A Sensitive Assay for Unbound Docetaxel Using Ultrafiltration plus HPLC-MS and Its Application to a Clinical Study.

INTRODUCTION: Docetaxel, a taxane used in the treatment of solid tumours, exerts pharmacological activity when in its unbound form. We report a sensitive assay to quantify unbound docetaxel after oral administration of docetaxel plus encequidar (oDox+E). Unbound drug quantification is important due to its direct correlation with drug-related toxicity and therapeutic efficacy. We improve on the sensitivity of current assay methods and demonstrate the utility of the assay on a novel formulation of oral docetaxel. METHODS: Ultrafiltration followed by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) was utilized. Long-term stability, precision, accuracy, and recovery experiments were conducted to validate the assay. Additionally, patient samples from a Phase I dose-escalation pharmacokinetic study were analyzed using the developed assay. RESULTS: The assay method exhibited long-term stability with an observed change between 0.8 and 6.9% after 131 days of storage at -60 °C. Precision and accuracy quality controls met the FDA acceptance criteria. An average recovery of 88% was obtained. Patient sample analysis demonstrated successful implementation of the assay. CONCLUSION: A validated sensitive assay was developed with an LLOQ of 0.084 ng/mL using 485 µL of human plasma. The sensitivity of the assay allowed quantification of unbound docetaxel concentrations in an early-phase oDox+E clinical study to compare it against IV docetaxel using pharmacokinetic modelling. Successful development of oDox+E represents an opportunity to replace the current IV docetaxel regimen with an oral regimen with lower cost, decreased side effects, and improve patient quality of life and experience.

Oral docetaxel plus encequidar - a phase 1 clinical trial.

PURPOSE: To determine the bioavailability, safety, and tolerability of a single dose of oral docetaxel plus encequidar (oDox + E) and compare its pharmacokinetic exposure with current standard of care IV docetaxel. INTRODUCTION: Docetaxel is a taxane widely used as an anti-neoplastic agent. Due to low oral bioavailability secondary to gut P-glycoprotein (P-gp) efflux, its current use is limited to intravenous administration. Oral docetaxel may provide a less resource intensive, more convenient, and tolerable alternative. Encequidar is a first in class, minimally absorbed, oral gut-specific P-gp inhibitor. We tested whether oDox + E can achieve comparable pharmacokinetic exposure to IV docetaxel. METHODS: A multicentre, phase I open-label, pharmacokinetic trial was undertaken to determine the bioavailability, safety, and tolerability of a single dose of oDox + E (at 75 mg/m2 + 15 mg, 150 mg/m2 + 15 mg, and 300 mg/m2 + 15 mg) in metastatic prostate cancer (mPC) patients compared to standard of care IV docetaxel as prescribed by their oncologists. The 15 mg of Encequidar at each dose level was given one hour prior to oral docetaxel. RESULTS: 11 patients were enrolled; 9 patients completed the study. Oral docetaxel exposure increased with dose, achieving the highest at 300 mg/m2 oDox + E (with AUC0 - infinity of 1343.3 ± 443.0 ng.h/mL compared to the IV docetaxel AUC0 - infinity of 2000 ± 325 ng.h/mL) and became non-linear at 300 mg/m2. The mean absolute bioavailability of oDox + E across all 3 dose levels was 16.14% (range: 8.19-25.09%). No patient deaths, dose limiting toxicity, treatment-related serious adverse event or grade 4 toxicity were observed. Maximal tolerated dose was not reached. CONCLUSION: oDox + E has a safe and tolerable adverse event profile in patients with metastatic prostate cancer. The increase in oral bioavailability of oDox + E suggests a multi-dose oDox + E regimen could theoretically achieve exposures comparable with standard of care IV docetaxel. Further development to examine the optimal multiple dose regimen of oDox + E is warranted. TRIAL REGISTRATION NUMBER: U1111-1173-5473.

Oral docetaxel plus encequidar - A pharmacokinetic model and evaluation against IV docetaxel.

