Plasma Of Patients Research Articles

First description of an IgM monoclonal antibody causing αIIbβ3 integrin activation and acquired Glanzmann thrombasthenia associated with macrothrombocytopenia

Essentials Acquired Glanzmann thrombasthenia (GT) is generally caused by anti-αIIb β3 autoantibodies. We report the case of a man with an acquired GT phenotype associated with macrothrombocytopenia. Perturbed platelet function were associated with an activating anti-αIIb β3 IgM autoantibody. This novel clinical entity raises interesting questions about the αIIb β3 integrin signaling. SUMMARY: Background Acquired Glanzmann thrombasthenia (GT) is a bleeding disorder generally caused by anti-αIIb β3 autoantibodies. Objectives We aimed to characterize the molecular mechanism leading to a progressive GT-like phenotype in a patient with chronic immune thrombocytopenia. Patient, Methods, and Results The patient suffered from repeated episodes of gastrointestinal bleeding; further studies indicated a moderate platelet aggregation defect. A few months later, platelet function showed abolished aggregation using all agonists, but normal agglutination with ristocetin. No platelet-bound antibodies were detected, but the presence of large amounts of an IgM type antibody detected together with αIIb β3 in the patient permeabilized platelets suggested that this IgM was an autoantibody causing the internalization of the complex. This was confirmed by the fact that the patient IgM bound to normal platelets but not to platelets from GT type I patients. Moreover, patient's plasma activated αIIb β3 on controls' platelets as evidenced by increased PAC-1 binding. We also demonstrated that the patient plasma triggered αIIb β3 outside-in signaling, as β3 Tyr773 and FAK were phosphorylated, and increased the rate of actin polymerization in resting platelets reflecting an impairment of cytoskeletal reorganization. Because different signs of dysmegakaryopoiesis were also observed in our patient, we evaluated the ability of its serum to impair proplatelets formation and showed that it significantly decreased the number of proplatelet-bearing megakaryocytes in controls' bone marrow stem cells culture compared with normal serum. Conclusions We present the case of a patient with a progressive and severely perturbed platelet function associated with the presence of an IgM activating autoantibody directed against αIIb β3 .

Diagnosis and the importance of early treatment of tyrosinemia type 1: A case report

Tyrosinemia type 1 is an autosomal recessive aminoacidopathy caused by fumarylacetoacetate hydrolase (FAH) deficiency. Consequently, tyrosine and its metabolites accumulate, resulting in liver and kidney toxicity. Symptoms of the disease usually manifest after three weeks of life and include vomiting, failure to thrive, hepatomegaly, jaundice, bleeding diathesis, rickets and renal tubular dysfunction. Untreated, the disease eventually progresses to liver or kidney failure and generally results in a fatal outcome. Expedient diagnosis is critical because an early start of treatment can increase the likelihood of a positive outcome. Here, we report on a male newborn with a family history positive for tyrosinemia type 1 who was subjected to a metabolic work-up immediately after birth. Amino acids were quantified by tandem mass spectrometry coupled with ultra performance liquid chromatography. Urinary organic acids were analyzed on capillary gas chromatography coupled with mass spectrometry. DNA analysis of the FAH gene was performed by Sanger sequencing. On the first day of life, the patient's plasma amino acids showed an increased tyrosine concentration, while urine organic acids detected succinylacetone, a tyrosine metabolite specific for tyrosinemia type 1. The patient's DNA analysis revealed homozygosity of the c.554-1G > T mutation in the FAH gene, which was consistent with the diagnosis. Nitisinone treatment, combined with a dietary restriction of tyrosine and phenylalanine, was introduced immediately. Regular visits and measurement of amino acid concentrations, which enables therapy adjustment and treatment efficiency monitoring in patients with tyrosinemia type 1, has continued over the past 4+ years, and is expected to continue.

Spontaneous remission of acquired factor XIII inhibitor concurrent to development of IgA-λ type multiple myeloma

Coagulation factor XIII is a fibrin-stabilizing factor that leads to the crosslinking of fibrin when activated by thrombin. Acquired factor XIII inhibitor is caused when antibodies are generated against factor XIII, reducing its activity. Here we report a case of acquired factor XIII inhibitor. Although prednisolone was administered, factor XIII activity was not recovered. Interestingly, the activity normalized following the onset of multiple myeloma. The presence of inhibitors was evaluated in the patient's plasma by absorption tests and enzyme-linked immunosorbent assay. Immunoglobulin G inhibitors of factor XIII were present at admission, but later decreased with the onset of the IgA-λ-type myeloma. Thus, it is possible that the level of factor XIII inhibitors and polyclonal immunoglobulins could have been suppressed by the progression of myeloma, resulting in the normalization of factor XIII activity.

