Wiebren A.A. Tjalma

Differences in human papillomavirus type distribution in high‐grade cervical intraepithelial neoplasia and invasive cervical cancer in Europe

Knowledge of differences in human papillomavirus (HPV)‐type prevalence between high‐grade cervical intraepithelial neoplasia (HG‐CIN) and invasive cervical cancer (ICC) is crucial for understanding the natural history of HPV‐infected cervical lesions and the potential impact of HPV vaccination on cervical cancer prevention. More than 6,000 women diagnosed with HG‐CIN or ICC from 17 European countries were enrolled in two parallel cross‐sectional studies (108288/108290). Centralised histopathology review and standardised HPV‐DNA typing were applied to formalin‐fixed paraffin‐embedded cervical specimens dated 2001–2008. The pooled prevalence of individual HPV types was estimated using meta‐analytic methods. A total of 3,103 women were diagnosed with HG‐CIN and a total of 3,162 with ICC (median ages: 34 and 49 years, respectively), of which 98.5 and 91.8% were HPV‐positive, respectively. The most common HPV types in women with HG‐CIN were HPV16/33/31 (59.9/10.5/9.0%) and in ICC were HPV16/18/45 (63.3/15.2/5.3%). In squamous cell carcinomas, HPV16/18/33 were most frequent (66.2/10.8/5.3%), and in adenocarcinomas, HPV16/18/45 (54.2/40.4/8.3%). The prevalence of HPV16/18/45 was 1.1/3.5/2.5 times higher in ICC than in HG‐CIN. The difference in age at diagnosis between CIN3 and squamous cervical cancer for HPV18 (9 years) was significantly less compared to HPV31/33/‘other’ (23/20/17 years), and for HPV45 (1 year) than HPV16/31/33/‘other’ (15/23/20/17 years). In Europe, HPV16 predominates in both HG‐CIN and ICC, whereas HPV18/45 are associated with a low median age of ICC. HPV18/45 are more frequent in ICC than HG‐CIN and associated with a high median age of HG‐CIN, with a narrow age interval between HG‐CIN and ICC detection. These findings support the need for primary prevention of HPV16/18/45‐related cervical lesions.

Comprehensive proteomic analysis of human cervical-vaginal fluid using colposcopy samples.

BackgroundCervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C4(RP)-LC protein separation) followed by C18(RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set.ResultsIn total, we were able to identify 339 proteins in human CVF of which 151 proteins were not identified in any other proteomics study on human CVF so far. Those included antimicrobial peptides, such as human beta-defensin 2 and cathelicidin, which were known to be present in CVF, and endometrial proteins such as glycodelin and ribonucleoprotein A. Comparison of our results with previously published data led to the identification of a common protein set of 136 proteins. This overlapping protein set shows increased fractions of immunological and extracellular proteins, confirming the extracellular immunological role of CVF.ConclusionWe demonstrated here that CVF colposcopy samples can be used in proteomics experiments and hence are applicable for biomarker discovery experiments. The delineation of an overlapping set of proteins that is identified in most proteomics studies on CVF may help in the description of a reference proteome when performing proteomics studies on human CVF.

The importance of biological factors (bcl-2, bax, p53, PCNA, MI, HPV and angiogenesis) in invasive cervical cancer

OBJECTIVE The present study was designed to analyse the relationship between apoptosis related proteins (bcl-2 and bax), tumour suppressor protein p53, proliferation markers (PCNA and mitotic index), human papillomavirus (HPV) and angiogenesis in cervical cancer and their impact on clinical outcome. STUDY DESIGN Tumours from 111 patients were assessed by immunohistochemistry for the expression of bcl-2, bax, p53 and PCNA, by PCR for the presence of HPV-DNA, for the quantification of the mitotic index and the microvessel density (CD 31). The results were correlated with various histopathologic characteristics and survival. RESULTS The multiple Coxs regression analysis for overall survival of all prognostic variables gave as best model: bcl-2 (P<0.001), lymphovascular permeation (P=0.004), mitotic index (P=0.019), tumour grade (P=0.048) and FIGO stage (P=0.070). Subanalysis was performed for the patients where the lymph node status was known (n=79). Adding the lymph node status gave as best model for overall survival bcl-2 (P=0.001), lymphovascular permeation (P=0.003) and mitotic index (P=0.044). However, they hardly influenced the association. CONCLUSION In the apoptotic pathway of cervical cancer, bcl-2 is one of most important proteins. It can probably not only mediate cell death but also regulate cell growth. A better understanding of their relations will probably provide the basis for more rational cancer therapies in the future.

Observations of an idiopathic granulomatous mastitis

Idiopathic granulomatous mastitis is a very rare benign breast disease, which mimics breast cancer both clinically and mammographically. Most cases have an unknown aetiology, however, we found an alpha-1-antitrypsin deficiency. A literature review is presented and the controversies in diagnosis and management are discussed.