The development of optimized dosing regimens plays a crucial role in oncology drug development. This study focused on the population pharmacokinetic modelling and simulation of docetaxel, comparing the pharmacokinetic exposure of oral docetaxel plus encequidar (oDox + E) with the standard of care intravenous (IV) docetaxel regimen. The aim was to evaluate the feasibility of oDox + E as a potential alternative to IV docetaxel. The article demonstrates an approach which aligns with the FDA's Project Optimus which aims to improve oncology drug development through model informed drug development (MIDD). The key question answered by this study was whether a feasible regimen of oDox + E existed. The purpose of this question was to provide an early GO / NO-GO decision point to guide drug development and improve development efficiency. METHODS:  A stepwise approach was employed to develop a population pharmacokinetic model for total and unbound docetaxel plasma concentrations after IV docetaxel and oDox + E administration. Simulations were performed from the final model to assess the probability of target attainment (PTA) for different oDox + E dose regimens (including multiple dose regimens) in relation to IV docetaxel using AUC over effective concentration (AUCOEC) metric across a range of effective concentrations (EC). A Go / No-Go framework was defined-the first part of the framework assessed whether a feasible oDox + E regimen existed (i.e., a PTA ≥ 80%), and the second part defined the conditions to proceed with a Go decision. RESULTS:  The overall population pharmacokinetic model consisted of a 3-compartment model with linear elimination, constant bioavailability, constant binding mechanics, and a combined error model. Simulations revealed that single dose oDox + E regimens did not achieve a PTA greater than 80%. However, two- and three-dose regimens at 600 mg achieved PTAs exceeding 80% for certain EC levels. CONCLUSION:  The study demonstrates the benefits of MIDD using oDox + E as a motivating example. A population pharmacokinetic model was developed for the total and unbound concentration in plasma of docetaxel after administration of IV docetaxel and oDox + E. The model was used to simulate oDox + E dose regimens which were compared to the current standard of care IV docetaxel regimen. A GO / NO-GO framework was applied to determine whether oDox + E should progress to the next phase of drug development and whether any conditions should apply. A two or three-dose regimen of oDox + E at 600 mg was able to achieve non-inferior pharmacokinetic exposure to current standard of care IV docetaxel in simulations. A Conditional GO decision was made based on this result and further quantification of the "effective concentration" would improve the ability to optimise the dose regimen.

Oral paclitaxel and encequidar in patients with breast cancer: a pharmacokinetic, safety, and antitumor activity study.

Background: Paclitaxel is widely used for the treatment of metastatic breast cancer (MBC). However, it has a low oral bioavailability due to gut extrusion caused by P-glycoprotein (P-gp). Oral paclitaxel (oPAC) may be more convenient, less resource-intensive, and more tolerable than its intravenous form. Encequidar (E) is a first-in-class, minimally absorbed, gut-specific oral P-gp inhibitor that facilitates the oral absorption of paclitaxel. Objectives: To investigate the pharmacokinetics (PK), overall response rate (ORR), and safety of weekly oral paclitaxel with encequidar (oPAC + E) in patients with advanced breast cancer. Design: This is a multicenter, single-arm, open-label study in six medical centers in Taiwan. Methods: Patients with advanced breast cancer were administered 205 mg/m2 oPAC and 12.9 mg E for 3 consecutive days weekly for up to 16 weeks. Plasma samples were collected at weeks 1 and 4. PK, ORR, and safety were evaluated. Results: In all, 28 patients were enrolled; 27 had MBC; 23 had prior chemotherapy; and 14 had ⩾2 lines of prior chemotherapy. PK were evaluable in 25 patients. Plasma paclitaxel area under the curve (AUC)(0-52 h) at week 1 (3419 ± 1475 ng h/ml) and week 4 (3224 ± 1150 ng h/ml) were equivalent. Best overall response in 28 evaluable patients was partial response (PR) in 11 (39.3%), 13 (46.4%) stable disease (SD), and 1 (3.6%) with progressive disease (PD). No patient achieved complete response (CR). The clinical benefit rate (CR + PR + SD) was 85.7%. Major adverse events among the 28 treated patients were grade 3 neutropenia (25%), grade 4 neutropenia (18%), with febrile neutropenia in 4%, and grade 3 diarrhea (4%). No treatment-related deaths occurred. Grade 2 peripheral neuropathy occurred in 1 (4%) patient and grade 3 peripheral neuropathy in 1 (4%) patient. Conclusions: oPAC + E produced a consistent therapeutic plasma paclitaxel exposure during treatment. There was a high rate of radiologically assessed clinical benefit, and a low rate of neurotoxicity which may provide advantages over IV paclitaxel. Registration: ClinicalTrials.gov Identifier: NCT03165955.