Adverse effects of intermittent flow therapeutic plasmapheresis in neurology patients in a resource constrained setting

Introduction: Therapeutic plasma exchange (TPE) aims at removing harmful substances from patient's plasma. It has become a treatment option in a variety of neurological disorders with good outcome. In the last decade, there has been an increase in the number and spectrum of indications for therapeutic apheresis. Aims and Objectives: To analyse the complications of plasmapheresis patients with varied neurological disorders in a tertiary care neurocentre to evaluate the safety of the procedure. Materials and Methods: A retrospective analysis of complications in all patients who underwent TPE for various neurological disorders in the Transfusion Medicine Department from April 2013 to May 2014 was carried out. Patient record files and TPE protocol forms were analysed to collect the data about various complications during the procedure. Results: A total of 1912 procedures were carried out on 399 patients. Guillain Barre Syndrome was the most common indication. A total of 145( 36.3%) patients had adverse reactions. Mild reactions like rash (40%), chill and rigor (21%), cramps (6.8%) were found in 98 cases (68%). Moderate reactions included hypotension (17.9%), access site pain and hematoma (7.5%), vasovagal attack (3.44%), seizure (1.37%) etc. Severe life threatning reactions were not seen in any of the cases. Conclusion: Our results advocate TPE as a safe and effective procedure in neurological disorders. Most complications are mild and can be managed effectively. Complications can be further reduced by careful patient monitoring, use of prophylactic calcium and use of albumin as replacement fluid.

Conformational fingerprint of blood and tissue ACEs: Personalized approach.

BackgroundThe pattern of binding of monoclonal antibodies (mAbs) to 18 epitopes on human angiotensin I-converting enzyme (ACE)–“conformational fingerprint of ACE”–is a sensitive marker of subtle conformational changes of ACE due to mutations, different glycosylation in various cells, the presence of ACE inhibitors and specific effectors, etc.Methodology/Principal findingsWe described in detail the methodology of the conformational fingerprinting of human blood and tissue ACEs that allows detecting differences in surface topography of ACE from different tissues, as well detecting inter-individual differences. Besides, we compared the sensitivity of the detection of ACE inhibitors in the patient’s plasma using conformational fingerprinting of ACE (with only 2 mAbs to ACE, 1G12 and 9B9) and already accepted kinetic assay and demonstrated that the mAbs-based assay is an order of magnitude more sensitive. This approach is also very effective in detection of known (like bilirubin and lysozyme) and still unknown ACE effectors/inhibitors which nature and set could vary in different tissues or different patients.Conclusions/SignificancePhenotyping of ACE (and conformational fingerprinting of ACE as a part of this novel approach for characterization of ACE) in individuals really became informative and clinically relevant. Appreciation (and counting on) of inter-individual differences in ACE conformation and accompanying effectors make the application of this approach for future personalized medicine with ACE inhibitors more accurate. This (or similar) methodology can be applied to any enzyme/protein for which there is a number of mAbs to its different epitopes.

Active and Ultra-Large Von Willebrand Factor Is Significantly Elevated in Patients with Immune Thrombotic Thrombocytopenic Purpura

