Search

Contains fulltext : 292872.pdf (Publisher’s version ) (Open Access) AIMS: Large B cell lymphoma with IRF4 rearrangement (LBCL-IRF4) is a new entity in the 2017 revised World Health Organisation (WHO) classification that was initially mainly reported in children. After identification of a 79-year-old patient, we assessed how often IRF4 rearrangements can be detected in adult diffuse large B cell lymphomas (DLBCLs) which have to be reclassified to LBCL-IRF4 based on fluorescence in-situ hybridisation (FISH) for IRF4. METHODS AND RESULTS: With FISH, we studied the presence of IRF4 rearrangements in 238 lymphomas that were diagnosed as DLBCL according to the previous WHO classification of 2008. CONCLUSIONS: In addition to the index patient, an IRF4 rearrangement was detected in another five of 237 patients (2%). The immunohistochemical profile of these five IRF4 rearranged lymphomas was consistent with previous reports of LBCL-IRF4. One case was recognised to represent transformation of follicular lymphoma rather than de-novo LBCL-IRF4. BCL6 rearrangements were found in two cases of LBCL-IRF4; BCL2 and MYC rearrangements were excluded. Patients presented with limited stage disease with involvement of the head and neck in three patients, and involvement of the lung and thyroid in two others. This study shows that, although rare, LBCL-IRF4 should also be considered in older patients and at localisations other than the head and neck region. 01 juni 2023

Objective High‐risk human papillomavirus (HrHPV)‐positive women detected by self‐sampling require an extra visit at the general practitioner for additional cytology testing, but the loss to follow up within this triage is substantial. The aim of this study was to evaluate the clinical utility of reflex cytology on hrHPV‐positive self‐samples for immediate stratification of women who need referral for colposcopy. Design A prospective cohort study. Setting Two Dutch cervical cancer‐screening laboratories. Population 1014 screenees who tested hrHPV‐positive on self‐samples between 1 December 2018 and 1 August 2019. Methods Self‐samples were directly used for cytological analysis. Cytological and histological outcomes during follow up were obtained from the Dutch Pathology Registry (PALGA). Main outcome measures Test performance of reflex cytology on self‐samples was determined for different thresholds and compared with physician‐taken cytology and histological outcomes. Results Reflex cytology on self‐samples for detecting abnormal cytology showed a sensitivity of 26.4% (95% CI 21.8–31.3) and specificity of 90.5% (95% CI 87.7–92.8). Of all ≥CIN2 cases, 29.4% (95% CI 22.5–37.1) were detected with reflex cytology on self‐samples. The positive predictive value for detection of ≥CIN2 was higher with cytology on self‐collected samples than on physician‐collected samples. Of women who were lost to follow up, 12.9% were found to have abnormal cytology on their self‐sampled material. Conclusion Cytology testing is achievable on hrHPV‐positive self‐samples, could decrease the loss to follow up in screening and is easily implementable in the current clinical practice. Of all hrHPV‐positive women with abnormal cytology on additional physician‐collected samples, 26.4% could have been directly referred for colposcopy if triage with reflex cytology on self‐sampled material had been performed. Tweetable abstract Reflex cytology for triage of hrHPV+ self‐samples is of added value for direct referral of women for colposcopy., Tweetable abstract Reflex cytology for triage of hrHPV+ self‐samples is of added value for direct referral of women for colposcopy.

Primary high-risk human papillomavirus (hrHPV) DNA testing has been introduced in several countries worldwide, including The Netherlands. The objective of this study was to compare three automated workflow procedures for hrHPV testing of which the hrHPV detection assays meet the international guidelines for HPV testing. To mimic a realistic screening situation, we aimed to process 15 000 residual PreservCyt cervical samples in a period of 3 months. During a 3 months period, four technicians were involved in processing 5000 specimens per month on three automated platforms, (1) Qiagen Digene HC2 HPV DNA test (HC2, signal amplification); (2) Roche Cobas HPV test (DNA amplification), and (3) Hologic Aptima HPV test (RNA amplification). We measured and scored general aspects (time-to-results, hands-on-time (HOT)), maintenance, pre-run, run and post-run aspects, inventory (orders, storage), and number of errors on a scale from 1 to 10. As determined for one complete workflow each, maximum processing capacity and HOT were 296 samples and 2 h:55 m, 282 samples and 3 h:20 m, and 264 samples and 4 h:15 m for Aptima, Cobas, and HC2, respectively. The mean throughput time per run was 5 h:51 m for Cobas in which 94 samples could be processed. For Aptima, the mean throughput time per run was 6 h:30 m for 60 samples. Mean throughput time for HC2 is longer since results were provided on day 2. In this study, the fully automated Aptima workflow scores best with a 7.2, followed by Cobas with a score of 7.1 and HC2 with a score of 5.8. Although all HPV tests used in this comparison meet the international test guidelines, the performance (workflow) characteristics of the assays vary widely. A specific choice of a laboratory for high-throughput testing can be different based on the laboratory’s demands, but also hands-on-time, time-to-results/ # samples, maintenance, pre-run, run and post-run parameters, consumables, technical support, and number of errors are important operational factors for the selection of a fully automated workflow for hrHPV testing.

Objective Several studies have shown that there is an upward shift in the classification of cervical cytology when high-risk human papillomavirus (hrHPV) status is known to be positive. The Netherlands implemented primary hrHPV screening with reflex cytology as the primary screening test in 2017. Prior to implementation of the new programme, we investigated whether knowledge of hrHPV status influences cytology rating. Methods Using a set of 200 cytology slides that had been previously tested, two pairs of cytotechnicians rated 100 slides per pair twice: first without knowledge of hrHPV status and then, after a wash-out period of two months, with knowledge of hrHPV status. Results We found that hrHPV positive slides were more likely to be rated up over the referral threshold (i.e. from negative for intraepithelial lesion or malignancy to atypical squamous cells of undetermined significance+) than hrHPV negative slides at the second review when hrHPV status was known (relative risk = 3.2; 95% confidence interval: 1.3–7.9). Conclusions If the same upward shift in ratings were to be observed in the national programme, it may have implications for referrals of women with low-grade lesions.

Background Based on their potential to analyze aberrant cellular signaling in relation to biological function, kinase activity profiling in tumor biopsies by peptide microarrays and mass spectrometry-based phosphoproteomics may guide selection of protein kinase inhibitors in patients with cancer. Variable tissue handling procedures in clinical practice may influence protein phosphorylation status and kinase activity and therewith may hamper biomarker discovery. Here, the effect of cold ischemia time (CIT) on the stability of kinase activity and protein phosphorylation status in fresh-frozen clinical tissue samples was studied using peptide microarrays and mass spectrometry-based phosphoproteomics. Methods Biopsies of colorectal cancer resection specimens from five patients were collected and snap frozen immediately after surgery and at 6 additional time points between 0 and 180 min of CIT. Kinase activity profiling was performed for all samples using a peptide microarray. MS-based global phosphoproteomics was performed in tumors from 3 patients at 4 time points. Statistical and cluster analyses were performed to analyze changes in kinase activity and phosphoproteome resulting from CIT. Results Unsupervised cluster analysis of kinase activity and phosphoproteome data revealed that samples from the same patients cluster together. Continuous ANOVA analysis of all 7 time points for 5 patient samples resulted in 4 peptides out of 210 (2%) with significantly (p 2) altered signal intensity in time. In 4 out of 5 patients tumor kinase activity was stable with CIT. MS-based phosphoproteomics resulted in the detection of 10,488 different phosphopeptides with on average 6044 phosphopeptides per tumor sample. 2715 phosphopeptides were detected in all samples at time point 0, of which 90 (3.3%) phosphopeptides showed significant changes in intensity with CIT (p 2. Conclusions The vast majority of the phosphoproteome as well as the activity of protein kinases in colorectal cancer resection tissue is stable up to 180 min of CIT and reflects tumor characteristics. However, specific changes in kinase activity with increasing CIT were observed. Therefore, stringent tissue collection procedures are advised to minimize changes in kinase activity during CIT.