Increased Expression of the Δ133p53ß Isoform Enhances Brain Metastasis.

The Δ133p53ß isoform is increased in many primary tumors and has many tumor-promoting properties that contribute to increased proliferation, migration and inflammation. Here we investigated whether Δ133p53ß contributed to some of the most aggressive tumors that had metastasized to the brain. Δ133p53ß mRNA expression was measured in lung, breast, melanoma, colorectal metastases and, where available, the matched primary tumor. The presence of Δ133p53ß expression was associated with the time for the primary tumor to metastasize and overall survival once the tumor was detected in the brain. Δ133p53ß was present in over 50% of lung, breast, melanoma and colorectal metastases to the brain. It was also increased in the brain metastases compared with the matched primary tumor. Brain metastases with Δ133p53ß expressed were associated with a reduced time for the primary tumor to metastasize to the brain compared with tumors with no Δ133p53ß expression. In-vitro-based analyses in Δ133p53ß-expressing cells showed increased cancer-promoting proteins on the cell surface and increased downstream p-AKT and p-MAPK signaling. Δ133p53ß-expressing cells also invaded more readily across a mock blood-brain barrier. Together these data suggested that Δ133p53ß contributes to brain metastases by making cells more likely to invade the brain.

Oral paclitaxel with encequidar compared to intravenous paclitaxel in patients with advanced cancer: A randomised crossover pharmacokinetic study.

AIMS: Paclitaxel is a widely used anti-neoplastic agent but has low oral bioavailability due to gut extrusion by P-glycoprotein (P-gp). Oral paclitaxel could be more convenient, less resource intensive, and more tolerable than intravenous administration. Encequidar (HM30181A) is a novel, minimally absorbed gut-specific P-gp inhibitor. We tested whether administration of oral paclitaxel with encequidar (oPac+E) achieved comparable AUC to intravenous paclitaxel (IVP) 80 mg/m2 . METHODS: We conducted a multi-centre randomised crossover study with two treatment periods. Patients (pts) with advanced cancer received either oral paclitaxel 615 mg/m2 divided over 3 days and encequidar 15 mg orally 1 hour prior, followed by IVP 80 mg/m2 , or the reverse sequence. PK blood samples were taken up to Day 9 for oPac+E and Day 5 for IVP. RESULTS: Forty-two patients were enrolled; 35 completed both treatment periods. AUC0-∞ was 5033.5 ± 1401.1 ng.h/mL for oPac+E and 5595.9 ± 1264.1 ng.h/mL with IVP. The geometric mean ratio (GMR) for AUC was 89.50% (90% CI 83.89-95.50). Mean absolute bioavailability of oPac+E was 12% (CV% = 23%). PK parameters did not change meaningfully after 4 weeks administration of oPac+E in an extension study. G3 treatment-emergent adverse events occurred in seven (18%) pts with oPac+E and two (5%) with IVP. Seventy-five per cent of patients preferred oPac+E over IVP. CONCLUSIONS: GMR for AUC was within the predefined acceptable range of 80-125% for demonstrating equivalence. oPac+E is tolerable and there is no evidence of P-gp induction with repeat administration. With further study, oPac+E could be an alternative to IVP.

Intronic TP53 Polymorphisms Are Associated with Increased Δ133TP53 Transcript, Immune Infiltration and Cancer Risk.

