C. J. L. M. Meijer

PCR-based high-risk HPV test in cervical cancer screening gives objective risk assessment of women with cytomorphologically normal cervical smears

Cervical‐cancer screening programmes using cytomorphological criteria could be more efficient if the screening included objective individual risk factors for women with normal cytology, such as a test for high‐risk human papillomavirus (HPV). The value of a PCR‐based test for high‐risk HPV types was studied in a cohort of 1622 women presenting in a routine biannual population‐based screening programme. Women were included in the study when they had no previous history of cervical dysplasia; and their initial Pap smear was read as normal (Pap 1 or 2). The mean age of the women was 42 years (range 34–54 years) and mean follow‐up time was 40 months (range 5–73 months). Women were referred for colposcopically directed biopsies if they had had 2 successive cervical smears read as Pap 3a (mild to moderate dyskaryosis) or one read as ≥ Pap 3b (severe dyskaryosis). Women with histologically confirmed cervical intraepithelial neoplasia grade III (CIN III) were considered positive cases. All women were tested for 14 high‐risk HPV genotypes. Of the 86 high‐risk HPV‐positive women, 6 developed CIN III, whereas only 1 of the 1536 HPV‐negative women did. The women with normal Pap smears containing high‐risk HPV genotypes were 116 times (95% Cl, 13–990) more at risk of developing CIN III, in contrast to women without high‐risk HPV. These results support the view that the interval between successive smears in cervical‐cancer screening can be increased considerably for women with cytomorphologically normal and high‐risk HPV‐negative cervical smears as determined by PCR.

Present evidence on the value of HPV testing for cervical cancer screening: a model-based exploration of the (cost-)effectiveness

Human papillomavirus (HPV) is the main risk factor for invasive cervical cancer. High risk ratios are found in cross-sectional data on HPV prevalence. The question raised is whether this present evidence is sufficient for making firm recommendations on HPV screening. A validated cervical cancer screening model was extended by adding HPV infection as a possible precursor of cervical intraepithelial neoplasia (CIN). Two widely different model quantifications were constructed so that both were compatible with the observed HPV risk ratios. One model assumed a much longer duration of HPV infection before progressing to CIN and a higher sensitivity of the HPV test than the other. In one version of the model, the calculated mortality reduction from HPV screening was higher and the (cost-)effectiveness was much better than for Pap smear screening. In the other version, outcomes were the opposite, although the cost-effectiveness of the combined HPV + cytology test was close to that of Pap smear screening. Although small follow-up studies and studies with limited strength of design suggest that HPV testing may well improve cervical cancer screening, only large longitudinal screening studies on the association between HPV infection and the development of neoplasias can give outcomes that would enable a firm conclusion to be made on the (cost-)effectiveness of HPV screening. Prospective studies should address women aged 30-60 years.

Peripheral primitive neuroectodermal tumour and extra-osseous Ewing's sarcoma: a histological, immunohistochemical and DNA flow cytometric study

Although peripheral primitive neuroectodermal tumour (pPNET) and extra-osseous Ewings sarcoma (EES) are thought to be closely related neoplasms, their clinical behaviour differs considerably. To determine the clinical relevance of the Schmidt classification scheme for differentiating pPNET and EES, 20 tumour specimens of poorly differentiated round cell tumours were evaluated. In addition, the diagnostic value of several neural markers and the prognostic value of quantitative morphological variables (DNA ploidy, S-phase fraction, and the mitotic activity) were assessed. Homer-Wright rosettes were present in 9 tumours. Neuron specific enolase (NSE) was expressed in 11 tumours, 8 of which expressed a second neural marker (CD57, S100, or neurofilament). According to the Schmidt classification, 11 pPNET and 5 EES were distinguished. HBA-71 was exclusively expressed in pPNET and EES. The remaining tumours were classified as sarcoma not otherwise specified (n=2), rhabdomyosarcoma (n=1), and desmoplastic tumour with divergent differentiation (n=1). EES611 patients fared significantly better than the pPNET patients (100% versus 42% 5-year survival). Neither DNA ploidy nor S-phase fraction assessed in 12 evaluative histograms (9 pPNET and 3 EES), nor mitotic activity yielded information of additional prognostic value. On the basis of this study and the Schmidt classification scheme, it can be concluded that if the diagnosis of EES and pPNET is based on light microscopy (Homer-Wright rosettes) and/or immunohistochemistry (at least two neural markers, i.e. NSE, S-100, CD57, and neurofilament), the classification provides important clinical information. Furthermore, positivity for HBA-71 is helpful in differentiating pPNET and EES from all other small round cell tumours.

