Temporal composition of the cervicovaginal microbiome associates with hrHPV infection outcomes in a longitudinal study.

BACKGROUND: Persistent infections with high-risk human papillomavirus (hrHPV) can cause cervical squamous intraepithelial lesions (SIL) that may progress to cancer. The cervicovaginal microbiome (CVM) correlates with SIL, but the temporal composition of the CVM after hrHPV infections has not been fully clarified. METHODS: To determine the association between the CVM composition and infection outcome, we applied high-resolution microbiome profiling using the circular probe-based RNA sequencing technology on a longitudinal cohort of cervical smears obtained from 141 hrHPV DNA-positive women with normal cytology at first visit, of whom 51 were diagnosed by cytology with SIL six months later. RESULTS: Here we show that women with a microbial community characterized by low diversity and high Lactobacillus crispatus abundance at both visits exhibit low risk to SIL development, while women with a microbial community characterized by high diversity and Lactobacillus depletion at first visit have a higher risk of developing SIL. At the level of individual species, we observed that a high abundance for Gardnerella vaginalis and Atopobium vaginae at both visits associate with SIL outcomes. These species together with Dialister micraerophilus showed a moderate discriminatory power for hrHPV infection progression. CONCLUSIONS: Our results suggest that the CVM can potentially be used as a biomarker for cervical disease and SIL development after hrHPV infection diagnosis with implications on cervical cancer prevention strategies and treatment of SIL.

Light-Responsive Elastin-Like Peptide-Based Targeted Nanoparticles for Enhanced Spheroid Penetration.

We describe here a near infrared light-responsive elastin-like peptide (ELP)-based targeted nanoparticle (NP) that can rapidly switch its size from 120 to 25 nm upon photo-irradiation. Interestingly, the targeting function, which is crucial for effective cargo delivery, is preserved after transformation. The NPs are assembled from (targeted) diblock ELP micelles encapsulating photosensitizer TT1-monoblock ELP conjugates. Methionine residues in this monoblock are photo-oxidized by singlet oxygen generated from TT1, turning the ELPs hydrophilic and thus trigger NP dissociation. Phenylalanine residues from the diblocks then interact with TT1 via π-π stacking, inducing the re-formation of smaller NPs. Due to their small size and targeting function, the NPs penetrate deeper in spheroids and kill cancer cells more efficiently compared to the larger ones. This work could contribute to the design of "smart" nanomedicines with deeper penetration capacity for effective anticancer therapies.

Targeted RNA next generation sequencing analysis of cervical smears can predict the presence of hrHPV-induced cervical lesions.

BACKGROUND: Because most cervical cancers are caused by high-risk human papillomaviruses (hrHPVs), cervical cancer prevention programs increasingly employ hrHPV testing as a primary test. The high sensitivity of HPV tests is accompanied by low specificity, resulting in high rates of overdiagnosis and overtreatment. Targeted circular probe-based RNA next generation sequencing (ciRNAseq) allows for the quantitative detection of RNAs of interest with high sequencing depth. Here, we examined the potential of ciRNAseq-testing on cervical scrapes to identify hrHPV-positive women at risk of having or developing high-grade cervical intraepithelial neoplasia (CIN). METHODS: We performed ciRNAseq on 610 cervical scrapes from the Dutch cervical cancer screening program to detect gene expression from 15 hrHPV genotypes and from 429 human genes. Differentially expressed hrHPV- and host genes in scrapes from women with outcome "no CIN" or "CIN2+" were identified and a model was built to distinguish these groups. RESULTS: Apart from increasing percentages of hrHPV oncogene expression from "no CIN" to high-grade cytology/histology, we identified genes involved in cell cycle regulation, tyrosine kinase signaling pathways, immune suppression, and DNA repair being expressed at significantly higher levels in scrapes with high-grade cytology and histology. Machine learning using random forest on all the expression data resulted in a model that detected 'no CIN' versus CIN2+ in an independent data set with sensitivity and specificity of respectively 85 ± 8% and 72 ± 13%. CONCLUSIONS: CiRNAseq on exfoliated cells in cervical scrapes measures hrHPV-(onco)gene expression and host gene expression in one single assay and in the process identifies HPV genotype. By combining these data and applying machine learning protocols, the risk of CIN can be calculated. Because ciRNAseq can be performed in high-throughput, making it cost-effective, it can be a promising screening technology to stratify women at risk of CIN2+. Further increasing specificity by model improvement in larger cohorts is warranted.

