Preoperative Mutational Analysis of Circulating Tumor Cells (CTCs) and Plasma-cfDNA Provides Complementary Information for Early Prediction of Relapse: A Pilot Study in Early-Stage Non-Small Cell Lung Cancer.

PURPOSE: We assessed whether preoperativemutational analyses of circulating tumor cells (CTCs) and plasma-cfDNA could be used as minimally invasive biomarkers and as complimentary tools for early prediction of relapse in early-stage non-small -cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Using ddPCR assays, hotspot mutations of BRAF, KRAS, EGFR and PIK3CA were identified in plasma-cfDNA samples and size-based enriched CTCs isolated from the same blood samples of 49 early-stage NSCLC patients before surgery and in a control group of healthy blood donors (n= 22). Direct concordance of the mutational spectrum was further evaluated in 27 patient-matched plasma-cfDNA and CTC-derived DNA in comparison to tissue-derived DNA. RESULTS: The prevalence of detectable mutations of the four tested genes was higher in CTC-derived DNA than in the corresponding plasma-cfDNA (38.8% and 24.5%, respectively).The most commonly mutated gene was PIK3CA, in both CTCs and plasma-cfDNA at baseline and at the time of relapse. Direct comparison of the mutation status of selected drug-responsive genes in CTC-derived DNA, corresponding plasma-cfDNA and paired primary FFPE tissues clearly showed the impact of heterogeneity both within a sample type, as well as between different sample components. The incidence of relapse was higher when at least one mutation was detected in CTC-derived DNA or plasma-cfDNA compared with patients in whom no mutation was detected (p =0.023). Univariate analysis showed a significantly higher risk of progression (HR: 2.716; 95% CI, 1.030-7.165; p =0.043) in patients with detectable mutations in plasma-cfDNA compared with patients with undetectable mutations, whereas the hazard ratio was higher when at least one mutation was detected in CTC-derived DNA or plasma-cfDNA (HR: 3.375; 95% CI, 1.098-10.375; p =0.034). CONCLUSIONS: Simultaneous mutational analyses of plasma-cfDNA and CTC-derived DNA provided complementary molecular information from the same blood sample and greater diversity in genomic information for cancer treatment and prognosis. The detection of specific mutations in ctDNA and CTCs in patients with early-stage NSCLC before surgery was independently associated with disease recurrence, which represents an important stratification factor for future trials.

Significant enhancement of ferromagnetism above room temperature in epitaxial 2D van der Waals ferromagnet Fe5-δGeTe2/Bi2Te3 heterostructures.

Two-dimensional (2D) van der Waals (vdW) ferromagnetic metals FexGeTe2 with x = 3-5 have raised significant interest in the scientific community. Fe5GeTe2 shows prospects for spintronic applications since the Curie temperature Tc has been reported near or higher than 300 K. In the present work, epitaxial Fe5-δGeTe2 (FGT) heterostructures were grown by Molecular Beam Epitaxy (MBE) on insulating crystalline substrates. The FGT films were combined with Bi2Te3 topological insulator (TI) aiming to investigate the possible beneficial effect of the TI on the magnetic properties of FGT. FGT/Bi2Te3 films were compared to FGT capped only with AlOx to prevent oxidation. SQUID and MOKE measurements revealed that the growth of Bi2Te3 TI on FGT films significantly enhances the saturation magnetization of FGT as well as the Tc well above room temperature (RT) reaching record values of 570 K. First-principles calculations predict a shift of the Fermi level and an associated enhancement of the majority spin (primarily) as well as the total density of states at the Fermi level suggesting that effective doping of FGT from Bi2Te3 could explain the enhancement of ferromagnetism in FGT. It is also predicted that strain induced stabilization of a high magnetic moment phase in FGT/Bi2Te3 could be an alternative explanation of magnetization and Tc enhancement. Ferromagnetic resonance measurements evidence an enhanced broadening in the FGT/Bi2Te3 heterostructure when compared to FGT. We obtain a large spin mixing conductance of g↑↓eff = 4.4 × 1020 m-2, which demonstrates the great potential of FGT/Bi2Te3 systems for spin-charge conversion applications at room temperature.

Metabolism-Related Gene Expression in Circulating Tumor Cells from Patients with Early Stage Non-Small Cell Lung Cancer.