Prognostic importance of DNA flow cytometric variables in rhabdomyosarcomas.

AIM--To determine whether DNA ploidy patterns and S phase fraction offer prognostic information in patients with rhabdomyosarcoma (RMS). METHODS--DNA flow cytometry was performed on formalin fixed, paraffin wax embedded samples from primary tumours, and metastatic deposits or recurrences in 70 patients. DNA histogram analysis was done using a semi-automated cell cycle analysis program. RESULTS--Of the 70 primary tumours, 23 were DNA diploid, 32 DNA aneuploid, eight DNA multiploid, and seven DNA tetraploid. The prognosis for DNA aneuploid patterns was favourable, intermediate within the group of DNA tetraploid tumours and poor among patients with DNA diploid and DNA multiploid tumours (p = 0.009). In multivariate analysis (Cox regression model) DNA ploidy was an important independent prognostic factor, along with TNM stage, localisation, and histopathological classification. Ten out of 32 patients with a high S phase fraction (> 15%) with primary RMS achieved long term survival in contrast to 20 out of 29 patients with a low S phase fraction (< or = 15%) (p = 0.008). In 24 cases the DNA ploidy of cases of relapse was analysed. Of the 15 cases, in which stem line changes had occurred, 13 died of disease. No stem line changes were noted in nine cases and in this group four patients died of disease (p = 0.02). CONCLUSIONS--Assessment of DNA ploidy and S phase fraction in primary RMS and evaluation of stem line changes in cases of relapse are important variables in predicting prognosis.

Prognostic value of morphometry in patients with normal breast tissue or usual ductal hyperplasia of the breast.

Women with usual ductal hyperplasia have a relative risk of 1.6–1.9 of subsequent breast cancer development. This slightly increased risk is generally not considered sufficiently high to justify (chemo)preventive therapy. It is therefore important to identify high‐risk ductal hyperplastic lesions that would benefit from such a treatment. Nuclear morphometric features have been shown in previous work to be useful for objectively describing morphologic features associated with high risk in (pre)invasive breast lesions. The aim of this study was to evaluate whether such morphometric features can also predict subsequent invasive cancer development in patients with the common pattern of usual ductal hyperplasia or a normal breast biopsy. The present case‐control study included 423 women with normal breast biopsies (n = 89) or biopsies containing usual ductal hyperplasia (n = 334). Of these 423 women, 132 developed invasive breast cancer during follow‐up (mean 16.7 ± 7.0 years). On the original hematoxylin and eosin‐stained sections, nuclear morphometry was performed with a digitizing video overlay system, and mitotic and apoptotic indices were assessed. Patients with mean nuclear feature values for area, perimeter, diameter or longest axis above the 75th percentile had 1.6–1.7 times the breast cancer risk of women with mean nuclear feature values below this value. Pairwise combinations of these features yielded slightly higher cancer risks for the fourth quartile patients, with the highest risk (1.9) for patients with SD of nuclear area and perimeter values above the 75th percentile. The number of apoptotic or mitotic cells had no prognostic value for patients with apparently normal tissue or usual ductal hyperplasia. Our results give a first indication that normal breast tissue or usual ductal hyperplasia harbor nuclear morphologic changes that, when assessed by morphometry, may be used to predict breast cancer development. It is worthwhile studying this further in independent groups of patients with long‐term follow‐up.

Extended duration of the detectable stage by adding HPV test in cervical cancer screening.

The human papillomavirus test (HPV) test could improve the (cost−) effectiveness of cervical screening by selecting women with a very low risk for cervical cancer during a long period. An analysis of a longitudinal study suggests that women with a negative Pap smear and a negative HPV test have a strongly reduced risk of developing cervical abnormalities in the years following the test, and that HPV testing lengthens the detectable stage by 2–5 years, compared to Pap smear detection alone.

