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.

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.

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.

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.

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.

Effects of the IDH1 R132H Mutation on the Energy Metabolism: A Comparison between Tissue and Corresponding Primary Glioma Cell Cultures.

The R132H mutation in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) is the most important prognostic factor for the survival of glioma patients. Subsequent studies led to the discovery of a panel of enzymes mainly involved in glutamate anaplerosis and aerobic glycolysis that change in abundance as a result of the IDH1 mutation. To further study these changes, appropriate glioma models are required that accurately mimic in vivo metabolism. To investigate how metabolism is affected by in vitro cell culture, we here compared surgically obtained snap-frozen glioma tissues with their corresponding primary glioma cell culture models with a previously developed targeted mass spectrometry proteomic assay. We determined the relative abundance of a panel of metabolic enzymes. Results confirmed increased glutamate use and decreased aerobic glycolysis in resected IDH1 R132H glioma tissue samples. However, these metabolic profiles were not reflected in the paired glioma primary cell cultures. We suggest that culture conditions and tumor microenvironment play a crucial role in maintaining the in vivo metabolic situation in cell culture models. For this reason, new models that more closely resemble the in vivo microenvironment, such as three-dimensional cell co-cultures or organotypic multicellular spheroid models, need to be developed and investigated.

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.

Genotyping and Characterization of HPV Status, Hypoxia, and Radiosensitivity in 22 Head and Neck Cancer Cell Lines.

To study head and neck squamous cell carcinomas (HNSCC) in vitro, a large variety of HNSCC cell lines have been developed. Here, we characterize a panel of 22 HNSCC cell lines, thereby providing a tool for research into tumor-specific treatment options in HNSCC. Both human papillomavirus (HPV) positive and HPV negative tumor cell lines were collected from commercial and collaborative sources. Short tandem repeat profiling was used to confirm or characterize the identity of the cell lines. Targeted sequencing was performed using a standard pathology single molecule Molecular Inversion Probe panel to detect mutations for 23 tumor suppressors and oncogenes. HPV status, p16 status, radiosensitivity data, and hypoxia data are summarized from all cell lines. We detected HPV transcripts in five cell lines, all of which overexpressed p16. One HPV negative cell line was also p16 positive. We detected mutations in KIT (SCCNij185), PIK3CA (SCCNij185), and CDKN2A (UT-SCC-5 and UT-SCC-38). TP53 mutations were the most frequent, occurring in 16/22 cell lines. HPV infection and TP53 mutations were almost mutually exclusive, with the exception of 93-VU-147T. The cell lines exhibited a wide range of sensitivities towards hypoxia and irradiation. Here, we provide a description of a set of frequently used HNSCC cell lines with diverse characteristics as found in HNSCC patients.

Multiplex RNA-based detection of clinically relevant MET alterations in advanced non-small cell lung cancer.

MET inhibitors have shown activity in non-small-cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very-high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very-high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very-high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA-based techniques can improve the selection of patients for MET-targeted therapies.

Tumor-Educated Platelet RNA for the Detection and (Pseudo)progression Monitoring of Glioblastoma.

Tumor-educated platelets (TEPs) are potential biomarkers for cancer diagnostics. We employ TEP-derived RNA panels, determined by swarm intelligence, to detect and monitor glioblastoma. We assessed specificity by comparing the spliced RNA profile of TEPs from glioblastoma patients with multiple sclerosis and brain metastasis patients (validation series, n = 157; accuracy, 80%; AUC, 0.81 [95% CI, 0.74-0.89; p < 0.001]). Second, analysis of patients with glioblastoma versus asymptomatic healthy controls in an independent validation series (n = 347) provided a detection accuracy of 95% and AUC of 0.97 (95% CI, 0.95-0.99; p < 0.001). Finally, we developed the digitalSWARM algorithm to improve monitoring of glioblastoma progression and demonstrate that the TEP tumor scores of individual glioblastoma patients represent tumor behavior and could be used to distinguish false positive progression from true progression (validation series, n = 20; accuracy, 85%; AUC, 0.86 [95% CI, 0.70-1.00; p < 0.012]). In conclusion, TEPs have potential as a minimally invasive biosource for blood-based diagnostics and monitoring of glioblastoma patients.

Monotherapy efficacy of blood-brain barrier permeable small molecule reactivators of protein phosphatase 2A in glioblastoma.