Novel high-resolution targeted sequencing of the cervicovaginal microbiome.

BACKGROUND: The cervicovaginal microbiome (CVM) plays a significant role in women's cervical health and disease. Microbial alterations at the species level and characteristic community state types (CST) have been associated with acquisition and persistence of high-risk human papillomavirus (hrHPV) infections that may result in progression of cervical lesions to malignancy. Current sequencing methods, especially most commonly used multiplex 16S rRNA gene sequencing, struggle to fully clarify these changes because they generally fail to provide sufficient taxonomic resolution to adequately perform species-level associative studies. To improve CVM species designation, we designed a novel sequencing tool targeting microbes at the species taxonomic rank and examined its potential for profiling the CVM. RESULTS: We introduce an accessible and practical circular probe-based RNA sequencing (CiRNAseq) technology with the potential to profile and quantify the CVM. In vitro and in silico validations demonstrate that CiRNAseq can distinctively detect species in a mock mixed microbial environment, with the output data reflecting its ability to estimate microbes' abundance. Moreover, compared to 16S rRNA gene sequencing, CiRNAseq provides equivalent results but with improved sequencing sensitivity. Analyses of a cohort of cervical smears from hrHPV-negative women versus hrHPV-positive women with high-grade cervical intraepithelial neoplasia confirmed known differences in CST occurring in the CVM of women with hrHPV-induced lesions. The technique also revealed variations in microbial diversity and abundance in the CVM of hrHPV-positive women when compared to hrHPV-negative women. CONCLUSIONS: CiRNAseq is a promising tool for studying the interplay between the CVM and hrHPV in cervical carcinogenesis. This technology could provide a better understanding of cervicovaginal CST and microbial species during health and disease, prompting the discovery of biomarkers, additional to hrHPV, that can help detect high-grade cervical lesions.

RNA-based high-risk HPV genotyping and identification of high-risk HPV transcriptional activity in cervical tissues.

Nearly all cervical cancers are initiated by a persistent infection with one of the high-risk human papillomaviruses (high-risk HPV). High-risk HPV DNA testing is highly sensitive but cannot distinguish between active, productive infections and dormant infections or merely deposited virus. A solution for this shortcoming may be the detection of transcriptional activity of viral oncogenes instead of mere presence of high-risk HPVs. In this study, fresh-frozen cervical tissues (n = 22) were subjected to high-risk HPV DNA detection using the line probe assay and to targeted RNA next-generation sequencing using single-molecule molecular inversion probes. Targeted RNA sequencing was applied for (1) RNA-based genotyping of high-risk HPV, giving information on specific HPV-subtype (2) discrimination of E2, E6, and E7 transcripts and (3) discovery of possible non-HPV cancer biomarkers. Data were analyzed using computational biology. Targeted RNA sequencing enabled reliable genotyping of high-risk HPV subtypes and allowed quantitative detection of E2, E6, and E7 viral gene expression, thereby discriminating cervical lesions from normal cervical tissues. Moreover, targeted RNA sequencing identified possible cervical cancer biomarkers other than high-risk HPV. Interestingly, targeted RNA sequencing also provided high-quality transcription profiles from cervical scrape samples, even after 1 week of dry storage or storage in Preservcyt fixative. This proof of concept study shows that targeted RNA sequencing can be used for high-risk HPV genotyping and simultaneous detection of high-risk HPV gene activity. Future studies are warranted to investigate the potential of targeted RNA sequencing for risk assessment for the development of cervical lesions, based on molecular analysis of cervical scrapes.

Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.

