Effects of depot-medroxyprogesterone acetate on the immune microenvironment of the human cervix and endometrium: implications for HIV susceptibility.

Depot-medroxyprogesterone acetate is a commonly used injectable contraceptive that has been associated with an increased risk of HIV acquisition. This study compares effects of depot-medroxyprogesterone acetate on immune parameters from several upper reproductive tract compartments relevant to HIV-1 susceptibility in repetitive samples from 15 depot-medroxyprogesterone acetate users and 27 women not on hormonal contraceptives. Compared with samples from unexposed women in the mid-luteal phase, depot-medroxyprogesterone acetate use was associated with: increased endocervical concentrations of MCP1 and IFNalpha2; decreased endocervical concentrations of IL1beta and IL6; increased proportions of endometrial CD4+ and CD8+ cells expressing the activation marker HLADR; increased density of endometrial macrophages; and decreased density of endometrial regulatory T cells. Unlike previous reports with samples from the vagina, we did not observe increased expression of the HIV co-receptor CCR5 on CD4+ T cells in the endocervix or endometrium. Our results indicate important differences in anatomic compartments regarding mechanisms by which depot-medroxyprogesterone acetate could be associated with increased risk of HIV acquisition, including increased recruitment of macrophages to the endometrium, decreased levels of pro-inflammatory cytokines in the endocervix possibly leading to enhanced susceptibility to viral infection, and activation of endometrial T cells.

Impact of loop electrosurgical excision procedure for cervical intraepithelial neoplasia on HIV-1 genital shedding: a prospective cohort study.

OBJECTIVE: We sought to examine the impact of the loop electrosurgical excision procedure (LEEP) on the rate and magnitude of HIV-1 genital shedding among women undergoing treatment for cervical intraepithelial neoplasia 2/3 (CIN2/3). DESIGN: Prospective cohort study. POPULATION: Women infected with HIV-1 undergoing LEEP for CIN2/3 in Kisumu, Kenya. METHODS: Participants underwent specimen collection for HIV-1 RNA prior to LEEP and at 1, 2, 4, 6, 10, and 14 weeks post-LEEP. HIV-1 viral load was measured in cervical and plasma specimens using commercial real-time polymerase chain reaction (PCR) assays, to a lower limit of detection of 40 copies per specimen. MAIN OUTCOME MEASURES: Presence and magnitude of HIV-1 RNA (copies per specimen or cps) in post-LEEP specimens, compared with baseline. RESULTS: Among women on highly active antiretroviral therapy (HAART), we found a statistically significant increase in cervical HIV-1 RNA concentration at week 2, with a mean increase of 0.43 log10 cps (95% CI 0.03-0.82) from baseline. Similarly, among women not receiving HAART, we found a statistically significant increase in HIV-1 shedding at week 2 (1.26 log10 cps, 95% CI 0.79-1.74). No other statistically significant increase in concentration or detection of cervical HIV-1 RNA at any of the remaining study visits were noted. CONCLUSIONS: In women infected with HIV undergoing LEEP, an increase in genital HIV shedding was observed at 2 but not at 4 weeks post-procedure. The current recommendation for women to abstain from vaginal intercourse for 4 weeks seems adequate to reduce the theoretical increased risk of HIV transmission following LEEP.

Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer.

BACKGROUND: Defects in BRCA1, BRCA2, and other members of the homologous recombination pathway have potential therapeutic relevance when used to support agents that introduce or exploit double-stranded DNA breaks. This study examines the association between homologous recombination defects and genomic patterns of loss of heterozygosity (LOH). METHODS: Ovarian tumours from two independent data sets were characterised for defects in BRCA1, BRCA2, and RAD51C, and LOH profiles were generated. Publically available data were downloaded for a third independent data set. The same analyses were performed on 57 cancer cell lines. RESULTS: Loss of heterozygosity regions of intermediate size were observed more frequently in tumours with defective BRCA1 or BRCA2 (P=10(-11)). The homologous recombination deficiency (HRD) score was defined as the number of these regions observed in a tumour sample. The association between HRD score and BRCA deficiency was validated in two independent ovarian cancer data sets (P=10(-5) and 10(-29)), and identified breast and pancreatic cell lines with BRCA defects. CONCLUSION: The HRD score appears capable of detecting homologous recombination defects regardless of aetiology or mechanism. This score could facilitate the use of PARP inhibitors and platinum in breast, ovarian, and other cancers.

Evolving immunosuppressive microenvironment during human cervical carcinogenesis.

