenesis of this virus, focusing on the action of the E6 and E7 viral oncoproteins. These oncoproteins function by inactivating cell cycle regulators p53 and retinoblastoma (Rb), thus providing the initial event in progression to malignancy. Cervical cancers develop from precursor lesions, which are termed squamous intraepithelial lesions (SIL) and are graded as high or low, depending on the degree of disruption of epithelial differentiation. Viral production occurs in low-grade lesions and is restricted to basal cells. In carcinomas, viral DNA is found integrated into the host genome, but no viral production is seen. The well-defined pre-invasive stages, as well as the viral factors involved at the molecular level, make cervical carcinoma a good model for investigating immune therapeutic alternatives or adjuvants to standard treatments.

Critical care issues in cervical cancer management.

Radical pelvic surgery in gynecologic oncology patients poses a challenge to the surgeon and the ancillary team in charge of the peri-operative care. The high frequency of medical problems observed in this patient population, in conjunction with the stresses of radical surgery, necessitates careful monitoring of patients' medical status. A comprehensive team approach in the perioperative period is critical to patient care. Early intervention and anticipation of potential problems for the patient at risk in the postoperative period minimizes morbidity and mortality. This article will review the essentials of critical care as it relates to patients undergoing radical pelvic operations.

p53 interference and growth inhibition in p53-mutant and overexpressing endometrial cancer cell lines.

BACKGROUND: The presence of p53 mutations and associated mutant p53 overexpression has been demonstrated in many cancer systems. Whether the overexpression of mutant p53 represents cause or effect, and whether p53 mutation contributes actively to the malignant phenotype is a matter of controversy. We examined the growth effects of oligonucleotides designed to interfere with p53 expression and/or activity in p53-mutant/overexpressing endometrial cancer cell lines. METHODS: Phosphorothioate oligonucleotides were used to target p53-related sequences in two p53-mutant/overexpressing endometrial cancer cell lines (KLE and RL95-2) and a normal fibroblast control. The ATP cell viability assay was used to measure growth effects after 6-day treatments with 27-mer and 14-mer sense (S) or antisense (AS) phosphorothioate oligodeoxyribonucleotides (oligos) targeting the promoter/ATG region of p53 and/or the p53 consensus (CON) DNA binding sequence. These sequences were designed to interfere with p53 expression and activity, respectively. Random sequences of the p53 27- and 14-mer were used as controls for nonspecific oligo effects, and a normal fibroblast cell line was used to compare oligo effects and serve as a negative p53 immunostaining control. RESULTS: Mean +/- SE IC50 (50% growth inhibition) of the S, AS p53, and p53 CON oligos were 4.2 +/- 1.3, 4.7 +/- 0.9, and 7.6 +/- 1.4 microM, respectively, for the two endometrial cell lines combined. The AS and S p53 oligos demonstrated dose-dependent inhibitory effects in both cell lines, while p53 CON produced variable effects alone and in combination with p53 AS. In KLE, a uniform inhibitory dose response was seen with p53 CON oligos. In RL95-2, the approximate IC50 for p53 CON was 0.5-1.0 microM, but at increasing doses above this, an inverse dose response was consistently observed. Combinations of p53 AS and p53 CON oligos produced predominantly synergistic growth inhibition. Although combinations of p53 AS and p53 CON in KLE were synergistic at low doses, antagonistic effects occurred at higher concentrations. Oligos had little effect on normal fibroblast growth, with calculated IC50 > 16 microM. Equimolar combinations of p53 S and AS were antagonistic, indicating that antiproliferative effects were sequence-specific. Random oligos demonstrated some nonspecific inhibitory effects, with >25% growth inhibition at 16 microM and beyond. Immunoperoxidase staining for mutant p53 after exposure to 16 microM concentrations of p53 AS oligos demonstrated reductions in p53 staining but persistent overexpression relative to wild-type (fibroblast) cells. CONCLUSION: Phosphorothioate oligos directed against p53 sequences in two p53-mutant endometrial cancer cell lines demonstrated antiproliferative effects. Combined anti-p53 and anti-p53 binding site oligos resulted in predominantly synergistic antiproliferative effects. The activity of sense oligos, the variable responses to p53 CON, and the persistent overexpression of mutant p53 at high concentrations of growth-inhibiting anti-p53 oligos suggest that, while promising, the antineoplastic effects of these oligos occur through complex and incompletely understood mechanisms.

In vitro antigene therapy targeting HPV-16 E6 and E7 in cervical carcinoma.

