Ki-67, cyclin E, and p16INK4 are complimentary surrogate biomarkers for human papilloma virus-related cervical neoplasia.

Prior studies of Ki-67, cyclin E, and p16 expression have suggested that these biomarkers may be preferentially expressed in cervical neoplasia. This study examined and compared the distribution of staining for these three antigens in 1) normal and reactive epithelial changes, 2) diagnostically challenging cases (atypical metaplasia and atypical atrophy), 3) squamous intraepithelial lesions (SIL), and 4) high-and low-risk human papilloma virus (HPV) type-specific SIL. One hundred four epithelial foci from 99 biopsies were studied, including low-grade squamous intraepithelial lesions (LSIL; 24), high-grade squamous intraepithelial lesions (HSIL; 36), mature or immature (metaplastic) squamous epithelium (29), and atrophic or metaplastic epithelium with atypia (15). Cases were scored positive for Ki-67 expression if expression extended above the basal one third of the epithelium, for cyclin E if moderate to strong staining was present, and for p16 if moderate to strong diffuse or focal staining was present. HPV status was scored by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of extracted DNA. Immunohistochemical findings were correlated with histologic and viral data. Overall, a histologic diagnosis of SIL correlated strongly with all of the biomarkers used (p <0.001). Positive scores for Ki-67, cyclin E, and p16 were seen in 68.4%, 96.7%, and 100% of LSILs and 94.7%, 91.6%, and 100% of HSILs, respectively. Positive predictive values of these three biomarkers for HPV were 82.4%, 89.5%, and 91.4%, respectively. The positive predictive value for HPV of either cyclin E or p16 was 88.7%. Strong diffuse staining for p16 was significantly associated with high-risk HPV-associated lesions. Normal or reactive epithelial changes scored positive for the three biomarkers in 7.7%, 8.0%, and 12%, respectively. Limitations in specificity included minimal or no suprabasal staining for Ki-67 in immature condylomas and occasional suprabasal staining of reactive epithelial changes (10%), diffuse weak nuclear cyclin E staining in some normal or metaplastic epithelia, and diffuse weak basal p16 staining and occasional stronger focal positivity in normal epithelia. Ki-67, cyclin E, and p16 are complementary surrogate biomarkers for HPV-related preinvasive squamous cervical disease. (Because cyclin E and p16 are most sensitive for LSIL and HSIL [including high-risk HPV], respectively, use of these biomarkers in combination for resolving diagnostic problems, with an appreciation of potential background staining, is recommended.)

Multimodality therapy in early-stage neuroendocrine carcinoma of the uterine cervix.

OBJECTIVE: Patients with early-stage neuroendocrine cervical carcinoma (NECC) have a high mortality rate despite aggressive therapy. The rarity of this tumor precludes initiation of a randomized, prospective trial. We reviewed our experience in early stage disease and performed a meta-analysis of the literature to identify prognostic factors and determine optimal multimodality therapy. METHODS: Eleven women with International Federation of Gynecology and Obstetrics (FIGO) early stage (IB--IIA) NECC were treated with surgery and chemotherapy at our institutions between 1978 and 1998. Administration of radiation therapy was recorded, but not required for inclusion in this study. A gynecologic pathologist reviewed all histopathologic sections. Medical records were retrospectively reviewed and clinical data obtained. Twenty-three early-stage NECC patients who were similarly treated during the study interval were identified by a Medline search of the English literature and included in the analysis. The Kaplan--Meier method and log-rank test were used for survival analysis. RESULTS: The overall 2-year survival rate for the 34 patients was 38%. The median age was 37 years (range, 20--75 years). Median cervical tumor diameter was 3.2 cm (range 0.5--11.0 cm). Lymphovascular space invasion was present in 21 (78%) of 27 patients (7 unknown). Fifteen (52%) of twenty-nine had lymph node metastases (5 unknown). Fifteen patients received postoperative platinum/etoposide (PE), seven received vincristine/adriamycin/cyclophosphamide (VAC), two received alternating cycles of VAC and PE, and ten received other chemotherapy regimens. Twenty women were treated with radiation therapy. The presence of lymph node metastases was a poor prognostic factor (P < 0.001). PE and VAC chemotherapy was associated with increased survival (P < 0.01). CONCLUSION: NECC is a highly lethal variant of cervical cancer. The presence of lymph node metastases is the most important prognostic variable. Postoperative VAC or PE appears most likely to improve chances for survival.