Chronic infection with human papillomavirus (HPV) can result in cervical cancer. To understand how HPV escapes immune eradication, we examined biophenotypes of immune cells in human normal cervix, cervical intraepithelial neoplasia (CIN), and cancer. Expression and cellular localization of Forkhead box protein-3 (FOXP3), indolamine 2,3-dioxygenase (IDO), interleukin (IL)-10, and interferon (IFN)-gamma were examined by immunofluorescence and immunohistochemistry. Mean cell densities of stromal FOXP3+ cells, IDO+ cells, IL-10+ cells, CD1a+ cells, and macrophages significantly increased from normal cervix to cancer, whereas densities of IFN-gamma+ and MMP-9+ cells increased from normal cervix to CIN but decreased in cancer. Flow cytometry confirmed significant elevation of cervical T cells expressing IFN-gamma and transforming growth factor-beta in CIN compared with normal cervix. Upon activation, a significantly increased proportion of cervical T cells expressed IFN-gamma in CIN than normal. A unique subset of morphologically immature stromal dendritic cells expressing IL-10 and IDO was more numerous in cancer than in normal cervix and CIN. The potentially suppressive immune milieu in the cervix may be permissive of HPV-associated cervical carcinogenesis.

Identification of a novel splice variant of AML1b in ovarian cancer patients conferring loss of wild-type tumor suppressive functions.

Acute myeloid leukemia (AML) 1 is often disrupted by chromosomal translocations generating oncogenic fusions in human leukemias. However, its role in epithelial cancers has not been extensively investigated. Herein, we show a marked accumulation of AML1 transcripts including a high frequency of a novel alternatively spliced AML1b transcript lacking exon 6 (AML1b(Del179-242)) in ovarian cancer patients. The increases in RNA transcripts for total wild-type AML1 and AML1b(Del179-242) are associated with poor patient outcomes. We have shown that although both wild-type AML1b and AML1b(Del179-242) are localized to nuclear speckles, AML1b(Del179-242) was observed to have dramatically reduced transactivation potential with the plasminogen activator inhibitor-1 promoters and behaved as a weak dominant negative of wild-type AML1b. Wild-type AML1b was found to inhibit the growth of immortalized ovarian epithelial cells (T29) decreasing colony-forming ability. Moreover, we have identified a novel function of AML1b where it inhibits ovarian cell migration. In contrast, AML1b(Del179-242) has lost the ability to inhibit both ovarian cell proliferation and migration indicating that the functional effects observed with wild-type AML1b are dependent on amino acids 179-242. Collectively, these studies suggest that deregulated alternative splicing of AML1b transcripts may potentially contribute to the pathophysiology of ovarian cancers.

Participants at the ASCCP 2000 Biennial Meeting adhere to published guidelines in their management of atypical squamous cells and atypical glandular cells on pap test.

OBJECTIVE: To determine practice patterns of the American Society for Colposcopy and Cervical Pathology (ASCCP) 2000 Biennial Meeting participants for management of women with atypical squamous cells of undetermined significance (ASCUS) and atypical glandular cells of undetermined significance (AGUS). MATERIALS AND METHODS: A three-page survey was developed to query participants about their standard practices for managing atypical squamous cells and atypical glandular cells on Pap tests; use of human papillomavirus testing; and use of vaginal estrogen cream in postmenopausal women before follow-up. RESULTS: There were 217 completed surveys (47.0% response rate). Responses of 194 qualified surveys are presented here. For women with first-time ASCUS (unqualified) and no previous history of cervical intraepithelial neoplasia (CIN), 16.4% of respondents would perform colposcopy. For ASCUS (favor squamous intraepithelial lesion [SIL]), a significantly higher percent, 74.9%, would proceed immediately to colposcopy (p < .001). For ASCUS (unqualified or favor SIL) in a woman with a previous history of CIN, 82.7% and 95.5% would perform colposcopy, respectively (p < .001). For a patient with AGUS, 97.5% would perform colposcopy (with or without endocervical curettage or endometrial biopsy). Human papillomavirus testing was seldom used in the management of ASCUS (10.4%) and AGUS (7.8%). CONCLUSIONS: Participants at the 2000 ASCCP Biennial Meeting made clear distinctions between ASCUS and AGUS in managing women with abnormalities on Pap, and management was consistent with guidelines published by the ASCCP, American College of Obstetricians and Gynecologists, and the National Cancer Institute.

Management of women with atypical Papanicolaou tests of undetermined significance by board-certified gynecologists: discrepancies with published guidelines.