Efficacy of the HPV-16/18 AS04-Adjuvanted Vaccine Against Low-Risk HPV Types (PATRICIA Randomized Trial): An Unexpected Observation

Background. Public Health England has reported a decrease of up to 20.8% in new diagnoses of external genital warts (GWs) among women aged <19 years since the national vaccination program with the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine began in 2008. A post hoc analysis of the phase III PATRICIA (PApilloma TRIal against Cancer In young Adults) trial (NCT00122681) was performed to ascertain whether protection against low-risk HPV types was apparent. Methods. Vaccine efficacy (VE) at 48 months was assessed against 6-month persistent infection (6MPI) with low-risk HPV types in the total vaccinated cohort (TVC) and in the TVC naive (for 25 HPV types tested) populations. Results. In the TVC naive cohort, VE against 6MPI (95% confidence interval) was 34.5% (11.3 to 51.8) for HPV-6/11, 34.9% (9.1 to 53.7) for HPV-6, 30.3% (−45.0 to 67.5) for HPV-11, and 49.5% (21.0 to 68.3) for HPV-74. Conclusions. The HPV-16/18 AS04-adjuvanted vaccine appears to have moderate efficacy against persistent infections with a number of low-risk HPV types (HPV-6/11/74), which are responsible for the majority of external GWs, and recently, antibody and cell-mediated immune response to HPV-6/11 have been observed. These findings may help to explain the decrease in external GW diagnoses seen in England.

Interval debulking surgery: an alternative for primary surgical debulking?

Retrospective analyses suggest that a subgroup of patients with Stage III and IV ovarian carcinoma can be treated with neo-adjuvant chemotherapy followed by interval debulking surgery. The absolute indications for neo-adjuvant chemotherapy appear to be Stage IV disease (excluding pleural fluid) or metastases of more than 1 g at sites where resection is impossible. In patients with an estimated total metastatic tumor load of >100 g, the presence of at least two of the following relative indications for neo-adjuvant chemotherapy are considered to be necessary: 1) uncountable (>100) peritoneal metastases, 2) estimated metastatic tumor load of >1000 g, 3) presence of large (>10 g) peritoneal metastatic plaques, 4) large volume ascites, and 5) World Health Organization (WHO) status II or III. Interval debulking surgery in patients with suboptimal primary debulking surgery has been proven effective in increasing overall survival and progression-free survival in a large prospective, randomized trial of the European Organization for Research and Treatment of Cancer (EORTC). The strategy of neo-adjuvant chemotherapy, followed by interval debulking surgery, should be confirmed in a prospective randomized trial. The EORTC 55971 trial is currently addressing this issue. We will review the studies on primary chemotherapy, interval debulking surgery, and the indications for primary chemotherapy followed by interval debulking surgery, and ongoing trials.

Use of cervicovaginal fluid for the identification of biomarkers for pathologies of the female genital tract.

Cervicovaginal fluid has an important function in the homeostasis and immunity of the lower female genital tract. Analysis of the cervicovaginal fluid proteome may therefore yield important information about the pathogenesis of numerous gynecological pathologies. Additionally, cervicovaginal fluid has great potential as a source of biomarkers for these conditions.This review provides a detailed discussion about the human cervicovaginal proteome and the proteomics studies performed to characterize this biological fluid. Furthermore, infection-correlated pathological conditions of the female genital tract are discussed for which cervicovaginal fluid has been used in order to identify potential biomarkers. Recent years, numerous studies have analyzed cervicovaginal fluid samples utilizing antibody-based technologies, such as ELISA or Western blotting, to identify biomarkers for preterm birth, premature preterm rupture of membranes, bacterial vaginosis and cervical cancer. The present article will discuss the importance of proteomic technologies as alternative techniques to gain additional meaningful information about these conditions. In addition, the review focuses on recent proteomic studies on cervicovaginal fluid samples for the identification of potential biomarkers. We conclude that the use of proteomic technology for analysis of human cervicovaginal fluid samples is promising and may lead to the discovery of new biomarkers which can improve disease prevention and therapy development.

Prognostic significance of oncogenic markers in ductal carcinoma in situ of the breast: a clinicopathologic study.