Expression of the p53 homologue p63 in early cervical neoplasia.

BACKGROUND: p63, a homologue of the tumor suppressor gene p53, is expressed in embryonic, adult murine, and human basal squamous epithelium and encodes both transactivating and dominant negative transcript isoforms. Mouse embryos functionally deficient in p63 fail to replenish basal squamous epithelial cells, resulting in multiple defects that include absent genital squamous epithelium. This study investigated the expression of p63 in the human cervical transformation zone and early cervical neoplasia. METHODS: Tissue localization of p63 was determined by immunohistochemistry in a wide range of epithelia. A correlation was also made between p63 expression and squamous basal cell (keratin 14), endocervical columnar cell (mucicarmine), and cell-cycle specific (Ki-67) markers. RESULTS: p63 expression by immunostaining delineated basal and parabasal cells of maturing ectocervical squamous mucosa, squamous metaplasia in the cervix, and basal and subcolumnar cells of the cervical transformation zone. In atrophic epithelia immunostaining for p63 was present in all cell strata. In early cervical neoplasia, p63 expression was inversely correlated with both squamous cell maturation and nonsquamous differentiation in CIN. This biomarker also identified basal cells in a subset of preinvasive cervical neoplasms with endocervical cell differentiation that were bcl-2 and keratin 14 negative. CONCLUSIONS: In the lower female genital tract, p63 is preferentially expressed in immature cells of squamous lineage and is not linked to cell proliferation. The broader range of p63 expression relevant to keratin 14 and bcl-2 indicates that p63 may identify additional subsets of benign and neoplastic epithelial basal cells in the cervical transformation zone and may be useful in studying cell differentiation in the early stages of neoplastic change in this region.

Reporting the potential benefits of new technologies for cervical cancer screening.

The AHCPR released its evidence-based report, "Evaluation of Cervical Cytology" in early 1999. This report represents the most comprehensive analysis available to date of Pap smears and new technologies designed to improve cervical cancer screening. Both the ACOG and the AHCPR have released simplified summaries of the results of the evidence report that may lead to misunderstandings of the potential clinical impact of these new technologies. This report reviews the 4 major statements in these summaries and discusses how they are either incorrect based on the full AHCPR report or may be misinterpreted because their ramifications are not fully discussed. New screening technology has the potential to finally bring the sensitivity of a new Pap test to an acceptable level. The increased sensitivity afforded by these new technologies can reduce negative outcomes at reasonable cost-effectiveness ratios and at an equivalent or superior specificity compared to the conventional Pap smear.

Stratified mucin-producing intraepithelial lesions of the cervix: adenosquamous or columnar cell neoplasia?

BACKGROUND: Squamous (CIN) and glandular (ACIS) intraepithelial lesions often coexist in the same cervical specimen. However, a less common and little studied variant consists of a stratified epithelium resembling CIN in which conspicuous mucin production is present (Stratified Mucin-producing Intraepithelial LEsions (SMILE). This report describes the phenotypic characteristics of the SMILE, its associated lesions, and its immunophenotype. METHODS: Eighteen SMILEs were identified by the presence of conspicuous cytoplasmic clearing or vacuoles in lesions otherwise resembling CIN. The morphologic spectrum of SMILEs was detailed; including associated intraepithelial and invasive cervical neoplasms. In addition, selected cases were stained for mucicarmine, markers of squamous cell/reserve cell differentiation (keratin-14 and p63), and proliferative activity (Mib-1). RESULTS: Stratified neoplastic epithelial cells with a high Mib-1 index and a rounded or lobular contour at the epithelialstromal interface characterized SMILEs. In contrast to CIN, in which mucin droplets are confined to surface cells, mucin was present throughout the epithelium, varying from indistinct cytoplasmic clearing to discrete vacuoles. SMILEs were distinguished from benign metaplasia by nuclear hyperchromasia and a high Mib-1 index. All but three coexisted with either a squamous (CIN) or glandular (ACIS) precursor lesion. Nine of nine coexisting invasive carcinomas contained glandular, adenosquamous differentiation, or both. SMILEs stained negative for keratin-14 and variably for p63. When present, staining with p63 was confined to basal areas of SMILEs and was absent in areas of columnar differentiation. CONCLUSIONS: SMILEs are unusual cervical intraepithelial lesions best classified as variants of endocervical columnar cell neoplasia based on immunophenotype. The distribution and immunophenotype of SMILEs are consistent with a neoplasm arising in reserve cells in the transformation zone. The coexistence of a wide spectrum of intraepithelial and invasive cell phenotypes suggests that SMILEs are a marker for phenotypic instability, emphasizing the importance of identifying SMILEs and ensuring a complete examination of specimens containing this unusual precursor lesion.