OBJECTIVE: The Bethesda System introduced 2 categories of atypia: atypical glandular cells of undetermined significance (AGUS) and atypical squamous cells of undetermined significance (ASCUS). Our objective was to test the hypothesis that there is lack of consensus regarding management of women with ASCUS and AGUS. STUDY DESIGN: A 2-page survey was mailed in April 1998 to a random sample of 491 fellows of the American College of Obstetricians and Gynecologists. RESULTS: There were 241 responses (50.6%). For first-time ASCUS, 23.0% of respondents would perform colposcopy and 24.4% would repeat the Papanicolaou test in <3 months, indicating aggressive management of ASCUS by 47.4% of respondents. For recurrent ASCUS, 88.7% followed recommendations to manage with colposcopy. For AGUS, 23% repeated the Papanicolaou test and only 43% would manage appropriately. For recurrent AGUS, only 25% would perform surgical excision. CONCLUSION: Compared with published guidelines, practitioners undermanage patients with AGUS and overmanage patients with ASCUS. Further physician education appears to be warranted.

Recent developments in understanding the immune response to human papilloma virus infection and cervical neoplasia.

PURPOSE/OBJECTIVES: To provide an overview of the immune system and describe the systemic and local immune response to human papilloma virus (HPV) infection in the cervix and to the development of cervical dysplasia. DATA SOURCES: Historical and current medical and nursing literature, current gynecologic oncology texts. DATA SYNTHESIS: The immune system provides protection against a wide variety of pathogens and plays a role in the response of the body to neoplastic cells. The immune system orchestrates the functions of various immune cells and proteins to fight against invading pathogens. Mucosal immunity is one of the key functions of the immune system and has unique features. Humoral and cytotoxic responses in the cervix and in the peripheral blood are seen during clearance of an HPV infection. CONCLUSIONS: The functional status of the immune system is associated closely with the development of cervical dysplasia and cancer in women with HPV infection. Clinicians must assist women in maintaining proper functioning of immune responses. IMPLICATIONS FOR NURSING PRACTICE: A better understanding of local and systemic immune responses to HPV infection may help nurses who provide care to women with cervical disease and women at high risk for cervical cancer to deliver better care and clearer information to patients.

Angiogenesis, proliferation, and apoptosis in anal high-grade squamous intraepithelial lesions.

PURPOSE: Management of anal high-grade squamous intraepithelial lesions is controversial. Anal and cervical high-grade squamous intraepithelial lesions are similar in that they occur in transitional squamous epithelium, are associated with human papilloma virus infection, and have increased incidence in the immunocompromised population. Ablation of cervical high-grade squamous intraepithelial lesions is preferred, but similar ablation or excision of anal high-grade squamous intraepithelial lesions may compromise bowel control; thus, there is a need to define the malignant potential of anal high-grade squamous intraepithelial lesions. METHODS: We analyzed 50 paraffin sections of normal anoderm, anal low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and anal squamous-cell carcinoma. Microvessels were detected immunohistochemically with von Willebrand factor and counted manually along the epithelial-stromal junction. Proliferation and apoptosis were determined in the epithelial cells with MIB-1 antibody immunostaining and the terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling, respectively. RESULTS: Microvascular density was significantly greater in anal high-grade squamous intraepithelial lesions (mean, 0.50 vessels/cm) vs. normal anoderm (mean, 0.21 vessels/cm; P = 0.0017, Mann-Whitney U test). The proliferative percentages were greater in low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and squamous-cell carcinoma (mean, 20.4, 21.8, and 23.6 percent) vs. normal anoderm (mean, 14.4 percent), although not significantly (P = 0.06, Kruskal-Wallis statistic). Although the mean proliferative proportions were similar in low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions, the apoptotic proportion was lower for high-grade squamous intraepithelial lesions than low-grade squamous intraepithelial lesions (10.13 vs. 19.96 percent, respectively; P = NS, Mann-Whitney U test). CONCLUSIONS: Angiogenesis, increased proliferation, and decreased apoptosis occur in anal high-grade squamous intraepithelial lesions as they do in the cervix before the development of malignancy. These biologic markers support the importance of anal high-grade squamous intraepithelial lesions as a potential premalignant lesion warranting surgical intervention.

Intranuclear localization of human papillomavirus 16 E7 during transformation and preferential binding of E7 to the Rb family member p130.

To study intracellular pathways by which the human papillomavirus 16 oncogene E7 participates in carcinogenesis, we expressed an inducible chimera of E7 by fusion to the hormone-binding domain of the estrogen receptor. The chimeric protein (E7ER) transformed rodent fibroblast cell lines and induced DNA synthesis on addition of estradiol. In coimmunoprecipitation experiments, E7ER preferentially bound p130 when compared to p107 and pRb. After estradiol addition, E7ER localization changed to a more intense intranuclear staining. Induction of E7 function was not correlated with binding to p130 or pRb but rather with intranuclear localization and modest induction of binding to p107.

Atypical squamous cells of undetermined significance: management patterns at an academic medical center.

