Functional Graphene for Peritumoral Brain Microenvironment Modulation Therapy in Glioblastoma.

Peritumoral brain invasion is the main target to cure glioblastoma. Chemoradiotherapy and targeted therapies fail to combat peritumoral relapse. Brain inaccessibility and tumor heterogeneity explain this failure, combined with overlooking the peritumor microenvironment. Reduce graphene oxide (rGO) provides a unique opportunity to modulate the local brain microenvironment. Multimodal graphene impacts are reported on glioblastoma cells in vitro but fail when translated in vivo because of low diffusion. This issue is solved by developing a new rGO formulation involving ultramixing during the functionalization with polyethyleneimine (PEI) leading to the formation of highly water-stable rGO-PEI. Wide mice brain diffusion and biocompatibility are demonstrated. Using an invasive GL261 model, an anti-invasive effect is observed. A major unexpected modification of the peritumoral area is also observed with the neutralization of gliosis. In vitro, mechanistic investigations are performed using primary astrocytes and cytokine array. The result suggests that direct contact of rGO-PEIUT neutralizes astrogliosis, decreasing several proinflammatory cytokines that would explain a bystander tumor anti-invasive effect. rGO also significantly downregulates several proinvasive/protumoral cytokines at the tumor cell level. The results open the way to a new microenvironment anti-invasive nanotherapy using a new graphene nanomaterial that is optimized for in vivo brain delivery.

Perilymph metabolomic and proteomic MALDI-ToF profiling with porous silicon chips: A proof-of-concept study.

INTRODUCTION: Sensorineural hearing losses (SNHLs) are a significant public health issue, and the hearing loss field is desperately in need of effective therapy. Pathophysiological mechanisms are not yet clearly understood in the absence of validated methods to assess the inner ear content. Proteomic and metabolomic analysis of perilymph is opening new research perspectives for SNHLs. We aimed to demonstrate the feasibility of an innovative mass spectrometry (MS) strategy using porous silicon chips (PSCs) to investigate the low molecular weight (LMW) protein and metabolite content of human perilymph. Our second objective was to stratify perilymph samples according to their MS profiles and compare these results with clinical data. MATERIAL AND METHODS: Perilymph samples obtained during cochlear implant surgery from patients with SNHLs were retrieved from a validated biobank. To focus on LMW entities, we used a PSC enrichment protocol before MALDI-ToF MS analysis. PSCs were used as a LMW molecular preanalytical stabilizer and amplifier. Patients' clinical data and SNHL characteristics were retrieved retrospectively from medical charts. RESULTS: We successfully acquired and compared 59 exploitable MS profiles out of 71 perilymph samples. There was a good correlation between duplicates. Comparing both ears from the same patient, we found good reproducibility even when there was a one-year interval between samplings. We identified three distinct groups when comparing the samples' metabolomic profiles and four homogeneous groups comparing their LMW proteome profiles. Clinical data analysis suggested that some groups shared clinical or preanalytical characteristics. CONCLUSION: This proof-of-concept study confirms that LMW proteome and metabolome content of perilymph can be analyzed with PSCs. Based on protein profiles, we managed to stratify perilymp samples according to their molecular composition. These results must be confirmed with a larger population, and sampling methods require improvement, but this approach seems promising. In the future, this approach may pave the way for companion test strategies to precisely diagnose and define potential molecular targets for audioprotective therapies.

Proteomic Imaging of Vestibular Schwannomas and Normal Nerves. Histopathologic Correlations.

