Alterations in classical and nonclassical HLA expression in recurrent and progressive HPV-induced usual vulvar intraepithelial neoplasia and implications for immunotherapy.

Immunotherapy of usual vulvar intraepithelial neoplasia (uVIN) is promising; however, many patients still fail to show clinical responses, which could be explained by an immune escape through alterations in human leukocyte antigen (HLA) expression. Therefore, we analyzed a cohort of patients with a primary (n = 43) and subsequent recurrent uVIN lesion (n = 20), vaccine-treated uVIN patients (n = 12), patients with human papillomavirus (HPV)-induced vulvar carcinoma (n = 21) and healthy controls (n = 26) for the expression of classical HLA-class I/II and nonclassical HLA-E/-G and MHC class I chain-related molecule A (MICA). HLA-class I was downregulated in 70% of uVIN patients, including patients with a clinical response to immunotherapy. Downregulation of HLA-class I is probably reversible, as only 15% of the uVIN cases displayed loss of heterozygosity (LOH) and HLA-class I could be upregulated in uVIN keratinocyte cultures by interferon γ. HLA-class I downregulation is more frequently associated with LOH in vulvar carcinomas (25-55.5%). HLA-class II was found to be focally expressed in 65% of uVIN patients. Of the nonclassical molecules, MICA was downregulated in 80% of uVIN whereas HLA-E and -G were expressed in a minority of cases. Their expression was more prominent in vulvar carcinoma. No differences were found between the alterations observed in paired primary and recurrent uVIN. Importantly, downregulation of HLA-B/C in primary uVIN lesions was associated with the development of recurrences and progression to cancer. We conclude that downregulation of HLA is frequently observed in premalignant HPV-induced lesions, including clinical responders to immunotherapy, and is associated with worse clinical outcome. However, in the majority of cases downregulation may still be reversible.

Spindle cell morphology is related to poor prognosis in vulvar squamous cell carcinoma.

BACKGROUND: Vulvar cancer is the fourth most common gynaecological malignancy, with an annual incidence of 2 out of 100,000 women. Although most cases of early stage vulvar cancer have a good prognosis, recurrence and rapid tumour progression can occur. We investigated the prevalence of spindle cell morphology in vulvar cancer and its association with survival. METHODS: This retrospective cohort study included 108 patients with primary vulvar squamous cell carcinoma who were treated at the Leiden University Medical Center during 2000-2009. Paraffin-embedded tissue was examined for the presence of spindle cell morphology. Survival and histology data were compared between cases with spindle and without spindle cell morphology. RESULTS: Twenty-two (20%) tumours showed spindle cells infiltrating the stromal tissue. All spindle cell tumours were human papillomavirus (HPV) negative. Spindle cell morphology was strongly associated with poor prognosis and with a high risk of lymph node involvement at the time of diagnosis (relative risk 2.26 (95% CI 1.47-3.47)). Five-year disease-specific survival was lower in patients with vs without spindle cell morphology (45.2% vs 79.7%, respectively; P=0.00057). CONCLUSION: Vulvar spindle cell morphology occurs frequently and seems to develop through the non-HPV pathway. It is associated with a worse prognosis than conventional vulvar squamous cell carcinoma.

Role of IL-12p40 in cervical carcinoma.

BACKGROUND: Previously, we have shown that low IL-12p40 mRNA expression by cervical cancer cells is associated with a poor survival of cervical cancer patients. As IL-12p40 is both a subcomponent of interleukin (IL)-12 and IL-23, the aim of this study was to elucidate the role of IL-12p40 in cervical cancer. METHODS: We have measured the expression of IL-23p19 mRNA, IL-12p35 mRNA and IL-12p40 mRNA using mRNA in situ hybridisation. The IL-1 and IL-6 were measured by immunohistochemistry. RESULTS: As IL-23 is a component of the IL-17/IL-23 pathway, a pathway induced by IL-1 and IL-6 in humans, we have studied IL-1 and IL-6 expression. Only a high number of stromal IL-6-positive cells was shown to associate with poor disease-specific survival. The worst disease-specific survival was associated with a subgroup of patients that displayed a high number of IL-6-positive cells and low IL-12p40 expression (P<0.001). Both a high number of IL-6-positive cells and a high number of IL-6-positive cells, plus low IL-12p40 expression were shown to be clinicopathological parameters independent of lymph node metastasis, parametrial involvement and Sedlis score (P=0.009 and P=0.007, respectively). CONCLUSION: Our results with IL-6 and IL-12p40 are in accordance with the hypothesis that the IL-17/IL-23 pathway has a suppressive role in cervical cancer.