▪Background: Immune thrombotic thrombocytopenic purpura (iTTP), a potentially fatal hematological emergency, is primarily caused by acquired deficiency of ADAMTS13 activity due to autoantibodies. Immunoglobulin G (IgG)-type autoantibodies bind ADAMTS13 and inhibit its ability to cleave endothelium-derived ultra large von Willebrand factor (ULVWF). However, it remains poorly understood whether plasma VWF status can be used as a disease marker for diagnosis and monitoring therapy in patients with acute iTTP.Objective: To address this question, we determined plasma levels of VWF antigen (VWF:Ag), collagen-binding activity (VWF:CB), active forms of VWF (VWF:Ac), and VWF multimers in iTTP patients during acute episode and in early remission.Patients and Methods: From the Alabama registry, we identified 69 unique patients with a confirmed diagnosis of iTTP in whom plasma ADAMTS13 activity was <10 U/dL with positive inhibitors and elevated anti-ADAMTS13 IgGs. Of 69 patients, 21 had longitudinal plasma samples collected. Plasma samples from 56 healthy individuals, who did not have a hematological disease, cancer, and infection, were recruited as controls. Plasma levels of VWF:Ag, VWF:CB, and VWF:Ac were determined by an ELISA-based assay. Plasma VWF multimer distribution was assessed by an in-gel Western blotting assay following electrophoresis on a 1% SDS-agarose gel.Results: The mean age for our cohort iTTP patients was 43.9 ± 13.4 years. Twenty-six patients were male and 43 were female with male to female ratio of 1 to 1.7. Fifty-three patients were African American descents, 14 Caucasians, 1 Hispanic, and 1 unknown race. Plasma levels of VWF:Ag in acute iTTP patients were 289.4 ± 17.7%, significantly increased compared with those in the healthy controls (144.9 ± 7.6%) (p<0.0001); plasma levels of VWF:CB in these patients were 241 ± 17.9%, also significantly elevated compared with those in the healthy controls (149.9 ± 12.01%) (p=0.0001); additionally, plasma levels of VWF:Ac (304.6 ± 23.2%), assessed by its ability to bind anti-VWF-A1 nanobody, were more dramatically elevated compared with those in the controls (101.6 ± 5.9%) (p<0.0001). More interestingly, while the ratios of VWF:CB to VWF:Ag in patients with acute iTTP (0.8 ± 0.04) were lower than those in the healthy controls (1.0 ± 0.05) (p=0.0036), the ratios of VWF:Ac to VWF:Ag were significantly higher in patients with acute episode (1.2 ± 0.1) than those in the controls (0.8 ± 0.05) (p=0.0003). Furthermore, there was no statistically significant difference in the patient plasma levels of VWF:Ag (p=0.69) and VWF:CB (p=0.08) during acute episode and during early remission. However, the plasma levels of VWF:Ac in patients with acute disease were significantly higher than those in the early remission (p=0.002). Surprisingly, 90% (36/40) of out iTTP patients during acute episode showed the presence of ULVWF in their plasma using in-gel Western blotting, which allows the ULVWF to be detected without the transfer step to avoid any potential loss of larger VWF multimers during protein transfer. These ULVWF multimers disappeared in 3/4 iTTP patients in remission when ADAMTS13 activity recovered. In 28 healthy control samples, only one showed ULVWF.Conclusion: Our results demonstrate, for the first time in a large cohort, that active forms of VWF and ultra large VWF multimers are present in iTTP patient's plasma during the acute period, which is reduced or disappears during the early remission. Therefore, measuring active forms of VWF and ultra large VWF multimers may aid in diagnosis of iTTP and help monitoring of disease processes following therapy. Our ongoing study is to determine whether these biomarkers can be used to predict responses to treatment and long-term outcome. DisclosuresZheng:Alexion: Research Funding, Speakers Bureau.

A Novel Approach to Immune Tolerance Induction in Hemophilia Α with Factor VIII Inhibitor

Factor VIII (FVIII) inhibitors are currently the most significant complication of hemophilia A therapy. Immune tolerance induction (ITI) is the treatment of choice for inhibitor eradication. However, the optimal ITI regimen has not been identified. We describe an ITI approach for an adolescent male with severe hemophilia A and recurrent and refractory high-titer FVIII inhibitor. This strategy is now being tested in a randomized, controlled trial (NCT03204539).A 1-year-old white, Hispanic male with severe hemophilia A, intron 22 inversion, developed a FVIII inhibitor while receiving on-demand treatment with recombinant FVIII (rFVIII). Due to life-threatening bleeding despite bypassing agents, he was immediately started on ITI with daily rFVIII along with intravenous immunoglobulin (IVIg), solumedrol, and rituximab. He achieved tolerance after 15 months and was switched to every-other-day dosing for prophylaxis. Unfortunately, on prophylaxis he had breakthrough bleeding and inhibitor recurrence, and has required additional ITI regimens over the past 14 years (Table 1).Most recently, the family agreed to switch to Wilate® [von Willebrand Factor/Coagulation Factor VIII Complex (Human); Octapharma USA, Inc.; Hoboken, NJ; U.S. License No. 1646] as an alternative von Willebrand factor (VWF)/FVIII concentrate since he was unable to achieve tolerance on prior plasma-derived (pd) VWF/FVIII concentrate at 100 international units (IU)/kg daily for ~4 years. Wilate® was started when inhibitor titer was 1.5 Bethesda Units (BU). Initial FVIII recovery was 30% and 24-hour trough level was 1%. After several months, blood samples were sent to a diagnostic laboratory at Haemophilie-Zentrum Rhein Main GmbH for lot selection. Lot selection entails measuring residual FVIII activity when patient plasma is mixed with different lots of VWF/FVIII in vitro. This involves using the modified New Oxford method to measure residual FVIII activity after incubation of a FVIII source (lot) with the inhibitor patient plasma. The inhibitor titer is the reciprocal of the dilution of patient plasma that results in 50% of residual FVIII, similar to the Bethesda Unit. Ideally, the lot providing the highest residual FVIII activity will more effectively challenge the immune system, provide better prevention and control of bleeding, and have shorter time to tolerance. Investigators have demonstrated the utility of lot section in in vitro studies. The patient's plasma was tested against 6 lots of Wilate® and the lot with the lowest inhibitor activity was selected for prescription. This lot was allocated to this patient, and the prescribing physician included the lot number on the factor prescription for distribution to the patient via the patient's specialty pharmacy. The patient received Wilate® from the same lot for ~11 months. During that time, his inhibitor decreased from 2.6 BU to 0 after 5 months. A new plasma sample was tested against an additional 5 lots of Wilate®. Inhibitor was negative at that time and all lots of Wilate® revealed a negative inhibitory activity. One lot was selected for ongoing treatment. After 18 months his 48-hour trough FVIII level was detectable. He had blood drawn for pharmacokinetic analysis, which showed FVIII recovery of 55% and an estimated half-life of 6.75 hours. He was switched to every-other-day dosing and has had only one trauma-induced soft-tissue bleed despite increased physical activity, with negative inhibitor and a 48-hour trough of 2%. DisclosuresThornburg:Shire: Research Funding; CSL Behring: Research Funding; ATHN: Research Funding; Bayer Pharmaceuticals: Research Funding; Octapharma: Research Funding; Bioverativ: Consultancy; Genentech: Speakers Bureau; Biomarin: Consultancy; Bluebird Bio: Consultancy; NovoNordisk: Research Funding; Johns Hopkins All Children's Hospital: Research Funding. Ducore:OPKO: Other: investigator; HemaBiologics: Consultancy, Other: investigator, travel support; Shire: Consultancy, Other: travel support, investigator; Biomarin: Other: investigator; Octapharma: Consultancy, Other: travel support, investigator , Research Funding; Bayer Healthcare: Consultancy, Other: travel support, investigator; Pfizer: Other: investigator; Spark Therapeutics: Consultancy, Other: investigator; CSL Behring: Other: investigator.

