Disease-specific Markers Research Articles

Bench to Bedside-Gene Therapy for Thalassemia and Sickle Cell Disease

The beta-hemoglobinopathies (beta-thalassemia and sickle cell disease) are the most prevalent inherited disorders worldwide and affect millions. Patients with beta-thalassemia major cannot survive without monthly, lifelong transfusions together with iron chelation therapy, and severe cases of sickle cell disease suffer from multiple life-threatening complications. Both categories of patients often have a shortened life expectancy in spite of supportive therapies, which impose an enormous financial burden on affected countries. The only available curative therapy is allogeneic hematopoietic stem cell transplantation, although most patients do not have an HLA-matched sibling donor, and those who do still risk graft rejection or graft-versus-host disease with associated morbidity and early mortality. This is why autologous gene therapy, by ex vivo transfer into the patient's own hematopoietic stem cells of a derivative of the normal beta-globin gene whose expression is appropriately regulated, is an attractive novel therapeutic modality. In addition, the very large number of known mutations causing beta-thalassemia makes gene therapy by gene addition ideally suited for regulatory product approval rather than the many mutation-specific products that would be required for site-specific gene correction. However, gene therapy of these disorders is especially challenging given the requirement for massive hemoglobin production in a lineage specific manner and the lack of selective advantage for corrected hematopoietic stem cells. During the past two decades, we and others have devised lentiviral vectors and applicable protocols to achieve the permanent correction of mouse models of the beta-hemoglobinopathies. The first approved human clinical trial worldwide resulted in the conversion to transfusion-independence of a patient with severe betaE/beta0-thalassemia, who required monthly transfusions since early childhood. This patient demonstrated prolonged transfusion-independent for over 8 years after gene therapy, and the initially identified partially dominant integration site (HMGA2) is no longer preeminent. A further optimized vector with high-grade purification is now being used in subsequent multi-center clinical trials in the USA, France, Australia, and Thailand both for beta-thalassemia major and severe sickle cell disease. As of the last public disclosure in June 2015, 34 subjects have been enrolled, of whom 12 and 2 patients with beta-thalassemia major and severe sickle cell disease, respectively, have undergone the gene therapy procedure. Currently analysable patients with beta-thalassemia major have rapidly decreased their transfusional needs or become completely transfusion-independent, often with near normal hemoglobin values and polyclonal distribution of vector bearing progenitors. Interestingly, conversion to transfusion-independence also occurred in a Cooley's anemia patient of the beta0/beta0 genotype. With regard to the first analysable patient with sickle cell disease, the anti-sickling globin variant utilized in the vector (βT87Q) is expressed at 40% level of all hemoglobin chains - a level well above the expected sickling inhibitory threshold - resulting in improvements in disease-specific biological markers and no hospitalization for sickle cell complications or acute episode despite weaning off transfusions, which this patient was receiving regularly for the prevention of stroke relapse. Prospects for bringing this novel therapeutic approach to medical practice and for complementary approaches to increase safety and efficacy will be discussed. DisclosuresLeboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Lab-on-a-chip systems in the point-of-care diagnostics

The need in modern medicine for near-patient diagnostics being able to accelerate therapeutic decisions and possibly replacing laboratory measurements is significantly growing. Reliable and cost-effective bioanalytical measurement systems are required which - acting as a micro-laboratory - contain integrated biomolecular recognition, sensing, signal processing and complex microfluidic sample preparation modules. These micro- and nanofabricated Lab-on-a-chip systems open new perspectives in the diagnostic supply chain, since they are able even for quantitative, high-precision and immediate analysis of special disease specific molecular markers or their combinations from a single drop of sample. Accordingly, crucial requirements regarding the instruments and the analytical methods are the high selectivity, extremely low detection limit, short response time and integrability into the healthcare information networks. All these features can make the hierarchical examination chain shorten, and revolutionize laboratory diagnostics, evolving a brand new situation in therapeutic intervention.