Single-visit approach of cervical cancer screening: see and treat in Indonesia.

BACKGROUND: We performed a cross-sectional study in Indonesia to evaluate the performance of a single-visit approach of cervical cancer screening, using visual inspection with acetic acid (VIA), histology and cryotherapy in low-resource settings. METHODS: Women having limited access to health-care facilities were screened by trained doctors using VIA. If the test was positive, biopsies were taken and when eligible, women were directly treated with cryotherapy. Follow-up was performed with VIA and cytology after 6 months. When cervical cancer was suspected or diagnosed, women were referred. The positivity rate, positive predictive value (PPV) and approximate specificity of the VIA test were calculated. The detection rate for cervical lesions was given. RESULTS: Screening results were completed in 22 040 women, of whom 92.7% had never been screened. Visual inspection with acetic acid was positive in 4.4%. The PPV of VIA to detect CIN I or greater and CIN II or greater was 58.7% and 29.7%, respectively. The approximate specificity was 98.1%, and the detection rate for CIN I or greater was 2.6%. CONCLUSION: The single-visit approach cervical cancer screening performed well, showing See and Treat is a promising way to reduce cervical cancer in Indonesia.

CXCR7 expression is associated with disease-free and disease-specific survival in cervical cancer patients.

BACKGROUND: The CXC chemokine receptor (CXCR)7 is involved in tumour development and metastases formation. The aim of the present study was to determine protein expression of CXCR7, its putative co-receptors epidermal growth factor receptor (EGFR) and CXCR4, its predominant ligand CXCL12, their co-dependency and their association with survival in cervical cancer patients. METHODS: CXC chemokine receptor 7, EGFR, CXCR4 and CXCL12 expression were determined immunohistochemically in 103 paraffin-embedded, cervical cancers. Subsequently, associations with patient characteristics were assessed and survival analyses were performed. RESULTS: CXC chemokine receptor 7 was expressed by 43% of tumour specimens, in a large majority of cases together with either EGFR or CXCR4 (double positive), or both (triple positive). The CXCR7 expression was associated with tumour size (P=0.013), lymph node metastasis (P=0.001) and EGFR expression (P=0.009). CXC chemokine receptor 7 was independently associated with disease-free survival (hazard ratio (HR)=4.3, 95% confidence intervals (CI) 1.7-11.0, P=0.002), and strongly associated with disease-specific survival (HR=3.9, 95% CI 1.5-10.2, P=0.005). CONCLUSION: CXC chemokine receptor 7 expression predicts poor disease-free and disease-specific survival in cervical cancer patients, and might be a promising new therapeutic marker. In a large majority of cases, CXCR7 is co-expressed with CXCR4 and/or EGFR, supporting the hypothesis that these receptors assist in CXCR7 signal transduction.

Expression of endoglin (CD105) in cervical cancer.

In this study, we have investigated the role of endoglin (CD105), a regulator of transforming growth factor (TGF)-beta(1) signalling on endothelial cells, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) in cervical cancer. We have measured the number and determined the location of both newly formed (CD105-positive) and the overall number of (CD31-positive) blood vessels, and bFGF and VEGF-A expression using immunohistochemistry in 30 cervical carcinoma specimens. Vascular endothelial growth factor-A mRNA expression was determined using RNA-in situ hybridisation. CD105- and CD31-positive vessels and bFGF- and VEGF-A-positive cells were predominantly present in the stroma. The presence of CD105- and CD31-positive vessels in the stroma did neither correlate with the number of VEGF-A-positive cells nor the number of bFGF-positive cells. However, the number of CD105- and CD31-positive vessels was associated with the expression of VEGF-A mRNA in the epithelial cell clusters (P=0.013 and P=0.005, respectively). The presence of CD105-positive and CD31-positive vessels was associated with the expression of alphavbeta6 (a TGF-beta(1) activator; P=0.013 and P=0.006, respectively). Clinically, the number of CD105-positive vessels associated with the number of lymph node metastasis (P<0.001). Furthermore, the presence of CD105-positive vessels within the epithelial cell clusters associated with poor disease-free survival (P=0.007).

