HPV E6/E7 mRNA testing is more specific than cytology in post-colposcopy follow-up of women with negative cervical biopsy

Background

In Norway, women with negative or low-grade cervical biopsies (normal/CIN1) are followed up after six months in order to decide on further follow-up or recall for screening at three-year intervals. A high specificity and positive predictive value (PPV) of the triage test is important to avoid unnecessary diagnostic and therapeutic procedures whereas a low risk of high-grade disease among triage negative women assures safety.

Materials and Methods

At the University Hospital of North Norway, cytology and the HPV mRNA test PreTect HPV-Proofer, detecting E6/E7 mRNA from HPV types 16, 18, 31, 33 and 45, are used in post-colposcopy follow-up of women with negative or low-grade biopsy. In this study, women with negative biopsy after high grade cytology (ASC-H/HSIL) and/or positive HPV mRNA test in the period 2005–2009 were included (n = 520). Histologically confirmed cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) was used as study endpoint.

Results

Of 520 women with negative or low-grade biopsy, 124 women (23.8%) had CIN2+ in follow-up biopsy. The sensitivity and specificity of the HPV mRNA test were 89.1% (95% CI, 80.1–98.1) and 92.5% (95% CI, 88.2–96.7), respectively. The ratios of sensitivity, specificity and PPV of HPV mRNA testing compared to repeat cytology for finding CIN2+ was 1.05 (95% CI: 0.92–1.21), 1.21 (95% CI: 1.12–1.32), and 1.49 (95% CI: 1.20–1.86), respectively. The PPV of mRNA was 77.3% (95% CI, 59.8–94.8) in women aged 40 or older.

Conclusion

Women with negative cervical biopsy require follow-up before resumption of routine screening. Post-colposcopy HPV mRNA testing was as sensitive but more specific than post-colposcopy cytology. In addition, the HPV mRNA test showed higher PPV. A positive mRNA test post-colposcopy could justify treatment in women above 40 years.     

Anti-dsDNA Antibodies Promote Initiation,and Acquired Loss of Renal Dnase1 Promotes Progression of Lupus Nephritis in Autoimmune (NZBxNZW)F1 Mice

Background

Lupus nephritis is characterized by deposition of chromatin fragment-IgG complexes in the mesangial matrix and glomerular basement membranes (GBM). The latter defines end-stage disease.

Methodology/Principals

In the present study we determined the impact of antibodies to dsDNA, renal Dnase1 and matrix metalloprotease (MMP) mRNA levels and enzyme activities on early and late events in murine lupus nephritis. The major focus was to analyse if these factors were interrelated, and if changes in their expression explain basic processes accounting for lupus nephritis.

Findings

Early phases of nephritis were associated with chromatin-IgG complex deposition in the mesangial matrix. A striking observation was that this event correlated with appearance of anti-dsDNA antibodies and mild or clinically silent nephritis. These events preceded down-regulation of renal Dnase1. Later, renal Dnase1 mRNA level and enzyme activity were reduced, while MMP2 mRNA level and enzyme activity increased. Reduced levels of renal Dnase1 were associated in time with deficient fragmentation of chromatin from dead cells. Large fragments were retained and accumulated in GBM. Also, since chromatin fragments are prone to stimulate Toll-like receptors in e.g. dendritic cells, this may in fact explain increased expression of MMPs.

Significance

These scenarios may explain the basis for deposition of chromatin-IgG complexes in glomeruli in early and late stages of nephritis, loss of glomerular integrity and finally renal failure.     

Trends in Food Habits and Their Relation to Socioeconomic Status among Nordic Adolescents 2001/2002-2009/2010

Background

In the Nordic countries, substantial policy and intervention efforts have been made to increase adolescents'' consumption of fruit and vegetables and to reduce their intake of sweets and soft drinks. Some initiatives have been formulated in a Nordic collaboration and implemented at national level. In recent years, social inequalities in food habits have been attracted particular governmental interest and several initiatives addressing the socioeconomic gradient in food habits have been highlighted. However, few internationally published studies have evaluated how trends in adolescents'' food habits develop in the context of Nordic nutrition policy, or have compared differences between the Nordic countries.