OBJECTIVES: Proteomic analysis of vestibular schwannoma (VS), non-vestibular schwannoma (NVS), and normal nerve (NN) using mass spectrometry and imaging of matrix assisted laser desorption ionization-time of flight (MALDI-TOF). METHODS: Retrospective, qualitative, and descriptive study on VS, NVS, and NN. Samples were provided by our Tumor Bank. They were analyzed histologically then sprayed by acid matrix. The laser beam of MALDI performed desorption-ionization of the sample. A mass spectrogram (MS) was drawn depending on time of flight of ionized peptides, and MALDI-imaging was obtained which is a summation color spectrum depending on sample's peptide content. The slice was reexamined histologically and results compared with MALDI-imaging. RESULTS: Fifty schwannomas were sampled, of which 27 exploitable: 22 VS (17 Antoni type A and five type B) and five NVS (all Antoni type B). Eleven NN were analyzed. Among the 22 VS, near-total correlation between MALDI-imaging and pathology was found in two cases (9.1%), partial correlation in four (18.2%), and no correlation in 16 (72.7%); correlations were more frequent in VS of the Antoni type B. MS showed a peptide spike at 2,000 m/z in 7 (31.8%) and 5,000 m/z in 21 (95.5%). Among the five NVS, near-total correlation was found in three cases (60%), partial correlation in one (20%), and no correlation in one (20%). MS showed a peptide spike at 2,000 m/z in two (40%) and 5,000 m/z in all (100%). Among the 11 NN, near-total correlation was found in nine cases (81.8%), partial correlation in one (9.1%), and no correlation in one (9.1%). MS showed no peptide spike at 2,000 or 5,000 m/z. Behind homogeneous areas on histology, there was great heterogeneity on MALDI-imaging and MS, regarding VS and NVS, but not NN. CONCLUSIONS: There was a lack of correlation between MALDI-imaging and pathology in VS (except Antoni type B) as compared with NVS and NN. The lack of correlation in VS of the type A as compared with type B VS and NVS could be attributed to the overexpression of degeneration-associated proteins/peptides in VS of the type B as well as NVS that are better correlated with histologic findings. The two peptide spikes detected in schwannoma and not in NN opens up the prospect of tumor biomarkers identifiable by sequencing. The proteomic polymorphism found in VS and NVS was absent on histology which is a new morphologic characteristic of schwannoma. Further studies should be performed in the future to confirm the benefit and usefulness of the MALDI in the analysis of VS and NVS.

Deep brain stimulation-associated brain tissue imprints: a new in vivo approach to biological research in human Parkinson's disease.

BACKGROUND: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) or the internal segment of the globus pallidus (GPi) has been established as a highly effective symptomatic therapy for Parkinson's disease (PD). An intriguing biological aspect related to the DBS procedure is that a temporary contact establishes between surgical instruments and the surrounding brain tissue. In this exploratory study, we took advantage of this unique context to harvest brain material adhering to the stylet routinely used during surgery, and to examine the biological value of these samples, here referred to as "brain tissue imprints" (BTIs). RESULTS: Nineteen BTIs from 12 STN- or GPi-electrode implanted patients were obtained in vivo during DBS surgery, without any modification of the surgical procedure. Immunofluorescence analyses confirmed that our approach allowed the harvesting of many neural cells including neurons harboring distinct neurotransmitter markers. Shotgun proteomic and transcriptomic analyses provided for the first time molecular information from DBS-associated brain samples, and confirmed the compatibility of this new type of sample with poly-omic approaches. The method appears to be safe and results consistent. CONCLUSIONS: We here propose BTIs as original and highly valuable brain samples, and DBS-related brain imprinting as a new conceptual approach to biological research in living patients with PD.

Accessing to the minor proteome of red blood cells through the influence of the nanoparticle surface properties on the corona composition.

Nanoparticle (NP)-protein interactions in complex samples have not yet been clearly understood. Nevertheless, several studies demonstrated that NP's physicochemical features significantly impact on the protein corona composition. Taking advantage of the NP potential to harvest different subsets of proteins, we assessed for the first time the capacity of three kinds of superparamagnetic NPs to highlight the erythrocyte minor proteome. Using both qualitative and quantitative proteomics approaches, nano-liquid chromatography-tandem mass spectrometry allowed the identification of 893 different proteins, confirming the reproducible capacity of NPs to increase the number of identified proteins, through a reduction of the sample concentration range and the capture of specific proteins on the three different surfaces. These NP-specific protein signatures revealed significant differences in their isoelectric point and molecular weight. Moreover, this NP strategy offered a deeper access to the erythrocyte proteome highlighting several signaling pathways implicated in important erythrocyte functions. The automated potentiality, the reproducibility, and the low-consuming sample demonstrate the strong compatibility of our strategy for large-scale clinical studies and may become a standardized sample preparation in future erythrocyte-associated proteomics studies.

Biomarker Signature Discovery from Mass Spectrometry Data.