Prevalence of human papillomavirus in Indonesia: a population-based study in three regions.

Cervical cancer is the most common cancer among women in the Indonesian population, yet little is known about the prevalence of human papillomavirus (HPV). We investigated age-specific prevalence of HPV types and possible risk factors of HPV positivity in a population-based sample of 2686 women, aged 15-70 years, in Jakarta, Tasikmalaya, and Bali, Indonesia. The overall HPV prevalence was 11.4%, age-standardized to the world standard population 11.6%. The most prevalent types found were HPV 52, HPV 16, HPV 18, and HPV 39, respectively, 23.2, 18.0, 16.1, and 11.8% of the high-risk HPV types. In 20.7% of infections, multiple types were involved. Different age-specific prevalence patterns were seen: overall high in Jakarta, and in Tasikmalaya, and declining with age in Bali. The number of marriages was most associated with HPV positivity (OR 1.81 95% CI 1.31-2.51)). Remarkably, in Indonesia HPV 16 and HPV 18 are equally common in the general population, as they are in cervical cancer. HPV 52 was the most prevalent type in the general population, suggesting that this type should be included when prophylactic HPV vaccination is introduced in Indonesia.

Elevated expression of SerpinA1 and SerpinA3 in HLA-positive cervical carcinoma.

In cervical cancer, an important mechanism by which tumour cells escape immune surveillance is loss of HLA class I, enabling tumours to evade recognition and lysis by cytotoxic T lymphocytes. Some tumours, however, escape from immune surveillance without accumulating defects in antigen presentation. We hypothesized that tumours with no or partial loss of HLA class I develop alternative mechanisms to prevent immune elimination. To investigate this hypothesis, genome-wide expression profiling using Illumina arrays was performed on cervical squamous cell carcinomas showing overall loss of HLA class I, partial, and normal HLA class I protein expression. Statistical analyses revealed no significant differences in gene expression between tumours with partial (n = 11) and normal HLA class I expression (n = 10). Comparison of tumours with normal/partial HLA class I expression (n = 21) with those with overall loss of HLA class I expression (n = 11) identified 150 differentially expressed genes. Most of these genes were involved in the defence response (n = 27) and, in particular, inflammatory and acute phase responses. Especially SerpinA1 and SerpinA3 were found to be up-regulated in HLA-positive tumours (3.6- and 8.2-fold, respectively), and this was confirmed by real-time PCR and immunohistochemistry. In a group of 117 tumours, high SerpinA1 and SerpinA3 expression in association with normal/partial HLA expression correlated significantly with poor overall survival (p = 0.035 and p = 0.05, respectively). Thus, HLA-positive tumours are characterized by higher expression of genes associated with an inflammatory profile. In addition, expression of the acute phase proteins SerpinA1 and SerpinA3 in HLA-positive tumours is associated with worse prognosis.

Identification of MEN1 and HRPT2 somatic mutations in paraffin-embedded (sporadic) parathyroid carcinomas.

OBJECTIVE: Parathyroid carcinoma remains difficult to diagnose. Recently, it has been shown that mutations in the HRPT2 gene (encoding parafibromin) are associated with the development of parathyroid carcinoma. Although MEN1 is not typically thought to be involved in carcinoma formation, parathyroid carcinoma may be an extremely rare feature of the multiple endocrine neoplasia type 1 (MEN1) syndrome. We recently concluded that loss of heterozygosity (LOH) of the MEN1 gene is present in a relatively large number of parathyroid carcinomas, often in combination with LOH at the HRPT2 locus. The aim of this study was to evaluate the role of MEN1 and HRPT2 mutations in sporadic parathyroid tumours fulfilling histological criteria for malignancy. PATIENTS AND DESIGN: Formalin-fixed, paraffin-embedded (FFPE) parathyroid carcinoma tissue from 28 cases identified in the period 1985-2000 in the Netherlands was studied. HRPT2 (27/28 cases) and MEN1 (23/28 cases) were analysed by direct sequencing. RESULTS: Somatic MEN1 mutations were found in three of 23 (13%) sporadic parathyroid carcinoma cases; these consisted of one missense and two frameshift mutations. One of the latter two cases displayed lymph-node and lung metastases during follow-up. Six HRPT2 mutations were found in 4/27 cases (15%): five were truncating mutations and one was a missense mutation. Consistent with previously published reports, we found double mutations (2x) and germline mutations (2x) in apparently sporadic parathyroid carcinomas. CONCLUSIONS: These results suggest that not only HRPT2 but also MEN1 mutations may play a role in sporadic parathyroid cancer formation.