GPIHBP1 autoantibody syndrome during interferon β1a treatment

Autoantibodies against glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) cause chylomicronemia by blocking the ability of GPIHBP1 to bind lipoprotein lipase (LPL) and transport the enzyme to its site of action in the capillary lumen. A patient with multiple sclerosis developed chylomicronemia during interferon (IFN) β1a therapy. The chylomicronemia resolved when the IFN β1a therapy was discontinued. Here, we sought to determine whether the drug-induced chylomicronemia was caused by GPIHBP1 autoantibodies. We tested plasma samples collected during and after IFN β1a therapy for GPIHBP1 autoantibodies (by western blotting and with enzyme-linked immunosorbent assays). We also tested whether the patient's plasma blocked the binding of LPL to GPIHBP1 on GPIHBP1-expressing cells. During IFN β1a therapy, the plasma contained GPIHBP1 autoantibodies, and those autoantibodies blocked GPIHBP1's ability to bind LPL. Thus, the chylomicronemia was because of the GPIHBP1 autoantibody syndrome. Consistent with that diagnosis, the plasma levels of GPIHBP1 and LPL were very low. After IFN β1a therapy was stopped, the plasma triglyceride levels returned to normal, and GPIHBP1 autoantibodies were undetectable. The appearance of GPIHBP1 autoantibodies during IFN β1a therapy caused chylomicronemia. The GPIHBP1 autoantibodies disappeared when the IFN β1a therapy was stopped, and the plasma triglyceride levels fell within the normal range.

Analysis of the distribution of rice allergens in brown rice grains and of the allergenicity of products containing rice bran.

To understand the allergenicity of rice bran, the distribution of rice allergens in brown rice grains was analysed and the allergenicity of cosmetics and health foods containing rice bran determined. RAG2 and a 19-kDa globulin were localized in polished rice, while a 52-kDa globulin was localized in rice bran. The 52-kDa globulin was also identified as the most likely causative allergen of rice bran allergy. Several products containing intact rice bran were found to contain the 52-kDa globulin. Our study provides the first data regarding cosmetics and health foods containing potential rice bran allergens. Western blot analysis using a rice-bran-allergic patient's plasma showed that 52-kDa globulin was detected as an IgE-binding protein of rice bran and some rice bran-containing cosmetics and health foods. Our results indicate that patients with rice bran allergy need to be careful about using products containing intact rice bran as a constituent.

Zero Diffusive Sodium Balance in Hemodialysis Provided by an Algorithm-Based Electrolyte Balancing Controller: A Proof of Principle Clinical Study.