Elevated serum CA 19-9 levels in patients with pulmonary nontuberculous mycobacterial disease

Increased serum CA 19-9 levels in patients with nonmalignant diseases have been investigated in previous reports. This study evaluates the clinical significance of serum CA 19-9 elevation in pulmonary nontuberculous mycobacterial disease and pulmonary tuberculosis. The median CA 19-9 level was higher in patients with pulmonary nontuberculous mycobacterial disease than in patients with pulmonary tuberculosis (pulmonary nontuberculous mycobacterial disease: 13.80, tuberculosis: 5.85, p<0.001). A multivariate logistic regression analysis performed in this study showed that Mycobacterium abscessus (OR 9.97, 95% CI: 1.58, 62.80; p=0.014) and active phase of pulmonary nontuberculous mycobacterial disease (OR 12.18, 95% CI: 1.07, 138.36, p=0.044) were found to be risk factors for serum CA 19-9 elevation in pulmonary nontuberculous mycobacterial disease. The serum CA 19-9 levels showed a tendency to decrease during successful treatment of pulmonary nontuberculous mycobacterial disease but not in pulmonary tuberculosis. These findings suggest that CA 19-9 may be a useful marker for monitoring therapeutic responses in pulmonary nontuberculous mycobacterial disease, although it is not pulmonary nontuberculous mycobacterial disease-specific marker.

Decreased plasma levels of neureglin-1 in drug naïve patients and chronic patients with schizophrenia

Although the neuregulin-1 (NRG1) gene is one of the susceptibility genes for schizophrenia and various other psychiatric diseases, it remains unclear how individual psychiatric diseases affect the expression of the NRG1 protein in patients. A previous study reported a schizophrenia-linked decrease in serum NRG1 levels. The present study aimed to replicate this initial finding and to assess its disease specificity for schizophrenia. We collected plasma samples from drug-naïve patients with first-episode schizophrenia (n=80), patients with chronic schizophrenia (n=86), patients with bipolar I disorder (n=60), patients with bipolar II disorder (n=60) and patients with major depressive disorder (n=60), we measured the plasma levels of NRG1β1 and compared the levels with those of age- and sex-matched healthy volunteers (n=82). One-way ANOVA and post hoc analyses detected specific NRG1β1 decreases in the participants with first-episode and chronic schizophrenia but not in those with bipolar I disorder, bipolar II disorder or major depressive disorder. The mean plasma levels of NRG1β1 immunoreactivity were 4.27±0.71ng/mL in the participants with first-episode schizophrenia, 4.08±0.64ng/mL in the participants with chronic schizophrenia and 7.21±0.91ng/mL in the healthy controls. Although we analyzed the pathological correlations of NRG1β1 immunoreactivity in terms of the clinical parameters of the sample, we observed only weak positive correlations with the age of the participants with chronic schizophrenia and the disease onset times of the participants with bipolar II disorder. We failed to identify correlations between other clinical parameters and plasma NRG1β1 immunoreactivity among all patient subjects. These findings suggest that NRG1 may serve as a relatively specific disease marker for schizophrenia. However, the pathological role of this decrease must be explored further.

The internist and the renal resistive index: truths and doubts.

The renal resistive index (RRI) is measured by Doppler sonography in an intrarenal artery, and is the difference between the peak systolic and end-diastolic blood velocities divided by the peak systolic velocity. The RRI is used for the study of vascular and renal parenchymal renal abnormalities, but growing evidence indicates that it is also a dynamic marker of systemic vascular properties. Renal vascular resistance is only one of several renal (vascular compliance, interstitial and venous pressure), and extrarenal (heart rate, aortic stiffness, pulse pressure) determinants that combine to determine the RRI values, and not the most important one. RRI cannot always be considered a specific marker of renal disease. To summarize from the literature: (1) hydronephrosis, abdominal hypertension, renal vein thrombosis and acute kidney injury are all associated with an acute increase in interstitial and venous pressure that determine RRI values. In all these conditions, RRI is a reliable marker of the severity of renal damage. (2) The hemodynamic impact of renal artery stenosis can be assayed by the RRI decrease in the homolateral kidney by virtue of decreasing pulse pressure. However, renal diseases that often coexist, increase renal vascular stiffness and hide the hemodynamic effect of renal stenosis. (3) In transplant kidney and in chronic renal disease, high RRI values (>0.80) can independently predict renal and clinical outcomes, but systemic (pulse pressure) rather than renal hemodynamic determinants sustain the predictive role of RRI. (4) Higher RRI detects target renal organ damage in hypertension and diabetes when renal function is still preserved, as a marker of systemic atherosclerotic burden. Is this the fact? We attempt to answer.