Background The introduction of population-based screening programs for colorectal cancer (CRC) results in less patients with advanced disease. There is an increase in the amount of node negative CRC, which makes adequate risk stratification for this particular group of patients necessary. The addition of more risk factors to the conventional histological high-risk factors is investigated in this retrospective study. Patients and Methods A cohort of 227 node negative (stage I and II) CRC patients who were not treated with adjuvant chemotherapy were selected from two previously conducted cohort studies. Detailed histopathological examination was performed by two independent observers and molecular background (BRAF/RAS mutations, microsatellite status (MSI)) was studied. Univariate analyses were used to analyse differences in histological and mutational characteristics between patients with and without recurrence. P-values below 0.05 were considered statistically significant. Results Poorly differentiated histology (p:0.002), BRAF mutation (p:0.002) and MSI status (p:0.006) were found significant relevant risk factors that were related to recurrent disease. Poorly differentiated histology was associated with intermediate/high tumor budding (TB) (p:0.001), a BRAF mutation (p:0.001) and MSI status (p:0.001). A combination of all three features (poorly differentiated histology, BRAF and MSI) was more often present in the recurrence group. Conclusions Recurrence in node negative CRC patients could be better predicted when molecular features such as, BRAF mutation and MSI status are incorporated into a model with poorly differentiated CRC. Therefore, these features might help in the selection of patients who possibly will benefit from adjuvant treatment.

Contains fulltext : 177357.pdf (Publisher’s version ) (Open Access) In 2017 the cervical cancer screening program in The Netherlands will be revised. Cervical smears will primarily be tested for the presence of high-risk human papillomavirus (hrHPV) instead of cytology, and vaginal self-sampling will be offered to non-responders. This includes a potential risk that part of the women who would otherwise opt for a cervical smear will wait for self-sampling. However, self-sampling for hrHPV in a responder population has never been studied yet. The aim of this study was to investigate the applicability and accuracy of self-sampling in detecting hrHPV in a screening responder population. A total of 2049 women, aged 30-60years, participating in the screening program in The Netherlands were included from April 2013 to May 2015. After they had their cervical smear taken, women self-collected a cervicovaginal sample with a brush-based device, the Evalyn Brush. Both the cervical smear and self-sample specimen were tested with the COBAS 4800 HPV platform. The hrHPV prevalence was 8.0% (95% CI 6.9-9.2) among the physician-taken samples, and 10.0% (95% CI 8.7-11.3) among the self-samples. There was 96.8% (95% CI 96.0-97.5) concordance of hrHPV prevalence between self-samples and physician-taken samples. Women in our study evaluated self-sampling as convenient (97.1%), user-friendly (98.5%), and 62.8% preferred self-sampling over a physician-taken sampling for the next screening round. In conclusion, self-sampling showed high concordance with physician-taken sampling for hrHPV detection in a responder screening population and highly acceptable to women. Implementation of HPV-self-sampling for the responder population as a primary screening tool may be considered.

Abstract Background In January 2017, the Dutch cervical cancer screening programme transitioned from cytomorphological to primary high-risk HPV (hrHPV) DNA screening, including the introduction of self-sampling, for women aged between 30 and 60 years. The Netherlands was the first country to switch to hrHPV screening at the national level. We investigated the health impact of this transition by comparing performance indicators from the new hrHPV-based programme with the previous cytology-based programme. Methods We obtained data from the Dutch nationwide network and registry of histo- and cytopathology (PALGA) for 454,573 women eligible for screening in 2017 who participated in the hrHPV-based programme between 1 January 2017 and 30 June 2018 (maximum follow-up of almost 21 months) and for 483,146 women eligible for screening in 2015 who participated in the cytology-based programme between 1 January 2015 and 31 March 2016 (maximum follow-up of 40 months). We compared indicators of participation (participation rate), referral (screen positivity; referral rate) and detection (cervical intraepithelial neoplasia (CIN) detection; number of referrals per detected CIN lesion). Results Participation in the hrHPV-based programme was significantly lower than that in the cytology-based programme (61% vs 64%). Screen positivity and direct referral rates were significantly higher in the hrHPV-based programme (positivity rate: 5% vs 9%; referral rate: 1% vs 3%). CIN2+ detection increased from 11 to 14 per 1000 women screened. Overall, approximately 2.2 times more clinical irrelevant findings (i.e. ≤CIN1) were found in the hrHPV-based programme, compared with approximately 1·3 times more clinically relevant findings (i.e. CIN2+); this difference was mostly due to a national policy change recommending colposcopy, rather than observation, of hrHPV-positive, ASC-US/LSIL results in the hrHPV-based programme. Conclusions This is the first time that comprehensive results of nationwide implementation of hrHPV-based screening have been reported using high-quality data with a long follow-up. We have shown that both benefits and potential harms are higher in one screening round of a well-implemented hrHPV-based screening programme than in an established cytology-based programme. Lower participation in the new hrHPV programme may be due to factors such as invitation policy changes and the phased roll-out of the new programme. Our findings add further to evidence from trials and modelling studies on the effectiveness of hrHPV-based screening.

Community-acquired pneumonia (CAP) is the most common form of pneumonia and is a leading infectious cause worldwide. Identification of patients that are at risk to develop severe disease has proven to be a major challenge. Soluble mannose receptor (sMR; sCD206) is a new serum marker for macrophage activation. Recent studies showed that sMR levels are increased in patients suffering from severe infections making it a potential biomarker for improved discrimination of disease severity. For measuring sMR, no standardized assay is available. Aim of this study is to develop an assay for standardized measurement of sMR. Next, this assay was used to assess sMR plasma levels for its ability to predict severe disease development in a patient cohort for community-acquired pneumonia.We developed a well-validated sandwich ELISA that enables standardized measurement of sMR in plasma and serum samples. Repeatability was tested by calculating the percentage coefficient of variation (%CV) within and between runs and within and between operators. sMR levels were assessed in a cohort of 100 patients with community-acquired pneumonia.All %CV values were10%, indicating low variation. Higher sMR levels were observed in patients with severe disease when compared to patients without severe disease development (p = 0.004). Patients with sMR levels between 100-430 ng/ml had 22.7% chance to develop severe disease whereas patients with levels between 430-1000 ng/ml had 33.3% chance to develop severe disease.We suggest that sMR has potential as a new biomarker for the prediction of disease severity in patients with community-acquired pneumonia.

In the era of personalized cancer medicine, identifying mutations within patient tumors plays an important role in defining high-risk stage II colon cancer patients. The prognostic role of BRAF V600E mutation, microsatellite instability (MSI) status, KRAS mutation and PIK3CA mutation in stage II colon cancer patients is not settled. We retrospectively analyzed 186 patients with stage II colon cancer who underwent an oncological resection but were not treated with adjuvant chemotherapy. KRAS mutations, PIK3CA mutation, V600E BRAF mutation and MSI status were determined. Survival analyses were performed. Mutations were found in the patients with each mutation in the following percentages: 23% (MSI), 35% (KRAS), 19% (BRAF) and 11% (PIK3CA). A trend toward worse overall survival (OS) was seen in patients with an MSI (5-year OS 74% versus 82%, adjusted hazard ratio [HR] 1.8, 95% confidence interval [CI] 0.6–4.9) and a KRAS-mutated tumor (5-year OS 77% versus 82%, adjusted HR 1.7, 95% CI 0.8–3.5). MSI and BRAF-mutated tumors tended to correlate with poorer disease-free survival (DFS) (5-year DFS 60% versus 78%, adjusted HR 1.6, 95% CI 0.5–2.1 and 5-year DFS 57% versus 77%, adjusted HR 1.1, 95% CI 0.4–2.6 respectively). In stage II colon cancer patients not treated with adjuvant chemotherapy, BRAF mutation and MSI status both tended to have a negative prognostic effect on disease-free survival. KRAS and MSI status also tended to be correlated with worse overall survival.