Overexpression of the alpha v beta 6 integrin in cervical squamous cell carcinoma is a prognostic factor for decreased survival.

Cervical squamous cell carcinomas are composed histologically of tumour cell islands surrounded by varying amounts of tumour stroma, the amount and composition of which are influenced by local TGF-beta(1). TGF-beta(1) is secreted in an inactive complex with latency-associated peptide (LAP). Both LAP and the extracellular matrix (ECM) protein fibronectin are important ligands for the integrin receptor alpha v beta 6. While alpha v beta 6 is only weakly expressed by normal epithelia, it is up-regulated in different carcinomas where it generally reflects a more aggressive phenotype. In cervical cancer, the expression of alpha v beta 6 has not thus far been investigated. Given the ability of alpha v beta 6 both to activate TGF-beta(1) and to interact with fibronectin, we studied correlations between the expression of these components and disease parameters in a large cohort of cervical cancer specimens. We analysed alpha v beta 6 expression using immunohistochemistry in primary cervical squamous carcinomas of FIGO stage IA to IIB patients and correlated the findings with formerly investigated fibronectin and TGF-beta(1) expression and clinico-pathological parameters. alpha v beta 6 expression was also examined in cervical intra-epithelial neoplasia (CIN) and lymph node metastases. alpha v beta 6 was only weakly expressed in normal epithelium but clearly up-regulated in CIN lesions. In carcinomas, strong expression of alpha v beta 6 in tumour cells correlated with different clinico-pathological parameters and with worse overall and disease-free survival. Furthermore, alpha v beta 6 expression correlated positively with TGF-beta(1) mRNA expression as well as with fibronectin expression. Overexpression of alpha v beta 6 in cervical squamous carcinomas is an unfavourable prognostic factor. This might reflect an increased capacity of alpha v beta 6-expressing tumour cells to migrate in a fibronectin-rich ECM and/or to activate TGF-beta(1) at the tumour/stroma interface, both of which processes may contribute to cervical cancer progression.

Decreased prevalence of dysplasia in high-risk population immigrants in a low-risk area for cervical cancer.

Incidence rates of cervical cancer and its precursors vary considerably, with the highest rates found in developing countries. Differences are influenced by endogenous and exogenous factors. Comparing cytologic abnormality incidence rates from a high-risk population in the original high-risk area, with those of women from this high-risk population who have immigrated to a low-risk area could give insight in the significance of endogenous versus environmental factors. Smears collected from Surinamese women attending the Surinamese screening program and smears collected from immigrant Surinamese women attending the Dutch screening program were cytologically analyzed using the Dutch microscopical coding system KOPAC. Statistical analysis was performed by using logistic regression to calculate (age-adjusted) odds ratios (ORs). The age-adjusted ORs of having dysplasia were higher for Surinamese women living in Suriname versus Surinamese immigrant women and increased with increasing P-scores: 0.77 (0.31-1.91) for borderline changes, 1.62 (0.58-4.57) for mild dysplasia, and 3.20 (1.55-6.60) for moderate to severe dysplasia/neoplasia. We conclude that fewer cases with dysplasia are present in a high-risk population that has immigrated to a low-risk area for cervical cancer than in the high-risk population continuously living in a high-risk area. This finding emphasizes the importance of environmental factors.

The number of pelvic lymph nodes in the quality control and prognosis of radical hysterectomy for the treatment of cervical cancer.

