Current cervical cancer screening is based on morphological assessment of Pap smears and associated with significant false negative and false positive results. Previously, we have shown that detection of hypermethylated genes in cervical scrapings using quantitative methylation-specific PCR (QMSP) is a promising tool for identification of squamous cell cervical cancer. Aim of the present pilot-study was to evaluate presence of hypermethylated genes in cervical carcinogenesis, both in squamous cell as well as adenocarcinomas. Cervical scrapings were obtained from 30 patients diagnosed with cervical cancer (20 squamous cell carcinomas and 10 adenocarcinomas) and 19 women with histologically normal cervices. The scraped cells were used for determination of promoter hypermethylation by QMSP for 12 genes and for morphological assessment. Overall, CALCA, DAPK, ESR1, TIMP3, APC and RAR-b 2 promoters were significantly more often hypermethylated in cancers than in controls, while adenocarcinomas were more often hypermethylated above the highest control ratio for APC, TIMP3 and RASSF1A promoters. Combining 4 genes (CALCA, DAPK, ESR1 and APC) yielded a sensitivity of 89% (with all adenocarcinomas identified), equal to cytomorphology (89%) and high-risk human papilloma virus (Hr-HPV; 90%). The 4-gene QMSP proved theoretically superior to cytomorphology as well as Hr-HPV in specificity (100% vs. 83 and 68%, respectively), because cytology identified 3 controls as moderate or severe dyskaryosis and 6 controls were positive for Hr-HPV. In conclusions, QMSP of 4 gene promoters combined appears to have comparable sensitivity and potentially better specificity in comparison to ''classic'' cytomorphological assessment and Hr-HPV detection. QMSP holds promise as a new diagnostic tool for both squamous cell carcinoma and adenocarcinoma of the cervix. ' 2006 Wiley-Liss, Inc. Key words: methylation; cervical cancer; QMSP; DAPK; ESR1Cervical cancer is an important cause of death in women worldwide. 1 Cervical carcinogenesis is highly associated with (high-risk) human papilloma virus (HPV) infections. 2 Cytomorphological examination of cervical smears is a widely applied, though not ideal screening method for cervical cancer and its precursors, since the Pap smear has false negative rates of 2-40%, due to a combination of sampling error, processing artifacts and the nature of subjective interpretation. 1,3,4 False-negative cytology can also be found in about 50% of cases when previous negative smears are reviewed from the small proportion of screened women who develop invasive cancer. 4 Moreover, as many as 20% of all Pap smears are interpreted as atypical squamous cells of undetermined significance (ASCUS) or borderline dyskaryotic, leading to increased surveillance and more invasive tests in many of these patients. 3,5 The incidence of squamous cell carcinoma of the cervix has decreased since introduction of nation wide screening programs, compared to a relative increased incidence of adenocarcinoma of the cervix. 6 The efficacy of ...

Purpose: Presence of pelvic lymph node metastases is the main prognostic factor in early-stage cervical cancer patients, primarily treated with surgery. Aim of this study was to identify cellular tumor pathways associated with pelvic lymph node metastasis in early-stage cervical cancer.Experimental Design: Gene expression profiles (Affymetrix U133 plus 2.0) of 20 patients with negative (N 0 ) and 19 with positive lymph nodes (N þ ), were compared with gene sets that represent all 285 presently available pathway signatures. Validation immunostaining of tumors of 274 consecutive early-stage cervical cancer patients was performed for representatives of the identified pathways.Results: Analysis of 285 pathways resulted in identification of five pathways (TGF-b, NFAT, ALK, BAD, and PAR1) that were dysregulated in the N 0 , and two pathways (b-catenin and Glycosphingolipid Biosynthesis Neo Lactoseries) in the N þ group. Class comparison analysis revealed that five of 149 genes that were most significantly differentially expressed between N 0 and N þ tumors (P < 0.001) were involved in b-catenin signaling (TCF4, CTNNAL1, CTNND1/p120, DKK3, and WNT5a). Immunohistochemical validation of two well-known cellular tumor pathways (TGF-b and b-catenin) confirmed that the TGF-b pathway (positivity of Smad4) was related to N 0 (OR: 0.20, 95% CI: 0.06-0.66) and the b-catenin pathway (p120 positivity) to N þ (OR: 1.79, 95%CI: 1.05-3.05).Conclusions: Our study provides new, validated insights in the molecular mechanism of lymph node metastasis in cervical cancer. Pathway analysis of the microarray expression profile suggested that the TGF-b and p120-associated noncanonical b-catenin pathways are important in pelvic lymph node metastasis in early-stage cervical cancer.

