Stratification of HPV-induced cervical pathology using the virally encoded molecular marker E4 in combination with p16 or MCM.

High-risk human papillomavirus (HPV) types cause cervical lesions of varying severity, ranging from transient productive infections to high-grade neoplasia. Disease stratification requires the examination of lesional pathology, and possibly also the detection of biomarkers. P16(INK4a) and MCM are established surrogates of high-risk HPV E6/E7 activity, and can be extensively expressed in high-grade lesions. Here we have combined these two cellular biomarkers with detection of the abundant HPV-encoded E4 protein in order to identify both productive and transforming lesions. This approach has allowed us to distinguish true papillomavirus infections from similar pathologies, and has allowed us to divide the heterogeneous CIN2 category into those that are CIN1-like and express E4, and those that more closely resemble nonproductive CIN3. To achieve this, 530 lesional areas were evaluated according to standard pathology criteria and by using a multiple staining approach that allows us to superimpose biomarker patterns either singly or in combination onto an annotated hematoxylin and eosin (H&E) image. Conventional grading of neoplasia was established by review panel, and compared directly with the composite molecular pathology visualized on the same tissue section. The detection of E4 coincided with the onset of vacuolation, becoming abundant in koilocytes as the MCM marker declined and cells lost their defined nuclear margins as visualized by standard H&E staining. Of the dual marker approaches, p16(INK4a) and E4 appeared most promising, with E4 generally identifying areas of low-grade disease even when p16(INK4a) was present. Extensive p16(INK4a) expression usually coincided with an absence of E4 expression or its focal retention in sporadic cells within the lesion. Our results suggest that a straightforward molecular evaluation of HPV life-cycle deregulation in cervical neoplasia may help improve disease stratification, and that this can be achieved using complementary molecular biomarker pairs such as MCM/E4 or, more promisingly, p16(INK4a)/E4 as an adjunct to conventional pathology.JD, HG, YS, and ZW were funded by the UK Medical Research Council. RvB was funded by the Stichting Pathologie Ontwikkeling en Onderzoek (SPOO) Foundation, The Netherlands.This is the author accepted manuscript. The final version is available via NPG at http://www.nature.com/modpathol/journal/vaop/ncurrent/full/modpathol201552a.html#ack

Investigating Diagnostic Problems of CIN1 and CIN2 Associated With High-risk HPV by Combining the Novel Molecular Biomarker PanHPVE4 With P16INK4a.

Grading cervical intraepithelial neoplasia (CIN) determines clinical management of women after abnormal cytology with potential for overdiagnosis and overtreatment. We studied a novel biomarker of human papillomavirus (HPV) life-cycle completion (panHPVE4), in combination with the minichromosome maintenance (MCM) protein cell-cycle marker and the p16INK4a transformation marker, to improve CIN diagnosis and categorization. Scoring these biomarkers alongside CIN grading by 3 pathologists was performed on 114 cervical specimens with high-risk (HR) HPV. Interobserver agreement for histopathology was moderate (κ=0.43 for CIN1/negative, 0.54 for CIN2/≤CIN1, and 0.36 for CIN3). Agreement was good or excellent for biomarker scoring (E4: κ=0.896; 95% confidence interval [CI]: 0.763-0.969; p16INK4a : κ=0.798; 95% CI: 0.712-0.884; MCM: κ=0.894; 95% CI: NC (this quantity cannot be calculated). Biomarker expression was studied by immunofluorescence and immunohistochemistry and was correlated with 104 final CIN diagnoses after histologic review. All 25 histologically negative specimens were p16INK4a and panHPVE4 negative, although 9 were MCM-positive. There were variable extents of p16INK4a positivity in 11/11 CIN1 and extensive panHPVE4 staining in 9/11. Ten CIN2 lesions expressed panHPVE4 and p16INK4a, and 13 CIN2 expressed only p16INK4a. CIN3 showed extensive p16INK4a positivity with no/minimal panHPVE4 staining. PanHPVE4, unlike MCM, distinguished CIN1 from negative. PanHPVE4 with p16INK4a separated CIN2/3 showing only expression of p16INK4a, indicating transforming HR-HPV E7 expression, from CIN1/2 showing completion of HR-HPV life cycle by E4 expression and variable p16INK4a expression. PanHPVE4 and p16INK4a staining are complementary markers that could provide simple, reliable support for diagnosing CIN. Their value in distinguishing CIN1/2 that supports HR-HPV life-cycle completion (and which might ultimately regress) from purely transforming CIN2/3 needing treatment warrants further research.This research was partly funded by the Stichting Pathologie Ontwikkeling en Onderzoek (SPOO) Foundation, The Netherlands. Funding was also provided from the UK Medical Research Council to HG, YS, ZW and JD.This is the final version of the article. It first appeared from Wolters Kluwer via http://dx.doi.org/10.1097/PAS.000000000000049