PURPOSE: Metabolic reprogramming is now characterized as one of the core hallmarks of cancer, and it has already been shown that the altered genomic profile of metabolically rewired cancer cells can give valuable information. In this study, we quantified three Metabolism-Related Gene (MRG) transcripts in the circulating tumor cells (CTCs) of early stage NSCLC patients and evaluated their associations with epithelial and EMT markers. EXPERIMENTAL DESIGN: We first developed and analytically validated highly sensitive RT-qPCR assays for the quantification of HK2, MCT1 and PHGDH transcripts, and further studied the expression of MRGs in CTCs that were isolated using a size-dependent microfluidic device (Parsortix, Angle) from the peripheral blood of: (a) 46 NSCLC patients at baseline, (b) 39/46 of these patients one month after surgery, (c) 10/46 patients at relapse and (d) 10 pairs of cancerous and adjacent non-cancerous FFPE tissues from the same NSCLC patients. Epithelial and EMT markers were also evaluated. RESULTS: MCT1 and HK2 were differentially expressed between HD and NSCLC patients. An overexpression of MCT1 was detected in 15/46 (32.6%) and 3/10 (30%) patients at baseline and at progression disease (PD), respectively, whereas an overexpression of HK2 was detected in 30.4% and 0% of CTCs in the same group of samples. The expression levels of all tested MRGs decreased in CTCs one month after surgery, but a significant increase was noticed at the time of relapse for PHGDH and MCT1 only. The expression levels of HK2 and MCT1 were associated with the overexpression of mesenchymal markers (TWIST-1 and VIM). CONCLUSION: An overexpression of MRGs was observed at a high frequency in the CTCs isolated from early NSCLC patients, thereby supporting the role of MRGs in metastatic processes. The glycolytic and mesenchymal subpopulation of CTCs was significantly predominant compared to CTCs that were glycolytic but not mesenchymal-like. Our data indicate that MRGs merit further evaluation through large and well-defined cohort studies.

The potential of liquid biopsy in the management of cancer patients.

Over the last decade, liquid biopsy has gained much attention as a powerful tool in personalized medicine, since it enables monitoring cancer evolution and follow-up of cancer patients in real time. Through minimally invasive procedures, liquid biopsy provides important information through the analysis of Circulating Tumor Cells (CTCs), and circulating tumor-derived material like circulating tumor DNA (ctDNA), circulating miRNAs (cfmiRNAs) and extracellular vehicles (EVs). CTCs and ctDNA analysis has already an important impact on the prognosis, detection of minimal residual disease (MRD), treatment selection and monitoring of cancer patients, while recent data show also its potential for early cancer diagnosis (Figure 1). Numerous clinical trials include now a liquid biopsy arm, and functional studies mainly based on CTC derived cell-lines and CTC derived explants (CDx) provide important insight on the metastatic process. The recent findings in the field of liquid biopsy and the benefits and main clinical applications of CTC and ctDNA analysis in solid tumors are summarized in this review.

Origin of the quasi-quantized Hall effect in ZrTe5.

The quantum Hall effect (QHE) is traditionally considered to be a purely two-dimensional (2D) phenomenon. Recently, however, a three-dimensional (3D) version of the QHE was reported in the Dirac semimetal ZrTe5. It was proposed to arise from a magnetic-field-driven Fermi surface instability, transforming the original 3D electron system into a stack of 2D sheets. Here, we report thermodynamic, spectroscopic, thermoelectric and charge transport measurements on such ZrTe5 samples. The measured properties: magnetization, ultrasound propagation, scanning tunneling spectroscopy, and Raman spectroscopy, show no signatures of a Fermi surface instability, consistent with in-field single crystal X-ray diffraction. Instead, a direct comparison of the experimental data with linear response calculations based on an effective 3D Dirac Hamiltonian suggests that the quasi-quantization of the observed Hall response emerges from the interplay of the intrinsic properties of the ZrTe5 electronic structure and its Dirac-type semi-metallic character.

Restoration of the degenerated cervical intervertebral space: how much should we distract? A magnetic resonance imaging study.