Methods

The study was based on Danish, Finnish, Norwegian and Swedish cross-sectional data from the international Health Behaviour in School-Aged Children (HBSC) study, collected via three nationally representative and comparable questionnaire surveys in 2001/2002, 2005/2006 and 2009/2010. Food habits were identified by students'' consumption of fruit, vegetables, sweets and sugar sweetened soft drink. Socioeconomic status (SES) was measured with the Family Affluence Scale (FAS). Multilevel logistic regression was used to analyze the data.

Results

Trends in fruit consumption developed differently across countries, characterized by an increase in Denmark and Norway and more stable trends in Sweden and Finland. Vegetable consumption increased particularly in Denmark and to a lesser extent in Norway, whereas Sweden and Finland displayed stable trends. Decreased trends were observed for sweet and soft drink consumption and were similar in Norway, Sweden and Finland. Sweet consumption decreased across all survey years, whereas soft drink consumption decreased between 2001/2002–2005/2006 and was stable thereafter. Denmark displayed an increase between 2001/2002–2005/2006 followed by a similar decrease between 2005/2006–2009/2010 for both sweet and soft drink consumption. Socioeconomic inequalities in fruit and vegetable consumption were observed in all countries, with no cross-country differences, and no changes over time. Small but not significant cross-country variation was identified for SES inequalities in sweet consumption. Reduced SES inequalities were observed in Sweden between 2005/2006 and 2009/2010. SES was not associated with soft drink consumption in this study population, with the exception of Denmark for the survey year 2009/2010.

Conclusion

Different trends resulted in increased country differences in food habits during the time of observations. In survey year 2009/2010, Danish students reported a higher intake of fruit and vegetable consumption than their counterparts in the other Nordic countries. Finnish students reported the lowest frequency of sweets and soft drink consumption. Despite the positive dietary trends documented in the present study, the majority of Nordic adolescents are far from meeting national dietary recommendations. Our findings underline the need for more comprehensive initiatives targeting young people''s food habits as well as a more deliberate and focused action to close gaps in social inequalities that affect food choices.     

Triage of Women with Minor Cervical Lesions: Data Suggesting a “Test and Treat” Approach for HPV E6/E7 mRNA Testing

Background

Human papillomavirus (HPV) testing is included in the cervical cancer screening program in the triage of women with equivocal (ASC-US) or low-grade (LSIL) cytological lesions. These women have an increased risk for developing high grade dysplasia and cancer (CIN2+) compared to women with normal cytology. However, in order to avoid unnecessary follow-up, as well as overtreatment, a high positive predictive value (PPV) of the triage test is important.

Methodology/Principal Findings

The HPV test PreTect HPV-Proofer, detecting E6/E7 mRNA from the HPV types 16, 18, 31, 33 and 45, is used as triage test together with repeat cytology. PPV data for HPV E6/E7 mRNA testing during the period from January 2006 up to June 2009 are reported. In total, 406 of 2099 women (19.3%) had a positive HPV test result. Of the women with a positive test result and with a histological diagnosis (n = 347), 243 women had histological high-grade dysplasia or cancer (CIN2+), giving a PPV of 70.0% (95% confidence interval [CI], 65.2%–74.8%). For HPV 16 or HPV 33 positive women above 40 years of age, the PPV was 83.7% (95% CI, 73.3%–94.0%) and 84.6% (95% CI, 65.0%–100.0%) respectively. The PPV of test positive women with HSIL cytology was 94.2% (95% CI, 88.7%–99.7%).

Conclusions

When the result in triage is HPV mRNA positive, our data suggest direct treatment for women above 40 years of age or for women with a concurrent cytological HSIL diagnosis, contributing to better clinical safety for these women. In addition, by decreasing the time to treatment, thereby reducing the number of recalls, the patient management algorithm will be considerably improved, in turn reducing follow-up costs as well as unnecessary psychological stress among patients.     