The aim of this study was to examine sexual inactivity and occurrence of selected sexually transmitted infections in relation to body mass index. We used data from two large Danish population-based cross-sectional studies conducted in 1991-1995 (HPV study: 6869 women, aged 22-32 years) and in 2004-2005 (Liva study: 19,484 women, aged 18-45 years). Data were collected using a structured interview and measured weight, height, high-risk human papillomavirus DNA, Chlamydia DNA for the HPV study and a structured questionnaire for the Liva study. Overweight and obese women were more likely to have had no lifetime sexual partner or no sexual partner in the last year, e.g., obese women had a threefold (95 percent CI: 1.95-5.04) odds ratio of having had no sexual partner in the last year compared to normal weight women. Additionally, overweight and obese women had a lower likelihood of genital warts and high-risk human papillomavirus infection. A similar tendency was found for self-reported Chlamydia, but not with presence of Chlamydia DNA. If higher likelihood of no lifetime or recent sexual partners among overweight and obese women reflects unmet sexual needs, it could give rise to concern because quality of sexual life is associated with general quality of life.

Objectives Some studies suggest that Chlamydia trachomatis (CT) enhances cervical carcinogenesis; however, a possible confounding effect of persistent human papillomavirus (HPV) infection was not addressed. We examined the potential role of CT infection in the development of subsequent cervical intraepithelial neoplasia grade 3 or worse (CIN3+) in women with prevalent HPV infection and in a subgroup of women with persistent HPV infection. Methods Participants in this population-based cohort study underwent a structured interview, including history of CT infection, and subsequently cervical exfoliated cells were obtained for HPV DNA and CT DNA testing. Women with high-risk HPV DNA infection and no prevalent cervical disease constituted the overall study population (n=1390). A subgroup of women with persistent HPV infection (n=320) was also identified. All women were passively followed for development of cervical lesions in the national Pathology Data Bank. HRs and 95% CIs for CIN3+ during follow-up (up to 19 years) were estimated in an accelerated failure time model. Results Women who reported more than one CT infection had a statistically significantly increased risk of CIN3+ (high-risk HPV-positive, HR=2.51, 95% CI 1.44 to 4.37) (persistent HPV infection, HR=3.65, 95% CI 1.53 to 8.70). We found no association between CT DNA and subsequent risk of CIN3+ among women who were HPV-positive or had a persistent HPV infection at baseline. Conclusions Repeated CT infections increased the risk of CIN3+ among women with prevalent as well as persistent high-risk HPV infection.

Background Tumour mutational burden (TMB) is a measurement of DNA variants in a tumour and is a potential biomarker of response to Immune Checkpoint Inhibitor (ICI) therapy. Patients with non-small cell lung cancer (NSCLC) whose tumours have a high TMB (≥10 mutations/Mb) might benefit from upfront ICI combination therapy. Clinical trials have measured TMB using Whole Exome Sequencing (WES) and/or the FoundationOne CDx (F1CDx) assay. In parallel, several commercial next-generation sequencing (NGS) assays have become available. Before implementation of TMB testing in clinical practice, technical and clinical validation are needed. Hence, a multi-center study was organised to establish concordance between several TMB assays. Methods Fifteen resection NSCLC formalin-fixed paraffin-embedded (FFPE) samples with broad TMB range were selected. Each participant received extracted DNA and was asked to report TMB values using their own protocol. In parallel, FFPE slides were analysed with F1CDx. Eight labs participated using five different methods: Oncomine TML assay (Thermofisher; n = 4), TSO500 assay (Illumina; n = 1), NEOplus assay (NEO New Oncology; n = 1), a 0,4 Mb targeted resequencing lab-developed assay (LDT; n = 1) and WES using three different TMB calculation methods (n = 1). Correlations between each platform and F1CDx were calculated. Also, the reported TMB category (high vs low) of the different platforms in comparison to F1CDx was evaluated for the fifteen samples. Results Assessment of TMB values obtained by the platforms and F1CDx demonstrated a high correlation (R2 between 0.81 and 0.94), except for the smaller LDT (R2 = 0.53). The TMB category (high vs low) reported by each platform showed concordance with the F1CDx category for eleven (73%) to thirteen (93%) of the fifteen samples. From the fifteen samples, the same category was reported by all different platforms for seven (47%) samples. Conclusion Our data show that assays from different providers can be used to predict TMB. However, samples with a TMB value around the cut-off of 10 mut/Mb are challenging and interpretation should occur with caution. Further studies are required before implementing these assays in routine clinical diagnosis. Legal entity responsible for the study The authors. Funding Bristol-Myers Squibb. Disclosure S. Lambin: Research grant / Funding (institution): Bristol-Myers Squibb. All other authors have declared no conflicts of interest.

582 Background: Left- and right-sided colorectal cancers (CRCs) are different in terms of prognosis, treatment response and underlying molecular mechanisms. The aim of this study was to unravel the phosphoproteomic profiles of left- and right-sided primary CRCs and to identify potential drug targets for both primary tumor sites. Methods: In total phosphoproteomic profiles of 69 fresh-frozen biopsies, including 34 left-sided and 35 right-sided primary CRCs, were obtained by LC-MS/MS after cell lysis, digestion and phosphopeptide enrichment using anti-phosphotyrosine antibodies. MS/MS spectra were searched against a UniProt human reference proteome FASTA file using MaxQuant software. Fold changes between normalized intensities were calculated. Group comparison was performed using the R package Limma. P values below 0.01 and fold changes > two were considered significant and biologically relevant. Results: In total 2,840 phosphopeptides were detected in at least 5% of the samples. Of these, 36 phosphopeptides were significantly upregulated and 14 were significantly downregulated in right-sided compared to left-sided CRCs using our stringent selection criteria. Since BRAF mutations and deficient DNA mismatch repair (dMMR) were more frequently observed in right-sided CRC (p < 0.01), group comparison was repeated with MMR proficient CRCs only, resulting in 21 phosphopeptides up- and one downregulated in left- versus right-sided CRC. The identified phosphoproteins from the phosphopeptides included three FDA approved drug targets. Conclusions: Differences in phosphoproteomic profiles were detected between left- and right-sided CRCs, besides molecular differences such as BRAF mutation and MMR status. These results indicate that the molecular biology of left-and right-sided CRCs may explain their differences in clinical behavior and response to treatment.

Community-acquired pneumonia (CAP) is mostly caused by Streptococcus pneumoniae. Identification of the pathogen causing CAP can be achieved by conventional culture techniques of sputum and/or blood, antigen detection from urine or molecular analysis. However, it remains difficult to determine patients who are at risk of severe disease development (intensive care unit [ICU] admittance and/or death). In this retrospective study, 121 patients admitted to the emergency department with pneumonia symptoms were included. Several markers of infection (pneumococcal DNA load in blood (real-time LytA PCR), white blood cell (WBC) count, C-reactive protein (CRP), procalcitonin (PCT) and soluble urokinase plasminogen activator receptor (suPAR) levels) were assessed for their ability to predict severe disease development. Of 121 patients, 6 were excluded from the study because of an alternative diagnosis, whereas 8 were excluded from biomarker analysis because of the presence of co-morbidities. Of the 115 patients analysed by the LytA PCR, 23 were positive. PCR detected S. pneumoniae DNA in 82% of patients with positive blood culture for S. pneumoniae. PCR missed three samples from patients in which S. pneumoniae was recovered by blood cultures. However, eight additional LytA PCR-positive samples were detected from patients whose blood cultures remained negative. Pneumococcal DNA load was also monitored in time for 31 patients, of whom 11 had positive PCR results. For 10 out of 11 (91%) positive PCR patients, a clear increase in Ct-values was observed, indicating a lower pneumococcal DNA load in the blood as a result of antibiotic therapy. Biomarker analysis was performed in 107 patients, of whom 29 showed severe disease development. Pneumococcal DNA load (p = 0.026), PCT (p = 0.046) and suPAR (p = 0.001) levels most reliably predicted severe disease development. In conclusion, in patients with CAP, higher pneumococcal DNA load, PCT and suPAR values are associated with severe disease development (ICU admission and/or death). These biomarkers may be useful tools for triage of patients suspected of having CAP in the emergency department.