The aim of the study was to quantify normal cervical disc space measurements and to generate a normal values' database. Furthermore, during operative restoration of a degenerated intervertebral disc, it is difficult to calculate the amount of distraction required to restore the collapsed space to its normal height. A secondary purpose is personalizing the anatomical correction. Therefore, we expressed disc height based on measurements of its neighboring disc as an equation, by using simple linear regression. We reviewed MRI studies from asymptomatic healthy subjects (16 men-24 women, mean age 27.25 years). We measured midsagittal anterior, middle and posterior vertebral body and disc height, and disc diameter from C3 to T1 vertebra. We calculated mean disc height, disc height index (DHI) and disc convexity index per spinal level. C6-7 mean and anterior disc height were significantly greater than all respective measurements, except C5-6 (p<0.01). Middle C6-7-disc height was significantly greater compared to respective measurements in every other level (p<0.01). C5-6, C6-7 and C7-T1 mean disc height is significantly greater in men. Middle disc height is the greatest among disc heights in every spinal level. DHI does not differ between sexes, it increases from C3-4 to C5-6 with a slight decrease in C6-7, while its value significantly decreases in C7-T1 (p<0.0001). These measurements could be used for anatomical, individualized restoration of the degenerated intervertebral disc; thus, avoiding overdistraction. Our data could improve preoperative templating or implant design.

Unconventional Hall response in the quantum limit of HfTe5.

Interacting electrons confined to their lowest Landau level in a high magnetic field can form a variety of correlated states, some of which manifest themselves in a Hall effect. Although such states have been predicted to occur in three-dimensional semimetals, a corresponding Hall response has not yet been experimentally observed. Here, we report the observation of an unconventional Hall response in the quantum limit of the bulk semimetal HfTe5, adjacent to the three-dimensional quantum Hall effect of a single electron band at low magnetic fields. The additional plateau-like feature in the Hall conductivity of the lowest Landau level is accompanied by a Shubnikov-de Haas minimum in the longitudinal electrical resistivity and its magnitude relates as 3/5 to the height of the last plateau of the three-dimensional quantum Hall effect. Our findings are consistent with strong electron-electron interactions, stabilizing an unconventional variant of the Hall effect in a three-dimensional material in the quantum limit.

Nicotine withdrawal-induced inattention is absent in alpha7 nAChR knockout mice.

RATIONALE: Smoking is the leading cause of preventable death in the USA, but quit attempts result in withdrawal-induced cognitive dysfunction and predicts relapse. Greater understanding of the neural mechanism(s) underlying these cognitive deficits is required to develop targeted treatments to aid quit attempts. OBJECTIVES: We examined nicotine withdrawal-induced inattention in mice lacking the α7 nicotinic acetylcholine receptor (nAChR) using the five-choice continuous performance test (5C-CPT). METHODS: Mice were trained in the 5C-CPT prior to osmotic minipump implantation containing saline or nicotine. Experiment 1 used 40 mg kg-1 day-1 nicotine treatment and tested C57BL/6 mice 4, 28, and 52 h after pump removal. Experiment 2 used 14 and 40 mg kg-1 day-1 nicotine treatment in α7 nAChR knockout (KO) and wildtype (WT) littermates tested 4 h after pump removal. Subsets of WT mice were killed before and after pump removal to assess changes in receptor expression associated with nicotine administration and withdrawal. RESULTS: Nicotine withdrawal impaired attention in the 5C-CPT, driven by response inhibition and target detection deficits. The overall attentional deficit was absent in α7 nAChR KO mice despite response disinhibition in these mice. Synaptosomal glutamate mGluR5 and dopamine D4 receptor expression were reduced during chronic nicotine but increased during withdrawal, potentially contributing to cognitive deficits. CONCLUSIONS: The α7 nAChR may underlie nicotine withdrawal-induced deficits in target detection but is not required for response disinhibition deficits. Alterations to the glutamatergic and dopaminergic pathways may also contribute to withdrawal-induced attentional deficits, providing novel targets to alleviate the cognitive symptoms of withdrawal during quit attempts.

Rosacea fulminans during pregnancy.

BACKGROUND: Rosacea fulminans (RF) is a severe form of facial dermatosis presenting with a sudden onset of numerous facial pustules, papules, and erythema. During pregnancy its treatment may be difficult and can have an impact on obstetrical outcomes. CASE: A 37-year-old woman during the 37th week of her fourth pregnancy presented RF that was associated with ocular manifestations. The usual treatment with isotretinoin was contraindicated during pregnancy and the patient started an alternative treatment with prednisone and azithromycin. After delivery at 38 weeks of gestational age, there was a significant improvement. CONCLUSION: RE is a severe dermatological disease with unknown etiology and with a rapid improvement in the immediate postpartum period.