Restoring and controlling fluid volume homeostasis is still a challenge in contemporary end-stage kidney disease patients treated by intermittent hemodialysis (HD) or hemodiafiltration (HDF). This primary target is achieved by ultrafiltration (dry weight probing) and control of intradialytic sodium transfer (dialysate-plasma Na gradient). The latter task is mostly ignored in clinical practice by applying a dialysate sodium prescription uniform for all patients of the dialysis center but unaligned to individual plasma sodium levels. Depending on the patient's natremia, a positive gradient gives rise to intradialytic diffusive sodium load and postdialytic thirst. On the contrary, a negative gradient may cause unwanted diffusive sodium removal and intradialytic symptoms. To overcome these challenges, a new conductivity-based electrolyte balancing algorithm embedded in a hemodialysis machine with the aim to achieve "zero diffusive sodium balance" in HD and online HDF treatments was tested in the form of a prospective clinical trial. The study comprised two phases: a first phase with a conventional fixed-sodium dialysate (standard care phase), followed by a phase with the electrolyte balancing control (EBC) module activated (controlled care phase). The results show a reduction in the variability of the intradialytic plasma sodium concentration shift, but it is overlain by a small but statistically significant increase in the mean plasma sodium levels. However, no clinical manifestations were observed. This sodium load can be explained by the design of the algorithm based on dialysate conductivity instead of sodium concentration. Furthermore, the increase in plasma sodium can be corrected by taking into account the potassium shift during the treatment. This study showed that the EBC module incorporated in the HD machine is able to automatically individualize the dialysate sodium to the patient's plasma sodium without measuring or calculating predialytic plasma levels from previous laboratory tests. This tool has the potential to facilitate fluid management, to control diffusive sodium flux, and to improve intradialytic tolerance in daily clinical practice.

Detection of mycobacterial small RNA in the bacterial culture supernatant and plasma of patients with active tuberculosis

Bacterial small RNA (sRNA) has been shown to play an important role in control of bacteria virulence, stress response and physiological metabolism by post-transcriptional regulation of gene expression. However, there were few reports about bacterial sRNA as a biomarker of infection. To test the potential role of sRNA in indicating infection of Mycobacterium tuberculosis, total RNA were extracted from the filtrated bacterial cultural supernatant. After synthesis of cDNA by reverse transcription, four Mycobacterial sRNAs including ASdes, ASpks, AS1726, and AS1890, which have been experimentally confirmed by Kristine B in the year of 2009, were detected by real time PCR. The specificity was verified by sequencing of the amplified products. Moreover, we demonstrate that the presence of sRNA Asdes in plasma of 55.56% (15/27) TB patients and 25.00% (6/24) normal controls with BCG vaccination (P < 0.05). Our results suggest that bacterial non-coding sRNA can be detected from either bacterial culture supernatants or patient's plasma. Detecting of Mycobacterial sRNA provides a rapid and relatively noninvasive approach for diagnosing disease and could be developed as a biomarker to identify patients with active tuberculosis to help make informed decisions about proper therapies.1

Abstract 5345: Somatic mutation status in cfDNA between therapy responders and non-responders of oropharyngeal squamous cell carcinoma patients

Abstract Circulating tumor DNA (ctDNA) could potentially be utilized as a biomarker in Oropharyngeal Squamous Cell Carcinoma (SCC) patients. The objective of this study was to determine if somatic variants could be detected in circulating cell-free DNA (cfDNA) of Oropharyngeal SCC patients using Next Generation Sequencing (NGS). Somatic mutations have been reported in Orophayngeal SCC cases. The identification and detection of somatic variants in circulating cell-free DNA in patient's plasma may predict response to therapy, help monitor disease progression, guide therapy, and determine likelihood of recurrence. In this study, we compared the somatic mutation status between therapy responders and non-responders among Orophayngeal SCC patients. IRB approval was obtained and 23 Oropharyngeal SCC patients were identified in our patient database. Genomic DNA was isolated from frozen tumor tissue using NucleoSpin Tissue kit by Clontech. Cell-Free (cfDNA) was then extracted from patient's plasma samples using Zymo's Quick-cfDNATM serum and plasma kit. The quality and size of DNA was determined by Agilent 2200 TapeStation. If genomic DNA contamination was identified it was removed with Zymo Select-a-Size DNA kit. Libraries were prepared with 10 to 25 ηg of cfDNA using Accel-Amplicon 56G Oncology Panel of Swift Biosciences. DNA sequencing was then performed on MiSeq platform with Miseq Reagent Kit. Variants were identified using Biomedical Genomic Workbench and Genialis's online data analysis platform for Swift Biosciences's Accel-amplicon panels. From the 23 matched samples, 8 somatic non-synonymous variants were found in both tissue and plasma, 7 of which were present in non-responders. The variants and their allele frequencies in the non-responder group were as follows (tumor DNA /cfDNA in %) HRAS Gly12Cys (36.54/0.45), TP53 Glu298* (5.48/0.21), CDKN2A Pro114Arg (48.11/1.52), TP53 Arg282Trp (46.47/1.77), FBXW7 Arg505Gly (30.17/0.58), FBXW7 Arg505Leu (30.46/0.64), TP53 Arg273Cys (38.91/2.51), and one variant in the responder group PTEN (21.07/0.67) Arg130Gln. In conclusion, somatic non-synonymous variants can be identified in cfDNA from Oropharyngeal SCC patients using NGS. The number of detected variants is far greater in the non-responder group suggesting somatic variant detection could be a prognostic marker for Oropharyngeal SCC patients. Citation Format: Alok R. Khandelwal, Adam Greer, Rhett Orgeron, Mickie Hamiter, Xiaohui Ma, Tara Moore-Medlin, Hong Yin, Glenn Mills, Cherie-Ann O. Nathan. Somatic mutation status in cfDNA between therapy responders and non-responders of oropharyngeal squamous cell carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5345.