A Breath of Fresh Air for Clinical Diagnoses

A Breath of Fresh Air for Clinical Diagnoses

P168: A unique glycoprotein; Wisteria floribunda agglutinin‐positive Mac‐2 binding protein (WFA+‐M2BP) is a disease‐specific serum marker for liver fibrosis

Journal of Viral HepatitisVolume 22, Issue S2 p. 102-103 Poster PresentationFree Access P168: A unique glycoprotein; Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+-M2BP) is a disease-specific serum marker for liver fibrosis First published: 25 June 2015 https://doi.org/10.1111/jvh.152_12425AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume22, IssueS2Special Issue: 15th International Symposium on Viral Hepatitis and Liver Diseases (ISVHLD) held from June 26th to 28th, 2015, in Berlin, GermanyJune 2015Pages 102-103 RelatedInformation

DNA-methylation and autoantibodies based cancer diagnosis from body fluids

Special focus and aim of our research activities at AIT, the Austrian Institute of Technology, is to define reliable biomarkers suitable for early and non-invasive disease diagnosis from body fluids such as serum/plasma and saliva. Along a selection of research projects, which are described in more detail underneath, we will present and introduce the broad portfolio of high throughput technologies we successfully apply for diagnostic biomarker discovery and validation. As a first show case of successful non-invasive disease biomarker discovery we will present a study where we investigated and compared the genome wide methylation levels of lung cancer patients, patients suffering from lung fibrosis, patients with COPD (chronic obstructive pulmonary disease), and DNA samples derived from healthy lungs. Along this study we could identify specific methylation patterns for each of these lung diseases. After quantitative PCR validation of 240 disease specific methylation markers in the discovery sample set, the 90 top markers were picked and applied for serum testing (n=204). When we applied gradient boosting classification for differential diagnosis of tested lung diseases and healthy controls an AUC value of 0.95 was reached here to separate cancer from all other non-cancer samples whereas in differential diagnosis of healthy-, COPD and fibrosis patients AUC values of 0.71 and 0.49 were obtained for fibrosis, respectively COPD. Thus in case of COPD the presented method may be used to monitor cancer risk within COPD patients. Our second show case comprises a study where we screened cancer patients’ sera for tumor-specific antibody profiles using an in-house developed 16k protein-microarray. This methodology, which will be described in detail, enabled us to define different tumor-associated antigen (TAA) classifier panels for the big 4 cancer entities (breast, colon, prostate and lung cancer) which all showed very promising classification successes in distinction of patients versus controls. We will further present preliminary data obtained when comparing serum and saliva autoantibody profiles of breast-cancer patients and healthy controls.

Application of Sensitive AlleleSEQR Chimerism PCR from Peripheral Blood in Allografted AML/MDS Patients Treated with Pre-emptive Posttransplant Immunomodulation

PCR technique based on short tandem repeats (STR, microsatellites) is currently most widely used to monitor chimerism after allogeneic hematopoietic stem cell transplantation. Analysis of the proportion of donor and recipient derived cells enables an early detection of graft rejection or secondary graft failure. In malignant diseases increasing recipient chimerism has also been used to predict relapse, particularly in patients who lack disease-specific PCR marker to detect minimal residual disease (MRD). In these patients serial chimerism analyses may guide the use of post-transplant pre-emptive immunomodulation with the goal of avoiding clinical relapse. However, the sensitivity of the recently standardized STR-PCR, ranging between 0.8 and 1.6%, may not be sufficient to allow detection of impending relapse early enough in rapidly proliferating diseases like acute myeloid leukemia (AML) [1]. Therefore, we have tested the Abbott AlleleSEQR chimerism analysis system in AML patients who received pre-emptive post-transplant immunomodulation. The patients were routinely investigated for chimerism with STR-PCR monthly from whole blood and from blood T-cell fraction during post-transplant period and from bone marrow CD34 positive precursor cell fraction at 2-3 month intervals. In high risk patients with AML or myelodysplastic syndrome (MDS), we have applied posttransplant immunomodulation with azacytidine, lenalidomide and/ or donor lymphocyte infusions (DLI) based on findings in MRD and chimerism analyses the target being MRD negativity and full donor chimerism. In this retrospective study the routinely collected samples were additionally analyzed by Abbott AlleleSEQR chimerism assay.