Idiopathic spontaneous hemoperitoneum during pregnancy.

Spontaneous hemoperitoneum is defined as bleeding within the peritoneal cavity of non-traumatic and non-iatrogenic etiology. It is a rare and life-threatening condition during pregnancy. Spontaneous hemoperitoneum is considered idiopathic when the source of bleeding is not detected during the exploratory laparotomy. The authors report two cases of spontaneous hemoperitoneum during pregnancy with sudden onset of abdominal pain during the third trimester of their pregnancy. Cesarean section was performed for fetal distress. In both cases, hemoperitoneum with a large quantity of blood was found, but the source of bleeding could not be identified during surgical exploration.

Development and validation of a multiplex methylation specific PCR-coupled liquid bead array for liquid biopsy analysis.

BACKGROUND: Liquid biopsy is based on minimally invasive blood tests and has the potential to characterize the evolution of a solid tumor in real time, by extracting molecular information from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Epigenetic silencing of tumor and metastasis suppressor genes plays a key role in survival and metastatic potential of cancer cells. Our group was the first to show the presence of epigenetic alterations in CTCs. METHODS: We present the development and analytical validation of a highly specific and sensitive Multiplex Methylation Specific PCR-coupled liquid bead array (MMSPA) for the simultaneous detection of the methylation status of three tumor and metastasis suppressor genes (CST6, SOX17 and BRMS1) in liquid biopsy material (CTCs, corresponding ctDNA) and paired primary breast tumors. RESULTS: In the EpCAM-positive CTCs fraction we observed methylation of: a) CST6, in 11/30(37%) and 11/30(37%), b) BRMS1 in 8/30(27%) and 11/30(37%) c) SOX17 in 8/30(27%) and 13/30(43%) early breast cancer patients and patients with verified metastasis respectively. In ctDNA we observed methylation of: a) CST6, in 5/30(17%) and 10/31(32%), b) BRMS1 in 8/30 (27%) and 8/31 (26%) c) SOX17 in 5/30(17%) and 13/31(42%) early breast cancer patients and patients with verified metastasis respectively. CONCLUSIONS: Our results indicate a high cancerous load at the epigenetic level in EpCAM-positive CTCs fractions and corresponding ctDNA in breast cancer. The main principle of the developed methodology has the potential to be extended in a large number of gene-targets and be applied in many types of cancer.

Postnatal Phencyclidine (PCP) as a Neurodevelopmental Animal Model of Schizophrenia Pathophysiology and Symptomatology: A Review.

Cognitive dysfunction and negative symptoms of schizophrenia remain an unmet clinical need. Therefore, it is essential that new treatments and approaches are developed to recover the cognitive and social impairments that are seen in patients with schizophrenia. These may only be discovered through the use of carefully validated, aetiologically relevant and translational animal models. With recent renewed interest in the neurodevelopmental hypothesis of schizophrenia, postnatal administration of N-methyl-D-aspartate receptor (NMDAR) antagonists such as phencyclidine (PCP) has been proposed as a model that can mimic aspects of schizophrenia pathophysiology. The purpose of the current review is to examine the validity of this model and compare it with the adult subchronic PCP model. We review the ability of postnatal PCP administration to produce behaviours (specifically cognitive deficits) and neuropathology of relevance to schizophrenia and their subsequent reversal by pharmacological treatments. We review studies investigating effects of postnatal PCP on cognitive domains in schizophrenia in rats. Morris water maze and delayed spontaneous alternation tasks have been used for working memory, attentional set-shifting for executive function, social novelty discrimination for selective attention and prepulse inhibition of acoustic startle for sensorimotor gating. In addition, we review studies on locomotor activity and neuropathology. We also include two studies using dual hit models incorporating postnatal PCP and two studies on social behaviour deficits following postnatal PCP. Overall, the evidence we provide supports the use of postnatal PCP to model cognitive and neuropathological disturbances of relevance to schizophrenia. To date, there is a lack of evidence to support a significant advantage of postnatal PCP over the adult subchronic PCP model and full advantage has not been taken of its neurodevelopmental component. When thoroughly characterised, it is likely that it will provide a useful neurodevelopmental model to complement other models such as maternal immune activation, particularly when combined with other manipulations to produce dual or triple hit models. However, the developmental trajectory of behavioural and neuropathological changes induced by postnatal PCP and their relevance to schizophrenia must be carefully mapped out. Overall, we support further development of dual (or triple) hit models incorporating genetic, neurodevelopmental and appropriate environmental elements in the search for more aetiologically valid animal models of schizophrenia and neurodevelopmental disorders (NDDs).