Mass spectrometry based high throughput proteomics are used for protein analysis and clinical diagnosis. Many machine learning methods have been used to construct classifiers based on mass spectrometry data, for discrimination between cancer stages. However, the classifiers generated by machine learning such as SVM techniques typically lack biological interpretability. We present an innovative technique for automated discovery of signatures optimized to characterize various cancer stages. We validate our signature discovery algorithm on one new colorectal cancer MALDI-TOF data set, and two well-known ovarian cancer SELDI-TOF data sets. In all of these cases, our signature based classifiers performed either better or at least as well as four benchmark machine learning algorithms including SVM and KNN. Moreover, our optimized signatures automatically select smaller sets of key biomarkers than the black-boxes generated by machine learning, and are much easier to interpret.

A micro-silicon chip for in vivo cerebral imprint in monkey.

Access to cerebral tissue is essential to better understand the molecular mechanisms associated with neurodegenerative diseases. In this study, we present, for the first time, a new tool designed to obtain molecular and cellular cerebral imprints in the striatum of anesthetized monkeys. The imprint is obtained during a spatially controlled interaction of a chemically modified micro-silicon chip with the brain tissue. Scanning electron and immunofluorescence microscopies showed homogeneous capture of cerebral tissue. Nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis of proteins harvested on the chip allowed the identification of 1158 different species of proteins. The gene expression profiles of mRNA extracted from the imprint tool showed great similarity to those obtained via the gold standard approach, which is based on post-mortem sections of the same nucleus. Functional analysis of the harvested molecules confirmed the spatially controlled capture of striatal proteins implicated in dopaminergic regulation. Finally, the behavioral monitoring and histological results establish the safety of obtaining repeated cerebral imprints in striatal regions. These results demonstrate the ability of our imprint tool to explore the molecular content of deep brain regions in vivo. They open the way to the molecular exploration of brain in animal models of neurological diseases and will provide complementary information to current data mainly restricted to post-mortem samples.

Induction of the fibrinolytic system by cartilage extract mediates its antiangiogenic effect in mouse glioma.

Both the antiangiogenic and antitumoral activity of shark cartilage extracts (SCE) have been demonstrated in animal models and clinical trials. Studies reported that SCE induces the expression of tissue plasminogen activator gene (PLAT) in endothelial cells and increases the activity of the protein (t-PA) in vitro. The aim of this study was to demonstrate the crucial role of t-PA induction in the antiangiogenic and antitumor activity of SCE in experimental glioma. This study showed antiangiogenic and antitumoral effects of SCE in three mice glioma models (C6, HGD and GL26). Histological examination suggested perivascular proteolysis and edema as well as important intratumoral necrosis, which artefactually increased the tumor volume at high doses. Thus, the antiangiogenic effect of SCE correlated with the presence of t-PA and angiostatin in degenerating vessels. Functional in vivo experiments were conducted to modulate the plasminogen pathway. No antiangiogenic effect was observed on tumors overexpressing the plasminogen activator inhibitor-1 (PAI-1). Moreover, therapeutical effects were neutralized in mice that were cotreated with ε-aminocaproic acid (EACA, 120 mg/kg p.o.), an inhibitor that blocks the high-affinity lysine binding sites of both plasminogen and plasmin. In contrast, cotreatment with N-acetylcysteine (NAC, 7,5mg/kg i.p.), a sulfhydril donor that reduces plasmin into angiostatin or other antiangiogenic fragments, increased the benefit of SCE on mice survival. In subcutaneous models, NAC prevented the increase in tumor volume caused by high doses of cartilage extract. In conclusion, this study indicates that induction of t-PA by shark cartilage extract plays an essential role in its antiangiogenic activity, but that control of excessive proteolysis by a plasmin reductor could prevent edema and uncover the full benefit of shark cartilage extract in the treatment of intracranial tumors.

Identification of proteomic signatures of mantle cell lymphoma, small lymphocytic lymphoma, and marginal zone lymphoma biopsies by surface enhanced laser desorption/ionization-time of flight mass spectrometry.

Mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), and marginal zone lymphoma (MZL) are small B-cell non-Hodgkin lymphomas (NHLs) that may be difficult to distinguish. In order to identify specific proteomic biomarkers, differential proteomic analysis of these three NHLs was performed using surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). Whole cell lysates obtained from 18 MCL, 20 SLL, and 20 MZL biopsies were applied on two different ProteinChips (cationic and anionic). Hierarchical clustering and discriminating scores combined with an innovative bio-informatics microdissection strategy allowed us to distinguish specific lymphoma proteomic signatures based on the expression of 37 protein peaks. SELDI-assisted protein purification combined with nano-liquid chromatography (LC) quadrupole-time of flight tandem mass spectrometry (Q-TOF MS/MS) was used to identify proteins overexpressed in both MCL and SLL tumors. Among them two histones, H2B and H4, were identified in MCL tumor biopsies and the signal recognition particle 9 kDa protein, SRP9, in SLL tumor biopsies.

The heterogeneity of meningioma revealed by multiparameter analysis: infiltrative and non-infiltrative clinical phenotypes.

Tumor invasion or infiltration of adjacent tissues is the source of clinical challenges in diagnosis as well as prevention and treatment. Among brain tumors, infiltration of the adjacent tissues with diverse pleiotropic mechanisms is frequently encountered in benign meningiomas. We assessed whether a multiparametric analysis of meningiomas based on data from both clinical observations and molecular analyses could provide a consistent and accurate appraisal of invasive and infiltrative phenotypes and help determine the diagnosis of these tumors. Tissue analyses of 37 meningiomas combined enzyme-linked immunosorbent assay (ELISA) and surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) assays of two different protein biomarkers (thrombospondin 1 and a phosphorylated form of vimentin) as well as gene expression analyses with oligonucleotide micro-arrays. Up to four different clinical and molecular parameters were then examined for tumor classification. From this study, we were able to cluster 36 out of the 37 tumors into two different subsets corresponding to infiltrative/invasive and non-infiltrative tumors. In addition, meningiomas that invade brain and those that infiltrate the neighboring skull bone exhibited no distinguishable molecular features. Our multi-parameter analysis that combines clinical data, transcriptomic and molecular assays clearly reveals the heterogeneity of meningiomas and distinguishes the intrinsically infiltrative/invasive tumors from the non-infiltrative meningiomas.

Nanodevices in diagnostics.

The real-time, personalized and highly sensitive early-stage diagnosis of disease remains an important challenge in modern medicine. With the ability to interact with matter at the nanoscale, the development of nanotechnology architectures and materials could potentially extend subcellular and molecular detection beyond the limits of conventional diagnostic modalities. At the very least, nanotechnology should be able to dramatically accelerate biomarker discovery, as well as facilitate disease monitoring, especially of maladies presenting a high degree of molecular and compositional heterogeneity. This article gives an overview of several of the most promising nanodevices and nanomaterials along with their applications in clinical practice. Significant work to adapt nanoscale materials and devices to clinical applications involving large interdisciplinary collaborations is already underway with the potential for nanotechnology to become an important enabling diagnostic technology.

Nanotexture Optimization by Oxygen Plasma of Mesoporous Silica Thin Film for Enrichment of Low Molecular Weight Peptides Captured from Human Serum.

This study investigated the optimization of mesoporous silica thin films by nanotexturing using oxygen plasma versus thermal oxidation. Calcination in oxygen plasma provides superior control over pore formation with regard to the pore surface and higher fidelity to the structure of the polymer template. The resulting porous film offers an ideal substrate for the selective partitioning of peptides from complex mixtures. The improved chemico-physical characteristics of porous thin films (pore size distribution, nanostructure, surface properties and pore connectivity) were systematically characterized with XRD, Ellipsometry, FTIR, TEM and N(2) adsorption/desorption. The enrichment of low molecular weight proteins captured from human serum on mesoporous silica thin films fabricated by both methodologies were investigated by comparison of their MALDI-TOF MS profiles. This novel on-chip fractionation technology offers advantages in recovering the low molecular weight peptides from human serum, which has been recognized as an informative resource for early diagnosis of cancer and other diseases.

Increased phosphorylation of vimentin in noninfiltrative meningiomas.