NMR-based urinalysis for rapid diagnosis of inborn errors of metabolism

Aims A rapid diagnosis for patients with suspected inborn errors of metabolism (IEM) is clinically important. However, most urine organic acid analysis (UOA) using GC-MS takes time. Here, we developed a rapid workflow using nuclear magnetic resonance (NMR) spectroscopy-based urinalysis as a complementary diagnostic tool to UOA. Methods Urine samples will be analysed using 1 H-NMR spectroscopy, including 100 urine controls without IEM and urine samples from patients with beta-ketothiolase deficiency (BKD), beta-ureidopropionase deficiency (UPD), propionic acadaemia (PA), multiple carboxylase deficiency (MCD) and succinic semialdehyde dehydrogenase (SSADH) deficiency. A 2-dimension ( 1 H- 13 C heteronuclear single quantum correlation, HSQC) will be performed to provide structural-based evidence on the positive identification of metabolites if necessary. Results Disease-specific markers were identified in all patients with IEM: butanone in BKD, β-ureidoisobutyric and β-ureidopropionic acids in UPD, 3-hydroxypropionic acid in PA, 3-hydroxyisovaleric acid in MCD, and 4-hydroxybutyric acid in SSADH deficiency, and they were not found in excess in unaffected controls. Discussion The operation of NMR is simple and fast; sample preparation is a 2-step procedure without the needs of derivatisation while a usual 1 H-NMR acquisition takes

Circulating biomarkers in pulmonary arterial hypertension: Update and future direction

Pulmonary arterial hypertension (PAH) is a complex disease with a poor prognosis. In recent years, great advances have occurred in our understanding of the pathophysiologic mechanisms underlying the characteristic vascular proliferative lesions, thus allowing the development of several specific drugs. Nevertheless, PAH still presents a high mortality; therefore, early diagnosis and prognostic stratification seem to be of paramount importance in order to choose the best therapeutic strategies. Circulating biomarkers have been proposed as potentially noninvasive and objective parameters for diagnosis, prognosis, and response to therapy. The molecules evaluated to date, including markers of dysfunction and neurohormonal activation, myocardial injury, inflammation and oxidative stress, vascular damage and remodelling, end-organ failure, and gene expression, reflect the complex pathophysiology of PAH. However, not one of these shows all the characteristics of the ideal biomarker; thus, a multiparameter approach is probably desirable. Moreover, future direction could be research of structural proteins specifically expressed in the pathologic tissue that act as disease-specific markers. This report presents an extensive review of circulating biomarkers in PAH and some consideration about potential future direction in this area.

Donor-Type Aplastic Anemia after Allogeneic Hematopoietic Stem Cell Transplantation: A Common Cause of Late Graft Failure in Patients with Complete Donor Chimerism

Late graft failure (LGF) without evidence of residual recipient cells is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-SCT). Various factors, such as infections and chronic graft-versus-host disease (GVHD), are involved in the development of such donor-type LGF, and it is therefore difficult to identify the exact cause and to restore the graft function without performing a second transplant. One exceptionally treatable condition is aplastic anemia (AA), which occurs during donor-derived hematopoiesis and can be diagnosed by the identification of disease-specific markers in the peripheral blood. These include glycosylphosphatidyl inositol-anchored protein (GPI-AP)-deficient blood cells (Mochizuki, el al. Blood, 2008) and HLA-allele lacking leukocytes (HLA-LLs) due to copy-number neutral loss of heterozygosity in the short arm of chromosome 6 (6pLOH, Katagiri, et al. Blood, 2011) or missense mutations in the HLA alleles, which can be found when the pancytopenia develops. During the six-year period from 2007 through 2013, 12 patients with LGF after allo-SCT were referred to our clinic for the diagnosis of the pathogenesis of LGF. Three of the 12 patients were diagnosed to have late graft rejection due to the residual lymphocytes of recipient origin. None of these patients and other three patients with complete chimerism showed either GPI-AP- cells or HLA-LLs. On the other hand, either of the markers were detected in the remaining six patients with complete donor chimerims who developed LGF two to 84 months after allo-SCT after they had achieved complete hematological recovery with a platelet count > 100 x109/L. The original diagnoses included severe AA in one patient and hematological malignancies in the other five patients (Table). A range of 0.016% to 0.2% GPI-AP- granulocytes were detected in four patients, while 6.5%-80% HLA-LLs were detected in two patients (Figure). All patients first developed thrombocytopenia and gradually progressed to pancytopenia. The donors of the three patients possessed one of the four HLA class I alleles (A*02:01 and A*02:06) that we previously identified as genes associated with a susceptibility to AA. The HLA-A allele (A*02:01) that was lost in the donor’s granulocytes in Case 6 was not shared by the recipient. ATG was given to two patients (cases 1 and 2) and induced complete resolution of the LGF. The thrombocytopenia of Case 6 stopped progressing at three months after allo-SCT following a dose-escalation of tacrolimus and became stable thereafter. These findings suggest that donor-type AA is a common cause of LGF with complete donor chimerism after allo-SCT. This complication occurs regardless of the recipient’s original disease, and is potentially curable by immunosuppressive therapy without the need for a second SCT. It is therefore important to screen the peripheral blood for the presence of GPI-AP- cells and HLA-LLs when donor-type LGF or thrombocytopenia occurs after a patient has achieved complete hematological recovery. Possessing HLA class I alleles associated with an AA susceptibility by donors may confer a risk of developing AA to recipients. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The association between hyperoxia and patient outcomes after cardiac arrest: analysis of a high-resolution database.