AIMS: To determine if the number of removed lymph nodes in radical hysterectomy with lymphadenectomy (RHL) influences survival of patients with early stage cervical cancer and to analyze the relation of different factors like patient age, tumour size and infiltration depth with the number of nodes examined in node-negative early stage cervical cancer patients. METHODS: Of consecutive patients, who underwent RHL between January 1984 and April 2005, 331 had negative nodes (group A) without adjuvant therapy and 136 had positive nodes (group B). The Kaplan-Meier method and Cox regression model were used to detect statistical significance. Factors associated with excision of nodes were confirmed with linear regression models. RESULTS: The median number of removed nodes was 19 and 18 for group A and group B, respectively. There was no significant relationship between the number of removed nodes and the cancer specific survival (CSS) or disease free survival (DSF) for patients of group A (p=0.625 and p=0.877, respectively). The number of removed nodes in group B was not significantly associated with the CSS (p=0.084) but it was for the DSF (p=0.014). Factors like patient age, tumour size and infiltration depth were not associated with the number of nodes. CONCLUSIONS: No relation was found between the number of negative nodes examined after RHL for the treatment of early stage cervical cancer and CSS or DFS. However, a higher amount of removed lymph nodes leaded to a better DFS for patients with positive nodes. It is suggested that patients with positive nodes benefit from a complete pelvic lymphadenectomy and a sufficient yield of removed nodes.

Clonal analysis favours a monoclonal origin for serous borderline tumours with peritoneal implants.

Serous borderline tumours (SBTs) of the ovary were originally classified as such because the vast majority behave in a remarkably indolent manner, even in the presence of widespread tumour deposits, termed implants, and/or lymph node involvement. The pathogenesis of the implants is currently unknown. Two major hypotheses have been proposed: the first favours a monoclonal origin, arguing that the peritoneal lesions derive from neoplastic cells that are shed from the primary ovarian tumour. The second hypothesis favours a polyclonal origin as a result of a field defect of susceptible Müllerian cells from which multiple independent tumours arise. To test both hypotheses, genome-wide allelotyping and B-RAF/K-RAS mutation analyses were employed to assess clonality in 25 metachronous or synchronous tumours from ten SBT patients. Loss of heterozygosity (LOH) profiling and K-RAS/B-RAF mutation analysis showed concordance of the genetic changes in all sites in 21 tumours from eight patients who were informative. These results favour a common origin, underscored by a likelihood ratio (probability of common origin/probability of independent origin) ranging from 2.43 to 7,662,850. In conclusion, this study strongly supports the hypothesis that both non-invasive and invasive implants arise as a consequence of spread from a single ovarian site.

Human papillomavirus type 18 and other risk factors for cervical cancer in Jakarta, Indonesia.

Infection with human papillomavirus (HPV) has now been established as a necessary cause of cervical cancer. Indonesia is a country with a high cervical cancer incidence and with the world's highest prevalence of HPV 18 in cervical cancer. No information exists about the prevalence of HPV 18 or other HPV types in the Indonesian population. We conducted a hospital-based case-control study in Jakarta, Indonesia. A total of 74 cervical carcinoma cases and 209 control women, recruited from the gynecological outpatient clinic of the same hospital, were included. All women were HPV typed by the line probe assay, and interviews were obtained regarding possible risk factors for cervical cancer. HPV was detected in 95.9% of the cases and in 25.4% of the controls. In the control group, 13.4% was infected with a high-risk HPV type. HPV 16 was detected in 35% of the case group and in 1.9% of the control group and HPV 18 was identified in 28% of the case group and in 2.4% of the control group, suggesting that the oncogenic potentials of HPV 16 and HPV 18 in Indonesia are similar. In addition to HPV infection, young age at first intercourse, having a history of more than one sexual partner, and high parity were significant risk factors for cervical cancer. Within the control group, we did not identify determinants of HPV infection. We hypothesize that the high prevalence of HPV 18 in cervical cancer in Indonesia is caused by the high prevalence of HPV 18 in the Indonesian population.

The absence of CCL2 expression in cervical carcinoma is associated with increased survival and loss of heterozygosity at 17q11.2.