BACKGROUND: Tissue invasion or tissue infiltration are clinical behaviors of a poor-prognosis subset of meningiomas. We carried out proteomic analyses of tissue extracts to discover new markers to accurately distinguish between infiltrative and noninfiltrative meningiomas. METHODOLOGY/PRINCIPAL FINDINGS: Protein lysates of 64 different tissue samples (including two brain-invasive and 32 infiltrative tumors) were submitted to SELDI-TOF mass spectrometric analysis. Mass profiles were used to build up both unsupervised and supervised hierarchical clustering. One marker was found at high levels in noninvasive and noninfiltrative tumors and appeared to be a discriminative marker for clustering infiltrative and/or invasive meningiomas versus noninvasive meningiomas in two distinct subsets. Sensitivity and specificity were 86.7% and 100%, respectively. This marker was purified and identified as a multiphosphorylated form of vimentin, a cytoskeletal protein expressed in meningiomas. CONCLUSIONS/SIGNIFICANCE: Specific forms of vimentin can be surrogate molecular indicators of the invasive/infiltrative phenotype in tumors.

Emerging applications of nanomedicine for the diagnosis and treatment of cardiovascular diseases.

Nanomedicine is an emerging field that utilizes nanotechnology concepts for advanced therapy and diagnostics. This convergent discipline merges research areas such as chemistry, biology, physics, mathematics and engineering. It therefore bridges the gap between molecular and cellular interactions, and has the potential to revolutionize medicine. This review presents recent developments in nanomedicine research poised to have an important impact on the treatment of cardiovascular disease. This will occur through improvement of the diagnosis and therapy of cardiovascular disorders as atherosclerosis, restenosis and myocardial infarction. Specifically, we discuss the use of nanoparticles for molecular imaging and advanced therapeutics, specially designed drug eluting stents and in vivo/ex vivo early detection techniques.

Enabling individualized therapy through nanotechnology.

Individualized medicine is the healthcare strategy that rebukes the idiomatic dogma of 'losing sight of the forest for the trees'. We are entering a new era of healthcare where it is no longer acceptable to develop and market a drug that is effective for only 80% of the patient population. The emergence of "-omic" technologies (e.g. genomics, transcriptomics, proteomics, metabolomics) and advances in systems biology are magnifying the deficiencies of standardized therapy, which often provide little treatment latitude for accommodating patient physiologic idiosyncrasies. A personalized approach to medicine is not a novel concept. Ever since the scientific community began unraveling the mysteries of the genome, the promise of discarding generic treatment regimens in favor of patient-specific therapies became more feasible and realistic. One of the major scientific impediments of this movement towards personalized medicine has been the need for technological enablement. Nanotechnology is projected to play a critical role in patient-specific therapy; however, this transition will depend heavily upon the evolutionary development of a systems biology approach to clinical medicine based upon "-omic" technology analysis and integration. This manuscript provides a forward looking assessment of the promise of nanomedicine as it pertains to individualized medicine and establishes a technology "snapshot" of the current state of nano-based products over a vast array of clinical indications and range of patient specificity. Other issues such as market driven hurdles and regulatory compliance reform are anticipated to "self-correct" in accordance to scientific advancement and healthcare demand. These peripheral, non-scientific concerns are not addressed at length in this manuscript; however they do exist, and their impact to the paradigm shifting healthcare transformation towards individualized medicine will be critical for its success.

Mesoporous silica chips for selective enrichment and stabilization of low molecular weight proteome.

The advanced properties of mesoporous silica have been demonstrated in applications, which include chemical sensing, filtration, catalysis, drug delivery and selective biomolecular uptake. These properties depend on the architectural, physical and chemical properties of the material, which in turn are determined by the processing parameters in evaporation-induced self-assembly. In this study, we introduce a combinatorial approach for the removal of the high molecular weight proteins and for the specific isolation and enrichment of low molecular weight species. This approach is based on mesoporous silica chips able to fractionate, selectively harvest and protect from enzymatic degradation, peptides and proteins present in complex human biological fluids. We present the characterization of the harvesting properties of a wide range of mesoporous chips using a library of peptides and proteins standard and their selectivity on the recovery of serum peptidome. Using MALDI-TOF-MS, we established the correlation between the harvesting specificity and the physicochemical properties of mesoporous silica surfaces. The introduction of this mesoporous material with fine controlled properties will provide a powerful platform for proteomics application offering a rapid and efficient methodology for low molecular weight biomarker discovery.