Abstract 3647: Prior DNA repair improves the read quality in next-generation sequencing of cell-free tumor DNA

Abstract The cell-free tumor DNA (ctDNA) in patient's plasma is a noninvasive tumor biomarker. The detection of ctDNA is helpful to evaluate tumor progression, tumor response to the therapy, and subtyping of tumor cells. Targeting next-generation sequencing (NGS) technology provides a sensitive and specific technique for ctDNA detection. It can detect multiple tumor-related gene mutations. However, collection of high quality of cell-free DNA (cfDNA) may not be available in some cases. The quality of cfDNA in plasma is impacted by multiple factors, such as plasma collection and storage conditions, DNase activity in the blood, and the apoptotic and necrotic status of the tumor. These factors affect the integrity of cfDNA, leading to full or partial degradation of cfDNA, including double-strand DNA breaks and single strand DNA nicking. Lesions on cfDNA decrease the usability of ctDNA by digital PCR and targeted NGS. Due to amount limitation of cfDNA in some cases, the repair of damaged cfDNA could be a necessary rescue step in producing viable libraries for NGS. CfDNA was extracted from the plasma with Zymo Research's Quick-cfDNA Serum and Plasma kit and was quantified by realtime PCR with Alu primer sets. The size of cfDNA was about 160 base pairs, as estimated by Agilent Tapestation 2200 with the High Sensitivity DNA kit. The DNA repair of cfDNA was conducted with PreCR Repair Mix of New England BioLabs. The sequencing library was prepared with Accel-Amplicon 56G Oncology Panel of Swift Biosciences and sequenced in Illumina's MiSeq. The quality of sequencing data was determined with FastQC. Compared to PreCR Repair Mix treated cfDNA samples, fastQ data from some of untreated cfDNA samples showed failed per base sequencing quality, about 50% of sequencing cycles with the mean quality score below 30. The per sequencing quality score of these cfDNA samples failed also to pass the quality filter. The read size distribution analysis indicated that about 60 to 70% of read sized between 30 to 45 bases and about 75 to 80% of read was smaller than 75 bases. However, sequencing data from same cfDNA samples treated with PreCR Repair Mix show a greatly improved sequencing quality. Both per base sequencing quality and per sequencing quality score passed quality filter. The above 97% of reads sized over 92 bases, complying with the requirement of 56G Oncology Panel. In conclusion, the DNA repair process before library preparation is necessary to rescue damaged cfDNA for amplicon-based NGS. Citation Format: Hong Yin, Adam H. Greer, Glenn Mills. Prior DNA repair improves the read quality in next-generation sequencing of cell-free tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3647.

Abstract 4542: Analytical validation of the SignateraTM RUO assay, a highly sensitive patient-specific multiplex PCR NGS-based noninvasive cancer recurrence detection and therapy monitoring assay

Abstract Introduction: Liquid biopsy-based detection and monitoring of tumor-specific somatic mutations in cell-free DNA has great potential to improve patient care by detecting cancer early, assisting adjuvant therapy decision-making, monitoring residual disease, determining treatment effects, assessing relapse and the consequent need for follow-up intervention. We have developed a novel highly sensitive and specific approach for the detection and quantification of circulating tumor DNA (ctDNA) by tracking personalized cancer signatures in plasma. We recently described our ability to detect ctDNA presence longitudinally in patients with non-small cell lung cancer (NSCLC) and have validated this personalized approach for detection of tumor DNA in plasma, and made it available for research use only (RUO). Summary of Experimental Procedures: This study describes results of the Signatera RUO assay's analytical validation test titrations of cancer cell lines and their matched normal counterparts. Multiplex-PCR (mPCR) assay pools specific to the tumor mutational signatures were derived from whole-exome data analysis of the tumor cell line and its corresponding matched normal cell line. Tumor DNA spike-in mixtures were amplified using the mPCR assays and then barcoded and sequenced at ultra-high depth on the Illumina HiSeq instrument (average &amp;gt; 100,000 reads per target). This study also demonstrates the ability to detect ctDNA using Signatera RUO assay's personalized somatic mutations in patients with epithelial and non-epithelial ovarian cancer. Results/Data Summary: The Signatera RUO analytical validation results demonstrate a variant-level sensitivity of ~60% at spiked-in tumor DNA concentrations of 0.03% along with an assay specificity of 99.9% in wild-type DNA. By targeting 16 somatic mutations present in the tumor, an estimated LOD of 0.01% tumor DNA in a patient's plasma is achieved. The Signatera RUO assay also demonstrates the ability to detect ctDNA in plasma down to less than 0.1% VAF at baseline (prior to treatment) in epithelial and non-epithelial ovarian cancer. Conclusion: The Signatera RUO assay provides a unique method to noninvasively detect ctDNA by assaying for personalized cancer signatures in plasma by ultra-high-depth sequencing of custom-made multiplex PCR assays, with high sensitivity and high specificity. Our study demonstrates the potential of this technology to improve the current standard of care by enabling early recurrence detection and the monitoring of treatment effectiveness across several tumor types. Citation Format: Himanshu Sethi, Raheleh Salari, Samantha Navarro, Prashanthi Natarajan, Ramya Srinivasan, Scott Dashner, Tony Tin, Mustafa Balcioglu, Ryan Swenerton, Bernhard Zimmermann. Analytical validation of the SignateraTM RUO assay, a highly sensitive patient-specific multiplex PCR NGS-based noninvasive cancer recurrence detection and therapy monitoring assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4542.