Previous observational studies have inconsistently associated early hyperoxia with worse outcomes after cardiac arrest, and have methodological limitations. We tested this association using a high-resolution database controlling for multiple disease-specific markers of severity of illness and care processes. This was a retrospective analysis of a single-center, prospective registry of consecutive cardiac arrest patients. We included patients who survived and were mechanically ventilated ≥24 h after arrest. Our main exposure was arterial oxygen tension (PaO2), which we categorized hourly for 24 h as severe hyperoxia (>300 mmHg), moderate or probable hyperoxia (101-299 mmHg), normoxia (60-100 mmHg) or hypoxia (<60 mmHg). We controlled for Utstein-style covariates, markers of disease severity and markers of care responsiveness. We performed unadjusted and multiple logistic regression to test the association between oxygen exposure and survival to discharge, and used ordered logistic regression to test the association of oxygen exposure with neurological outcome and Sequential Organ Failure Assessment (SOFA) score at 24 h. Of 184 patients, 36 % were exposed to severe hyperoxia and overall mortality was 54 %. Severe hyperoxia, but not moderate or probable hyperoxia, was associated with decreased survival in both unadjusted and adjusted analysis [adjusted odds ratio (OR) for survival 0.83 per hour exposure, P = 0.04]. Moderate or probable hyperoxia was not associated with survival but was associated with improved SOFA score 24 h (OR 0.92, P < 0.01). Severe hyperoxia was independently associated with decreased survival to hospital discharge. Moderate or probable hyperoxia was not associated with decreased survival and was associated with improved organ function at 24 h.

Comprehensive analysis of human small RNA sequencing data provides insights into expression profiles and miRNA editing

MicroRNAs (miRNAs) play key regulatory roles in various biological processes and diseases. A comprehensive analysis of large scale small RNA sequencing data (smRNA-seq) will be very helpful to explore tissue or disease specific miRNA markers and uncover miRNA variants. Here, we systematically analyzed 410 human smRNA-seq datasets, which samples are from 24 tissue/disease/cell lines. We tested the mapping strategies and found that it was necessary to make multiple-round mappings with different mismatch parameters. miRNA expression profiles revealed that on average ∼70% of known miRNAs were expressed at low level or not expressed (RPM < 1) in a sample and only ∼9% of known miRNAs were relatively highly expressed (RPM > 100). About 30% known miRNAs were not expressed in all of our used samples. The miRNA expression profiles were compiled into an online database (HMED, http://bioinfo.life.hust.edu.cn/smallRNA/). Dozens of tissue/disease specific miRNAs, disease/control dysregulated miRNAs and miRNAs with arm switching events were discovered. Further, we identified some highly confident editing sites including 24 A-to-I sites and 23 C-to-U sites. About half of them were widespread miRNA editing sites in different tissues. We characterized that the 2 types of editing sites have different features with regard to location, editing level and frequency. Our analyses for expression profiles, specific miRNA markers, arm switching, and editing sites, may provide valuable information for further studies of miRNA function and biomarker finding.