Whole-Genome Sequencing and Variant Analysis of Human Papillomavirus 16 Infections

ABSTRACT Human papillomavirus (HPV) is a strongly conserved DNA virus, high-risk types of which can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A to D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole-genome level. We analyzed HPV16 major variant diversity and conservation in persistent infections and performed a single nucleotide polymorphism (SNP) comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to 3 years. Our analysis shows a remarkably large variant diversity in the population. Whole-genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified where the initial and follow-up samples clustered differently. Non-A1/A2 variants seemed to clear preferentially (P = 0.02). Our analysis shows that population-wide HPV16 sequence diversity is very large. In persistent infections, the HPV16 sequence was fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strongly acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to 3 years of follow-up. These findings suggest the progression of an early HPV16 infection could be host related. IMPORTANCE Human papillomavirus 16 (HPV16) is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge of variants circulating in European populations, especially with longitudinal follow-up, is limited. By sequencing a number of infections with known follow-up for up to 3 years, we gained initial insights into the genetic diversity of HPV16 and the effects of the viral genome on the persistence of infections. A SNP comparison between sequences obtained from clearing and persistent infections did not identify strongly acting DNA variations responsible for these infection outcomes. In addition, we identified an HPV16 reinfection event where sequencing of initial and follow-up samples showed different HPV16 variants. Based on conventional genotyping, this infection would incorrectly be considered a persistent HPV16 infection. In the context of vaccine efficacy and monitoring studies, such infections could potentially cause reduced reported efficacy or efficiency.

Extent and location of tumor infiltrating lymphocytes in microsatellite stable colon cancer predict outcome to adjuvant active specific immunotherapy

Meeting abstracts To determine the prognostic and predictive value of tumor infiltrating lymphocytes (TIL) in colon cancer in a cohort of patients who previously took part in a trial on adjuvant Active Specific Immunotherapy (ASI). Vermorken et al.[[1][1]] conducted a multicenter clinical trial on

Abstract 757: Transfection of dendritic cells with IL-21 mRNA gives rise to CD8+ effector T cells with a higher proliferation rate and increased cytotoxic capacity

Interleukin -21, mainly produced by activated CD4+ T cells, has been shown to play a central role in the differentiation and proliferation of B and T cells. Here, we investigated the utility of IL-21 to generate high numbers of functional CD8+ effector T cells for the immunotherapy of cancer. To this end, we made use of IL-21 mRNA transfected dendritic cells (DCs) to prime and activate CD8+ T cells recognizing the melanoma antigen Mart-1. Expansion rates and functional avidity were assessed. mRNA was transcribed in vitro from pGEM encoding eGFP/2A/IL-21 or IL-21. IL-21 transfection of cytokine-matured DCs was performed, which did not alter the expression of co-stimulatory markers (CD40, CD80, CD83 and CD86). Translation of the mRNA and secretion of the IL-21 protein were confirmed by ELISA. Initially, CD28+ T cell bulks were stimulated with allogeneic DCs transfected with IL-21 or eGFP (i.e. negative control) mRNA. The T cells primed in the presence of IL-21 showed a higher proliferation rate and cytotoxic capacity (by Granzyme B expression) than controls. Subsequently, IL-21 mRNA was co-transfected with mRNA encoding a MART-1 minigene, consisting of four copies of the MART-126L-35 immunodominant epitope preceded by a Ubiquitin sequence, into mature DCs. These DCs were used for the priming of isolated autologous CD8β+ T cells. After two rounds of stimulation, a slight, but not significant, increase in the frequencies of HLA-A2-tetramer binding T cells was observed for the MART-126L-35 epitope in comparison to control cultures with MoDCs transfected with the Ubi-MART-1 minigene mRNA without IL-21 mRNA. Of note, these specifically primed tetramer+ T cells displayed an equivalent functional avidity by IFNγ release as compared to their counterparts in control cultures, but did express significantly higher levels of Granzyme-B (P=0.05), indicative of a higher cytotoxic capacity. To assess the effects of IL-21 stimulation on effector-memory T cells, we made use of an established MART-126L-35 specific T cell clone. In multiple parallel cultures, significantly increased proliferation rates and Granzyme-B expression levels were observed when the T cells were stimulated by MoDCs co-transfected with both Ubi-MART-1 and IL-21 mRNA, as compared to Ubi-MART-1 mRNA only. We conclude that the inclusion of IL-21 in active vaccination approaches, such as mRNA-based DC vaccines, or indeed in adoptive T cell therapies, may increase their efficacy through the accelerated expansion of anti-tumor CD8+ effector T cells with increased cytolytic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 757. doi:10.1158/1538-7445.AM2011-757