To investigate risk factors for incident and redetected Chlamydia trachomatis (CT) infection in women, including the role of high-risk human papillomavirus (HPV).In this population-based, prospective cohort study conducted in Copenhagen, Denmark, 10,729 women aged 20 to 29 years were tested for CT and HPV DNA and provided information on sexual and health behavior at baseline. Of these, 7998 (74.5%) participated in a follow-up visit 2 years later with identical data collection. We used logistic regression to investigate risk factors for incident and redetected CT infection at follow-up.Among CT DNA negative women at baseline (n = 7529), 106 (1.4%) were CT DNA positive at follow-up (incident infection). Increasing number of sexual partners during follow-up (odds ratio [OR], 1.07 per partner; 95% confidence interval (CI), 1.02-1.11), low educational level (OR, 1.69; 95% CI, 1.11-2.56; for basic education vs. high school or higher), and high-risk HPV positivity at baseline (OR, 1.66; 95% CI, 1.06-2.58) were risk factors for incident infection, whereas older age (OR, 0.86 per year increase; 95% CI, 0.80-0.93) and condom use (OR, 0.60; 95% CI, 0.38-0.94) were associated with reduced risk. Among CT DNA positive women at baseline (n = 469), 108 (23.0%) tested positive at follow-up (redetected infection). We found no statistically significant associations between age, educational level, sexual behavior, smoking, or high-risk HPV status and the risk for redetected CT.Young age, low educational level, high number of sexual partners, failure to use condoms, and high-risk HPV positivity are associated with increased risk for incident CT infection. These findings may guide the development of targeted CT prevention strategies, including screening and information campaigns.

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria from agar media. Direct identification from positive blood cultures should decrease the time to obtaining the result. In this study, three different processing methods for the rapid direct identification of bacteria from positive blood culture bottles were compared. In total, 101 positive aerobe BacT/ALERT bottles were included in this study. Aliquots from all bottles were used for three bacterial processing methods, i.e. the commercially available Bruker’s MALDI Sepsityper kit, the commercially available Molzym’s MolYsis Basic5 kit and a centrifugation/washing method. In addition, the best method was used to evaluate the possibility of MALDI application after a reduced incubation time of 7 h of Staphylococcus aureus- and Escherichia coli-spiked (1,000, 100 and 10 colony-forming units [CFU]) aerobe BacT/ALERT blood cultures. Sixty-six (65%), 51 (50.5%) and 79 (78%) bottles were identified correctly at the species level when the centrifugation/washing method, MolYsis Basic 5 and Sepsityper were used, respectively. Incorrect identification was obtained in 35 (35%), 50 (49.5%) and 22 (22%) bottles, respectively. Gram-positive cocci were correctly identified in 33/52 (64%) of the cases. However, Gram-negative rods showed a correct identification in 45/47 (96%) of all bottles when the Sepsityper kit was used. Seven hours of pre-incubation of S. aureus- and E. coli-spiked aerobe BacT/ALERT blood cultures never resulted in reliable identification with MALDI-TOF MS. Sepsityper is superior for the direct identification of microorganisms from aerobe BacT/ALERT bottles. Gram-negative pathogens show better results compared to Gram-positive bacteria. Reduced incubation followed by MALDI-TOF MS did not result in faster reliable identification.

Aim: Although the predictive and prognostic value of thymidylate synthase (TS) expression and gene polymorphism in colon cancer has been widely studied, the results are inconclusive probably because of methodological differences. With this study, we aimed to elucidate the role of TS gene polymorphisms genotyping in therapy response in stage III colon carcinoma patients treated with 5-FU adjuvant chemotherapy.Patients and Methods: 251 patients diagnosed with stage III colon carcinoma treated with surgery followed by 5-FU based adjuvant therapy were selected. The variable number of tandem repeats (VNTR) and the single nucleotide polymorphism (SNP) in the 5′-untranslated region of the TS gene were genotyped.Results: There was a positive association between tumor T stage and the VNTR genotypes (p=0.05). In both univariate and multivariate survival analysis no effects of the studied polymorphisms on survival were found. However, there was an association between both polymorphisms and age. Among patients younger than 60 years, the patients homozygous for 2R seemed to have a better overall survival, whereas among the patients older than 67 this longer survival was seen by the carriers of other genotypes.Conclusion: We conclude that the TS VNTR and SNP do not predict response to 5-FU therapy in patients with stage III colon carcinoma. However, age appears to modify the effects of TS polymorphisms on survival.

Although direct sequencing is the gold standard for KRAS mutation detection in routine diagnostics, it remains laborious, time consuming, and not very sensitive. Our objective was to evaluate SNaPshot and the KRAS StripAssay as alternatives to sequencing for KRAS mutation detection in daily practice. KRAS exon 2-specific PCR followed by sequencing or by a SNaPshot reaction was performed. For the StripAssay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. To test sensitivities, dilution series of mutated DNA in wild-type DNA were made. Additionally, direct sequencing and SNaPshot were evaluated in 296 colon cancer samples. Detection limits of direct sequencing, SNaPshot, and StripAssay were 20%, 10%, and 1% tumor cells, respectively. Direct sequencing and SNaPshot can detect all 12 mutations in KRAS codons 12 and 13, whereas the StripAssay detects 10 of the most frequent ones. Workload and time to results are comparable for SNaPshot and direct sequencing. SNaPshot is flexible and easy to multiplex. The StripAssay is less time consuming for daily laboratory practice. SNaPshot is more flexible and slightly more sensitive than direct sequencing. The clinical evaluation showed comparable performances between direct sequencing and SNaPshot. The StripAssay is rapid and an extremely sensitive assay that could be considered when few tumor cells are available. However, found mutants should be confirmed to avoid risk of false positives.

IntroductionThe aim of this study was to compare the reproducibility of epidermal growth factor receptor (EGFR) immunohistochemistry (IHC),EGFRgene amplification analysis, andEGFRandKRASmutation analysis among different laboratories performing routine diagnostic analyses in pathology in The Netherlands, and to generate normative data.MethodsIn 2008, IHC, in-situ hybridisation (ISH) forEGFR, and mutation analysis forEGFRandKRASwere tested. Tissue microarray sections were distributed for IHC and ISH, and tissue sections and isolated DNA with known mutations were distributed for mutation analysis. In 2009, ISH and mutation analysis were evaluated. False-negative and false-positive results were defined as different from the consensus, and sensitivity and specificity were estimated.ResultsIn 2008, eight laboratories participated in the IHC ring study. In only 4/17 cases (23%) a consensus score of ≥75% was reached, indicating that this analysis was not sufficiently reliable to be applied in clinical practice. ForEGFRISH, andEGFRandKRASmutation analysis, an interpretable result (success rate) was obtained in ≥97% of the cases, with mean sensitivity ≥96% and specificity ≥95%. For small sample proficiency testing, a norm was established defining outlier laboratories with unsatisfactory performance.ConclusionsThe result of EGFR IHC is not a suitable criterion for reliably selecting patients for anti-EGFR treatment. In contrast, molecular diagnostic methods forEGFRandKRASmutation detection andEGFRISH may be reliably performed with high accuracy, allowing treatment decisions for lung cancer.