Conservative management of adenocarcinoma in situ of the cervix.

OBJECTIVE: The purpose of this study was to determine the method of treatment and outcome of women with cervical adenocarcinoma in situ (AIS). METHODS: Following institutional review board approval, all women diagnosed with cervical AIS from 1987 to 1999 were identified. Data were retrospectively collected by record review and correspondence with medical providers. RESULTS: Of 132 women treated with cone biopsy for AIS, 95 (72%) were managed conservatively after cold knife cone or loop electrical excisional procedure alone; 37 (28%) eventually underwent hysterectomy. The median age of diagnosis was 29 years (range, 17-47) in the conservative management group and 40 years (range, 25-72) in the hysterectomy group (P < 0.0001). Seventy-four percent were nulliparous in the conservative group compared with 27% in the hysterectomy group (P < 0.0001). Of the 95 conservatively managed patients, 92 obtained negative margins; three were followed despite positive or unevaluable margins. During a median follow-up of 30 months, 9 women required evaluation for follow-up abnormalities after cone biopsy with negative margins. None had pathologic evidence of recurrent AIS. Twenty-three infants were delivered. Hysterectomy was generally performed for undesired fertility or persistently positive cone margins. One woman required hysterectomy for recurrent AIS. Thirteen (62%) of twenty-one hysterectomy specimens had residual AIS following cone biopsy with positive or unevaluable margins; 1 (6%) of 16 had residual AIS following cone biopsy with negative margins (P < 0.0001). No patient developed invasive adenocarcinoma. CONCLUSIONS: Younger women with cervical AIS may be effectively treated with cone biopsy alone if negative margins can be achieved.

Prospective management of stage IA(1) cervical adenocarcinoma by conization alone to preserve fertility: a preliminary report.

OBJECTIVE: Microinvasive cervical adenocarcinoma has an excellent prognosis and emerging data support the hypothesis that it should be treated in the same way as its squamous counterpart. We report our preliminary experience prospectively treating stage IA(1) cervical adenocarcinoma by conization alone in women who strongly desired to preserve their fertility. METHODS: Since May 1998, all patients with stage IA(1) cervical adenocarcinoma who expressed a strong desire to preserve fertility have been offered cold knife conization (CKC) and careful surveillance without hysterectomy. Women with lesions identifiable only microscopically, up to 3 mm invasive depth, up to 7 mm tumor width, and a conization specimen including the entire lesion with negative margins were eligible for conservative management. Postconization surveillance consisted of a Pap smear and endocervical curettage every 4 months. Medical records were reviewed for clinical data, follow-up, and disease status. RESULTS: Five women ages 26-33 elected CKC and surveillance. Four were nulliparous and one primiparous. Four tumors were endocervical cell type; one was adenosquamous. Three were grade 1, one was grade 2, and one grade 3. None had lymph-vascular space invasion. None of the patients has developed recurrent disease after 6-20 months of follow-up. CONCLUSIONS: Our preliminary data suggest that patients with FIGO stage IA(1) cervical adenocarcinoma who strongly desire to preserve their fertility may be treated by conization alone if they are fully informed of the unknown risks for disease recurrence and are carefully followed. A multicenter trial is the next logical step to test the efficacy of this approach.