Human papillomavirus (HPV) infection is believed to play a central role in cervical carcinogenesis. Specifically, two viral oncoproteins, E6 and E7, possess transforming ability and have been shown to interact with the cellular tumor suppressors p53 and p105, the retinoblastoma (Rb) gene product. To test the hypothesis that E6 and E7 play an active role in the maintenance of the malignant phenotype and may be ideal targets for antigene therapy, we tested the antiproliferative effects of phosphorothioate oligodeoxynucleotides (oligos) targeting HPV-16 E6 and E7 in cervical cancer cell lines and primary tumor explants. The ATP cell viability assay was used to measure growth effects of 27-mer antisense oligos targeting the ATG translational start region of HPV-16 E6 and E7 sequences in HPV-16-positive cell lines SiHa and CaSki and four advanced, primary cervical tumor explants. A random oligo sequence, an HPV-18-positive and HPV-negative cell line, one histologically confirmed endometrial and two ovarian tumors were used as negative controls. HPV type was confirmed by hybrid capture techniques. Cell lines and sterile (staging laparotomy) tumor cells were plated at 5000 cells/0.1 ml and 100,000 cells/0.5 ml in 96-well plates or soft agar, respectively, and incubated at 37 degrees C with a single treatment of oligos at 0-16 microM. E6/E7 combinations at a fixed ratio of 1:1 were used at 0-8 microM for each oligo. Cellular ATP was measured by luciferin/luciferase fluorescence on Day 6. HPV-16 E6 and E7 oligos showed antiproliferative effects in all HPV-16-positive cell lines and primary tumor explants (IC50s 6.9-9.5 microM for cell lines, 9.1-12.1 microM primary cervical tumors), while the HPV-negative C33-A cell line and HPV-18-positive cell line HeLa were relatively insensitive to the HPV-16 oligos (IC50s > 30 microM extrapolated). The endometrial and two ovarian primary tumors were also insensitive to the HPV E6 and E7 oligos (IC50s > 25 microM extrapolated). Random oligos had little effect on cell growth at concentrations up to 16 microM (< 25% inhibition), except in CaSki (@50% inhibition at 16 microM). Combinations of E6 and E7 demonstrated mixed synergistic and antagonistic effects as determined by combination indices (CI) derived from median effect parameters. In the HPV-16-positive primary cervical tumors and the cell line SiHa, E6/E7 combinations were synergistic at low doses (< 25% growth inhibitory dose range) and antagonistic at doses above this. For the HPV-16-positive cell line CaSki, however, E6/E7 combinations were antagonistic at all dose ranges. Phosphorothioate oligos directed against the viral oncogenes E6 and E7 were shown to have antiproliferative effects specific to HPV-containing cancer cells. These specific antiproliferative effects suggest that HPV-16 E6 and E7 sequences play an active role in the malignant growth properties of cervical cancer cells and may be ideal targets for antigene therapy.

Combination anti-gene therapy targeting c-myc and p53 in ovarian cancer cell lines.

Gene therapy clinical trials targeting p53 and other genes are underway in nongynecologic cancer systems. To explore the potential for antigene therapy in gynecologic oncology, we examined the in vitro effects of oligonucleotides targeting c-myc and p53 in the ovarian cancer cell lines CAOV-3, SKOV-3, and BG-1. The ATP cell viability assay was used to measure growth effects after 6-day treatments with 27-mer antisense phosphorothioate oligodeoxyribonucleotides (oligos) targeting the Puf/nm23 binding region of c-myc and promoter/ATG region of p53. A random sequence of the p53 27-mer was used as a control, and an untransformed fibroblast cell line was used for comparison. IC50 was defined as the oligo concentration required for 50% growth reduction compared to untreated controls. Synergistic vs antagonistic effects of oligo combinations were quantitated by combination indexes (CI) as calculated from median effect parameters by the methods of Chou and Talalay. Mean +/- SE IC50's of c-myc and p53 antisense oligos in CAOV-3 and SKOV-3 ranged from 1.0 +/- 0.2 to 9.7 +/- 1.3 microM. The IC50's of c-myc oligos were consistently lower than corresponding p53 oligos in all cell lines (P < 0.034, t test). The fibroblast cell line was sensitive to anti-c-myc and combination anti-c-myc/p53 oligos (IC50 = 1.5 +/- 0.6 and 1.4 +/- 0.2 microM, respectively), but not to anti-p53 oligos alone (IC50 > 16 microM). Nonspecific toxicity was observed at concentrations of 16 microM for all cell lines except in BG-1, where maximal growth stimulation occurred at this concentration with anti-p53 oligos. Growth stimulation was also observed in BG-1 with anti-c-myc and anti-c-myc/p53 combinations at intermediate doses, with inhibition at higher doses. While c-myc/p53 combinations in CAOV-3 were synergistic (CI < 0.8), they were antagonistic in SKOV-3 (CI > 3.2). Phosphorothioate oligos directed against c-myc and p53 in different cell lines were shown to have both antiproliferative and stimulatory activity, as single agents and in combination, at concentrations that are achievable in vivo. Because of the complex patterns of effects, further in vitro studies are warranted before considering clinical trials with these agents in gynecologic cancers.