We investigated the influence of selected TP53 SNPs in exon 4 and intron 4 on cancer risk, clinicopathological features and expression of TP53 isoforms. The intron 4 SNPs were significantly over-represented in cohorts of mixed cancers compared to three ethnically matched controls, suggesting they confer increased cancer risk. Further analysis showed that heterozygosity at rs1042522(GC) and either of the two intronic SNPs rs9895829(TC) and rs2909430(AG) confer a 2.34-5.35-fold greater risk of developing cancer. These SNP combinations were found to be associated with shorter patient survival for glioblastoma and prostate cancer. Additionally, these SNPs were associated with tumor-promoting inflammation as evidenced by high levels of infiltrating immune cells and expression of the Δ133TP53 and TP53ß transcripts. We propose that these SNP combinations allow increased expression of the Δ133p53 isoforms to promote the recruitment of immune cells that create an immunosuppressive environment leading to cancer progression.

The oncogene BCL6 is up-regulated in glioblastoma in response to DNA damage, and drives survival after therapy.

The prognosis for people with the high-grade brain tumor glioblastoma is very poor, due largely to low cell death in response to genotoxic therapy. The transcription factor BCL6, a protein that normally suppresses the DNA damage response during immune cell maturation, and a known driver of B-cell lymphoma, was shown to mediate the survival of glioblastoma cells. Expression was observed in glioblastoma tumor specimens and cell lines. When BCL6 expression or activity was reduced in these lines, increased apoptosis and a profound loss of proliferation was observed, consistent with gene expression signatures suggestive of anti-apoptotic and pro-survival signaling role for BCL6 in glioblastoma. Further, treatment with the standard therapies for glioblastoma-ionizing radiation and temozolomide-both induced BCL6 expression in vitro, and an in vivo orthotopic animal model of glioblastoma. Importantly, inhibition of BCL6 in combination with genotoxic therapies enhanced the therapeutic effect. Together these data demonstrate that BCL6 is an active transcription factor in glioblastoma, that it drives survival of cells, and that it increased with DNA damage, which increased the survival rate of therapy-treated cells. This makes BCL6 an excellent therapeutic target in glioblastoma-by increasing sensitivity to standard DNA damaging therapy, BCL6 inhibitors have real potential to improve the outcome for people with this disease.

Human Papillomavirus E6/E7 Expression in Preeclampsia-Affected Placentae.

Whether HPV is causative of pregnancy complications is uncertain. E6 and E7 affect functions underling preeclampsia (PET) in cultured trophoblasts, but whether E6 and E7 is produced in the placenta is uncertain. Here, we investigated whether E6/E7 was expressed in the placentae from pregnancies with PET, other pregnancy complications (fetal growth restriction (FGR) and diabetes mellitus), and uncomplicated pregnancies. Placental tissues collected from two geographical locations were subjected to RNAscope analyses of high- and low- risk E6/E7, and individual HPV types identified using an HPV array. High-risk E6/E7 expression was increased in both PET cohorts, (81% and 86% of patients positive for high-risk HPV DNA compared to 13% of control patients). Various HPV types were identified. Although HPV 18 was the most frequent in all cohorts, the majority of individuals had multiple HPV types (55% of the PET compared to 25% of the control cohort). Further evidence that E6 and E7 is present early when placental pathology underlying preeclampsia is established, is provided with the finding of high-risk E6/E7 in the first-trimester placenta anchoring trophoblast. In conclusion, E6/E7 expression and multiple HPV types were frequent in placentae from preeclampsia-complicated pregnancies.

Long Non-coding RNA ANRIL in the Nucleus Associates With Periostin Expression in Breast Cancer.

The long non-coding RNA (LncRNA) antisense RNA in the INK4 locus (ANRIL) is overexpressed in several cancers including breast cancer. To better understand the role of ANRIL in breast cancer this study investigated where ANRIL was expressed in breast tumors using in situ hybridization by RNAscope. Additional RNAscope assays for IL6, CCL2, and POSTN were used to establish whether ANRIL correlated with increased tumor promoting cytokines. Breast tumors with ANRIL over expressed from real-time quantitative (RT-q) PCR assays were selected for analysis using RNAscope. All tumors showed ANRIL expression in malignant cells, but amongst tumors ANRIL showed different subcellular locations with 56% of tumors with ANRIL only in the nucleus, 16% with ANRIL only in the cytoplasm and 28% with ANRIL in both the nucleus and cytoplasm. Cases with nuclear ANRIL were positively correlated with POSTN expression in malignant cells (ρ = 0.57, P = 0.0086), and no correlation was found between ANRIL and IL6 or CCL2. Reduced POSTN was also found using siRNA to ANRIL in MDA-MB-231 and MCF7 breast cancer cells. These data indicate that ANRIL is expressed in malignant breast cells, and suggest its subcellular location may indicate its function in cancer progression.