The prevalence of human papillomavirus (HPV) genotypes in relation to age was investigated by the polymerase chain reaction (PCR) method in cytologically normal smears from 4 different groups of women. Group A consisted of young women from a district population, aged 15-34 years, using oral contraceptives and visiting general practitioners for a check-up (n = 156); group B were asymptomatic women, aged 35-55, in a district population participating in a triennial screening program for cervical cancer (n = 1555); group C and D consisted of women, seen at the gynecological outpatient department for a wide spectrum of gynecological complaints or for control of their hormonal contraception, aged 15-34 years (n = 2320), and aged 35-55 years (n = 1826) respectively. An HPV (all types) prevalence of 14.1%, 4.1%, 13.9% and 6.6% and an HPV 16/18 prevalence of 3.8%, 0.9%, 3.3% and 1.5% were found in groups A, B, C and D respectively. Statistically significant differences (p value < 0.001) in HPV prevalence were found between women aged 15-34 years and women aged 35-55 years in the district population and in the hospital population. No statistically significant differences in HPV 16/18 were observed after age-matching between women in corresponding age-classes of both populations. In a 5-year interval analysis a strong age-dependent relationship was demonstrated, with a maximum between 20 and 24 years. After the age of 35 a constant level of 1-2% HPV 16/18 was observed. These results indicate that genital HPV infections are age-dependent and suggest that HPV infections at young age can be transient. The implications of these findings in the context of cervical cancer screening are discussed.

Item does not contain fulltext BACKGROUND: Neoadjuvant radiochemotherapy (RCT) is thought to result in a favorable oncological outcome in esophageal cancer patients. Unfortunately, it also implies that adjacent healthy tissue is preoperatively exposed to the potential damaging influence of RCT. Here, the impact of preoperative RCT on matrix metalloproteinase (MMP) expression in healthy esophageal tissue aligned with the tumor at the time of surgery is examined. PATIENTS AND METHODS: 23 patients participating in a clinical trial were randomized to either the control (n = 12) or the neoadjuvant RCT group (n = 11). In the latter group, surgery was performed 5 weeks after the last course of RCT. Full-thickness biopsies were taken from healthy esophageal tissue at the proximal border of the resection specimen and more distally next to the tumor. MMP-2 and MMP-9 activity in the samples was assessed by quantitative gelatin zymography and immunohistochemistry. RESULTS: In the proximal segment, the activities of the MMP-9-dimer (135 kDa) and proMMP-9 (92 kDa) were significantly increased in the RCT group as compared with the control group: 28.5 versus 3.0 (p = 0.025) and 87.7 versus 13.0 (p = 0.015) arbitrary units for 135 kDa and 92 kDa, respectively. In the distal part, RCT resulted in a significant increase of proMMP-2 (72 kDa: 35.8 versus 17.8, p = 0.005) and proMMP-9 (81.2 versus 23.3, p = 0.03). CONCLUSION: In esophageal cancer patients, neoadjuvant RCT results in increased MMP expression in healthy esophageal tissue as measured at the time of surgery. Since increased levels of MMPs are associated with severe postoperative complications including anastomotic leakage this finding necessitates further clinical research. 6 p.

Few etiologic studies of squamous cell carcinoma (SCC) of the penis have been carried out in populations where childhood circumcision is rare. A total of 71 patients with invasive (n = 53) or in situ (n = 18) penile SCC, 86 prostate cancer controls, and 103 population controls were interviewed in a population-based case-control study in Denmark. For 37 penile SCC patients, tissue samples were PCR examined for human papillomavirus (HPV) DNA. Overall, 65% of PCR-examined penile SCCs were high-risk HPV-positive, most of which (22 of 24; 92%) were due to HPV16. Penile SCC risk was positively associated with measures of early and high sexual activity, including lifetime number of female sex partners, number of female sex partners before age 20, age at first intercourse, penile-oral sex, a history of anogenital warts, and never having used condoms. Histories of phimosis and priapism at least 5 years before diagnosis were also significant risk factors, whereas alcohol abstinence was associated with reduced risk. Our study confirms sexually transmitted HPV16 infection and phimosis as major risk factors for penile SCC and suggests that penile-oral sex may be an important means of viral transmission. The association with priapism was unexpected and needs replication. (Cancer Epidemiol Biomarkers Prev 2008;17(10):2683–91)

Contains fulltext : 71387.pdf (Publisher’s version ) (Open Access) BACKGROUND: Not all patients with locally advanced rectal cancer (LARC) respond equally to neo-adjuvant radiochemotherapy (RCT). Patients with highly apoptotic less advanced rectal cancers do not benefit from short-term radiotherapy. This study investigates whether this is also the case in the setting of RCT for LARC. PATIENTS AND METHODS: Tissue microarrays were constructed of biopsy and resection specimens of 201 LARC patients. Apoptosis (M30) and several apoptosis-regulating proteins [p53, Bcl-2, Bax, cyclooxygenase-2 (Cox-2) and mamma serine protease inhibitor (maspin)] were studied with immunohistochemistry. Subsequently, predictive values for local recurrence (LR), overall survival (OS) and histological tumour regression were analysed. RESULTS: Apoptotic levels, quantified as the number of apoptotic cells/mm(2) tumour epithelium, were higher in posttherapy tissues compared with biopsies (P < 0.001). Biopsies from clinical T4 stage tumours demonstrated significantly higher levels of apoptosis than clinical T3 stage tumours (P = 0.020). Therapy-induced apoptosis was higher when the interval between the last day of irradiation and surgery increased (P < 0.001, correlation coefficient = 0.355). Pre- and posttherapy apoptosis, p53, Bcl-2, Bax and Cox-2 were not associated with LR, OS or tumour regression. Intense pretherapy cytoplasmatic staining of maspin indicated a higher risk on LR (P = 0.009) only. CONCLUSION: Combined RCT is also successful in highly apoptotic tumours and is therefore independent of intrinsic apoptosis.

Better information on the prevalence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infection is needed in many world areas.Cross-sectional study of population-based samples of nonpregnant women aged 15 to 44 years in Nigeria, Colombia, Argentina, Vietnam (2 areas), China, Thailand (2 areas), Korea, and Spain. 5,328 consenting women aged 15 to 44 years participated. Exfoliated cervical cells were collected and testing for CT and NG and human papillomavirus (HPV) was done using PCR-based assays.Age-standardized CT prevalence ranged between 0.2% (95% confidence interval, CI: 0.0-0.7%) in Spain and 5.6% (95% CI: 3.4-7.8%) in Nigeria. NG ranged between 0% (with broad CIs) in several areas and 2.6% (95% CI: 1.0-4.2%) in Nigeria. Prevalence of CT in all areas combined was greater in women aged 15 to 24 (4.5; 95% CI: 3.4-5.8%) than 25 to 44 (2.6; 95% CI: 2.1-3.1%), whereas NG prevalence was similar in the 2 age groups (0.3%). The only significant risk factors were NG infection (for CT), CT infection (for NG) and infection with high-risk HPV types (for both).The prevalence of CT and, most notably, NG was relatively low in a variety of countries. Our findings, however, do not apply to subsets of high-risk women who are likely to be underrepresented in our population-based samples.

Recently, we showed that Epstein-Barr virus (EBV)-positive gastric carcinoma (GC) forms a distinct clinicopathologic entity with a better prognosis due to lower incidence of lymph node metastases (LN+). Here we investigated whether in EBV-positive GC more pronounced activation of cellular immune responses is associated with absence of (micro)metastases. Twenty EBV-positive primary tumors (PT) (9 LN+) were matched with 28 EBV-negative GC (11 LN+) for T- and N-stage, gender, and age. The PT (n = 28) and its LNs were analyzed by EBER RNA in situ hybridization and by immunohistochemistry for MHC class I and II expression, for CD3, CD8, CD4, CD20, CD56, CD83, and Granzyme B (GzB) expression. In LN metastases of EBV-positive GC, the EBV genome is maintained, excluding tumor escape by virus deletion. All GC express MHC class I independently of EBV status. In comparison with EBV-negative GC, EBV-positive GC have higher expression of MHC class II on the tumor cells (P = 0.029) and a more extensive infiltrate (P < 0.0001) of activated GzB+ CD8+ T cells (P = 0.028), which is most abundant in those EBV-positive tumors that do not metastasize (P < 0.0001). In addition, in EBV-positive GC without metastases, the infiltrate contains higher numbers of mature dendritic cells (DC) (P = 0.018). At present, the antigenic target has to be determined. These data support the notion that local triggering of cellular immune responses in EBV-positive GC prevents lymph node metastasis formation.