Thiopurine-induced toxicity is associated with dysfunction variant of the human molybdenum cofactor sulfurase gene (xanthinuria type II)

BackgroundThe aim of our study was to identify the genetic background of thiopurine-induced toxicity in a patient with a wild-type thiopurine methyltransferase genotype and activity. A 38-year-old Caucasian woman presented with cutaneous necrotizing vasculitis pancytopenia one month after starting azathioprine therapy. MethodsDuring a routine biochemical follow-up of the patient, undetectable serum uric acid (<10 μl) was observed. A high performance liquid chromatography analysis of urinary purines revealed increased levels of xanthine (137 mmol/mol creatinine). The suspected diagnosis of hereditary xanthinuria, a rare autosomal recessive disorder of the last two steps of purine metabolism, was confirmed by sequence analysis. ResultsAn analysis of XDH/XO and AOX1 revealed common polymorphisms, while analysis of the MOCOS gene identified a rare homozygous variant c.362C > T. Dysfunction of this variant was confirmed by significantly decreased xanthine dehydrogenase/oxidase activity in the patient's plasma (<2% of control mean activity). ConclusionsWe present a biochemical, enzymatic, and molecular genetic case study suggesting an important association between a hitherto undescribed dysfunction variant in the MOCOS gene and thiopurine-induced toxicity. The identified variant c.362C > T results in slower thiopurine metabolism caused by inhibition of 6-mercaptopurine oxidation (catabolism) to 6-thioxanthine and 6-thiouric acid, which increases the formation of the nucleotide 6-thioguanine, which is toxic. This is the first clinical case to identify the crucial role of the MOCOS gene in thiopurine intolerance and confirm the impact of genetic variability of purine enzymes on different therapeutic outcomes in patients undergoing thiopurine treatment.

Similarities and Differences of Blood N-Glycoproteins in Five Solid Carcinomas at Localized Clinical Stage Analyzed by SWATH-MS

Cancer is mostly incurable when diagnosed at a metastatic stage, making its early detection via blood proteins of immense clinical interest. Proteomic changes in tumor tissue may lead to changes detectable in the protein composition of circulating blood plasma. Using a proteomic workflow combining N-glycosite enrichment and SWATH mass spectrometry, we generate a data resource of 284 blood samples derived from patients with different types of localized-stage carcinomas and from matched controls. We observe whether the changes in the patient's plasma are specific to a particular carcinoma or represent a generic signature of proteins modified uniformly in a common, systemic response to many cancers. A quantitative comparison of the resulting N-glycosite profiles discovers that proteins related to blood platelets are common to several cancers (e.g., THBS1), whereas others are highly cancer-type specific. Available proteomics data, including a SWATH library to study N-glycoproteins, will facilitatefollow-up biomarker research into early cancer detection.

Whole-Exome Sequencing Identifies an Intronic Cryptic Splice Site in SERPINF1 Causing Osteogenesis Imperfecta Type VI.

ABSTRACTThe heritable disorder osteogenesis imperfecta (OI) is characterized by bone fragility and low bone mass. OI type VI is an autosomal recessive form of the disorder with moderate to severe bone fragility. OI type VI is caused by mutations in the serpin peptidase inhibitor, clade F, member 1 (SERPINF1), the gene coding for pigment epithelium‐derived factor (PEDF). Here, we report a patient with OI type VI caused by a novel homozygous intronic variant in SERPINF1 identified by whole‐exome sequencing (WES). The mutation was not identified using a low bone mass gene panel based on next‐generation sequencing. This variant creates a novel consensus splice donor site (AGGC to AGGT) in intron 4. Analysis of cDNA generated from fibroblasts revealed retention of a 32‐bp intronic fragment between exons 4 and 5 in the cDNA, a result of alternative splicing from the novel splice‐donor site. As a result, the aberrant insertion of this intronic fragment generated a frameshift pathogenic variant and induced nonsense‐mediated decay. Furthermore, gene expression by quantitative PCR showed SERPINF1 expression was dramatically reduced in patient fibroblasts, and PEDF level was also significantly reduced in the patient's plasma. In conclusion, we report a novel homozygous variant that generates an alternative splice‐donor in intron 4 of SERPINF1 which gives rise to severe bone fragility. The work also demonstrates clinical utility of WES analysis, and consideration of noncoding variants, in the diagnostic setting of rare bone diseases. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

RP-HPLC-UV Method for Estimation of Fluorouracil-Epirubicin-Cyclophosphamide and Their Metabolite Mixtures in Human Plasma (Matrix).