Diffuse gliomas often carry point mutations in isocitrate dehydrogenase ( IDH1mut), resulting in metabolic stress. Although IDHmut gliomas are difficult to culture in vitro, they thrive in the brain via diffuse infiltration, suggesting brain-specific tumor-stroma interactions that can compensate for IDH-1 deficits. To elucidate the metabolic adjustments in clinical IDHmut gliomas that contribute to their malignancy, we applied a recently developed method of targeted quantitative RNA next-generation sequencing to 66 clinical gliomas and relevant orthotopic glioma xenografts, with and without the endogenous IDH-1R132H mutation. Datasets were analyzed in R using Manhattan plots to calculate distance between expression profiles, Ward's method to perform unsupervised agglomerative clustering, and the Mann Whitney U test and Fisher's exact tests for supervised group analyses. The significance of transcriptome data was investigated by protein analysis, in situ enzymatic activity mapping, and in vivo magnetic resonance spectroscopy of orthotopic IDH1mut- and IDHwt-glioma xenografts. Gene set enrichment analyses of clinical IDH1mut gliomas strongly suggest a role for catabolism of lactate and the neurotransmitter glutamate, whereas, in IDHwt gliomas, processing of glucose and glutamine are the predominant metabolic pathways. Further evidence of the differential metabolic activity in these cancers comes from in situ enzymatic mapping studies and preclinical in vivo magnetic resonance spectroscopy imaging. Our data support an evolutionary model in which IDHmut glioma cells exist in symbiosis with supportive neuronal cells and astrocytes as suppliers of glutamate and lactate, possibly explaining the diffuse nature of these cancers. The dependency on glutamate and lactate opens the way for novel approaches in the treatment of IDHmut gliomas.-Lenting, K., Khurshed, M., Peeters, T. H., van den Heuvel, C. N. A. M., van Lith, S. A. M., de Bitter, T., Hendriks, W., Span, P. N., Molenaar, R. J., Botman, D., Verrijp, K., Heerschap, A., ter Laan, M., Kusters, B., van Ewijk, A., Huynen, M. A., van Noorden, C. J. F., Leenders, W. P. J. Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.

Effects of the Green Tea Polyphenol Epigallocatechin-3-Gallate on Glioma: A Critical Evaluation of the Literature.

The review discusses the effects of Epigallocatechin-3-gallate Gallate (EGCG) on glioma as a basis for future research on clinical application of EGCG. Epidemiological studies on the effects of green tea or EGCG on the risk of glioma is inconclusive due to the limited number of studies, the inclusion of all tea types in these studies, and the focus on caffeine rather than EGCG. In vivo experiments using EGCG monotherapy are inconclusive. Nevertheless, EGCG induces cell death, prevents cellular proliferation, and limits invasion in multiple glioma cell lines. Furthermore, EGCG enhances the efficacy of anti-glioma therapies, including irradiation, temozolomide, carmustine, cisplatin, tamoxifen, and TNF-related apoptosis-inducing ligand, but reduces the effect of bortezomib. Pro-drugs, co-treatment, and encapsulation are being investigated to enhance clinical applicability of EGCG. Mechanisms of actions of EGCG have been partly elucidated. EGCG has both anti-oxidant and oxidant properties. EGCG inhibits pro-survival proteins, such as telomerase, survivin, GRP78, PEA15, and P-gp. EGCG inhibits signaling of PDGFR, IGF-1R, and 67LR. EGCG reduces invasiveness of cancer cells by inhibiting the activities of various metalloproteinases, cytokines, and chemokines. Last, EGCG inhibits some NADPH-producing enzymes, thus disturbing redox status and metabolism of glioma cells. In conclusion, EGCG may be a suitable adjuvant to potentiate anti-glioma therapies.

A Conjugate of an Anti-Epidermal Growth Factor Receptor (EGFR) VHH and a Cell-Penetrating Peptide Drives Receptor Internalization and Blocks EGFR Activation.

Overexpression of (mutated) receptor tyrosine kinases is a characteristic of many aggressive tumors, and induction of receptor uptake has long been recognized as a therapeutic modality. A conjugate of a synthetically produced cell-penetrating peptide (CPP), corresponding to amino acids 38-59 of human lactoferrin, and the recombinant llama single-domain antibody (VHH) 7D12, which binds the human epidermal growth factor receptor (EGFR), was generated by sortase A mediated transpeptidation. The conjugate blocks EGF-mediated EGFR activation with higher efficacy than that of both modalities alone; a phenomenon that is caused by both effective receptor blockade and internalization. Thus, the VHH-CPP conjugate shows a combination of activities that implement a highly powerful new design principle to block receptor activation by its clearance from the cell surface.

Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates.

Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation. First, sortase A mediated transPEGylation is used for introduction of a chemical click moiety. The resulting clickable VHHs are then used for conjugation to other groups employing the Cu+-independent strain-promoted alkyne-azide cycloadition (SPAAC) reaction. Using this approach, tail-to-tail bispecific VHHs and VHH-targeted nanoparticles are generated without affecting VHH functionality. Furthermore, this approach allows the bioconjugation of multiple moieties to VHHs for simple and convenient production of VHH-based theranostics.

Self-Assembling VHH-Elastin-Like Peptides for Photodynamic Nanomedicine.

Recombinant llama heavy-chain antibody fragments (VHHs) are promising tools in the field of targeted nanomedicine. 7D12, a VHH against the epidermal growth factor receptor (EGFR) that is overexpressed in various cancers, has been evaluated as an effective cancer-targeting VHH in multiple studies. The small size of VHHs (15-20 kDa) results in a low circulation half-life, which can be disadvantageous for certain applications. A solution to this problem is to attach VHHs to the surface of nanoparticles to increase the hydrodynamic radius of the conjugate. This approach simultaneously allows the incorporation of different VHHs and other targeting moieties and therapeutic components into one structure, creating multispecificity and versatility for therapy and diagnosis. Here, we present the construction of highly defined 7D12-containing nanoparticles by utilizing thermoresponsive diblock elastin-like peptides that reversibly self-assemble into micellar structures. The resulting particles have a hydrodynamic radius of 24.3 ± 0.9 nm and retain full EGFR-binding capacity. We present proof of concept of the usability of such particles by controlled incorporation of a photosensitizer and show that the resulting nanoparticles induce EGFR-specific light-induced cell killing. This approach is easily extended to the controlled incorporation of various functional modules, improving therapy and diagnosis with targeted nanomedicine.

Targeting Cyclin-Dependent Kinases in Synovial Sarcoma: Palbociclib as a Potential Treatment for Synovial Sarcoma Patients.

BACKGROUND: In synovial sarcomas alterations in the cyclin D1-CDK4/6-Rb axis have been described. Also, ß-catenin, a cyclin D1 regulator, is often overexpressed. Additionally, studies have shown that the t(X;18) translocation influences tumor behavior partly through cyclin D1 activation. We investigated how alterations in the cyclin D1-CDK4/6-Rb axis impact prognosis and studied effects of targeting this axis with the CDK4/6 inhibitor palbociclib. METHODS: Synovial sarcoma samples (n = 43) were immunohistochemically stained for ß-catenin, cyclin D1, p16, p21, p27, Rb, and phospho-Rb. Fluorescent in situ hybridization (FISH) was performed to detect CCND1 amplification or translocation. In 4 synovial sarcoma cell lines sensitivity to palbociclib was investigated using cell viability assays, and effects on the sensitive cell lines were evaluated on protein level and by cell cycle arrest. RESULTS: Expression of nuclear phospho-Rb and nuclear ß-catenin in the patient samples was associated with poor survival. FISH showed a sporadic translocation of CCND1 in a subset of tumors. An 8-fold CCND1 amplification was found in 1 cell line, but not in the patient samples investigated. Palbociclib effectively inhibited Rb-phosphorylation in 3 cell lines, resulting in an induction of a G1 arrest and proliferation block. CONCLUSIONS: In this series nuclear phospho-Rb and nuclear ß-catenin expression were negative prognostic factors. In vitro data suggest that palbociclib may be a potential treatment for a subset of synovial sarcoma patients. Whether this effect can be enhanced by combination treatment deserves further preclinical investigations.

Glutamate as chemotactic fuel for diffuse glioma cells: are they glutamate suckers?