OBJECTIVE: Our intent was to compare the management of patients with atypical squamous cells of undetermined significance on cytologic screening at an academic center to published guidelines. STUDY DESIGN: We reviewed the management of 223 atypical squamous cells of undetermined significance cervical smears. Patients with a history of dysplasia were excluded. The time interval to and nature of follow-up testing was determined, and the influence of atypical squamous cells of undetermined significance qualifiers and provider specialty analyzed. RESULTS: Initial follow-up consisted of repeat cytologic examination alone in 94% of cases. Of patients with follow-up, 29% were retested within 2 months and 68% within 4 months. No conclusive differences in management were found by qualifier type or by provider specialty. Subsequent high-grade dysplasia was found in 2.6% of patients. CONCLUSIONS: A discrepancy exists between published guidelines and actual management of patients with atypical squamous cells of undetermined significance smears at this medical center. Patients often undergo follow-up testing at shorter intervals than those suggested despite a low likelihood of finding high-grade disease.

Angiogenesis in histologically benign squamous mucosa is a sensitive marker for nearby cervical intraepithelial neoplasia.

Cervical intraepithelial neoplasia (CIN) lesions elicit a neovascularization response at the stromal-epithelial junction. In this study, the angiogenic properties of histologically benign cervical epithelium adjacent to CIN were compared to samples of normal cervix from patients with no evidence of CIN. Vessels were detected by immunohistochemical staining for von Willebrand factor (vWf). Expression of a potent angiogenic factor, vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), was measured by in situ hybridization. Statistical comparisons were made using the Student t-test. In histologically benign cervix adjacent to CIN, the microvessel count was significantly increased (164.5 versus 47.3 vessels/cm; p<0.004) and vessels were more closely apposed to the epithelium (15.3 versus 22.4 microm to the basal cell nuclei; p<0.0001) than in normal cervix. Expression of VEGF/VPF was rare in normal cervix but was detectable in more than 90% of sample s of benign epithelium adjacent to CIN. Therefore, vWf staining and VEGF/VPF expression are more sensitive at identifying nearby CIN than conventional histology.

Cross-species comparison of angiogenesis during the premalignant stages of squamous carcinogenesis in the human cervix and K14-HPV16 transgenic mice.

Infection of the human cervix with certain papillomavirus subtypes is associated with the development of neoplastic squamous lesions that can progress to overt cervical malignancies. Recently, multistage squamous carcinogenesis has been achieved in K14-HPV16 transgenic mice, wherein expression of the human papillomavirus (HPV) type 16 early genes is targeted to basal squamous epithelial cells by regulatory elements of the human keratin-14 (K14) promoter. Immunostaining of the endothelial marker vWf revealed a parallel upregulation of angiogenesis during the early neoplastic stages in both human cervix and the epidermis of K14-HPV16 transgenic mice. Moreover, high-grade premalignant lesions and cancers in humans and transgenic mice were characterized by an additional increment in the number of new capillaries and close apposition of the microvasculature to the overlying neoplastic epithelium. Expression of the potent angiogenic factor VEGF was progressively up-regulated during carcinogenesis in both species, correlating with the increased density and altered distribution of the microvasculature. Thus, angiogenesis occurs during the premalignant stages of squamous carcinogenesis in both human cervical disease and a relevant transgenic model and may be controlled by similar molecular mechanisms in both species. These results validate the use of the transgenic model to elucidate the role of angiogenesis during HPV-associated neoplastic progression.

Demonstration and characterization of the angiogenic properties of cervical dysplasia.

Cervical dysplasia, or cervical intraepithelial neoplasia (CIN), is a premalignant precursor to cervical cancer. This study was designed to determine whether dysplastic lesions are angiogenic. Tissue sections from 23 surgical specimens were immunohistochemically stained for factor VIII antigen, a marker for endothelial cells. The results demonstrate that a region of neovascularization develops along the basement membrane subtending dysplastic epithelium when compared to adjacent normal epithelium. Comparison of microvessel counts underlying low grade lesions (condyloma and CIN I) with microvessel counts of CIN III lesions shows a statistically significant increase in the more advanced lesions. In a subset of the high grade lesions, large vascular structures are also noted in the upper layers of the epithelium, suggesting that a second stage of neovascularization consists of extension of stromal vascular papillae into the dysplastic lesions toward the surface of the epithelium. There is no statistical correlation between the amount of inflammation and the angiogenic ratio for each lesion, implying that angiogenesis is not secondary to the inflammatory response evoked by the lesion. The human papillomavirus type present in four CIN III lesions was determined by in situ hybridization; the amount of angiogenesis appears to be independent of the human papillomavirus type.