Disruption of mGluR5 in parvalbumin-positive interneurons induces core features of neurodevelopmental disorders.

Alterations in glutamatergic transmission onto developing GABAergic systems, in particular onto parvalbumin-positive (Pv(+)) fast-spiking interneurons, have been proposed as underlying causes of several neurodevelopmental disorders, including schizophrenia and autism. Excitatory glutamatergic transmission, through ionotropic and metabotropic glutamate receptors, is necessary for the correct postnatal development of the Pv(+) GABAergic network. We generated mutant mice in which the metabotropic glutamate receptor 5 (mGluR5) was specifically ablated from Pv(+) interneurons postnatally, and investigated the consequences of such a manipulation at the cellular, network and systems levels. Deletion of mGluR5 from Pv(+) interneurons resulted in reduced numbers of Pv(+) neurons and decreased inhibitory currents, as well as alterations in event-related potentials and brain oscillatory activity. These cellular and sensory changes translated into domain-specific memory deficits and increased compulsive-like behaviors, abnormal sensorimotor gating and altered responsiveness to stimulant agents. Our findings suggest a fundamental role for mGluR5 in the development of Pv(+) neurons and show that alterations in this system can produce broad-spectrum alterations in brain network activity and behavior that are relevant to neurodevelopmental disorders.

Plasma-assisted nanoscale protein patterning on Si substrates via colloidal lithography.

Selective immobilization of proteins in well-defined patterns on substrates has recently attracted considerable attention as an enabling technology for applications ranging from biosensors and BioMEMS to tissue engineering. In this work, a method is reported for low-cost, large scale and high throughput, selective immobilization of proteins on nanopatterned Si, based on colloidal lithography and plasma processing to define the areas (<300 nm) where proteins are selectively immobilized. A close-packed monolayer of PS microparticles is deposited on oxidized Si and, either after microparticle size reduction or alternatively after metal deposition through the PS close-packed monolayer, is used as etching mask to define SiO2 nanoislands (on Si). C4F8 plasma was used to selectively etch and modify the SiO2 nanoislands while depositing a fluorocarbon layer on the Si surface. The plasma-treated surfaces were chemically characterized in terms of functional group identification through XPS analysis and reaction with specific molecules. Highly selective protein immobilization mainly through physical adsorption on SiO2 nanoislands and not on surrounding Si was observed after C4F8 plasma-induced chemical modification of the substrate. The thickness of the immobilized protein monolayer was estimated by means of AFM image analysis. The method reported herein constitutes a cost-efficient route toward rapid, large surface, and high-density patterning of biomolecules on solid supports that can be easily applied in BioMEMS or microanalytical systems.

Assessment of reward responsiveness in the response bias probabilistic reward task in rats: implications for cross-species translational research.

Mood disorders, such as major depressive disorder, are characterized by abnormal reward responsiveness. The Response Bias Probabilistic Reward Task (hereafter referred to as probabilistic reward task (PRT)) quantifies reward responsiveness in human subjects, and an equivalent animal assessment is needed to facilitate preclinical translational research. Thus, the goals of the present studies were to develop, validate and characterize a rat analog of the PRT. Adult male Wistar and Long-Evans rats were trained in operant testing chambers to discriminate between two tone stimuli that varied in duration (0.5 and 2 s). During a subsequent test session consisting of 100 trials, the two tones were made ambiguous (0.9 and 1.6 s) and correct identification of one tone was reinforced with a food pellet three times more frequently than the other tone. In subsequent experiments, Wistar rats were administered either a low dose of the dopamine D2/D3 receptor agonist pramipexole (0.1 mg kg(-1), subcutaneous) or the psychostimulant amphetamine (0.5 mg kg(-1), intraperitoneal) before the test session. Similar to human subjects, both rat strains developed a response bias toward the more frequently reinforced stimulus, reflecting robust reward responsiveness. Mirroring prior findings in humans, a low dose of pramipexole blunted response bias. Moreover, in rats, amphetamine potentiated response bias. These results indicate that in rats, reward responsiveness can be quantified and bidirectionally modulated by pharmacological manipulations that alter striatal dopamine transmission. Thus, this new procedure in rats, which is conceptually and procedurally analogous to the one used in humans, provides a reverse translational platform to investigate abnormal reward responsiveness across species.

