Effectiveness of self-sampling human papillomavirus test on precancer detection and screening uptake in Japan: The ACCESS randomized controlled trial.

Japan is lagging in cervical cancer prevention. The effectiveness of a self-sampling human papillomavirus (HPV) test, a possible measure to overcome this situation, has not yet been evaluated. A randomized controlled trial was performed to evaluate the effectiveness of a self-sampling HPV test on detection of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and screening uptake. Women between 30 and 58 years old who did not participate in the cervical cancer screening program for ≥3 years were eligible and assigned to the intervention group (cytology or self-sampling HPV test) or control group (cytology). Participants assigned to the intervention group were sent a self-sampling kit according to their ordering (opt-in strategy). A total of 7337 and 7772 women were assigned to the intervention and control groups, respectively. Screening uptake in the intervention group was significantly higher than that in the control group (20.0% vs. 6.4%; risk ratio: 3.10; 95% confidence interval [CI]: 2.82, 3.42). The compliance rate with cytology triage for HPV-positive women was 46.8% (95% CI: 35.5%, 58.4%). CIN2+ was detected in five and four participants in the intervention and control groups, respectively; there was no difference for intention-to-screen analysis (risk ratio: 1.32; 95% CI: 0.36, 4.93). Self-sampling of HPV test increased screening uptake; however, no difference was observed in the detection of CIN2+, probably due to the low compliance rate for cytology triage in HPV-positive women. Efforts to increase cytology triage are essential to maximize precancer detections.

Acceptability of self-sampling human papillomavirus test for cervical cancer screening in Japan: A questionnaire survey in the ACCESS trial.

PURPOSE: In terms of medical policy for cervical cancer prevention, Japan lags far behind other industrialized countries. We initiated a randomized controlled trial to evaluate the self-sampling human papillomavirus (HPV) test as a tool to raise screening uptake and detection of pre-cancer. This study was conducted to explore the acceptability and preference of self-sampling using a subset of the data from this trial. METHODS: A pre-invitation letter was sent to eligible women, aged 30-59 years who had not undergone cervical cancer screening for three or more years. After excluding those who declined to participate in this trial, the remaining women were assigned to the self-sampling and control groups. A second invitation letter was sent to the former group, and those wanting to undergo the self-sampling test ordered the kit. A self-sampling HPV kit, consent form, and a self-administered questionnaire were sent to participants who ordered the test. RESULTS: Of the 7,340 participants in the self-sampling group, 1,196 (16.3%) administered the test, and 1,192 (99.7%) answered the questionnaire. Acceptability of the test was favorable; 75.3-81.3% of participants agreed with positive impressions (easy, convenient, and clarity of instruction), and 65.1-77.8% disagreed with negative impressions (painful, uncomfortable, and embarrassing). However, only 21.2% were confident in their sampling procedure. Willingness to undergo screening with a self-collected sample was significantly higher than that with a doctor-collected sample (89.3% vs. 49.1%; p<0.001). Willingness to undergo screening with a doctor-collected sample was inversely associated with age and duration without screening (both p<0.001), but that with a self-collected sample was not associated. CONCLUSIONS: Among women who used the self-sampling HPV test, high acceptability was confirmed, while concerns about self-sampling procedures remained. Screening with a self-collected sample was preferred over a doctor-collected sample and the former might alleviate disparities in screening rates.

Implementation of a self-sampling HPV test for non-responders to cervical cancer screening in Japan: secondary analysis of the ACCESS trial.

A self-sampling human papillomavirus (HPV) test could improve the morbidity and mortality of cervical cancer in Japan. However, its effectiveness and feasibility have not been demonstrated sufficiently. Hence, we launched a randomized controlled trial, which is ongoing, and report the results of a secondary analysis. To ensure autonomous participation with a minimum selection bias, opt-out consent was obtained from women who met the inclusion criteria, and written consent was obtained from those who underwent a self-sampling test. The number of women who met the inclusion criteria was 20,555; 4283 and 1138 opted out before and after the assignment, respectively. Of the 7340 women in the self-sampling arm, 1372 (18.7%) ordered and 1196 (16.3%) underwent the test. Younger women in their 30 s and 40 s tended to undertake the test more frequently than older women in their 50 s (P for trend < 0.001). Invalid HPV test results were rare (1.3%), and neither adverse events nor serious complaints were reported. Despite adopting the opt-out procedure, more women than expected declined to participate, suggesting the need for a waiver of consent or assignment before consent to reduce selection bias. A self-sampling HPV test can be implemented in Japan and would be more accessible to young women, the predominant group affected by cervical cancer.