A combination of 5-fluorouracil (FU), epirubicin (EP) and cyclophosphamide (CP) is routinely employed in the treatment of breast cancer. The objective of this study was to develop a reverse phase high-performance liquid chromatography (HPLC)-UV method for simultaneous quantitative analysis of the triple-drug and their metabolites in plasma. RP-HPLC system with a C18 column and a diode array detector was employed. The plasma samples were precipitated with acetonitrile and the supernatant was dried under a flow of nitrogen gas. The mobile phase comprised of two combinations, water (pH 4.0) and methanol (98:2 v/v), and water (pH 4.0):methanol:acetonitrile (70:13:17 v/v/v). The retention times for the compounds were determined and the parameters of validation established in plasma indicated the robustness and reliability. The corresponding HPLC peaks were confirmed using electron spray ionization mass spectrometry. FU and metabolites had a recovery of >93%; EP, epirubicinol and CP were >78% from plasma. Stability at 28-30°C in water (pH 4.0) of FU, 5,6-dihydro-5-fluorouracil and EP were higher followed by CP, EPol, fluorodeoxyuridine and fluorouridine (FUR). Storage of the drug-spiked plasma at -80°C assessed for 72 h showed a small but significant (P < 0.05) change in the recovery of FUR and EP. The method was validated in patient's plasma samples (n = 6).

Therapeutic plasma exchange: Experience in a third level hospital, 2013-2016, Lima (Peru).

Therapeutic plasma exchange (TPE) is an extracorporeal procedure which consists of removing the patient's plasma and replacing it with an appropriate replacement fluid. Plasma and blood cells are separated by a centrifugation process. Our department has used TPE for several years, and in 2013 we introduced an institutional apheresis protocol. The main objective of this report is to describe the TPE procedures performed between 2013 and 2016 in the Peruvian population. We analyzed the technical and clinical aspects of 864 centrifugal TPE procedures as well as the associated complications. We evaluated 230 patients treated in our institution. The therapeutic indications included 16 different diseases: 89.5% (N = 206/230) neurological, 7.3% (N = 17/230) hematological, 1.7% (N = 4/230) rheumatologic, 0.8% (N = 2/230) dermatological and 0.4% (N = 1/230) nephrological. 70.4% (N = 142/230) of patients were diagnosed with Guillain-Barré syndrome. Albumin 5% solution was the most frequent replacement solution, used in 65.8% (N = 569/864) of the procedures. The mean plasma volume (PV) replaced was 2451.73 ml, corresponding to 1.0 PV in all procedures. Complications occurred in 10.9% (N = 95/864) of the sessions. Allergic reactions were the most common events and cardiopulmonary arrests were recorded in two patients. This is the first report of TPE performed in the Peruvian population. The use of an institutional apheresis protocol was beneficial to improve registries in our service and our professional health attention. This study reports a low rate of complications, suggesting that TPE is safe. There is a need to create a multicenter Peruvian apheresis registry to assess the benefits and risks of TPE in Peru.

Saibokuto as a Possible Therapy for Type B Insulin Resistance Syndrome: The Disappearance of Anti-insulin Receptor Antibody and a Marked Amelioration of Glycemic Control by Saibokuto Treatment

Type B insulin resistance syndrome is a rare autoimmune disease and no effective therapy has yet been established. On the other hand, it is known that Saibokuto, one type of Japanese Kampo medicine, may have beneficial effects on various symptoms associated with this disease and it is therefore occasionally prescribed for various immune disorders. We herein describe a case of type B insulin resistance syndrome in which anti-insulin receptor antibody disappeared and the patient's glycemic control markedly improved after the administration of Saibokuto. At first, we administered various anti-oral diabetic drugs and insulin therapy, but the patient's glycemic control became further aggravated. In addition, Helicobacter pylori eradication therapy was not effective, although its benefit has been reported. Interestingly, after the patient started taking Saibokuto, her glycemic control markedly improved. In addition, the patient's plasma insulin levels markedly decreased and anti-insulin receptor antibody became negative after taking Saibokuto. Taken together, there is a possibility that Saibokuto may one of the options for type B insulin resistance syndrome therapy.