The National Cancer Data Base report on ovarian cancer. American College of Surgeons Commission on Cancer and the American Cancer Society.

BACKGROUND: Reports generated from the National Cancer Data Base (NCDB), a joint project of the American College of Surgeons Commission of Cancer and the American Cancer Society, have described trends in demographics, stage, treatment patterns, and survival for a variety of cancers. In this report, the most current (1991) data for ovarian cancer are presented and include some comparisons with 1985/1986 data. METHODS: Three calls for data from hospital registries across the United States have yielded 17,114 ovarian cancer cases for 1985, 1986, and 1991 combined. These data represent approximately 23%, 23%, and 43%, respectively, of the annual number of cases of ovarian cancer in the United States for those years. RESULTS: One-fourth of the reported cases of ovarian cancer were diagnosed in women less than 50 years of age. Younger patients (< 40 years) were more likely to have received conservative therapy (unilateral oophorectomy), consistent with their high prevalence (59%) of Stage I disease. The number of patients reported with an unknown American Joint Committee on Cancer (AJCC) stage decreased from 49% in 1985/1986 to 17% in 1991, although the distribution within stages was unchanged. Increases in important staging procedures were reported in 1991, with threefold increase in the proportion of debulking procedures and a 50% increase in omentectomies accompanying hysterectomy compared with 1985/1986. More advanced disease was reported for those of older age, lower income, African Americans, and patients in smaller hospitals. Relative 5-year survival rates were 74% for patients with Stage I disease, 58% for Stage II, 30% for Stage III, and 19% for Stage IV. Asians and Hispanics presented with a relatively high rate of Stage I-II disease (45%) compared with non-Hispanic whites and African Americans (38% and 33%, respectively). Hispanics presented with the most favorable Stage I/IV ratio (1.5) and had an overall 5-year survival of 50% compared with 41% and 37% for non-Hispanic whites and African Americans (Stage I/IV ratios of 1.0 and 0.7, respectively). There was little difference reported in the use of multimodality treatment between 1985/1986 and 1991. CONCLUSIONS: A trend toward more complete surgery with full surgical/pathologic staging was observed in 1991, but there was not yet evidence to indicate significant improvements in ovarian cancer survival compared with published figures during the past 10-15 years. Important ethnic and demographic differences in type of surgery and survival were noted but could not be differentiated from differences in tumor stage.

Invasive endometrial cancer in uteri resected for atypical endometrial hyperplasia.

Endometrial hyperplasia (EH) includes a spectrum of lesions with unclear malignant potential. To examine the association between the most advanced forms of hyperplasia and the occurrence of endometrial cancer, we compared the findings of endometrial biopsies or curettings with the subsequent hysterectomy specimens in 44 women who underwent hysterectomy for "atypical" EH in the 39-month period from January 1, 1989 through April 1, 1992. Endometrial cancer was found in 19 (43%) of 44 hysterectomy specimens obtained within a mean of 10 weeks of uterine sampling. Myometrial invasion was present in 17 (89%) of the 19 specimens with cancer, while significant myometrial invasion (FIGO Stage IC or higher) was present in 7 (37%), and 4 (21%) were Grade 2 or higher. Preoperative sampling method and type of atypical hyperplasia (simple vs complex) were not significantly associated with the finding of cancer at hysterectomy. Although over one-third of the cancers found were in the less than 50 age group, an age of 70 or greater was significantly associated with cancer at hysterectomy (P < 0.05, Fisher's exact test). In a limited set of hysterectomy specimens for which estrogen and progesterone receptor status was assayed (n = 11), there was no significant difference between the invasive cancer and EH subsets. Our findings suggest that women who are candidates for hysterectomy on the basis of atypical EH should be carefully evaluated for the possibility of advanced disease. The specimens in these circumstances should be opened upon removal to determine if myometrial invasion is present and if further surgical staging is indicated.

Malondialdehyde precursors in gamma-irradiated DNA, deoxynucleotides and deoxynucleosides.

Gamma-irradiation of DNA, deoxynucleosides, or deoxynucleotides produces material that reacts with thiobarbituric acid to form a chromophore with maximum absorbance at 532 nm. This material is not malondialdehyde. We have identified a new radiation product (thymin-1'-yl)-propenal as the TBA-reactive product of gamma-irradiation of thymidine. Thymine-propenal has been described by other investigators as a product of bleomycin-treatment of DNA. Irradiation of thymidine nucleotides produces phosphorylated precursors to thymine-propenal. Studies of the requirements for formation of TBA-reactivity indicate a mechanism involving reaction of a free radical with the deoxyribose moiety and molecular oxygen. On the basis of these results it is proposed that gamma-irradiation produces TBA-reactive material in DNA by the same reaction sequence in which bleomycin catalyzes the formation of base-propenals in DNA. Bleomycin and gamma-irradiation differ in the extent to which the sequence proceeds to completion with release of free base-propenals.