Study protocol of the ACCESS trial: a randomised trial to evaluate the effectiveness of human papillomavirus testing by self-sampling in cervical cancer screening uptake and precancer detection.

INTRODUCTION: Recently, the incidence of cervical cancer has increased in Japan, probably because of an interruption in human papillomavirus (HPV) vaccination and a low cervical cancer screening rate. There is a lack of evidence for self-sampling HPV testing as a cervical cancer screening tool in Japan. The Accelerating Cervical Cancer Elimination by Self-Sampling test trial aims to compare the effectiveness of screening using the self-sampling HPV test with that of routine screening concerning screening uptake and precancer detection. METHODS AND ANALYSIS: This trial has a single-municipality, open-label, parallel, superiority and randomised design. Approximately 20 000 women who have not undergone cervical cancer screening for at least 3 years will be assigned randomly to the self-sampling arm and the control arm using a 1:1 ratio. Participants assigned to the control arm will undergo routine cervical cancer screening (cytology test) provided by Ichihara City, while those assigned to the self-sampling arm will choose the routine screening or self-sampling HPV test. HPV tests will be performed using the cobas 8800 system (Roche Diagnostics, Rotkreuz, Switzerland). Participants who will undergo the self-sampling HPV testing will be recommended to undergo routine screening. The results of the cytology test and further tests, such as colposcopy and biopsy, will be collected and used for this trial. The risk ratio and risk difference in the proportion of participants with cervical intraepithelial neoplasia two or worse between the two arms will be calculated. The test for the null hypothesis (the detection rates are equal between the two arms) will be performed using Pearson's χ2 test. ETHICS AND DISSEMINATION: This trial was approved by the Research Ethics Committees of the Chiba Foundation for Health Promotion and Disease Prevention and the collaborating research institutes. The results will be disseminated through peer-reviewed journals and conference presentations. TRIAL REGISTRATION NUMBER: jRCT1030200276. Pre-results.

Allergic TH2 Response Governed by B-Cell Lymphoma 6 Function in Naturally Occurring Memory Phenotype CD4+ T Cells.

Transcriptional repressor B-cell lymphoma 6 (Bcl6) appears to regulate TH2 immune responses in allergies, but its precise role is unclear. We previously reported that Bcl6 suppressed IL-4 production in naïve CD4+ T cell-derived memory TH2 cells. To investigate Bcl6 function in allergic responses in naturally occurring memory phenotype CD4+ T (MPT) cells and their derived TH2 (MPTH2) cells, Bcl6-manipulated mice, highly conserved intron enhancer (hcIE)-deficient mice, and reporter mice for conserved noncoding sequence 2 (CNS2) 3' distal enhancer region were used to elucidate Bcl6 function in MPT cells. The molecular mechanisms of Bcl6-mediated TH2 cytokine gene regulation were elucidated using cellular and molecular approaches. Bcl6 function in MPT cells was determined using adoptive transfer to naïve mice, which were assessed for allergic airway inflammation. Bcl6 suppressed IL-4 production in MPT and MPTH2 cells by suppressing CNS2 enhancer activity. Bcl6 downregulated Il4 expression in MPTH2 cells, but not MPT cells, by suppressing hcIE activity. The inhibitory functions of Bcl6 in MPT and MPTH2 cells attenuated allergic responses. Bcl6 is a critical regulator of IL-4 production by MPT and MPTH2 cells in TH2 immune responses related to the pathogenesis of allergies.

Development of chronic allergic responses by dampening Bcl6-mediated suppressor activity in memory T helper 2 cells.

Mice deficient in the transcriptional repressor B-cell CLL/lymphoma 6 (Bcl6) exhibit similar T helper 2 (TH2) immune responses as patients with allergic diseases. However, the molecular mechanisms underlying Bcl6-directed regulation of TH2 cytokine genes remain unclear. We identified multiple Bcl6/STAT binding sites (BSs) in TH2 cytokine gene loci. We found that Bcl6 is modestly associated with the BSs, and it had no significant effect on cytokine production in newly differentiated TH2 cells. Contrarily, in memory TH2 (mTH2) cells derived from adaptively transferred TH2 effectors, Bcl6 outcompeted STAT5 for binding to TH2 cytokine gene loci, particularly Interleukin4 (Il4) loci, and attenuated GATA binding protein 3 (GATA3) binding to highly conserved intron enhancer regions in mTH2 cells. Bcl6 suppressed cytokine production epigenetically in mTH2 cells to negatively tune histone acetylation at TH2 cytokine gene loci, including Il4 loci. In addition, IL-33, a pro-TH2 cytokine, diminished Bcl6's association with loci to which GATA3 recruitment was inversely augmented, resulting in altered IL-4, but not IL-5 and IL-13, production in mTH2 cells but no altered production in newly differentiated TH2 cells. Use of a murine asthma model that generates high levels of pro-TH2 cytokines, such as IL-33, suggested that the suppressive function of Bcl6 in mTH2 cells is abolished in severe asthma. These findings indicate a role of the interaction between TH2-promoting factors and Bcl6 in promoting appropriate IL-4 production in mTH2 cells and suggest that chronic allergic diseases involve the TH2-promoting factor-mediated functional breakdown of Bcl6, resulting in allergy exacerbation.