NMR-based urinalysis for rapid diagnosis of β-ureidopropionase deficiency in a patient with Dravet syndrome

BackgroundBeta-ureidopropionase deficiency is a rare inborn error of metabolism (IEM) affecting pyrimidine metabolism. To-date, about 30 genetically confirmed cases had been reported. The clinical phenotypes of this condition are variable; some patients were asymptomatic while some may present with developmental delay or autistic features. In severe cases, patients may present with profound neurological deficit including hypotonia, seizures and mental retardation. Using NMR-based urinalysis, this condition can be rapidly diagnosed within 15min. CaseAn 11-month-old Chinese boy had dual molecular diagnoses, β-ureidopropionase deficiency and Dravet syndrome. He presented with intractable and recurrent convulsions, global developmental delay and microcephaly. Urine organic acid analysis using GC–MS and NMR-based urinalysis showed excessive amount of β-ureidopropionic acid and β-ureidoisobutyric acid, the two disease-specific markers for β-ureidopropionase deficiency. Genetic analysis confirmed homozygous known disease-causing mutation UPB1 NM_016327.2: c.977G>A; NP_057411.1:p.R326Q. In addition, genetic analysis for Dravet syndrome showed the presence of heterozygous disease-causing mutation SCN1A NM_001165963.1:c.4494delC; NP_001159435.1:p.F1499Lfs*2. ConclusionsThe differentiation between Dravet syndrome and β-ureidopropionase deficiency is clinically challenging since both conditions share overlapping clinical features. The detection of urine β-ureidoisobutyric and β-ureidopropionic acids using NMR or GC–MS is helpful in laboratory diagnosis of β-ureidopropionase deficiency. The disease-causing mutation, c.977G>A of β-ureidopropionase deficiency, is highly prevalent in Chinese population (allele frequency=1.7%); β-ureidopropionase deficiency screening test should be performed for any patients with unexplained neurological deficit, developmental delay or autism.

Combination of individualized local control and target-specific agent to improve unresectable liver cancer managements: a matched case-control study.

Management of late-stage hepatocellular carcinoma is difficult. A direct comparison of clinical data is needed in order to demonstrate the survival benefits of different therapies. We directly compared various therapies in a retrospective matched case-control study. A total of 79 patients with unresectable tumors greater than 10 cm in size were included in the study between 2008 and 2012. Thirty-five patients were treated with transarterial chemoembolization for local control, 20 were treated with sorafenib systemic chemotherapy, and 24 received combination treatment. The total follow-up time after initial therapy was 4.5 years. Survival time after treatment was significantly longer in the combination therapy group (P < 0.0001). The median survival times for combination, local control, and systemic chemotherapy were 15 (12-21), 10 (9-13), and 3.5 (2.5-9.0) months (95 % confidence interval), respectively. The hazard ratios for local control and systemic chemotherapy were 1.985 and 5.102, respectively, with combination treatment as the reference. There was no observed difference in combination therapy from the side effects of the individual therapies. In conclusion, the limited availability of therapeutic options for late-stage liver cancer necessitates reliance on multidisciplinary personalized medicine approaches with target-specific medications to increase survival time. Combining individualized local control therapy and drugs that target specific disease markers provides more benefits to patients.

Immunodiagnostics and immunosensor design (IUPAC Technical Report)

Abstract This work compiles information on the principles of diagnostic immunochemical methods and the recent advances in this field. It presents an overview of modern techniques for the production of diagnostic antibodies, their modification with the aim of improving their diagnostic potency, the different types of immunochemical detection systems, and the increasing diagnostic applications for human health that include specific disease markers, individualized diagnosis of cancer subtypes, therapeutic and addictive drugs, food residues, and environmental contaminants. A special focus lies in novel developments of immunosensor techniques, promising approaches to miniaturized detection units and the associated microfluidic systems. The trends towards high-throughput systems, multiplexed analysis, and miniaturization of the diagnostic tools are discussed. It is also made evident that progress in the last few years has largely relied on novel chemical approaches.

Abstract 2064: Mass Spectrometry Imaging of therapeutic antibodies: Distribution of unlabeled trastuzumab in CB.17 SCID mice implanted with the human breast BT474 xenograft