The Δ133p53ß isoform promotes an immunosuppressive environment leading to aggressive prostate cancer.

Prostate cancer is the second most common cancer in men, for which there are no reliable biomarkers or targeted therapies. Here we demonstrate that elevated levels of Δ133TP53ß isoform characterize prostate cancers with immune cell infiltration, particularly T cells and CD163+ macrophages. These cancers are associated with shorter progression-free survival, Gleason scores ≥ 7, and an immunosuppressive environment defined by a higher proportion of PD-1, PD-L1 and colony-stimulating factor 1 receptor (CSF1R) positive cells. Consistent with this, RNA-seq of tumours showed enrichment for pathways associated with immune signalling and cell migration. We further show a role for hypoxia and wild-type p53 in upregulating Δ133TP53 levels. Finally, AUC analysis showed that Δ133TP53ß expression level alone predicted aggressive disease with 88% accuracy. Our data identify Δ133TP53ß as a highly accurate prognostic factor for aggressive prostate cancer.

Breast milk paclitaxel excretion following intravenous chemotherapy-a case report.

Breast cancer can be diagnosed during pregnancy and in the peri-partum period, and the potential exposure of a foetus or neonate to chemotherapy is of concern to mothers and clinicians. Paclitaxel is a commonly used agent in breast cancer, but little is known about its excretion in breast milk. Breastfeeding during chemotherapy has been traditionally cautioned against due to the risk of neonatal exposure to chemotherapy agents, however, data are limited. We measured serum and breast milk concentrations of paclitaxel in a 33-year-old woman with an early breast cancer diagnosed during pregnancy and treated with weekly paclitaxel 80 mg/m2. We found breast milk paclitaxel levels drop below the minimum quantifiable dose at 72 h following chemotherapy, with a relative infant dose of 0.091%. Breast milk excretion of paclitaxel following a dose of 80 mg/m2 is negligible at 72 h, and this may be a safe time to recommence breastfeeding following exposure.

A mouse model of the Δ133p53 isoform: roles in cancer progression and inflammation.

This review paper outlines studies on the Δ122p53 mouse, a model of the human Δ133p53 isoform, together with studies in other model organisms, cell culture, and where available, clinical investigations. In general, these studies imply that, in contrast to the canonical p53 tumor suppressor, Δ133p53 family members have oncogenic capability. Δ122p53 is multi-functional, conferring survival and proliferative advantages on cells, promoting invasion, metastasis and vascularization, as does Δ133p53. Cancers with high levels of Δ133p53 often have poor prognosis. Δ122p53 mediates its effects through the JAK-STAT and RhoA-ROCK signaling pathways. We propose that Δ133p53 isoforms have evolved as inflammatory signaling molecules to deal with the consequent tissue damage of p53 activation. However, if sustained expression of the isoforms occur, pathologies may result.

Elevation of the TP53 isoform Δ133p53ß in glioblastomas: an alternative to mutant p53 in promoting tumor development.

As tumor protein 53 (p53) isoforms have tumor-promoting, migration, and inflammatory properties, this study investigated whether p53 isoforms contributed to glioblastoma progression. The expression levels of full-length TP53α (TAp53α) and six TP53 isoforms were quantitated by RT-qPCR in 89 glioblastomas and correlated with TP53 mutation status, tumor-associated macrophage content, and various immune cell markers. Elevated levels of Δ133p53ß mRNA characterised glioblastomas with increased CD163-positive macrophages and wild-type TP53. In situ-based analyses found Δ133p53ß expression localised to malignant cells in areas with increased hypoxia, and in cells with the monocyte chemoattractant protein C-C motif chemokine ligand 2 (CCL2) expressed. Tumors with increased Δ133p53ß had increased numbers of cells positive for macrophage colony-stimulating factor 1 receptor (CSF1R) and programmed death ligand 1 (PDL1). In addition, cells expressing a murine 'mimic' of Δ133p53 (Δ122p53) were resistant to temozolomide treatment and oxidative stress. Our findings suggest that elevated Δ133p53ß is an alternative pathway to TP53 mutation in glioblastoma that aids tumor progression by promoting an immunosuppressive and chemoresistant environment. Adding Δ133p53ß to a TP53 signature along with TP53 mutation status will better predict treatment resistance in glioblastoma. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