Udgivelsesdato: 2006-null BACKGROUND: Infection with human papillomavirus is considered a necessary factor in developing high-grade squamous intraepithelial lesions of the cervix. However, most human papillomavirus positive women do not develop high-grade squamous intraepithelial lesions and other factors may be important for this transition. The objective of the present study was to examine if smoking and alcohol intake are associated with the risk of developing high-grade squamous intraepithelial lesions in women positive for high-risk human papillomavirus types. METHODS: We used baseline information on exposures on 548 high-risk human papillomavirus positive women with normal cytology, comparing 94 women who developed high-grade squamous intraepithelial lesions with 454 women who remained cytologically normal. Logistic regression was applied for statistical analysis. RESULTS: Compared with never smokers, the odds ratio for high-grade squamous intraepithelial lesions among current smokers was 1.99 (95% CI: 1.21-3.28). Among current smokers, number of cigarettes, years of smoking, and early age at smoking initiation were associated with increased risk. However, when modeled simultaneously, it seemed that smoking duration and age at smoking initiation were more associated with risk of high-grade squamous intraepithelial lesions than amount of smoking. Alcohol intake was not associated with risk of high-grade squamous intraepithelial lesions among these women. CONCLUSION: Smoking is associated with an increased risk of developing high-grade squamous intraepithelial lesions in women who are infected with oncogenic human papillomavirus.

Coinfection with multiple types of human papillomavirus (HPV) and its implications for the development of efficacious HPV vaccines is a subject of great interest. To describe the occurrence of concurrent infection with multiple HPV types and to determine whether genital HPV infection modifies the risk of acquiring a new HPV infection with another HPV type, 1610 subjects were monitored for an average of 4.1 years in Bogota, Colombia. Information on risk factors for HPV infection and cervical cells was collected for detection of HPV DNA of 36 types at study entry and at 6 consecutive 6-month follow-up visits. Clustering or the concurrent acquisition of multiple types occurred more often than would be expected by chance. Subjects with incident HPV-16 or -18 infection had 5-7 times higher odds of acquiring a subsequent HPV-58 infection than subjects not infected with HPV-16 or -18. This might affect the protection conferred by effective HPV vaccines.

BACKGROUND: Genital human papillomavirus (HPV) infections are generally accepted to be sexually transmitted, but studies of HPV infections in sex partners are limited. We investigated HPV type-specific concordance and viral load in 238 heterosexual couples. Women with cervical intraepithelial neoplasia were the index patients in these couples.METHODS: GP5+/6+ polymerase chain reaction (PCR), followed by reverse-line blot analysis, was used for the detection of 45 HPV types in cervical and penile scrape samples. Viral loads were subsequently determined in scrape samples positive for HPV types 16, 18, 31, and 33 by LightCycler-based real-time PCR assays.RESULTS: A total of 89.9% of the women and 72.9% of their male partners were HPV positive. Predominantly high-risk HPV types were found in persons of both sexes, but infections with multiple and non-high-risk HPV types were more common in men. Of the HPV-positive couples, 57.8% of the men had the same HPV type as their partners; this rate was significantly higher than that expected by chance (P < .001). Moreover, these HPV-concordant men had higher penile scrape viral loads than did the non-HPV-concordant men. For HPV type 16-positive women, higher cervical viral loads were predictive of presence of HPV type 16 in their sex partners.CONCLUSIONS: In sexually active couples, HPV type concordance was more prevalent than expected by chance and was associated with increased viral loads. These data provide biological support for HPV transmission between sex partners.

Two conventional PCR-enzyme immunoassays (PCR-EIAs) and two real-time PCR assays (LightCycler system; Roche Diagnostics) were evaluated as confirmation assays with cppB and 16S rRNA genes as targets. Of 765 male and female genitourinary and nasopharyngeal specimens positive for Neisseria gonorrhoeae in the COBAS AMPLICOR Chlamydia trachomatis / Neisseria gonorrhoeae PCR test (Roche Diagnostics), 229 (30%) were confirmed positive; 13 of these (5.7%) were lacking the cppB gene. Of the 534 samples (70%) that could not be confirmed, 81 (15%) showed a positive crossing point. However, melting curve analysis revealed an aberrant melting temperature in the LightCycler 16S rRNA assay; therefore, these samples were considered non- N. gonorrhoeae Neisseria species. Both of the 16S rRNA assays performed well, with positive predictive values of 99.1% and 100% for the PCR-EIAs and the real-time assays, respectively, and a negative predictive value of 99.8% for both. The cppB assays were compromised by the absence of the cppB gene in 5.7% of the N. gonorrhoeae -positive samples, resulting in negative predictive values of 96.8% and 97.6% for the PCR-EIAs and the real-time assays, respectively. Therefore, the 16S rRNA gene is preferable to the cppB gene as a target for confirmation assays. The melting curve analysis of the real-time assays provides useful additional information.

Human papillomavirus (HPV) infections and HPV-associated penile lesions are frequently found in male sexual partners of women with cervical intraepithelial neoplasia (CIN). To determine the significance of these findings, we studied the prevalence of HPV and HPV associated penile lesions in a male hospital population with non-STD complaints. Penoscopy was performed after application of acetic acid to identify flat lesions, papular lesions, condylomata acuminata and pearly penile papules (PPPs). Presence of HPV DNA in penile scrapes was tested by GP5+6+ PCR. In case of HPV 16 positivity, viral loads were quantified using a LightCycler based real-time PCR method. Comparing the non-STD male hospital population (n = 118) with the male sexual partners of women with CIN (n = 238), flat penile lesions were found in 14% vs. 60% and penile HPV in 25% vs. 59% of the men, respectively. We found that the presence of penile HPV and, in case of HPV 16 positivity, higher viral loads were associated with the presence of flat penile lesions. Amongst the HPV-positive men, flat penile lesions were more common and larger in size in male sexual partners of women with CIN than in the non-STD hospital population. HPV infections and HPV-associated flat penile lesions are commonly found in the non-STD male population. However, these lesions are less frequently present and smaller in size than in male sexual partners of women with CIN. Higher viral loads in penile scrapes of male sexual partners of women with CIN are reflected by a higher prevalence of flat penile lesions and a larger size of these lesions.

To determine whether Chlamydia trachomatis infection is consistently associated with an increased risk of invasive cervical carcinoma (ICC) after accounting for the strong effect of human papillomavirus (HPV) infection, a case-control study of 1,238 cases of ICC and 1,100 control women from 7 countries was carried out (hospital-based studies in Thailand, the Philippines, Morocco, Peru, Brazil and population-based studies in Colombia and Spain, all coordinated by the International Agency for Research on Cancer, Lyon, France). C. trachomatis serum antibody detection was made by means of a microfluorescence assay. Among HPV DNA-positive cases and controls, the risk of squamous cell ICC was elevated in C. trachomatis seropositive women (OR = 1.8; 95% CI = 1.2-2.7) after adjustment for age, center, oral contraceptive use, history of Pap smears, number of full-term pregnancies and herpes simplex virus 2 seropositivity. The effect of C. trachomatis seropositivity on squamous cell ICC risk increased with increasing C. trachomatis antibody titers and was higher in women under 55 years of age. C. trachomatis antibodies were not associated with adeno- or adenosquamous cell carcinoma (OR = 1.0; 95% CI = 0.53-1.9) in HPV DNA-positive women. An association of C. trachomatis with squamous cell ICC was found among all cases and control women with or without adjustment for HPV.