Clinical evaluation of microRNA expression profiling in non small cell lung cancer.

Deregulation of miRNAs expression levels has been detected in many human tumor types, and recent studies have demonstrated the critical roles of miRNAs in cancer pathogenesis. Numerous recent studies have shown that miRNAs are rapidly released from tissues into the circulation in many pathological conditions. The high relative stability of miRNAs in biofluids such as plasma and serum, and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNAs as promising non-invasive new tumor biomarkers. In this study, we used liquid bead array technology (Luminex) to profile the expression of 320 mature miRNAs in a pilot testing group of 19 matched fresh frozen cancerous and non-cancerous tissues from NSCLC patients. We further validated our results by RT-qPCR for differentially expressed miRNAs in an independent group of 40 matched fresh frozen tissues, 37 plasma samples from NSCLC patients and 28 healthy donors. We found that eight miRNAs (miR-21, miR-30d, miR-451, miR-10a, miR-30e-5p and miR-126*, miR-126, miR-145) were differentially expressed by three different statistical analysis approaches. Two of them (miR-10a and miR-30e-5p) are reported here for the first time. Bead-array results were further verified in an independent group of 40 matched fresh frozen tissues by RT-qPCR. According to RT-qPCR miR-21 was significantly up-regulated (P = 0.010), miR-126* (P = 0.002), miR-30d (P = 0.012), miR-30e-5p (P < 0.001) and miR-451 (P < 0.001) were down-regulated, while miR-10a was not differentiated (P = 0.732) in NSCLC tissues. However, in NSCLC plasma samples, only three of these miRNAs (miR-21, miR-10a, and miR-30e-5p) displayed differential expression when compared to plasma of healthy donors. High expression of miR-21 was associated with DFI and OS both in NSCLC tissues (P = 0.022 and P = 0.037) and plasma (P = 0.045 and P = 0.065), respectively. Moreover, we report for the first time that low expression of miR-10a in NSCLC plasma samples was associated with worse DFI (P = 0.050) and high expression of miR-30e-5p was found to be associated with shorter OS (P = 0.048). In conclusion, circulating miR-21, miR-10a and miR-30e-5p in plasma should be further evaluated as potential non-invasive biomarkers in NSCLC.

Response to the article: "primary aldosteronism: are we missing the wood for the trees?".

This letter is a short response to Dr Funder's recent commentary on primary aldosteronism under the title "Primary aldosteronism: are we missing the wood for the trees?". We briefly discuss recent data from our department on autonomous aldosterone secretion (AAS) and the efficacy of mineralocorticoid receptor antagonists in a group of patients with AAS, that would otherwise remain undiagnosed.

Development of a multiplexed PCR-coupled liquid bead array assay for vascular endothelial growth factor (VEGF) splice variants.

OBJECTIVES: To develop a multiplex PCR-coupled liquid bead array assay for the expression of VEGF splice variants. DESIGN AND METHODS: The assay was based on the combination of multiplex PCR with liquid bead array technology, and optimized and evaluated in terms of analytical sensitivity, specificity, and reproducibility using the MCF-7 cell line. Clinical performance was evaluated in 16 pairs of fresh frozen cancerous and corresponding noncancerous adjacent tissues from NSCLC patients. RESULTS: The assay is highly sensitive, reproducible and can detect specifically VEGF splice variants in clinical samples. When applied in 32 clinical samples it gave comparable results to RT-qPCR (concordance of 81%, 75%, 88% and 81% for PBGD, VEGF(121), VEGF(165), and VEGF(189) respectively). CONCLUSIONS: The developed assay can simultaneously detect three VEGF splice variants with high specificity and sensitivity and can be further used to evaluate the role of each individual VEGF splice variant in molecular therapies targeted against VEGF.