To determine whether CCL2 mRNA expression is beneficial or detrimental for cervical cancer patients, the association between the expression of this molecule by cervical tumour cells, the number of tumour-associated macrophages, and clinicopathological parameters such as recurrence, relapse-free survival, and overall patient survival was investigated. In cervical cancer samples from 93 untreated cervical cancer patients, the CCL2 mRNA expression level was quantified using RNA in situ hybridization and verified using real-time quantitative RT-PCR. The number of tumour-associated macrophages was determined using immunohistochemistry. Furthermore, the study investigated whether lack of CCL2 expression was due to genetic alterations near the 17q11.2 (CCL2 genomic) region. CCL2 mRNA expression by cervical tumour cells was associated with the number of tumour-associated macrophages (p < 0.001). Lack of CCL2 mRNA expression (15 samples; 16%) was associated with increased cumulative relapse-free survival (log rank test, p = 0.030), increased cumulative overall survival (log rank test, p = 0.024), less post-operative surgery, reduced local and distant recurrence, reduced vascular invasion, and smaller tumour size (<40 mm). The absence of CCL2 mRNA expression corresponded with loss of heterozygosity (LOH) at 17q11.2 in five of six samples. The increased cumulative relapse-free survival and cumulative overall survival of cervical cancer patients lacking tumour cell-associated CCL2 mRNA suggest that the tumour-associated macrophages support tumour progression, presumably by promoting angiogenesis and production of growth factors.

Psychosocial factors and the course of cervical intra-epithelial neoplasia: a prospective study.

OBJECTIVE: To investigate the influence of psychosocial factors on the course of cervical intra-epithelial neoplasia (CIN). METHODS: A group of 93 patients with CIN 1 or 2 was followed for 2.25 years by half-yearly colposcopy and cytology. Negatively-rated life events, social support, and coping style were studied in relation to distress during follow-up and in relation to time till progression and regression of CIN. Human papillomavirus (HPV) infection was controlled for as well as sick role bias caused by suspicion of having cervical cancer and distress due to the abnormal cervical smear. RESULTS: During follow-up, progression was found in 20 patients (22%), stable disease in 22 patients (24%), and regression in 51 patients (55%). Negatively-rated life events and lack of social support predicted distress longitudinally. No association was found between progression or regression of CIN and negatively-rated life events, lack of social support, coping style, and distress. CONCLUSION: We found no evidence that psychosocial factors influence the course of CIN.

Prognostic difference of surgical treatment of exophytic versus barrel-shaped bulky cervical cancer.

OBJECTIVE: The aim of the study was to evaluate the prognostic significance of tumor geography, defined as exophytic or barrel-shaped growth, in bulky (>4 cm) cervical cancer. METHODS: Four hundred women with cervical cancer, treated by primary radical hysterectomy between January 1984 and November 2000, were followed in a prospective cohort study. Clinical and pathology data were stored in a databank and the clinical protocol was unchanged during the study except for the amendment of additional indications of postoperative radiation in 1997. The assessment of tumor geography was based on pelvic examination at the time of tumor staging or radical hysterectomy or from the pathology report. Survival probabilities were calculated by the Kaplan-Meier method and compared with the log-rank test. RESULTS: The mean age of the patients was 45 years and the mean follow-up duration 48 months. Tumors were of squamous cell type in 291 patients (73%). Lymph node metastases were present in 91 patients (24%) and postoperative radiation was given in 179 patients (45%). In 291 patients, tumor diameter was <4 cm; in 58 patients, the tumor was defined as bulky exophytic and in 51 patients as bulky barrel shaped. There were no differences among these three groups in terms of operating time, blood loss during surgery or complications at 3 or 6 months postoperatively. Bulky exophytic tumors had an identical overall survival as compared to small-diameter (<4 cm) tumors. The overall survival (OS) of bulky barrel-shaped tumors was significantly worse (P < 10(-4)). The same was found for disease-free survival (DFS). CONCLUSION: Bulky exophytic cervical cancer has an identical surgical morbidity, overall and disease-free survival as compared to nonbulky (<4 cm) cervical cancer. In view of these identical characteristics, primary surgical treatment should be considered for patients with bulky exophytic cervical cancer.