Background. Cost-effectiveness analyses of screening programmes for asymptomatic Chlamydia trachomatis infection suggest that screening at low prevalences in the population is cost-effective. However, the decision models in these studies are based on assumptions about the risk of complications, which are derived from the literature. Incorrect assumptions may lead to under- or overestimation of the effectiveness of screening. The first objective of this paper is to evaluate the assumptions about the probability of complications after an asymptomatic C. trachomatis infection. The second objective is to calculate alternative rates by using available data on the incidence of complications. Methods. We identified cost-effectiveness studies via Medline, and evaluated these for the evidence for the quoted probabilities. In addition, the probability of complications was calculated for Amsterdam from available registration data. Results. In the three studies that were identified, the assumptions for the rates of pelvic inflammatory disease (PID) (clinical and subclinical) after C. trachomatis infection varied from 15% to 80%, and for ectopic pregnancy, tubal factor infertility, and chronic pelvic pain after PID from 5-25%, 10-20%, and 18-30%, respectively. The assumptions were based on data from high-risk populations, case-control data, and data not accounting for misdiagnoses. Using data obtained from local registrations, we estimated the probability of a clinical PID (0.43%), ectopic pregnancy (0.07%), and tubal factor infertility (0.02%) for women with a current infection. These estimates were consistently lower than the estimates based on the literature. Conclusions. We argue that an overestimation of the current complication rates is likely. The effect of overestimation is potentially the greatest in populations with a low prevalence, since the currently assumed cost savings associated with screening may disappear when using more realistic estimates for complications. © International Epidemiological Association 2004; all rights reserved.

BACKGROUND The performance of two commercially available detection systems for high-risk HPV (hrHPV), Hybrid Capture 2 (HC2) and in situ hybridization (ISH), were compared on cervical scrapings. METHODS Using general primer (GP)-mediated GP5+/6+-polymerase chain reaction (PCR)-enzyme immunoassay and reverse line blot genotyping, 76 liquid-based cervical samples were identified with ≥ 1 of the 12 hrHPV types present in the probes of the HC2 and ISH assays. The positivity rate of the assays and the HC2 viral load were determined and related to cytologic findings (n = 76 samples) and histologic findings (n = 43 samples). RESULTS Overall, HC2 scored significantly more samples positive compared with ISH (P < 0.01). Seventy-four of 76 samples (97%) were positive according to HC2. Forty-six of 76 samples (61%) were positive according to ISH, including 80% and 70% of samples that were classified cytologically as moderate dysplasia and severe dysplasia, respectively. All women with underlying cervical intraepithelial neoplasia (CIN) lesions and 67% of women without CIN had positive HC2 samples. ISH scored 33%, 66%, 88%, and 73% of samples positive of women with no CIN, Grade 1 CIN (CIN 1), CIN 2, and CIN 3, respectively. The HC2 viral load was significantly higher in women who had a cytologic diagnosis of dysplasia (P < 0.01) and in women who had an underlying diagnosis of CIN (P < 0.01) compared with women who had neither. In addition, the viral load was significantly higher in ISH positive samples compared with ISH negative samples (P < 0.01). CONCLUSIONS An increased HC2 viral load was associated with an increased chance of underlying high-grade CIN disease in women who tested hrHPV GP5+/6+-PCR positive. Moreover, although positive ISH results were associated with an increased overall viral load in the sample, the analytic sensitivity of ISH was too low to detect all women with prevalent high-grade CIN. Cancer (Cancer Cytopathol) 2004;102:11–8. © 2003 American Cancer Society.

In this study we developed and evaluated a new PCR-based typing assay, directed to the VD2 region of the omp1 gene, for the detection and typing of urogenital Chlamydia trachomatis infections. A nested VD2 PCR-reverse line blot (RLB) assay was developed for the typing of nine different urogenital serovars of C. trachomatis . The assay developed was tested with reference strains of C. trachomatis serovars and cervical scrapes of 86 Colombian women previously found to be positive for C. trachomatis by using plasmid PCR. Two sets of primers directed to the VD2 region of the omp1 gene of C. trachomatis were designed, and fragments of 220 and 166 bp were generated in the primary and nested PCRs, respectively. In addition, an RLB assay was developed to identify nine different urogenital serovars of C. trachomatis (Ba, D, E, F, G, H, I, J, and K) and group controls, including group B (Ba, D, and E), group C (I, J, K, and H), and an intermediate group (F and G). Using this assay, we were able to type 81 of the 86 samples (94.2%). Of these samples, 91.3% were single C. trachomatis infections, and 8.7% were multiple infections. The most common serovars identified were serovars D (22.2%), F (18.5%), G (13.6%), and E (12.3%). Of the women with multiple C. trachomatis infections, >50% contained both serovars D and E. The nested VD2 PCR-RLB developed is a simple, fast, and specific method for the identification of individual urogenital C. trachomatis serovars previously detected by using plasmid PCR. Moreover, it is an appropriate method for studying multiple C. trachomatis infections and for use in large epidemiological studies.

Cytological cervical screening is rather inefficient because of relatively high proportions of false negative and false positive smears. To evaluate the efficiency of high-risk human papillomavirus (hrHPV) testing, by GP5+/6+ PCR-enzyme immunoassay (EIA), in conjunction with cytology (Intervention Group) to that of the classical cytology (Control Group), we initiated the Population Based Screening Study Amsterdam (POBASCAM). POBASCAM is a population-based randomized controlled trial for implementation of hrHPV testing in cervical screening. The outcome measure is the proportion of histologically confirmed ≥CIN3 lesions in each study arm up to and including the next screening round after 5 years. We present the design, methods and baseline data of POBASCAM. When, in the next 5 years, the follow-up will be completed, the data obtained will be used in model studies, including a cost-effectiveness study, to advise the Dutch Ministry of Public Health in deciding whether cervical screening should be based on combined hrHPV and cytology testing instead of cytology alone. Between January 1999 and September 2002, 44,102 women (mean age = 42.8 years; range = 29-61) that participated in the regular Dutch screening program were included in our study. In the Intervention Group the distribution of cytology and hrHPV by cytology class was as follows: normal cytology 96.6% (3. 6% hrHPV positive); borderline and mild dyskaryosis (BMD) 2.5% (34.6% hrHPV positive); and moderate dyskaryosis or worse (>BMD) 0.8% (88.3% hrHPV positive), i.e., 0.4% moderate dyskaryosis (82.9% hrHPV positive), 0.3% severe dyskaryosis (92.5% hrHPV positive), 0.1% carcinoma in situ (95.2% hrHPV positive), BMD, i.e., 0.4% moderate dyskaryosis, 0.3% severe dyskaryosis, 0.1% carcinoma in situ, BMD of 13.7% among women with a positive hrHPV test was not age-dependent. Our study indicates that large-scale hrHPV testing by GP5+/6+ PCR-EIA in the setting of population-based cervical screening is practically feasible, is accepted by both participating women and general practitioners and yields highly reproducible results.

Penile HPV-associated lesions are frequently seen in male sexual partners of women with CIN. The natural course and clinical significance of these lesions are unclear. Women with CIN and their male sexual partners were randomized for condom use (condom group n = 68, noncondom group n = 68). Males were screened for the presence of penile lesions, i.e., flat lesions, papular lesions and condylomata acuminata, and of HPV in their penile swabs by PCR testing. Median follow-up time was 13.1 months (range 2.9-57.4). The outcome of our study was clinical regression of penile lesions defined as disappearance of lesions at penoscopy. Potentially prognostic factors, i.e., HPV status, lesion type and age, were studied as well. Outcomes were assessed in 57 men of the condom group and in 43 men of the noncondom group. Condom use shortened the median time to regression of flat penile lesions (7.4 months condom group vs. 13.9 months noncondom group; HR = 2.1, 95% CI 1.2-3.7). This effect was not found for papular lesions (HR = 0.5, 95% CI 0.1-2.8). HPV-negative men showed a significantly shorter median time to regression of flat lesions (3.8 months) compared to men with either HPV-positive status (8.5 months; HR = 0.4, 95% CI 0.2-0.9) or inconsistent HPV status (13.1 months; HR = 0.2, 95% CI 0.1-0.6). Regression of flat penile lesions is HPV-dependent and accelerated by condom use. This effect is probably the result of blocking viral transmission between sexual partners.