More radical surgery in patients with (minimal) central residual disease identified by routine biopsy 8 to 10 weeks after (chemo)radiation does not improve survival and should not be recommended.

Cervical cancer is the possible outcome of a genital infection with high-risk human papillomavirus type 16 (HPV16) and is preceded by a phase of persistent HPV infection during which the host immune system fails to eliminate the virus. Our previous work showed that failure is reflected by the absence of type 1 T-cell immunity against HPV16 early antigens E2 and E6 in patients with HPV161 cervical lesions. We now show that a majority of both patients with cervical lesions and healthy subjects display HPV16 L1 peptide-specific type 1 T-cell responses with similar magnitude. The T-cell response in patients was directed at a broad range of peptides within L1, suggesting that during persistent or repeated exposure to HPV16 L1, the immune system maximizes its efforts to counter the viral challenge. Unlike the type 1 T-cell responses against HPV16 early antigens E2 and E6, type 1 T-cell immunity against L1 does not correlate with health or disease. This argues that T-cell responses against early and late HPV16 antigens essentially differ in the manner in which they are induced and regulated, as well as in their impact on the subsequent stages of HPV16-induced cervical disease. ' 2005 Wiley-Liss, Inc.Key words: HPV16; T cell; L1; VLP; cervical cancer; healthy individuals Infection of both men and women with oncogenic human papillomavirus (HPV) types, such as HPV type 16 (HPV16), is quite common 1-3 and leads to progressive disease in only a minor fraction of infected subjects. [4][5][6] The majority of the infections and HPV-induced epithelial lesions spontaneously resolve, most likely through intervention by the adaptive immune response. [7][8][9] In a majority of healthy subjects (approximately 60%) strong type 1 Tcell reactivity directed at the nonstructural proteins HPV16 E2 and E6 can be detected, [10][11][12] suggesting that these proteins are important target antigens for a protective immune response in humans, similar to what was found in animal models. [13][14][15] These animal models also revealed that L1-specific immunity can protect against infection with the cottontail rabbit papilloma virus 16 or the canine oral papilloma virus. [17][18][19] A large HPV16 L1-VLP vaccination trial in humans revealed that vaccine-induced immunity could prevent persistent HPV16 infections. 20 Although the immunologic evaluation of this trial was focused on the role of neutralizing antibodies in preventing viral infection, VLP vaccination must also have affected the underlying CD41 Th responses and possibly Th-dependent cell-mediated effector functions.Previous work has shown that patients diagnosed with HPV16 1 CIN display proliferative responses against HPV16 L1 21,22 and that there was no difference in such proliferative responses (measured by IL-2 production) between patients who cleared their lesions or in whom lesions persisted. 23 Immunity in such subjects may differ not so much in the quantitative aspects of the immune response but more in the quality of the response, as is indicated by our studies on immunity against...