Identification of a novel phosphorylation site of acyl-CoA binding protein (ACBP) in nodularin-induced apoptotic hepatocytes

The liver specific protein phosphatase inhibiting toxin nodularin (from Nodularia spumigena) rapidly induces hepatocyte apoptosis. Incubation of freshly isolated hepatocytes with this toxin results in hyperphosphorylation of cellular proteins before any morphological signs of apoptosis appear. These phosphorylated proteins may play key roles in the early stage of apoptosis. Here, we identified one of the phosphoproteins to be acyl-CoA binding protein (ACBP), a highly conserved and ubiquitously expressed protein. Phosphorylation-site analysis by matrix-assisted laser desorption ionization time-of-flight MS/MS revealed that the observed phosphorylation is positioned on Ser1 in the N-terminal tryptic peptide Ac-SQADFDKAAE EVKRLK of the rat liver protein. Additionally, we observed a translocation of ACBP towards the cellular membrane in the apoptotic hepatocytes. Moreover, nodularin-induced apoptosis was highly dependent on calpain activation, an event that has previously been shown to be regulated by ACBP. Our findings introduce the possibility that reversible phosphorylation of ACBP regulates its ability to activate calpain in phosphatase inhibitor-induced apoptosis and controls the cellular accessibility of long-chain fatty acid-CoAs for cellular signaling.     

Simulated diving after heat stress potentiates the induction of heat shock protein 70 and elevates glutathione in human endothelial cells

Heat stress prior to diving has been shown to confer protection against endothelial damage due to decompression sickness. Several lines of evidence indicate a relation between such protection and the heat shock protein (HSP)70 and HSP90 and the major cellular red-ox determinant, glutathione (GSH). The present study has used human endothelial cells as a model system to investigate how heat stress and simulated diving affect these central cellular defense molecules. The results demonstrated for the first time that a simulated dive at 2.6 MPa (26 bar) had a potentiating effect on the heat-induced expression of HSP70, increasing the HSP70 concentration on average 54 times above control level. In contrast, a simulated dive had no significant potentiating effect on the HSP90 level, which might be due to the higher baseline level of HSP90. Both 2 and 24-h dive had similar effects on the HSP70 and HSP90, suggesting that the observed effects were independent of duration of the dive. The rapid HSP response following a 2-h dive with a decompression time of 5 min might suggest that the effects were due to compression or pressure per se rather than decompression and may involve posttranslational processing of HSP. The exposure order seemed to be critical for the HSP70 response supporting the suggestion that the potentiating effect of dive was not due to de novo synthesis of HSP70. Neither heat shock nor a simulated dive had any significant effect on the intracellular GSH level while a heat shock and a subsequent dive increased the total GSH level approximately 62%. Neither of these conditions seemed to have any effect on the GSH red-ox status.     

Silencing of renal DNaseI in murine lupus nephritis imposes exposure of large chromatin fragments and activation of Toll like receptors and the Clec4e

Recent studies demonstrate that transformation of mild lupus nephritis into end-stage disease is imposed by silencing of renal DNaseI gene expression in (NZBxNZW)F1 mice. Down-regulation of DNaseI results in reduced chromatin fragmentation, and in deposition of extracellular chromatin-IgG complexes in glomerular basement membranes in individuals that produce IgG anti-chromatin antibodies. The main focus of the present study is to describe the biological consequences of renal DNaseI shut-down and reduced chromatin fragmentation with a particular focus on whether exposed large chromatin fragments activate Toll like receptors and the necrosis-related Clec4e receptor in murine and human lupus nephritis. Furthermore, analyses where performed to determine if matrix metalloproteases are up-regulated as a consequence of chromatin-mediated Toll like receptors/Clec4e stimulation. Mouse and human mRNA expression levels of DNaseI, Toll like receptors 7-9, Clec4e, pro-inflammatory cytokines and MMP2/MMP9 were determined and compared with in situ protein expression profiles and clinical data. We demonstrate that exposure of chromatin significantly up-regulate Toll like receptors and Clec4e in mice, and also but less pronounced in patients with lupus nephritis treated with immunosuppresants. In conclusion, silencing of renal DNaseI gene expression initiates a cascade of inflammatory signals leading to progression of both murine and human lupus nephritis. Principal component analyses biplot of data from murine and human lupus nephrits demonstrate the importance of DNaseI gene shut down for progression of the organ disease.     