Metformin potentiates the anticancer effects of cisplatin under normoxic conditions in vitro.

Metformin is a diabetes drug with anticancer properties. Several studies have investigated the effects of metformin combined with chemotherapeutic agents, with controversial results. This study evaluated the efficacy of combined metformin/cisplatin treatment in an endometrial cancer cell line. Ishikawa cells were treated with metformin, cisplatin or both types of treatment. Cell proliferation was evaluated by quantification and colorimetric and thymidine incorporation assays, cell cycle progression was assessed by flow cytometry, and apoptosis by the caspase-3 activity assay. The effects of metformin and cisplatin used in combination were assessed under normoxic (21% O2) and hypoxic (1% O2) conditions. Mitochondrial morphology was examined using the MitoTracker dye, while function was assayed by lactate production. Discrepant results were obtained from the different assays of cell proliferation, with the value obtained from the colorimetric assay being higher than that from cell counts after drug treatment. Combined treatment with metformin (≥2 mM) and cisplatin (1 µM) had additive anti-proliferative effects on cells under normoxic conditions. However, the additive effect of metformin was attenuated under hypoxia. Metformin caused morphological and functional changes in mitochondria, which appeared shortened after exposure to metformin, while the connections between individual mitochondria appeared weaker. Additionally, decreased MitoTracker staining was observed after an 8-h exposure to metformin. The colorimetric assay did not accurately determine the effects of metformin and cisplatin on cell proliferation. The additive effects of metformin on cisplatin-induced inhibition of cell proliferation were attenuated under hypoxic conditions, while metformin compromised mitochondrial structure and function. Additional studies are needed to determine the efficacy of this drug combination in vivo.

ADAR1 protein induces adenosine-targeted DNA mutations in senescent Bcl6 gene-deficient cells.

Somatic mutations accumulate in senescent cells. Bcl6, which functions as a transcriptional repressor, has been identified as a potent inhibitor of cell senescence, but a role of Bcl6 in the accumulation of somatic mutations has remained unclear. Ig class-switch recombination simultaneously induces somatic mutations in an IgM class-switch (Ig-Sµ) region of IgG B cells. Surprisingly, mutations were detected in the Ig-Sµ region of Bcl6-deficient IgM B cells without class-switch recombination, and these mutations were mainly generated by conversion of adenosine to guanosine, suggesting a novel DNA mutator in the B cells. The ADAR1 (adenosine deaminase acting on RNA1) gene was overexpressed in Bcl6-deficient cells, and its promoter analysis revealed that ADAR1 is a molecular target of Bcl6. Exogenous ADAR1 induced adenosine-targeted DNA mutations in IgM B cells from ADAR1-transgenic mice and in wild-type mouse embryonic fibroblasts (MEFs). These mutations accumulated in senescent MEFs accompanied with endogenous ADAR1 expression, and the frequency in senescent Bcl6-deficient MEFs was higher than senescent wild-type MEFs. Thus, Bcl6 protects senescent cells from accumulation of adenosine-targeted DNA mutations induced by ADAR1.

Effective collaboration between IL-4 and IL-21 on B cell activation.

Although IL-4 and IL-21 synergistically promote proliferation and differentiation of activated B cells, the mutual role in their collaboration is not known. When splenic B cells were sequentially stimulated with anti-IgM Ab and anti-CD40 Ab plus IL-4 and then with IL-21 at a 2-day interval, proliferation, frequency of class switching to IgG1 and plasma cell differentiation were continuously enhanced until day 5 of culture. Amounts of AID and Blimp1 mRNA in sequentially activated B cells with IL-4 and IL-21 increased more than those in activated B cells without IL-21. However, sequential stimulation of B cells with anti-IgM Ab and anti-CD40 Ab plus IL-21 and then with IL-4 at more than 1-day interval did not display the synergistic effect. Furthermore, sequential stimulation of activated B cells with a low dose of IL-4, which did not induce Ig class switching, at the beginning of culture and with IL-21 or IL-4 on day 2 of culture induced proliferation and differentiation of CXCR4(-) or CXCR4(+) B cells, respectively. Thus, IL-21 effectively promotes proliferation and differentiation of CXCR4(-) B cells pre-activated with anti-IgM Ab and anti-CD40 Ab plus IL-4.