Abstract:  Ductal carcinoma in situ (DCIS) is a heterogeneous malignant condition of the breast with an excellent prognosis. Until recently mastectomy was the standard treatment. As the results of the National Surgical Adjuvant Breast and Bowel Project‐17 trial and the introduction of the Van Nuys Prognostic Index (VNPI) less radical therapies are used. Objectives are to identify clinicopathologic and biologic factors that may predict outcome. Cases of DCIS diagnosed in two Belgian University Centers were included. Paraffin‐embedded material and Hematoxylin and Eosin stained slides of DCIS cases were reviewed and tumor size, margin width, nuclear grade, and comedo necrosis were assessed. Molecular markers (estrogen receptor, progesterone receptor, HER1‐4, Ki67, and c‐myc) were assayed immunohistochemically. Applied treatment strategies were correlated with the prospective use of the VNPI score. Kaplan–Meier survival plots were generated with log‐rank significance and multiple regression analysis was carried out using Cox proportional hazards regression analysis; 159 patients were included with a median age of 54 years (range 29–78); 141 had DCIS and 18 DCIS with microinvasion. The median time of follow‐up was 54 months (range 5–253). Twenty‐three patients developed a recurrence (14.5%). The median time to recurrence was 46 months (range 5–253). Before the introduction of the VNPI, 37.5% of the DCIS patients showed a recurrence while thereafter 6.7% recurred (p < 0.005). Two recurrences occurred in the VNPI group I (7.1%); seven in the VNPI group II (8.5%) (median time to recurrence 66.3 months) and 14 in the VNPI group III (28.5%) (median time to recurrence 40.2 months) (disease‐free survival [DFS]: p < 0.05). A Cox proportional hazards regression analysis indicated that tumor size, margin width, pathologic class, and age were independent predictors of recurrence, but none of the studied molecular markers showed this. Overexpression of HER4 in the presence of HER3 was found to be associated with a better DFS (p < 0.05). This study confirms the value of the VNPI score and questions the benefit of an aggressive approach in the low‐risk DCIS lesions. Independent predictors for recurrence included size, margin width, pathologic class, and age, but none of the molecular markers were part of it. Overexpression of HER4 in the presence of HER3 was associated with a better DFS.

Can cervical cancer screening be stopped at 50? The prevalence of HPV in elderly women

Although the relation between cervical cancer and the human papillomavirus (HPV) has been established beyond doubt, the introduction of HPV detection in cervical cancer screening is halted, primarily by the high rate of false positivity in relation to morbidity, since the majority of women infected with HPV will not develop lesions. To counteract overconsumption of cervical cancer screening in elderly women, we wanted to test the hypothesis that women of 50 years or older who are HPV‐negative and have a cytologically normal smear might be encouraged to refrain from further screening. As a first step, the prevalence of high‐risk HPV in a population of 1,936 women of 50 years and older was investigated. After an initial decline, a slightly higher prevalence can be seen with increasing age. There is a decrease in the prevalence of multiple infections with age, paralleled by an increase in single infections, especially of HPV type 16 in the eldest‐age group. However, neither the decrease in multiple infections nor the increase in single infections is statistically significant. The data obtained in this study show that, even in the presence of a slight increase in the HPV prevalence in elderly women, approximately 94% of the elderly women can be withdrawn from the cervical cancer screening. However, a follow‐up study will be necessary to determine the frequency of (re)infection as well as the course of an HPV infection in elderly women.

Low CD10 mRNA expression identifies high-risk ductal carcinoma in situ (DCIS).

Purpose Optimal management of breast ductal carcinoma in situ (DCIS) is controversial, and many patients are still overtreated. The local death of myoepithelial cells (MECs) is believed to be a pre-requisite to tumor invasion. We thus hypothesized that loss of CD10 expression, a MEC surface peptidase, would signify basement membrane disruption and confer increased risk of relapse in DCIS. The aim of our study was to retrospectively evaluate the prognostic value of CD10 in DCIS. Experimental Design CD10 expression was evaluated by quantitative RT-PCR and immunohistochemistry using paraffin-embedded samples of normal breast tissue (n = 11); of morphologically normal ducts associated with DCIS (n = 10); and of DCIS without an invasive component (n = 154). Results CD10 immunostaining was only observed in MECs in normal tissue and in DCIS. Normal tissue showed high mRNA expression levels of CD10, whereas DCIS showed a variable range. After a median follow-up of 6 years, DCIS with CD10 expression below the levels observed in normal tissue (71%) demonstrated a higher risk of local relapse (HR = 1.88; [95CI:1.30–2.70], p = 0.001) in univariate analysis. No relapse was observed in patients expressing high CD10 mRNA levels (29%) similar to the ones observed in normal tissue. In multivariate analysis including known prognostic factors, low CD10 mRNA expression remained significant (HR = 2.25; [95%CI:1.24–4.09], p = 0.008), as did the recently revised Van Nuys Prognostic Index (VNPI) score (HR = 2.03; [95%CI:1.23–3.35], p = 0.006). Conclusion The decrease of CD10 expression in MECs is associated with a higher risk of relapse in DCIS; this knowledge has the potential to improve DCIS management.