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It has been suggested that local invasive procedures may alter the natural course of (pre)malignant cervical disease. This could be due to partial excision of the lesions, or via induction of cellular immunity against human papillomavirus (HPV) by the local invasive procedures. We studied the influence of local invasive procedures on HPV-16 E7 specific immune responses in patients with different grades of cervical intra-epithelial neoplasia (CIN) and different stages of cervical cancer. Blood was obtained at intake and after invasive procedures from patients with CIN or cervical cancer. Antigen specific T-cell responses were measured by IFN-c ELISPOT analysis, after stimulation with recombinant HPV-16 E7 protein. As expected, HPV-16 E7 specific IFN-c T cell responses were more frequent in HPV-16 DNA positive patients compared with that in HPV-16 DNA negative patients (39/50 vs. 16/36, (p 5 0.006, v 2 test). After invasive procedures, a small number of HPV-16 DNA positive CIN patients, but a considerable proportion of HPV-16 DNA positive cervical cancer patients, showed an enhancement of T cell responses against HPV-16 E7. Induction of T cell reactivity was most pronounced in cervical cancer patients who had undergone previous invasive procedures. Both CD4 1 and CD8 1 T cells showed E7 specific IFN-c production upon in-vitro stimulation. Our study shows that invasive procedures may enhance HPV-specific cell-mediated immunity in a considerable number of patients with cervical cancer, but in only a minority of CIN patients. Our data indicate that invasive procedures should be considered as possible confounding factors when analyzing the effectiveness of therapeutic immunization studies, especially, when induction of HPV-specific immune responses is used as intermediate end-point. ' 2005 Wiley-Liss, Inc.Key words: cervical cancer; IFN-g; HPV; CD4 1 T cell; CD8 1 T cell Infection with oncogenic human papillomavirus (HPV) plays an important role in cervical carcinogenesis, and HPV DNA can be detected in 90-100% of all cervical cancers. 1,2 The majority of women infected with oncogenic HPV types do not develop cervical intraepithelial neoplasia (CIN) or cervical cancer but clear their HPV infection. The immune system plays an important role herein, as demonstrated by the observation that immunocompromised women, such as AIDS-patients, more often fail to clear an HPV infection and have an increased risk to develop cervical cancer. 3 The E6 and E7 transforming oncoproteins of HPV play a crucial role in the transformation and maintenance of the malignant phenotype, and therefore, these proteins are the ideal candidates for tumor-specific cervical cancer immunotherapy. Cytotoxic T cell (CTL) and T helper activity specific for E6 and E7 of HPV-16 and HPV-18 (the 2 most common HPV types) have been demonstrated in the peripheral blood of patients with (pre)malignant cervical neoplasia and healthy controls. [4][5][6][7][8][9][10][11][12] It has been suggested that spontaneous regression of CIN lesions might be associated with...

Background: Loss of mismatch repair (MMR) gene expression has been associated with fewer metastases and improved prognosis in various tumour types. Aims: To evaluate the predictive and prognostic significance of loss of MMR protein MSH2 in early stage cervical cancer. Methods: Specimens from 218 consecutive patients with early stage, surgically treated cervical cancer were analysed. Median age was 42 years (interquartile range 35-53). International Federation of Gynecology and Obstetrics (FIGO) stages were IB1 (57%), IB2 (25%) and IIA (18%). Histology was 70% squamous cell, 6% adenosquamous and 24% adenocarcinoma. Pelvic lymph node metastasis was present in 66 (30%) patients. Median follow-up was 5.2 years (interquartile range 2.5-7.9). Tissue microarrays (TMAs) were constructed containing three cores of paraffin-embedded tumour per case. MSH2 expression was assessed by immunohistochemistry on TMAs and full sections. Results: In TMAs MSH2 expression could be analysed in 184/218 (84%) tumours. Loss of MSH2 was observed in 58/184 (32%) tumours, with a moderately strong concordance between TMAs and full sections (k = 0.47). In tumours with loss of MSH2, pelvic lymph node metastasis and cancer invasion beyond 10 mm were more frequent (48% vs 25%, and 59% vs 37%, respectively). However, loss of MSH2 expression was not related to recurrence or survival. Conclusion: TMAs are powerful tools for high throughput screening of biological markers for prognostic value in cervical cancer. Absence of MSH2 expression is associated with a high-risk profile in early stage cervical cancer, but does not predict lymph node status with sufficient accuracy to be used in the clinic.

Although current cytomorphology-based cervical cancer screening has reduced the incidence of cervical cancer, Papsmears are associated with high false positive and false negative rates. This has spurred the search for new technologies to improve current screening. New methodologies are automation of Pap-smear analysis, addition of new biological or molecular markers to traditional cytology or using these new markers to replace the current screening method. In this overview we will summarize data on cervical cancer epidemiology and etiology and the current cervical cancer screening approach. Available data on new screening approaches, such as quantitative cytochemistry, detection of loss of heterozygosity (LOH) and hypermethylation analysis will be reviewed.We discuss the potential of these approaches to replace or augment current screening. When available, data on cost– effectiveness of certain approaches will be provided. In short, Human Papillomavirus (HPV) DNA detection stands closest to implementation in nation-wide screening programs of all markers reviewed. However, specificity is low in women aged <35 years and the psychological effects of knowledge of HPV positivity in absence of cervical (pre) malignant disease are important drawbacks. In our opinion the results of large clinical trials should be awaited before proceeding to implement HPV DNA detection. New technologies based on molecular changes associated with cervical carcinogenesis might result in comparable sensitivity, but improved specificity. Hypermethylation analysis is likely to be more objective to identify patients with high grade squamous intra-epithelial lesions (HSIL) or invasive cancer with a higher specificity than current cytomorphology based screening.