Diffuse gliomas comprise a group of primary brain tumors that originate from glial (precursor) cells and present as a variety of malignancy grades which have in common that they grow by diffuse infiltration. This phenotype complicates treatment enormously as it precludes curative surgery and radiotherapy. Furthermore, diffusely infiltrating glioma cells often hide behind a functional blood-brain barrier, hampering delivery of systemically administered therapeutic and diagnostic compounds to the tumor cells. The present review addresses the biological mechanisms that underlie the diffuse infiltrative phenotype, knowledge of which may improve treatment strategies for this disastrous tumor type. The invasive phenotype is specific for glioma: most other brain tumor types, both primary and metastatic, grow as delineated lesions. Differences between the genetic make-up of glioma and that of other tumor types may therefore help to unravel molecular pathways, involved in diffuse infiltrative growth. One such difference concerns mutations in the NADP(+)-dependent isocitrate dehydrogenase (IDH1 and IDH2) genes, which occur in >80% of cases of low grade glioma and secondary glioblastoma. In this review we present a novel hypothesis which links IDH1 and IDH2 mutations to glutamate metabolism, possibly explaining the specific biological behavior of diffuse glioma.

Identification of a novel MET mutation in high-grade glioma resulting in an auto-active intracellular protein.

MET has gained interest as a therapeutic target for a number of malignancies because of its involvement in tumorigenesis, invasion and metastasis. At present, a number of inhibitors, both antibodies against MET or its ligand hepatocyte growth factor, and small molecule MET tyrosine kinase inhibitors are in clinical trials. We here describe a novel variant of MET that is expressed in 6% of high-grade gliomas. Characterization of this mutation in a glioma cell line revealed that it consists of an intronic deletion, resulting in a splice event connecting an intact splice donor site in exon 6 with the next splice acceptor site being that of exon 9. The encoded protein lacks parts of the extracellular IPT domains 1 and 2, encoded by exons 7 and 8, resulting in a novel pseudo-IPT and is named MET(Δ7-8). MET(Δ7-8) is located predominantly in the cytosol and is constitutively active. The auto-activating nature of MET(Δ7-8), in combination with a lack of transmembrane localization, renders MET(Δ7-8) not targetable using antibodies, although the protein is efficiently deactivated by MET-specific tyrosine kinase inhibitors. Testing of MET-expressing tumors for the presence of this variant may be important for treatment decision making.

Longitudinal analysis on the ecological dynamics of the cervicovaginal microbiome in hrHPV infection.

The cervicovaginal microbiome (CVM) is a dynamic continuous microenvironment that can be clustered in microbial community state types (CSTs) and is associated with women's cervical health. Lactobacillus-depleted communities particularly associate with an increased susceptibility for persistence of high-risk human papillomavirus (hrHPV) infections and progression of disease, but the long-term ecological dynamics of CSTs after hrHPV infection diagnosis remain poorly understood. To determine such dynamics, we examined the CVM of our longitudinal cohort of 141 women diagnosed with hrHPV infection at baseline with collected cervical smears at two timepoints six-months apart. Here we describe that the long-term microbiome dissimilarity has a positive correlation with microbial diversity at both visits and that women with high abundance and dominance for Lactobacillus iners at baseline exhibit more similar microbiome composition at second visit than women with Lactobacillus-depleted communities at baseline. We further show that the species Lactobacillus acidophilus and Megasphaera genomosp type 1 associate with CST changes between both visits. Lastly, we also observe that Gardnerella vaginalis is associated with the stability of Lactobacillus-depleted communities while L. iners is associated with the instability of Megasphaera genomosp type 1-dominated communities. Our data suggest dynamic patterns of cervicovaginal CSTs during hrHPV infection, which could be potentially used to develop microbiome-based therapies against infection progression towards disease.

PET Imaging and Protein Expression of Prostate-Specific Membrane Antigen in Glioblastoma: A Multicenter Inventory Study.