Glioblastoma is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumour suppressors has not been thoroughly studied as yet as a glioblastoma therapeutic strategy. Tumour suppressor protein phosphatase 2A is inhibited by non-genetic mechanisms in glioblastoma, and thus, it would be potentially amendable for therapeutic reactivation. Here, we demonstrate that small molecule activators of protein phosphatase 2A, NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood-brain barrier, and in vivo partition to mouse brain tissue after oral dosing. In vitro, small molecule activators of protein phosphatase 2A exhibit robust cell-killing activity against five established glioblastoma cell lines, and nine patient-derived primary glioma cell lines. Collectively, these cell lines have heterogeneous genetic background, kinase inhibitor resistance profile and stemness properties; and they represent different clinical glioblastoma subtypes. Moreover, small molecule activators of protein phosphatase 2A were found to be superior to a range of kinase inhibitors in their capacity to kill patient-derived primary glioma cells. Oral dosing of either of the small molecule activators of protein phosphatase 2A significantly reduced growth of infiltrative intracranial glioblastoma tumours. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested glioblastoma cell lines, also significantly increased survival of mice bearing orthotopic glioblastoma xenografts. In summary, this report presents a proof-of-principle data for blood-brain barrier-permeable tumour suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background. These results also provide the first indications that protein phosphatase 2A reactivation might be able to challenge the current paradigm in glioblastoma therapies which has been strongly focused on targeting specific genetically altered cancer drivers with highly specific inhibitors. Based on demonstrated role for protein phosphatase 2A inhibition in glioblastoma cell drug resistance, small molecule activators of protein phosphatase 2A may prove to be beneficial in future glioblastoma combination therapies.

EpCAM-Binding DARPins for Targeted Photodynamic Therapy of Ovarian Cancer.

Ovarian cancer is the most lethal gynecological malignancy due to late detection associated with dissemination throughout the abdominal cavity. Targeted photodynamic therapy (tPDT) aimed at epithelial cell adhesion molecule (EpCAM), overexpressed in over 90% of ovarian cancer metastatic lesions, is a promising novel therapeutic modality. Here, we tested the specificity and activity of conjugates of EpCAM-directed designed ankyrin repeat proteins (DARPins) with the photosensitizer IRDye 700DX in in vitro and in vivo ovarian cancer models. EpCAM-binding DARPins (Ec1: Kd = 68 pM; Ac2: Kd = 130 nM) and a control DARPin were site-specifically functionalized with fluorophores or IRDye 700DX. Conjugation of anti-EpCAM DARPins with fluorophores maintained EpCAM-specific binding in cell lines and patient-derived ovarian cancer explants. Penetration of DARPin Ec1 into tumor spheroids was slower than that of Ac2, indicative of a binding site barrier effect for Ec1. DARPin-IRDye 700DX conjugates killed EpCAM-expressing cells in a highly specific and illumination-dependent fashion in 2D and 3D cultures. Furthermore, they effectively homed to EpCAM-expressing subcutaneous OV90 xenografts in mice. In conclusion, the high activity and specificity observed in preclinical ovarian cancer models, combined with a high specificity in patient material, warrant a further investigation of EpCAM-targeted PDT for ovarian cancer.

p120-catenin-dependent collective brain infiltration by glioma cell networks.

Diffuse brain infiltration by glioma cells causes detrimental disease progression, but its multicellular coordination is poorly understood. We show here that glioma cells infiltrate the brain collectively as multicellular networks. Contacts between moving glioma cells are adaptive epithelial-like or filamentous junctions stabilized by N-cadherin, ß-catenin and p120-catenin, which undergo kinetic turnover, transmit intercellular calcium transients and mediate directional persistence. Downregulation of p120-catenin compromises cell-cell interaction and communication, disrupts collective networks, and both the cadherin and RhoA binding domains of p120-catenin are required for network formation and migration. Deregulating p120-catenin further prevents diffuse glioma cell infiltration of the mouse brain with marginalized microlesions as the outcome. Transcriptomics analysis has identified p120-catenin as an upstream regulator of neurogenesis and cell cycle pathways and a predictor of poor clinical outcome in glioma patients. Collective glioma networks infiltrating the brain thus depend on adherens junctions dynamics, the targeting of which may offer an unanticipated strategy to halt glioma progression.

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.

ACLY (ATP Citrate Lyase) Mediates Radioresistance in Head and Neck Squamous Cell Carcinomas and is a Novel Predictive Radiotherapy Biomarker.

Radiotherapy is an important treatment modality of head and neck squamous cell carcinomas (HNSCC). Multiple links have been described between the metabolic activity of tumors and their clinical outcome. Here we test the hypothesis that metabolic features determine radiosensitivity, explaining the relationship between metabolism and clinical outcome. Radiosensitivity of 14 human HNSCC cell lines was determined using colony forming assays and the expression profile of approximately 200 metabolic and cancer-related genes was generated using targeted RNA sequencing by single molecule molecular inversion probes. Results: Correlation between radiosensitivity data and expression profiles yielded 18 genes associated with radiosensitivity or radioresistance, of which adenosine triphosphate (ATP) citrate lyase (ACLY) was of particular interest. Pharmacological inhibition of ACLY caused an impairment of DNA damage repair, specifically homologous recombination, and lead to radiosensitization in HNSCC cell lines. Examination of a The Cancer Genome Atlas (TCGA) cohort of HNSCC patients revealed that high expression of ACLY was predictive for radiotherapy failure, as it was only associated with poor overall survival in patients who received radiotherapy (hazard ratio of 2.00, 95% CI: 1.12-3.55; p = 0.0184). These data were further validated in an independent cohort of HNSCC patients treated with chemoradiation. Furthermore, patients with poor locoregional control after radiotherapy have significantly higher nuclear ACLY protein levels. Together, we here show that ACLY affects DNA damage repair, and is a predictive factor for radiotherapy outcome in HNSCC.