Thein vivoexpression of the Epstein–Barr virus (EBV)BamHI-A rightward transcripts (BARTs) as well as the putative BART-encoded BARF0 and RK-BARF0 proteins in various EBV-associated malignancies was investigated. RT-PCRs specific for the different splice variants of the BARTs and both a nucleic acid sequence-based amplification assay and an RT-PCR specific for the BARF0 ORF were used. Abundant transcription of BARTs was found in EBV-associated Hodgkin's lymphomas, Burkitt's lymphomas (BL), T-cell non-Hodgkin's lymphomas, post-transplant lymphoproliferative disorders, AIDS-related lymphomas and gastric carcinomas. Using RNAin situhybridization (RISH), BARTs were detected within the neoplastic cells of these malignancies. BARTs encoding RK-BARF0 were not detected. The BARTs detected were shown possibly to encode the RPMS1 and BARF0 proteins, based on their splicing. However, BARTs actually harbouring the BARF0 ORF were detected only in specimens containing a relatively large number of EBV-positive cells. New monoclonal antibodies against the BARF0 protein were generated that efficiently recognized prokaryotic and eukaryotic recombinant BARF0. However, the BARF0 protein was not detected in clinical samples, nor in EBV-positive cell lines, even though these were positive for BARTs by RISH and/or BARF0 RNAin vitroanalysis. Using immunoblot analysis, no antibodies against baculovirus-expressed BARF0 protein were detected in the sera of nasopharyngeal carcinoma patients, BL patients and Hodgkin's disease patients, patients with chronic EBV infection, infectious mononucleosis patients or EBV-positive healthy donors. Thus, BARTs containing the BARF0 ORF are expressedin vivobut the BARF0 protein cannot be detected and may be expressed only marginally. It is concluded that the BARF0 protein is unlikely to play a rolein vivoin EBV-positive malignancies.

Given the fact that infection with high-risk human papillomavirus (HPV) is causally involved in cervical cancer, addition of high-risk HPV testing to a cervical smear may improve the efficacy of cervical cancer screening programmes, the triage of women with equivocal or borderline Pap smears, and the monitoring of women who have been treated for cervical intraepithelial neoplasia grade 3 (CIN 3). Compared to a cervical smear HPV tests revealed a superior sensitivity (ie clinical sensitivity) for lesions >/= CIN 3, and a negative predictive value approaching 100%. However, a potential complication is the availability of several HPV testing methods, all displaying a different sensitivity and specificity to detect HPV-positive women (ie analytical sensitivity and specificity). There is now compelling evidence that the clinical sensitivity and specificity of HPV tests are not simply synonymous to their analytical sensitivity and specificity, respectively. In fact, a distinction between so-called clinically relevant and irrelevant high-risk HPV infections should be made when considering HPV tests for primary screening, triage policies, or post-treatment monitoring. Here, we discuss the potential importance of HPV load in the context of currently widely applied HPV detection methods, to distinguish clinically relevant from irrelevant HPV infections. From this it can be concluded that it is of utmost importance to define criteria, involving viral load threshold and the type of HPV detection method that should be fulfilled by an HPV test before implementation of such a test in clinical practice and population-based cervical cancer screening programmes.

Little is known about the factors that influence clearance of human papillomavirus (HPV), the primary cause of cervical carcinoma. A total of 227 women cytologically normal and HPV positive at baseline were identified from a population-based cohort of 1,995 Bogota, Colombia, women aged 13-85 years followed between 1993 and 2000 (mean follow-up, 5.3 years). HPV DNA detection and viral load determination were based on a GP5+/GP6+ polymerase chain reaction enzyme immunoassay. Rate ratio estimates for HPV clearance were calculated by using methods for interval-censored survival time data. Analyses were based on 316 type-specific HPV infections. HPV 16 had a significantly lower clearance rate than infections with low-risk types (rate ratio (RR) = 0.47, 95% confidence interval (CI): 0.32, 0.72), HPV types related to HPV 16 (types 31, 33, 35, 52, 58) had intermediate clearance rates (RR = 0.62, 95% CI: 0.47, 0.94), and other high-risk types did not show evidence of slower clearance compared with low-risk types. Infections with single and multiple HPV types had similar clearance rates. There was no evidence of a dose-response relation between clearance and viral load. Observed was slower clearance in parous women (RR = 0.64, 95% CI: 0.47, 0.89) and faster clearance in ever users of oral contraceptives (RR = 1.38, 95% CI: 1.07, 1.77).

Five methods were compared to determine the most accurate method for identification of coagulase-negative staphylococci (CoNS) ( n = 142 strains). Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) showed the best results for rapid and accurate CoNS differentiation (99.3% of strains correctly identified). An alternative to this approach could be Vitek2 combined with partial tuf gene sequencing (100% of strains correctly identified when both methods are performed simultaneously).

Low grade squamous intra-epithelial lesions could be considered as a manifestation of human papillomavirus exposition, however the discrepancy between rates of infection with human papillomavirus and development of low grade squamous intra-epithelial lesions is notable. Here we report a cross-sectional three-armed case–control study in the Colombian population, to compare the risk factors of women with low grade squamous intra-epithelial lesions with that of human papillomavirus DNA-negative and positive women with normal cytology. British Journal of Cancer (2002) 87, 1417–1421. doi:10.1038/sj.bjc.6600650 www.bjcancer.com © 2002 Cancer Research UK

Human papillomavirus is the principal risk factor associated with cervical cancer, the most common malignancy among women in Colombia. We conducted a survey, aiming to report type specific prevalence and determinants of human papillomavirus infection in women with normal cytology. A total of 1859 women from Bogota, Colombia were interviewed and tested for human papillomavirus using a general primer GP5+/GP6+ mediated PCR–EIA. The overall HPV DNA prevalence was 14.8%; 9% of the women were infected by high risk types, 3.1% by low risk types, 2.3% by both high risk/low risk types and 0.4% by uncharacterized types (human papillomavirus X). Thirty-two different human papillomavirus types were detected, being human papillomavirus 16, 58, 56, 81(CP8304) and 18 the most common types. The human papillomavirus prevalence was 26.1% among women younger than 20 years, 2.3% in women aged 45–54 years, and 13.2% in women aged 55 years or more. For low risk types the highest peak of prevalence was observed in women aged 55 years or more. Compared to women aged 35–44 years, women aged less than 20 years had a 10-fold increased risk of having multiple infections. Besides age, there was a positive association between the risk of human papillomavirus infection and number of regular sexual partners and oral contraceptive use. In women aged below 25 years, high educational level and having had casual sexual partners predicted infection risk. In conclusion, there was a broad diversity of human papillomavirus infections with high risk types being the most common types detected. In this population multiplicity of sexual partners and, among young women, high educational level and casual sexual partners seem to determine risk. British Journal of Cancer (2002) 87, 324–333. doi:10.1038/sj.bjc.6600442 www.bjcancer.com © 2002 Cancer Research UK

We followed 353 women referred with abnormal cervical cytology in a non-intervention cohort study. In 91 pregnant women we compared high-risk human papilloma virus rates in the subsequent trimesters and postpartum in comparison to 262 non-pregnant women. High-risk human papilloma virus clearance was compared with 179 high-risk human papilloma virus positive non-pregnant women. Our main questions were: (1) do high-risk human papilloma virus rates change during pregnancy?; and (2) is there any difference between high-risk human papilloma virus clearance in pregnant and non-pregnant women? Women were monitored 3–4 monthly by cytology, colposcopy, and high-risk human papilloma virus testing. The median follow-up time was 33 months (range 3–74). Non-pregnant women showed prevalence rates of high-risk human papilloma virus of 64, 57, 53, and 50%, respectively, in four subsequent 3-months periods since the start of the study. These high-risk human papilloma virus rates were higher than in the three trimesters of pregnancy, and during the first 3 months postpartum, i.e. 50, 44, 45, and 31%, respectively. Postpartum only, this difference was statistically significant (P=0.004). Paired comparisons of high-risk human papilloma virus prevalence rates of the different trimesters with the postpartum rate showed (McNemar test) decreased rates: first trimester: 18% (P=0.02), second trimester: 13% (P=0.02) and third trimester: 23% (P