Adenoid basal carcinomas of the cervix: a unique morphological evolution with cell cycle correlates.

Adenoid basal carcinoma (ABC) is a rare cervical carcinoma of postmenopausal women composed of small basal-type (basaloid) cells with focal endocervical ("adenoid") differentiation. ABCs are associated with high-grade squamous intraepithelial lesions (HSIL) and contain integrated human papillomavirus type 16 DNA. However, ABCs have a favorable prognosis and do not metastasize. Five (5) ABCs were analyzed histologically for a marker distinguishing basal/ squamous from columnar (adenoid) differentiation (p63) and cell cycle activity (Ki-67), and compared with 20 cervical (CC) carcinomas. In contrast to other CCs, ABCs contained 4 distinct components, including (1) a classic HSIL; (2) a limited invasive component with squamoid maturation, often with a discrete layer of peripheral basal cells; (3) outgrowth of small basal cells from either HSIL or squamoid areas; (4) focal endocervical (adenoid) differentiation. ABCs showed distinct differences in cell cycle activity relative to CCs. Ki-67 positivity was high in associated HSILs but remained high and concentrated in the suprabasal cells of the invasive squamoid component of ABC. Moreover, proliferative index was variable to sharply reduced in areas of basaloid and adenoid differentiation, in contrast to conventional CCs. ABC is a unique neoplasm, not only by its transition through multiple phenotypes during invasion, but also by a proliferative index that is high in more mature neoplastic cells during the infiltrative process and reduced with progressive basal differentiation. The precise mechanism underlying this unique process of tumor evolution is unclear. However, the postmenopausal status of these patients suggests that host factors related to aging may influence tumor evolution and morphology after HPV 16 infection.

Allelic imbalance in lichen sclerosus, hyperplasia, and intraepithelial neoplasia of the vulva.

BACKGROUND: Few studies have addressed in detail the genetic alterations that occur in vulvar squamous carcinomas (VSCC) and their precursor lesions. In a previous study, we determined the most common chromosomal loci for allelic imbalance (AI) in HPV-positive and -negative VSCCs. The present study was designed to determine whether AI and the microsatellite instability phenotype (MIN) were present in epithelial lesions known to be associated with VSCC. DESIGN: Fifty-seven epithelial loci were analyzed, including HPV-positive (classic) and -negative (differentiated) vulvar intraepithelial neoplasms (VINs), lichen sclerosus (LS), and nonatypical hyperplasias. Thirty-one epithelial loci (55%) were obtained from patients with associated invasive vulvar carcinoma. HPV status was determined by polymerase chain reaction analysis. AI and MIN were determined by comparisons of microdissected target tissues with stromal controls, targeting 11 chromosomal loci. RESULTS: AI was identified in all epithelial categories, involving at least one chromosomal locus in 67, 53, 50, and 43% of classic VIN, differentiated VIN, hyperplasia, and LS. MIN was infrequent (10-13%), but confined to HPV-negative epithelial changes. HPV-positive lesions generally scored for AI more frequently, but certain loci scored nearly equally in both HPV-positive and -negative lesions, including 8p, 11q, and 17p. There were no differences in frequency of AI between epithelia with and without associated invasive carcinoma. CONCLUSIONS: The presence of allelic imbalance in vulvar hyperplasia and LS supports the hypothesis that these alterations are at greater risk for neoplasia despite the absence of conspicuous cellular atypia. A model is proposed in which these changes represent monoclonal expansion and are at empirically greater risk for subsequent "critical events" leading to morphologic atypia (VIN). The possibility that these early genetic changes influence both HPV-positive and -negative pathways merits further study.

Preclinical feasibility study of NMP179, a nuclear matrix protein marker for cervical dysplasia.