RETRACTED: Association of prior HPV vaccination with reduced preterm birth: A population based study.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This paper is being retracted at the request of the authors. The authors report that there was an incorrect interpretation of the odds ratio meaning that instead of HPV vaccination potentially being protective, there may be an associated increased risk of preterm delivery. The authors believe that an increased risk of preterm delivery is unlikely and not consistent with the evidence to date. Further, the authors have not been able to access the original source data as per protocol to check the data validity. The authors wish to repeat the study to reassure themselves that there were no data processing or other errors in the databases in order to reach definitive conclusions. Lastly, it is of serious concern to the Editor-in-Chief that the Conflict of Interest statement was only added to the paper by the authors after acceptance and was not made visible to the editor or reviewers prior to acceptance. The authors state that there was no input to the methodology, implementation and results of this study by any commercial entity. The pharma distribution company CSL mentioned in the conflict of interest statement only knew about the study after publication.

Tumor protein 53 mutations are enriched in diffuse large B-cell lymphoma with irregular CD19 marker expression.

Accumulating evidence suggests tumor protein 53 (p53) promotes correct cellular differentiation. Thus, mutant TP53 may be more frequent in tumors with irregular differentiation. This study investigated whether TP53 mutations were more frequent in diffuse large B cell lymphoma (DLBCL) that lacked the B cell lineage marker CD19. Sixteen CD19 negative and 78 CD19 positive DLBCL were sequenced for TP53 mutations. Twenty nine tumors had TP53 mutations and were associated with poorer survival. Mutant TP53 was more frequent in CD19 negative lymphomas (81% versus 21%, p < 0.0001). Analysis of other B cell markers revealed a lack of paired box 5 (PAX5) in CD19 positive lymphomas with mutant TP53 (50%), which was more frequent compared to tumors with wild-type TP53 (15%, p = 0.002). In summary, DLBCL lacking CD19 or PAX5 expression were more likely to have mutant TP53, suggesting irregular B cell marker phenotypes are associated with TP53 mutation.

Telomere profiles and tumor-associated macrophages with different immune signatures affect prognosis in glioblastoma.

Telomere maintenance is a hallmark of cancer and likely to be targeted in future treatments. In glioblastoma established methods of identifying telomerase and alternative lengthening of telomeres leave a significant proportion of tumors with no defined telomere maintenance mechanism. This study investigated the composition of these tumors using RNA-Seq. Glioblastomas with an indeterminate telomere maintenance mechanism had an increased immune signature compared with alternative lengthening of telomeres and telomerase-positive tumors. Immunohistochemistry for CD163 confirmed that the majority (80%) of tumors with an indeterminate telomere maintenance mechanism had a high presence of tumor-associated macrophages. The RNA-Seq and immunostaining data separated tumors with no defined telomere maintenance mechanism into three subgroups: alternative lengthening of telomeres like tumors with a high presence of tumor-associated macrophages and telomerase like tumors with a high presence of tumor-associated macrophages. The third subgroup had no increase in tumor-associated macrophages and may represent a distinct category. The presence of tumor-associated macrophages conferred a worse prognosis with reduced patient survival times (alternative lengthening of telomeres with and without macrophages P=0.0004, and telomerase with and without macrophages P=0.013). The immune signatures obtained from RNA-Seq were significantly different between telomere maintenance mechanisms. Alternative lengthening of telomeres like tumors with macrophages had increased expression of interferon-induced proteins with tetratricopeptide repeats (IFIT1-3). Telomerase-positive tumors with macrophages had increased expression of macrophage receptor with collagenous structure (MARCO), CXCL12 and sushi-repeat containing protein x-linked 2 (SRPX2). Telomerase-positive tumors with macrophages were also associated with a reduced frequency of total/near total resections (44% vs >76% for all other subtypes, P=0.014). In summary, different immune signatures are found among telomere maintenance mechanism-based subgroups in glioblastoma. The reduced extent of surgical resection of telomerase-positive tumors with macrophages suggests that some tumor-associated macrophages are more unfavorable.