Upregulation of prostate-specific membrane antigen (PSMA) in neovasculature has been described in glioblastoma multiforme (GBM), whereas vasculature in nonaffected brain shows hardly any expression of PSMA. It is unclear whether PSMA-targeting tracer uptake on PET is based on PSMA-specific binding to neovasculature or aspecific uptake in tumor. Here, we quantified uptake of various PSMA-targeting tracers in GBM and correlated this with PSMA expression in tumor biopsy samples from the same patients. Methods: Fourteen patients diagnosed with de novo (n = 8) or recurrent (n = 6) GBM underwent a preoperative PET scan after injection of 1.5 MBq/kg [68Ga]Ga-PSMA-11 (n = 7), 200 MBq of [18F]DCFpyl (n = 3), or 200 MBq of [18F]PSMA-1007 (n = 4). Uptake in tumor and tumor-to-background ratios, with contralateral nonaffected brain as background, were determined. In a subset of patients, PSMA expression levels from different regions in the tumor tissue samples (n = 40), determined using immunohistochemistry (n = 35) or RNA sequencing (n = 13), were correlated with tracer uptake on PET. Results: Moderate to high (SUVmax, 1.3-20.0) heterogeneous uptake was found in all tumors irrespective of the tracer type used. Uptake in nonaffected brain was low, resulting in high tumor-to-background ratios (6.1-359.0) calculated by dividing SUVmax of tumor by SUVmax of background. Immunohistochemistry showed variable PSMA expression on endothelial cells of tumor microvasculature, as well as on dispersed individual cells (of unknown origin), and granular staining of the neuropil. No correlation was found between in vivo uptake and PSMA expression levels (for immunohistochemistry, r = -0.173, P = 0.320; for RNA, r = -0.033, P = 0.915). Conclusion: Our results indicate the potential use of various PSMA-targeting tracers in GBM. However, we found no correlation between PSMA expression levels on immunohistochemistry and uptake intensity on PET. Whether this may be explained by methodologic reasons, such as the inability to measure functionally active PSMA with immunohistochemistry, tracer pharmacokinetics, or the contribution of a disturbed blood-brain barrier to tracer retention, should still be investigated.

Effects of targeting the VEGF and PDGF pathways in diffuse orthotopic glioma models.

Currently available compounds that interfere with VEGF-A signalling effectively inhibit angiogenesis in gliomas, but influence diffuse infiltrative growth to a much lesser extent. Development of a functional tumour vascular bed not only involves VEGF-A but also requires platelet-derived growth factor receptor-ß (PDGFRß), which induces maturation of tumour blood vessels. Therefore, we tested whether combined inhibition of VEGFR and PDGFRß increases therapeutic benefit in the orthotopic glioma xenograft models E98 and E473, both displaying the diffuse infiltrative growth that is characteristically observed in most human gliomas. We used bevacizumab and vandetanib as VEGF(R) inhibitors, and sunitinib to additionally target PDGFRß. We show that combination therapy of sunitinib and vandetanib does not improve therapeutic efficacy compared to treatment with sunitinib, vandetanib or bevacizumab alone. Furthermore, all compounds induced reduction of vessel leakage in compact E98 tumour areas, resulting in decreased detectability of these mostly infiltrative xenografts in Gd-DTPA-enhanced MRI scans. These data show that inhibition of VEGF signalling cannot be optimized by additional PDGFR inhibition and support the concept that diffuse infiltrative areas in gliomas are resistant to anti-angiogenic therapy.

Assessing the Cervicovaginal Microbiota in the Context of hrHPV Infections: Temporal Dynamics and Therapeutic Strategies.

Cervical cancer is the third leading cause of female cancers globally, resulting in more than 300,000 deaths every year. The majority of all cervical cancers are caused by persistent infections with high-risk human papillomaviruses (hrHPV) that can progress to cancer via a series of premalignant lesions. Most women, however, clear this infection within a year, concomitant with disease regression. Both hrHPV clearance and disease regression have been associated with the composition of the cervicovaginal microenvironment, which is defined by the host immune system and the cervicovaginal microbiome (CVM). A healthy microbiome is generally characterized by a high abundance of Lactobacillus species, and a change in the composition may cause bacterial vaginosis (BV), which is associated with an increased susceptibility to persistent hrHPV infections and disease. In this review, the composition of the CVM is discussed, with emphasis on the possible causes that drive changes in the cervicovaginal microbiota in relation to hrHPV infections, disease progression, and disease regression. The literature search focused on the composition of the CVM and its correlation with hrHPV infections and neoplastic lesions as well as the current efforts to adjust the microbiome against adverse viral outcomes.

In-depth insights into cervicovaginal microbial communities and hrHPV infections using high-resolution microbiome profiling.