βTrCP-mediated proteolysis of NF-kB1 p105 requires phosphorylation of p105 serines 927 and 932

This work was supported by the U.K. Medical Research Council, the Arthritis Research Campaign (project grant L0536 to V.L.), and the AINP consortium, EC—5th framework.NF-κB1 p105 functions both as a precursor of NF-κB1 p50 and as a cytoplasmic inhibitor of NF-κB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-α), the IκB kinase (IKK) complex rapidly phosphorylates NF-κB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-α to trigger p105 degradation, which releases the associated Rel/NF-κB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser927-Gly-Val-Glu-Thr-Ser932) homologous to the IKK target sequence in IκBα. In this study, TNF-α-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1−/− and IKK2−/− double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-α to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105758-967 fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for βTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of βTrCP by RNA interference blocks TNF-α-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that βTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, βTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IκBα. Thus, it appears that reduced p105 recruitment of βTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-α stimulation relative to that for IκBα.Publisher PDFPeer reviewe

Investigating Diagnostic Problems of CIN1 and CIN2 Associated With High-risk HPV by Combining the Novel Molecular Biomarker PanHPVE4 With P16INK4a

This is the final version of the article. It first appeared from Wolters Kluwer via http://dx.doi.org/10.1097/PAS.0000000000000498Grading cervical intraepithelial neoplasia (CIN) determines clinical management of women after abnormal cytology with potential for over-diagnosis and overtreatment. We studied a novel biomarker of HPV life-cycle completion (panHPVE4), in combination with the MCM cell-cycle marker and the p16INK4a transformation marker to improve CIN diagnosis and categorization. Scoring these biomarkers alongside CIN grading by three pathologists was performed on 114 cervical specimens with high-risk (HR-) HPV. Inter-observer agreement for histopathology was moderate (kappa (ĸ): 0.43 for CIN1/negative, 0.54 for CIN2/≤CIN1, and 0.36 for CIN3). Agreement was good or excellent for biomarker scoring (E4: ĸ=0.896; 95%CI: 0.763-0.969, p16INK4a: ĸ=0.798; 95%CI: 0.712-0.884, MCM: ĸ=0.894; 95%CI: n.c.). Biomarker expression was studied by immunofluorescence and immunohistochemistry and correlated with 104 final CIN diagnoses following histological review. All 25 histologically negative specimens were p16INK4a and panHPVE4 negative although 9 were MCM positive. There were variable extents of p16INK4a positivity in 11/11 CIN1, and extensive panHPVE4 staining in 9/11. Ten CIN2 lesions expressed panHPVE4 and p16INK4a and 13 CIN2 expressed only p16INK4a. CIN3 showed extensive p16INK4a positivity with no/minimal panHPVE4 staining. PanHPVE4, unlike MCM, distinguished CIN1 from negative. PanHPVE4 with p16INK4a separated CIN2/3 showing only expression of p16INK4a indicating transforming HR-HPV E7 expression, from CIN1/2 showing completion of HR-HPV life-cycle by E4 expression and variable p16INK4a expression. PanHPVE4 and p16INK4a staining are complementary markers that could provide simple, reliable support for diagnosing CIN. Their value in distinguishing CIN1/2 that supports HR-HPV life cycle completion (and which might ultimately regress), from purely transforming CIN2/3 needing treatment warrants further research.This research was partly funded by the Stichting Pathologie Ontwikkeling en Onderzoek (SPOO) Foundation, The Netherlands. Funding was also provided from the UK Medical Research Council to HG, YS, ZW and JD