OBJECTIVE: To evaluate, in a preclinical feasibility study, the efficacy of NMP179, a monoclonal antibody recognizing a cervical tumor-associated nuclear matrix antigen, for the early detection of high and low grade cervical intraepithelial neoplasia. STUDY DESIGN: In a blind study involving two clinical sites, NMP179 immunocytochemical staining data from 261 cervicovaginal Thin-Prep specimens were evaluated. Assay sensitivity and specificity were calculated based upon a positive threshold of > 10 immunostained cells per case, using cytologic diagnosis as an end point. RESULTS: Based upon the examination of squamous epithelial cells, NMP179 detected 96.7% of cases with cytologically diagnosed high grade squamous intraepithelial lesions (HSIL) and 70.5% of low grade squamous intraepithelial lesions. The antibody also reacted with 29.6% of normal (within normal limits or benign cellular changes) smears. CONCLUSION: The NMP179 assay detected HSIL with very high accuracy (96.7%). The assay was 79.3% sensitive for the detection of low and high grade cervical intraepithelial neoplasia (grades 1-3), with a specificity of 70.4%. NMP179 may be an effective marker for the early detection of preneoplastic squamous intraepithelial lesions of the cervix and may be useful as an adjunctive tool for better management of cervical intraepithelial neoplasia.

Early cervical adenocarcinoma: selection criteria for radical surgery.

OBJECTIVE: To identify selection criteria for radical surgery in early cervical adenocarcinoma based on pretreatment clinical stage and correlation with high-risk surgical-pathologic factors. METHODS: One hundred seventy-five women with International Federation of Gynecology and Obstetrics (FIGO) clinical stage IB1 (n = 132) and IB2-IIA (n = 43) cervical adenocarcinoma were treated primarily at our institutions from 1982 to 1996. Histopathologic sections were reviewed by a gynecologic pathologist. Medical records were reviewed retrospectively and clinical follow-up was done. RESULTS: The overall 5-year survival rate was 87% (95% confidence interval [CI] 81%, 93%) for stage IB1 and 61% (95% CI 46%, 77%) for stage IB2-IIA (P<.001). Adenosquamous cell type, deep cervical invasion, and lymph-vascular space invasion were significant independent high-risk surgical-pathologic factors that affected disease-free survival (each P<.002). One hundred fourteen (86%) of 132 stage IB1 patients and 19 (44%) of 43 stage IB2-IIA subjects were treated primarily with radical surgery. Lymph node metastases, lymph-vascular space invasion, adenosquamous cell type, deep cervical invasion, and positive surgical margins were more than twice as frequent in stage IB2-IIA patients who had radical surgery than in stage IB1 patients (each P <.05). Based on high-risk surgical-pathologic factors in 133 subjects who had radical surgery, postoperative radiotherapy was recommended for 18 (16%) of 114 stage IB1 patients and 18 (95%) of 19 stage IB2-IIA subjects (P<.001). CONCLUSION: Radical surgery for FIGO clinical stage IB1 cervical adenocarcinoma and primary radiotherapy for stage IB2-IIA disease would largely avoid combined-modality therapy, thereby reducing treatment-related toxicity and cost.

Stage IA1 cervical adenocarcinoma: definition and treatment.

OBJECTIVE: To propose a definition for stage IA1 cervical adenocarcinoma, based on the International Federation of Gynecology and Obstetrics (FIGO) staging system, and to determine if patients meeting criteria might be candidates for conservative surgery. METHODS: Two hundred women were diagnosed with early-stage cervical adenocarcinoma from 1982 to 1996. Histopathologic sections were reviewed by a gynecologic pathologist. Medical records were reviewed, and patients included in this study had microscopically identifiable lesions, up to 3 mm invasive depth, up to 7 mm tumor width, and negative margins if cone biopsy was performed. RESULTS: Twenty-one patients with microinvasive adenocarcinoma met criteria for FIGO stage IA1 carcinoma of the cervix. The median (range) follow-up was 76 (30-172) months and median (range) patient age was 38 (24-75) years. Definitive treatment included type II or III radical hysterectomy in 16 cases, simple abdominal or vaginal hysterectomy in four cases, and loop electrosurgical excision procedure in one case; one patient received adjuvant pelvic radiation. The histologic subtypes were endocervical adenocarcinoma in 18 cases, adenosquamous carcinoma in two cases, and clear-cell adenocarcinoma in one case. There was no evidence of parametrial invasion or lymph node metastases in any patient who had radical surgery, and there were no disease recurrences. CONCLUSION: Patients with microinvasive adenocarcinoma who met criteria for FIGO stage IA1 cervical carcinoma had disease limited to the cervix, and conservative surgery, such as cone biopsy or simple hysterectomy, might offer them definitive treatment.