Tailoring of the nanotexture of mesoporous silica films and their functionalized derivatives for selectively harvesting low molecular weight protein.

We present a fast, efficient, and reliable system based on mesoporous silica chips to specifically fractionate and enrich the low molecular weight proteome. Mesoporous silica thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different templates and concentrations in the precursor solution, various pore size distributions, pore structures, and connectivity were obtained and applied for selective recovery of low mass proteins. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. In addition, to overcome the limitations of the prefunctionalization method in polymer selection, plasma ashing was used for the first time for the treatment of the mesoporous silica surface prior to chemical modification. Surface charge modifications by different functional groups resulted in a selective capture of the low molecular weight proteins from serum sample. In conclusion, our study demonstrates that the ability to tune the physicochemical properties of mesoporous silica surfaces, for a selective enrichment of the low molecular weight proteome from complex biological fluids, has the potential to promote proteomic biomarker discovery.

A minimally invasive microdevice for molecular sampling and analysis.

In this paper, we present a new minimally invasive biopsy microdevice adapted to proteomic mass spectrometry analysis. The concept is born from a multidisciplinary collaboration in fields of proteomics, cancer research, and microtechnology. In mixing different skills, we have developed and manufactured a miniaturized biopsy device using microtechnology techniques in order to minimize tissue damage during surgical gesture. Dedicated chemically functionalized areas were added to the device in order to increase capture yield and specificity during tissue contact. Fields of application range from cancer research to the study of neurodegenerative diseases.

Proteomic study of the impact of the JAK2-V617F mutation on the phenotype of essential thrombocythemia.

OBJECTIVE: Previous studies have suggested that two subtypes of essential thrombocythemia (ET) could be separated on the basis of their JAK2 status (V617F-positive or V617F-negative), with a continuum between V617F-positive ET and polycythemia vera (PV). Nevertheless, increasingly contradictory data on the impact of JAK2-V617F (presence and load) on ET phenotype highlight the need for further investigations. MATERIALS AND METHODS: We investigated the influence of JAK2-V617F on ET phenotype using mass spectrometry-based analysis of serum protein profiles of ET patients, comparatively with PV patients. RESULTS: V617F-positive ET and PV displayed significant differences in their serum protein profiles. Furthermore, JAK2-V617F presence did not impact significantly the serum proteome of ET patients: we observed very few differences in serum protein profiles of V617F-positive and -negative ET. Reciprocally, clustering of ET patients on the basis of their serum protein profiles did not correlate with JAK2-V617F presence. Finally, the JAK2-V617F load did not influence serum apolipoprotein A-1 levels in ET, a previously validated marker of JAK2-V617F allele burden in PV. CONCLUSION: Serum proteome of ET patients was not influenced by the presence of JAK2-V617F or by high V617F allelic ratio (up to 50%) suggesting that ET phenotype is, at best, only partially influenced by the JAK2-V617F mutation.

Apolipoprotein A1: A new serum marker correlated to JAK2 V617F proportion at diagnosis in patients with polycythemia vera.

Polycythemia vera (PV) is a myeloproliferative disorder (MPD) characterized by an acquired gain-of-function mutation of the JAK2 protein (JAK2 V617F). Allele-specific quantitative PCR has showed a JAK2 V617F dosage effect on haematological and clinical parameters of PV at diagnosis, but it is unknown whether the level of certain serum proteins might correlate with the proportion of mutated JAK2. Taking into account that such proteins could represent useful prognostic marker, we investigated the serum protein profile of PV patients by SELDI-TOF MS. We identified apolipoprotein A1 (Apo-A1) as a serum marker correlated to the percentage of JAK2 V617F alleles; Apo-A1 expression being the highest for PV patients with more than 75% of mutated alleles. Immuno-assay on an automated random immuno-analyser confirmed the correlation between Apo-A1 concentrations and JAK2 V617F percentages, and showed that serum Apo-A1 assay allowed the specific discrimination of PV patients with high levels of mutated alleles (≥75%). These data suggest that Apo-A1 assay could be a useful assay for the stratification of PV patients at diagnosis.