Bcl6 is required for the IL-4-mediated rescue of the B cells from apoptosis induced by IL-21.

IL-21 has a pro-apoptotic effect on freshly isolated B cells stimulated with LPS, and also induces Bcl6 expression in the activated B cells. However, a role for Bcl6 in the activated B cells is not known. When naive B cells from Bcl6-deficient mice were stimulated with LPS plus IL-21, those B cells died by apoptosis as wild-type B cells. Co-stimulation of those B cells with IL-4 partially rescued the wild-type B cells but not the Bcl6-deficient B cells from the IL-21-induced apoptosis. Bcl-2 was not up-regulated in both B cells stimulated with LPS plus IL-21 and IL-4. Bcl-X(L) and Bax were up-regulated in both B cells stimulated with LPS plus IL-4, and the co-stimulation with IL-21 did not modulate these up-regulations in wild-type B cells. However, the co-stimulation clearly suppressed the Bcl-X(L) up-regulation but not the Bax up-regulation in Bcl6-deficient B cells. Thus, Bcl6 is required for maintaining the Bcl-X(L) up-regulation in B cells stimulated with LPS plus IL-21 and IL-4, and the up-regulation may partially rescue the B cells from apoptosis induced by IL-21.

JunD/AP-1 and STAT3 are the major enhancer molecules for high Bcl6 expression in germinal center B cells.

The Bcl6 proto-oncogene, which encodes a transcriptional repressor, is ubiquitously expressed and predominantly in germinal center (GC) B cells. Although the promoter region of the human Bcl6 gene has been reported, enhancer molecules for its high expression in GC B cells were largely unknown. Here we show that transcriptional start sites of the murine Bcl6 gene were different from the reported human one. DNA sequence around the new promoter region is highly conserved between mice and humans and has no canonical TATA or CCAAT box. Two AP-1-binding elements in the promoter region were the major enhancer elements in GC-derived B lymphoma cells, and JunD/AP-1 was detected in GC B cells. In addition, we identified the silencer region with three Bcl6-binding elements around the start site. Bcl6 bound to the silencer elements and its over-expression repressed the promoter activity through the elements. Activated STAT factors (STATs), especially activated STAT3, also bound to the silencer elements in GC B cells and competed with Bcl6 for the binding, suggesting that JunD/AP-1 and activated STATs drive high Bcl6 expression in GC B cells. Since stimulation of splenic B cells with IL-4 or IL-21 induced high Bcl6 expression with induction of junD and activation of STATs, these cytokines may be inducers for its high expression in GC B cells. However, IL-21 but not IL-4 stimulation activated STAT3 in splenic B cells. Thus, IL-21 may be a major inducer for high Bcl6 expression in GC B cells.

Bcl6 is a transcriptional repressor for the IL-18 gene.

Bcl6 functions as a sequence-specific transcriptional repressor, and Bcl6-deficient (Bcl6(-/-)) mice have been reported to display Th2-type inflammatory diseases in multiple organs. Since IL-18 is a potent stimulator of Th2 cells, we examined the expression of IL-18 mRNA in bone marrow-derived macrophages from Bcl6(-/-) mice after LPS stimulation. Here we show that the expression was strikingly up-regulated after stimulation. The expression was also up-regulated in RAW264 cells, a murine macrophage cell line, by transfection with the dominant negative type of Bcl6 gene. We identified a putative Bcl6-binding DNA sequence (IL-18BS) upstream of exon 1 of the murine IL-18 gene and three IL-18BSs in the promoter region of human IL-18 gene. Binding of Bcl6 in nuclear protein from resting RAW264 cells to murine IL-18BS was detected by gel retardation assay and chromatin immunoprecipitation assay. The binding activity was diminished gradually in RAW264 cells after LPS stimulation. However, the amount of Bcl6 protein in these cells was constant over the period examined, suggesting the functional modification of Bcl6 protein after stimulation. Furthermore, murine IL-18BS was required for Bcl6 to repress the expression of the luciferase reporter gene under control of the IL-18 promoter. Taken together, Bcl6 is a key regulator of IL-18 production by macrophages.