Papillary immature metaplasia (immature condyloma) of the cervix: a clinicopathologic analysis and comparison with papillary squamous carcinoma.

Papillary immature metaplasia (PIM) is a variant of human papillomavirus (HPV) 6 or 11 infection. PIM resembles an immature metaplasia but has filiform papillae, variable cytological atypia, and, frequently, extension into the endocervical canal. Because the unusual morphology and presentation of PIM may cause confusion between this and other benign and malignant papillary neoplasms, we conducted a clinicopathologic analysis of PIM and compared expression of Ki-67 between PIM, condyloma, and papillary carcinoma. Data on patient age, duration of the lesions, and procedures, including cone biopsy, were obtained. The distribution and intensity of staining for Ki-67 in the epithelium was recorded and compared with both condyloma and papillary carcinoma. HPV typing was performed by polymerase chain reaction (PCR) and restriction fragment length pleomorphism analysis (RFLP). Ten of 13 PIMs were HPV 6/11 positive. Three cases contained areas closely resembling condyloma. Eleven cone biopsies were performed on nine cases. Three were found to have a coexisting high-grade squamous intraepithelial lesion that was either HPV 6/11 negative or contained another HPV type. All PIMs displayed variable staining for Ki-67 with a low index of staining in the mid and upper epithelial layers. In contrast, areas of condyloma had significantly stronger staining in areas with viral cytopathic effect (koilocytosis). Six papillary carcinomas were analyzed and displayed moderate to diffuse staining, including staining of the superficial cell nuclei. PIM is a distinct pathological subset of cervical condyloma that frequently is managed by cone biopsy and may persist. The marked reduction in Ki-67 staining in superficial cell layers distinguishes PIM from some condylomata and most HSILs and papillary carcinomas. Immunostaining thus may be helpful in distinguishing PIM from papillary carcinoma, although the differentiation of the two is best made on morphological grounds.

Disparities in mean age and histopathologic grade between human papillomavirus type-specific early cervical neoplasms.

Noninvasive squamous and glandular precursor lesions associated with human papillomavirus (HPV) types 16 and 18 have been reported to vary in morphology. HPV 16 is associated predominantly with high-grade squamous intraepithelial lesions (HSIL; cervical intraepithelial neoplasia (CIN 2 and 3), and HPV 18 is associated with low-grade squamous intraepithelial lesions (condyloma/CIN 1) and CIN 3/adenocarcinoma in situ (ACIS). This study explored the relationship of morphologic growth pattern in these precursor groups with age of presentation. One hundred fourteen CIN lesions (including those with ACIS), associated with HPV 16 or 18, were subdivided into well-differentiated low- and high-grade SIL (CIN 1 and 2, respectively), poorly differentiated HSIL (CIN 3) with or without ACIS. HPV was detected by polymerase chain reaction (PCR) amplification with L1 consensus or type-specific E7 primers and typed by restriction fragment length polymorphism (RFLP) analysis. Age of the patient was obtained from the pathology report. Mean age for each group was as follows: Low-risk HPVs, 25 years; HPV 18 CIN 1-2, 21.6 yrs; HPV 18 CIN 3/ACIS, 35.2 yrs; HPV 16 CIN 1,2, 25.9 yrs; and HPV 16 CIN 3, 29.8 yrs. There were significant differences in mean ages between HPV 18 CIN 1 and 2 and HPV 16 CIN 1 to 2 (P = .04), HPV 16 CIN 1-2 and CIN 3 (P = .01) and HPV 18 CIN 1 to 2 and HPV 18 CIN 3/ACIS (P = .00001). None of the cases of HPV 18-associated CIN3/ACIS was associated with a CINI lesion. The disparity in mean ages between well and poorly differentiated HPV 16/18 related that precursor lesions could reflect factors such as morphologic progression with increasing age, different rates of lesion persistence, depending on grade, or efficiency of detection between the two groups. The marked difference in mean age between HPV 18-associated CIN 1-2 and CIN 3/ACIS, combined with their lack of coexistence in the same cervix, raises alternate possibilities that specific viral or host factors may determine the morphological phenotype associated with some HPV 18 infections. In the latter, the possibility that age independently confers an increased risk for higher-grade lesions should be considered.