Triage of women with low-grade cervical lesions--HPV mRNA testing versus repeat cytology

Background

In Norway, women with low-grade squamous intraepithelial lesions (LSIL) are followed up after six months in order to decide whether they should undergo further follow-up or be referred back to the screening interval of three years. A high specificity and positive predictive value (PPV) of the triage test is important to avoid unnecessary diagnostic and therapeutic procedures.

Materials and Methods

At the University Hospital of North Norway, repeat cytology and the HPV mRNA test PreTect HPV-Proofer, detecting E6/E7 mRNA from HPV types 16, 18, 31, 33 and 45, are used in triage of women with ASC-US and LSIL. In this study, women with LSIL cytology in the period 2005–2008 were included (n = 522). Two triage methods were evaluated in two separate groups: repeat cytology only (n = 225) and HPV mRNA testing in addition to repeat cytology (n = 297). Histologically confirmed cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) was used as the study endpoint.

Results

Of 522 women with LSIL, 207 had biopsies and 125 of them had CIN2+. The sensitivity and specificity of repeat cytology (ASC-US or worse) were 85.7% (95% confidence interval (CI): 72.1, 92.2) and 54.4 % (95% CI: 46.9, 61.9), respectively. The sensitivity and specificity of the HPV mRNA test were 94.2% (95% CI: 88.7, 99.7) and 86.0% (95% CI: 81.5, 90.5), respectively. The PPV of repeat cytology was 38.4% (95% CI: 29.9, 46.9) compared to 67.0% (95% CI: 57.7, 76.4) of the HPV mRNA test.

Conclusion

HPV mRNA testing was more sensitive and specific than repeat cytology in triage of women with LSIL cytology. In addition, the HPV mRNA test showed higher PPV. These data indicate that the HPV mRNA test is a better triage test for women with LSIL than repeat cytology.     

HPV mRNA Is More Specific than HPV DNA in Triage of Women with Minor Cervical Lesions

Background

In Norway, repeat cytology and HPV testing comprise delayed triage of women with minor cytological lesions. The objective of this study was to evaluate HPV DNA and HPV mRNA testing in triage of women with an ASC-US/LSIL diagnosis.

Materials and Methods

We used repeat cytology, HPV DNA testing (Cobas 4800) and HPV mRNA testing (PreTect HPV-Proofer) to follow up 311 women aged 25–69 years with ASC-US/LSIL index cytology.

Results

Of 311 women scheduled for secondary screening, 30 women (9.6%) had ASC-H/HSIL cytology at triage and 281 women (90.4%) had ASC-US/LSIL or normal cytology. The HPV DNA test was positive in 92 (32.7%) of 281 instances, and 37 (13.2%) were mRNA positive. Of the 132 women with repeated ASC-US/LSIL, we received biopsies from 97.0% (65/67) of the DNA-positive and 92.9% (26/28) of the mRNA-positive cases. The positive predictive values for CIN2+ were 21.5% (14/65) for DNA positive and 34.6% (9/26) for mRNA positive (ns). The odds ratio for being referred to colposcopy in DNA-positive cases were 2.8 times (95% CI: 1.8–4.6) higher that of mRNA-positive cases. Compared to the mRNA test, the DNA test detected four more cases of CIN2 and one case of CIN3.

Conclusions

The higher positivity rate of the DNA test in triage leads to higher referral rate for colposcopy and biopsy, and subsequent additional follow-up of negative biopsies. By following mRNA-negative women who had ASC-US/LSIL at triage with cytology, the additional cases of CIN2+ gained in DNA screening can be discovered. Our study indicates that in triage of repeated ASC-US/LSIL, HPV mRNA testing is more specific and is more relevant in clinical use than an HPV DNA test.