A clinicopathological study of episomal papillomavirus infection of the human placenta and pregnancy complications.

Viral infections are known to adversely affect pregnancy, but scant attention has been given to human papilloma virus (HPV) infection. We aimed to determine the molecular and histopathological features of placental HPV infection, in association with pregnancy complications including fetal growth restriction, pre-maturity, pre-eclampsia, and diabetes. Three hundred and thirty-nine placentae were selected based on the presence or absence of pregnancy complications. Five independent methods were used to identify HPV in the placenta, namely, immunohistochemistry for L1 viral capsid, in situ hybridization to high-risk HPV DNA, PCR, western blotting, and transmission electron microscopy. Pregnancy complications and uterine cervical smear screening results were correlated with placental HPV histopathology. In this study, which was deliberately biased towards complications, HPV was found in the decidua of 75% of placentae (253/339) and was statistically associated with histological acute chorioamnionitis (P<0.05). In 14% (35/253) of the HPV positive cases, HPV L1 immunoreactivity also occurred in the villous trophoblast where it was associated with a lymphohistiocytic villitis (HPV-LHV), and was exclusively of high-risk HPV type. HPV-LHV significantly associated with fetal growth restriction, preterm delivery, and pre-eclampsia (all P<0.05). All cases of pre-eclampsia (20/20) in our cohort had high-risk placental HPV. A further 55 cases (22%, 55/253) of HPV positive placentae had minimal villous trophoblast HPV L1 immunoreactivity, but a sclerosing pauci-immune villitis, statistically associated with diabetes (49.1%, 27/55, P<0.05). For women with placental HPV, 33% (69/207) had an HPV-related positive smear result before pregnancy compared with (9.4% 8/85) of women with HPV-negative placentae (P=0.0001). Our findings support further investigations to determine if vaccination of women and men will improve pregnancy outcomes.

Alpha-enolase is upregulated on the cell surface and responds to plasminogen activation in mice expressing a ∆133p53α mimic.

The p53 protein is a master regulator of the stress response. It acts as a tumor suppressor by inducing transcriptional activation of p53 target genes, with roles in apoptosis, cell cycle arrest and metabolism. The discovery of at least 12 isoforms of p53, some of which have tumor-promoting properties, has opened new avenues of research. Our previous work studied tumor phenotypes in four mouse models with different p53 backgrounds: wild-type p53, p53 null, mutant p53 lacking the proline domain (mΔpro), and a mimic for the human Δ133p53α p53 isoform (Δ122p53). To identify the major proteins affected by p53 function early in the response to DNA damage, the current study investigated the entire proteome of bone marrow, thymus, and lung in the four p53 models. Protein extracts from untreated controls and those treated with amsacrine were analyzed using two-dimensional fluorescence difference gel electrophoresis. In the bone marrow, reactive proteins were universally decreased by wild-type p53, including α-enolase. Further analysis of α-enolase in the p53 models revealed that it was instead increased in Δ122p53 hematopoietic and tumor cell cytosol and on the cell surface. Alpha-enolase on the surface of Δ122p53 cells acted as a plasminogen receptor, with tumor necrosis factor alpha induced upon plasminogen stimulation. Taken together, these data identified new proteins associated with p53 function. One of these proteins, α-enolase, is regulated differently by wild-type p53 and Δ122p53 cells, with reduced abundance as part of a wild-type p53 response and increased abundance with Δ122p53 function. Increased cell surface α-enolase on Δ122p53 cells provides a possible explanation for the model's pro-inflammatory features and suggests that p53 isoforms may direct an inflammatory response by increasing the amount of α-enolase on the cell surface.