The role of apoptosis in gynaecological malignancies.

Apoptosis is a process of single-cell deletion requiring active participation of the cell in its own demise. First described in 1972, it is now known to play a major role in embryogenesis, tissue homeostasis and neoplasia. Apoptosis can be initiated when DNA damage occurs causing the cell to pause in its reproductive cycle. If the DNA damage is beyond repair, the cell proceeds to apoptotic cell death. When the genetic mechanism(s) involved in the pathway of apoptosis is altered, the cell does not die. Further mutations occur by proliferation and such multiple mutational events can lead to a malignant phenotype and cancer growth. The tumour suppressor gene p53 causes a DNA-damaged cell to rest and attempt repair. If damage is irreparable, p53 levels will continue to increase, initiating apoptosis. Mutation of p53, found in approximately 50% of cancers, can stop the apoptotic process. Increased bcl-2 expression, an apoptosis inhibitor, also plays a role in cellular transformation and cancer growth. Its altered expression occurs in the presence of oncogene expression. This paper reviews the role of apoptosis in malignant transformation, cancer growth, and response to therapy for gynaecological cancers. For cervical cancer and its precursors, data on apoptotic index, bcl-2 and Bax expression are presented and discussed in relationship to human papillomavirus expression. In ovarian epithelial malignancies, the role that apoptosis plays in chemotherapeutic responses is reviewed. The data for endometrial cancer are currently limited to apoptotic index.

Association between cervical neoplasia and apoptosis as detected by in situ nuclear labeling.

Invasive cervical cancer is thought to arise from the progression of precancerous lesions. How these lesions proceed from precancers to cancer remains unknown. Data regarding other tissues indicate that altered programmed cell death (apoptosis), in addition to cellular proliferation, is associated with the development of neoplasia. Therefore, in order to better understand the development of cervical neoplasia, we investigated the rate of apoptosis in cervical precancer and cancer. Archival cervical samples from normal epithelium (n = 11), low-grade squamous intraepithelial lesions (LSIL, n = 11), high-grade squamous intraepithelial lesions (HSIL, n = 10), and squamous cancers (n = 10) were evaluated for chromatin cleavage, a hallmark of programmed cell death. We used in situ end-labeling of DNA strand breaks by terminal deoxynucleotidyltransferase incorporation of biotinylated deoxyuridine to 3'-OH ends of DNA, identified by a nickel-avidin-peroxidase. The apoptotic index (sum of apoptotic bodies divided by the total nuclei times 100) significantly decreased (P < 0.001) as the degree of neoplasia increased: 3.5% (+/-0.4) in normal cervical epithelium (4.8 +/- 0.4) in LSIL, 1.4% (+/-0.4) in HSIL, and 0.4% (+/-0.1) in squamous cancers. Compared to normal epithelium, the total cell number per 10 mm2 increased significantly (P < 0.001): 124 (+/-12) in normal epithelium, 162 (+/-9.7) in LSIL, 315 (+/-31) in HSIL, and 413 (+/-32) in squamous cancers. We conclude that increasing cervical atypicality is associated with a decrease in apoptosis. We hypothesize from our data that one factor involved in the progression of neoplasia in the uterine cervix is a decrease in the rate of normal cellular deletion.

A comparison of early (E7) and late (L1) primer-mediated amplification of papillomaviral DNA in cervical neoplasia.