The cervicovaginal microbiome (CVM) correlates with women's cervical health, and variations in its composition are associated with high-risk human papillomavirus (hrHPV) infection outcomes. Cervicovaginal microbes have been grouped into five community state types (CSTs) based on microbial community composition and abundance. However, studying the impact of CSTs in health and disease is challenging because the current sequencing technologies have limited confident discrimination between closely related and yet functionally different bacterial species. Circular probe-based RNA sequencing (ciRNAseq) achieves high-resolution microbiome profiling and therefore provides in-depth and unambiguous knowledge about the composition of the CVM. Based on ciRNAseq profiling of a large cohort of cervical smears (n = 541), we here define subgroups of CSTs I, III, and IV based on intra-CST differences with respect to abundances of Lactobacillus acidophilus (CSTs I-A vs. I-B and CSTs III-A vs. III-B), Lactobacillus iners (CSTs I-A vs. I-B and CSTs III-A vs. III-B), and Megasphaera genomosp type 1 (CSTs IV-A vs. IV-B). Our results further support the existence of subgroups of CST IV-C that are dominant for non-Lactobacillus species and have intermediate microbial diversity. We also show that CST V is associated with uninfected conditions, and CST IV-A associates with hrHPV-induced cervical disease. In conclusion, we characterized new subdivisions of cervicovaginal CSTs, which may further advance our understanding of women's cervical health and hrHPV-related progression to disease.

Detection and localization of early- and late-stage cancers using platelet RNA.

Cancer patients benefit from early tumor detection since treatment outcomes are more favorable for less advanced cancers. Platelets are involved in cancer progression and are considered a promising biosource for cancer detection, as they alter their RNA content upon local and systemic cues. We show that tumor-educated platelet (TEP) RNA-based blood tests enable the detection of 18 cancer types. With 99% specificity in asymptomatic controls, thromboSeq correctly detected the presence of cancer in two-thirds of 1,096 blood samples from stage I-IV cancer patients and in half of 352 stage I-III tumors. Symptomatic controls, including inflammatory and cardiovascular diseases, and benign tumors had increased false-positive test results with an average specificity of 78%. Moreover, thromboSeq determined the tumor site of origin in five different tumor types correctly in over 80% of the cancer patients. These results highlight the potential properties of TEP-derived RNA panels to supplement current approaches for blood-based cancer screening.

Effect of tyrosine kinase inhibitor treatment of renal cell carcinoma on the accumulation of carbonic anhydrase IX-specific chimeric monoclonal antibody cG250.

OBJECTIVE: To investigate the effect of three different tyrosine kinase inhibitors (TKIs) on the biodistribution of chimeric monoclonal antibody (mAb) cG250, which identifies carbonic anhydrase IX (CAIX), in nude mice bearing human renal cell carcinoma (RCC) xenografts. TKIs represent the best, but still suboptimal treatment for metastatic RCC (mRCC) and combined therapy or sequential therapy might be beneficial. CAIX is abundantly over expressed in RCC and clinical trials have shown abundant and specific tumour accumulation of cG250. Combining a TKI with mAb cG250, involved in a different effector mechanism, might lead to improved tumour responses and survival in patients with mRCC. MATERIALS AND METHODS: Nude mice bearing human RCC xenografts were treated orally with 0.75 mg/day sunitinib, 1 mg/day vandetanib, 1 mg/day sorafenib or vehicle control for 7 or 14 days. At 7 days, mice were injected i.v. with 185 kBq/5 µg (125) I-cG250. Mice were killed at predetermined days and cG250 biodistribution was determined. Tumours were analysed by immunohistochemistry for the presence of endothelial cells, laminin, smooth muscle actin, CAIX expression and uptake of mAb cG250. RESULTS: While on TKI treatment, tumour uptake of cG250 decreased dramatically, tumour growth was slightly inhibited and vascular density decreased considerably as judged by various markers. When treatment was stopped at 7 days, there was robust neovascularization, mainly at the tumour periphery. Consequently, cG250 uptake also recovered, albeit cG250 uptake appeared to be restricted to the tumour periphery where vigorous neovascularization was visible. CONCLUSIONS: Simultaneous administration of a TKI and mAb cG250 severely compromised mAb accumulation. However, shortly after discontinuation of TKI treatment mAb accumulation was restored. Combined treatment strategies with TKI and mAb should be carefully designed.