Abstract Introduction Mass Spectrometry Imaging (MSI) is an emerging technique in preclinical analysis that allows for analysis of the distribution and the quantification of target molecules in tissue sections. The main advantage of this imaging technology is the detection of the molecule of interest in tissues without labelling and with high specificity. MSI is now routinely used for small molecule distribution analysis in lead optimization process, and in PK/PD or toxicity studies. Due to the mass range limitation of the instrument analyzer one of the main challenges is the detection of administered large molecules such as therapeutic antibodies. . In this study, we describe the detection and the distribution of trastuzumab, a monoclonal antibody that recognizes the HER2/neu receptor, in a BT474 human breast xenograft model. Experimental procedures BT474 tumor fragments are implanted in female CB.17 SCID mice. When the tumors reach 80-120 mm3, the trastuzumab treatment of 20mg/kg with eight bi-weekly intraperitoneal injections was initiated. The endpoint was fixed at day 30 post first administration. At 50 hours post 1st, 5th and 8th doses vehicle and trastuzumab treated animals were sacrificed and tumor, plasma and carcass were collected. One section of each tumor tissue was first digested with a proteolytic enzyme deposited with a dedicated device onto the tissue. After the proteolytic digestion, the selected MALDI matrix is sprayed onto the tissue and then the distribution of the Trastuzumab is determined based on its specific peptide mass fingerprint (PMF) by high resolution mass spectrometry imaging with a MALDI-FTICR instrument (SolariX FTICR 7.0T from Bruker). The images are obtained with Quantinetix software dedicated to the MSI data set. Novel Aspect We describe a process which combines MSI and bottom-up proteomics approach directly on tissue to follow the distribution of a therapeutic antibody without any labelling. Until now, mass spectrometry imaging was used to study some biomarkers' (proteins) distribution on tissue. This work describes a novel MSI application for analysis of a dosed therapeutic antibody. Data collection is ongoing. Conclusions We have adapted the use of Mass Spectrometry Imaging to follow the distribution of a non-labeled therapeutic antibody based on its peptide mass fingerprint in dosed tissues. This opens the use of this technology to other large molecules, such as therapeutic proteins or Antibody-Drug Conjugates (ADCs) in a variety of therapeutic fields (Oncology, Immunology, CNS, etc.) In addition to the distribution of the target therapeutic antibody, Mass Spectrometry Imaging could also allow the detection of other important endogenous molecules in the same or adjacent tissue section, such as specific disease markers, thus allowing further understanding of PK/PD relationships. Citation Format: David Bonnel, Chassidy Hall, Robert J. Mullin, Kathryn A. Simon, Jonathan Stauber. Mass Spectrometry Imaging of therapeutic antibodies: Distribution of unlabeled trastuzumab in CB.17 SCID mice implanted with the human breast BT474 xenograft. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2064. doi:10.1158/1538-7445.AM2014-2064

Physiology of Saliva: An Overview

Physiology of saliva evolves in supporting many important activities in human life. Changes in age, environmental and living condition as well as eating habit would influence salivary function. Saliva palys a role in determining the development pattern and oral health. Saliva has a unique function in the defense mechanism to microorganism in the oral environment. Research focuses on the salivary physiology is limited and seems to be separated from medical and dental professional education. Better and deeper comprehensive understanding of saliva in health and disease is needed. Nowadays, many studies have used saliva to find diagnosis markers for specific diseases. Salivary analysis is intended to give descriptive information on disease status for prevention, monitoring and treatment purposes. This brief review aimed to give understanding on salivary physiology in order to add new views of its application in the field of medicine and dentistry.

NMR-based urinalysis for beta-ketothiolase deficiency

BackgroundBeta-ketothiolase deficiency is a rare inborn errors of metabolism (IEM) affecting the catabolism of isoleucine, characterized by severe ketoacidosis in children of 6 to 24months old. A prompt diagnosis is of paramount importance as the metabolic decompensation can be effectively reverted by glucose infusion and health outcomes are improved on a protein-restricted diet. Currently, majority of the laboratory diagnosis were made based on mass-spectrometry and molecular genetics while little is mentioned on the advancement of nuclear magnetic resonance (NMR) spectroscopy for the diagnosis of this condition. CaseWe report a case of beta-ketothiolase deficiency in a 1-y-old Chinese boy who presented with repeated vomiting, impaired consciousness and severe ketoacidosis. NMR urinalysis detected excessive amount of butanone (a disease specific marker of beta-ketothiolase deficiency), tiglylglycine, (intermediate of isoleucine catabolism) and ketones. Diagnosis of beta-ketothiolase deficiency was further established by molecular genetic studies of ACAT1 gene of the proband. ConclusionsThis case illustrated that NMR-based urinalysis is complementary to organic acid analysis for diagnosis of beta-ketothiolase deficiency. The operation of NMR is simple and fast; sample preparation is a two-step procedure while the NMR acquisition is automatic and usually takes <15min. We envisage that NMR analysis will become more available in clinical laboratories and will play an important role in acute pediatric care.