Studies have demonstrated that in 50-90% of cervical carcinomas, human papillomavirus (HPV) DNA sequences are covalently bound (integrated) to the chromosomal DNA. All evidence shows that when integration takes place disruption of the viral genome occurs downstream to the E7 open reading frame, which is invariably retained in functional form. Theoretically, this phenomenon could result in loss of HPV sequences (L1) not critical to the presumed tumourigenic functions and if so, could influence primer selection for HPV DNA detection in these tumours. A series of cervical carcinomas (CA, n = 133), adenocarcinomas in situ (ACIS, n = 28) and high grade squamous intraepithelial lesions (HSIL, n = 30) were analysed for HPV nucleic acids using primers designed to amplify the E7 and L1 regions. Primer sizes and sensitivities were adjusted to produce equivalent amplification efficiency. Of 191 cases studied, 134 (70%) scored positive for HPV16 or 18 with either the E7 or L1 primer set. Of these, 116 (87%) were positive with both primer pairs. There were no significant differences in proportions of HPV 16/18 positives or lesion types scoring positive exclusively with the E7 vs the L1 primer sets. However, HPV18 associated, E7 positive carcinomas were slightly less likely than HPV16 associated carcinomas to be L1 positive (P = 0.07). Although a high proportion of HPV16 and particularly HPV18 positive carcinomas have been associated with exclusively integrated HPV DNA, there is little evidence that this influences detection sensitivity with E7 vs L1 primers. The combination of E7 and L1 primers provided the maximum sensitivity in this study, with 18 of 134 cases scoring positive with only one primer set.

Absence of papillomavirus DNA in normal tissue adjacent to most cervical intraepithelial neoplasms.

OBJECTIVE: To determine if human papillomavirus (HPV) DNA is present in the normal mucosa adjacent to cervical intraepithelial neoplasia (CIN). METHODS: Serial sections of 28 CIN lesions were studied. Lesional and normal epithelia and stroma were microdissected; the DNA was extracted and amplified by polymerase chain reaction (PCR) using primers designed to amplify both HPV late (L1) and human beta-globin sequences. Human papillomavirus was typed by restriction fragment length polymorphism analysis following digestion of PCR products. RESULTS: Twenty-five of 28 (89%) lesional epithelia scored positive for HPV nucleic acids. In' four of 25 (16%) HPV-positives, the normal squamous epithelium scored positive for HPV nucleic acids, two of which (8%) also scored positive in the stroma. Repeat microdissection and PCR analysis of three of these cases was performed and all were negative in both normal epithelium and stroma, suggesting laboratory contamination. CONCLUSION: Human papillomavirus nucleic acids are present uncommonly in normal-appearing squamous epithelium adjacent to CIN. This does not exclude occult infection in the natural history of CIN but indicates that when lesions develop, occult infection is not normally maintained in the normal mucosa. This is consistent with the low recurrence rates following ablation as well as low indices of HPV positivity in normal cervices during follow-up. This finding should be taken into consideration when counseling patients and is relevant to the concept of HPV testing during follow-up after cone biopsy.

Viral and histopathologic correlates of MN and MIB-1 expression in cervical intraepithelial neoplasia.

A recently studied tumor antigen, MN, has been associated with cervical carcinomas and cervical intraepithelial neoplasms (CIN), suggesting that it may serve as a marker for cervical cancer or cancer risk. To determine if expression of the MN antigen paralleled parameters reflecting viral or biological events in precursor epithelium, MN expression was correlated with MIB-1 expression, morphological phenotype, and human papillomavirus (HPV) distribution and type in a series of CINs. Seventy-three percent, 62% and 83% of CIN I, II, and III, respectively, were MN antigen positive. The proportion of neoplastic cells immunoreactive for MN did not correlate with the CIN grade or with HPV types stratified by their association with cancer. Evaluation of serial sections showed no correlation between the frequency of MN antigen staining, the proportion of MIB-1 immunoreactive cells, or the proportion of HPV positive cells detected by in situ hybridization (ISH). CINs associated with prototypical high risk (HPV 16) types exhibited increased immunostaining for the MIB-1 antigen and were more often classified as HSIL in contrast to the other types. Thus, although MN expression previously has been associated strongly with squamous carcinoma, it did not emerge as a specific marker for either cancer-associated HPV types or high grade CIN. CIN I lesions associated with low and high risk HPV types were not distinguished by MIB-1 expression and viral replication. This emphasizes the interrelationship between vegetative viral functions (including viral replication) and morphological phenotype, irrespective of HPV type.