Mapping actionable pathways and mutations in brain tumours using targeted RNA next generation sequencing.

Many biology-based precision drugs are available that neutralize aberrant molecular pathways in cancer. Molecular heterogeneity and the lack of reliable companion diagnostic biomarkers for many drugs makes targeted treatment of cancer inaccurate for many individuals. Identifying actionable hyperactive biological pathways in individual cancers may improve this situation.To achieve this we applied a novel targeted RNA next generation sequencing (t/RNA-NGS) technique to surgically obtained glioma tissues. The test combines mutation detection with analysis of biological pathway activities that are involved in tumour behavior in many cancer types (e.g. tyrosine kinase signaling, angiogenesis signaling, immune response, metabolism), via quantitative measurement of transcript levels and splice variants of hundreds of genes. We here present proof of concept that the technique, which uses molecular inversion probes, generates a histology-independent molecular diagnosis and identifies classifiers that are strongly associated with conventional histopathology diagnoses and even with patient prognosis. The test not only confirmed known glioma-associated molecular aberrations but also identified aberrant expression levels of actionable genes and mutations that have so far been considered not to be associated with glioma, opening up the possibility of drug repurposing for individual patients. Its cost-effectiveness makes t/RNA-NGS to an attractive instrument to aid oncologists in therapy decision making.

Isocitrate dehydrogenase 1-mutated cancers are sensitive to the green tea polyphenol epigallocatechin-3-gallate.

BACKGROUND: Mutations in isocitrate dehydrogenase 1 (IDH1) occur in various types of cancer and induce metabolic alterations resulting from the neomorphic activity that causes production of D-2-hydroxyglutarate (D-2-HG) at the expense of α-ketoglutarate (α-KG) and NADPH. To overcome metabolic stress induced by these alterations, IDH-mutated (IDH mut ) cancers utilize rescue mechanisms comprising pathways in which glutaminase and glutamate dehydrogenase (GLUD) are involved. We hypothesized that inhibition of glutamate processing with the pleiotropic GLUD-inhibitor epigallocatechin-3-gallate (EGCG) would not only hamper D-2-HG production, but also decrease NAD(P)H and α-KG synthesis in IDH mut cancers, resulting in increased metabolic stress and increased sensitivity to radiotherapy. METHODS: We performed 13C-tracing studies to show that HCT116 colorectal cancer cells with an IDH1 R132H knock-in allele depend more on glutaminolysis than on glycolysis for the production of D-2-HG. We treated HCT116 cells, HCT116-IDH1 R132H cells, and HT1080 cells (carrying an IDH1 R132C mutation) with EGCG and evaluated D-2-HG production, cell proliferation rates, and sensitivity to radiotherapy. RESULTS: Significant amounts of 13C from glutamate accumulate in D-2-HG in HCT116-IDH1 wt/R132H but not in HCT116-IDH1 wt/wt . Preventing glutamate processing in HCT116-IDH1 wt/R132H cells with EGCG resulted in reduction of D-2-HG production. In addition, EGCG treatment decreased proliferation rates of IDH1 mut cells and at high doses sensitized cancer cells to ionizing radiation. Effects of EGCG in IDH-mutated cell lines were diminished by treatment with the IDH1mut inhibitor AGI-5198. CONCLUSIONS: This work shows that glutamate can be directly processed into D-2-HG and that reduction of glutamatolysis may be an effective and promising new treatment option for IDH mut cancers.

Molecular Profiling of Druggable Targets in Clear Cell Renal Cell Carcinoma Through Targeted RNA Sequencing.

Clear cell renal cell carcinoma (ccRCC) comprises more than 80% of all renal cancers and when metastasized leads to a 5-year survival rate of only 10%. The high rate of therapy failure and resistance development calls for reliable methods that provide information on the actionable biological pathways and predict optimal treatment protocols for individual patients. We here applied targeted RNA sequencing (t/RNA-NGS) using single molecule Molecular Inversion Probes on tumor nephrectomy samples of five ccRCC patients, comparing tumor with healthy kidney tissues. Transcriptome profiling focused on expression of genes with involvement in ccRCC biology that can be targeted with clinically available drugs. Results confirm high expression of vascular endothelial growth factor-A (VEGF-A) in tumor tissue relative to healthy-appearing kidney, in line with the angiogenic nature of ccRCC. PDGFRα and KIT, targets of the multi-kinase inhibitor sunitinib which is one of the current choices of first-line drug in metastasized ccRCC patients, were expressed at relatively low levels in tumor tissues, whereas significantly increased in normal kidney. Of all measured druggable tyrosine kinases, MET, AXL, or EGFR were expressed at higher levels in tumors than in normal kidney tissues, although intertumor differences were observed. Using cancer cell lines we show that t/RNA-NGS gene expression profiles can be used to predict in vitro sensitivity to targeted drugs. In conclusion, t/RNA-NGS analysis may provide insights into the (druggable) molecular make-up of individual renal cancers, and may guide personalized therapy of renal cell cancers.