Partial Rearrangements Research Articles

DEVELOPMENT OF ULTRASTRUCTURAL CHANGES IN DIAPHRAGM MUSCLE FIBERS DURING PARTIAL TRACHEAL STENOSIS IN YOUNG SEXUALLY IMMATURE RATS

Introduction. The study of congenital pathology of the respiratory tract in children is of great importance for theoretical and clinical pediatric otolaryngology. Currently, in the scientific literature there are single works concerning the study of the pathogenesis and clinic of laryngomalacia. First of all, this concerns the effect of acute and chronic hypoxia on the muscles of the respiratory system, in particular, the diaphragm. The aim of thіs work is to reveal the effect of hypoxia on the development of pronounced structural and functional changes in the diaphragm muscles of sexually immature animals when modeling partial tracheal occlusion. Materials and methods. Surgical partial occlusion of the trachea was performed on in sexually immature Wistar rats; we applied electron microscopy, morphometric and statistical research methods.
 Results. The obtained data demonstrated that the muscle fibers of the diaphragm are characterized by significant structural, functional and metabolic resistance to the development of acute or chronic hypoxia due to limited lung ventilation in rats. The density of muscle fibers did not change in any of the used terms of the experiment (7 and 21 days). On the 7th day of hypoxic exposure, a change in the state of the matrix of mitochondria of muscle fibers was observed, which was accompanied by partial ultrastructural rearrangements of the organization of these organelles. At the same time, no significant changes were observed during histological studies. Electrongrams demonstrated the ultrastructural damages were mainly limited to focal reduction of myofibrils and an increase in the mitochondria's diameter and found as especially pronounced on day 21 (a state of chronic hypoxia) on the muscular component of the respiratory system in rats. These changes are accompanied by the development of dystrophic disorders in the diaphragm myofibrils. On day 21 changes in the transendothelial transport of substance molecules were also detected. This was based on establishing the fact of a significant reduction in the number of pinocytosis vesicles in the cytoplasm of endothelial cells of diaphragm hemocapillaries in animals with partial tracheal occlusion. Disruption of the structural organization of actomyosin complexes was revealed in muscle fibers due to pathological changes in the mitochondria of myofibrils. It should be noted the development of destruction or complete loss of an important ultrastructural component of muscle fibers, Z-lines. These changes were completely absent on the 7th day following the influence of partial tracheal occlusion and the development of acute hypoxia. Moreover, on day 21, partial lysis of a significant amount of myofilaments of muscle fibers was observed. In addition, the development of the process of fiber swelling was detected against the background of which the above-mentioned ultrastructural destruction of the muscular component in the diaphragm was observed. These changes indicate the need for earlier treatment of laryngomalacia in children. The delay in surgical intervention leads to the development of severe or irreversible structural changes not only in the respiratory organs, but also in their muscular system.
 Conclusion. The muscular elements of the diaphragm have the pronounced and significant resistance to the state of hypoxia under conditions of limited ventilation of the respiratory tract. Ultrastructural and morphometric data, found on the 21st day after the modeling of partial tracheal stenosis, are not only of a compensatory and adaptive in nature, but also signs of pronounced disorders in the muscle fibers of the central part of the diaphragm in test animals.

Novel bimodal TRBD1-TRBD2 rearrangements with dual or absent D-region contribute to TRB V-(D)-J combinatorial diversity.

T-cell receptor (TR) diversity of the variable domains is generated by recombination of both the alpha (TRA) and beta (TRB) chains. The textbook process of TRB chain production starts with TRBD and TRBJ gene rearrangement, followed by the rearrangement of a TRBV gene to the partially rearranged D-J gene. Unsuccessful V-D-J TRB rearrangements lead to apoptosis of the cell. Here, we performed deep sequencing of the poorly explored pool of partial TRBD1-TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human partial TRBD1-TRBD2 rearrangements using novel sequencing and validated them by detecting V-D-J recombination-specific byproducts: excision circles containing the recombination signal (RS) joint 5'D2-RS - 3'D1-RS. Identified rearrangements were in compliance with the classical 12/23 rule, common for humans, rats, and mice and contained typical V-D-J recombination footprints. Interestingly, we detected a bimodal distribution of D-D junctions indicating two active recombination sites producing long and short D-D rearrangements. Long TRB D-D rearrangements with two D-regions are coding joints D1-D2 remaining classically on the chromosome. The short TRB D-D rearrangements with no D-region are signal joints, the coding joint D1-D2 being excised from the chromosome. They both contribute to the TRB V-(D)-J combinatorial diversity. Indeed, short D-D rearrangements may be followed by direct V-J2 recombination. Long D-D rearrangements may recombine further with J2 and V genes forming partial D1-D2-J2 and then complete V-D1-D2-J2 rearrangement. Productive TRB V-D1-D2-J2 chains are present and expressed in thousands of clones of human antigen-experienced memory T cells proving their capacity for antigen recognition and actual participation in the immune response.

Combining MYD88 L265P mutation detection and clonality determination on CSF cellular and cell-free DNA improves diagnosis of primary CNS lymphoma.

Diagnosis of primary central nervous system lymphoma (PCNSL) is challenging, and although brain biopsy remains the gold standard, cerebrospinal fluid (CSF) constitutes a less invasive source of lymphomatous biomarkers. In a retrospective cohort of 54 PCNSL cases tested at diagnosis or relapse, we evaluated the contribution of immunoglobulin heavy chain (IGH) gene clonality and MYD88 L265P detection on both CSF cell pellets and supernatants, in comparison with cytology, flow cytometry, interleukin (IL)-10 and IL-6 quantification. Clonality assessment included a new assay to detect partial IGH-DJ rearrangements. Clonal IGH rearrangements and/or MYD88 L265P mutation were detected in 27 (50%) cell pellets and 24 (44%) supernatant cell-free (cf) DNA. Combining analyses on both compartments, 36 (66%) cases had at least one detectable molecular marker, present only in cfDNA for 9 (16%) of them. While cytology and flow cytometry were positive in only 7 (13.0%) and 9 (17.3%) cases respectively, high IL-10 levels were observed in 36 (66.7%) cases. Overall, taking into account molecular and cytokine results, 46/54 (85%) cases had at least one lymphomatous biomarker detectable in the CSF. These results show that this combination of biomarkers evaluated on both cell pellet and supernatant CSF fractions improves significantly the biological diagnosis of PCNSL.

Aneuploidy promotes intraspecific diversification of the endemic East Asian herb Lycoris aurea complex.

Polyploidy has received considerable interest in the past, but aneuploidy and partial rearrangements may also influence genomic divergence. In this study, we reported a comprehensive cytogeographic, morphological and genetic analysis of Lycoris aurea complex throughout its range and attempted to explore the association between aneuploidy and species diversification. The karyotypes of this complex presented aneuploidy variations mainly divided into four cytotypes: I (2n = 10m + 2T), II (2n = 8m + 6T), III (2n = 7m + 8T), and IV (2n = 6m + 10T). Cytotype distributions were highly structured geographically. Two main cytotypes, II and IV, are geographically allopatric. The populations with cytotype II are mainly distributed in central China and the southern islands of Japan. Cytotypes IV is disjunctly distributed in southwestern and southeastern China. The cytotypes with fewer chromosome numbers tend to occur at high latitudes. For analyzing the phylogeographic pattern and genetic structure of this complex, we sequenced four chloroplast DNA fragments (4,748 bp in total) of 241 individuals from 42 populations. Extremely high diversity of cpDNA haplotypes was found, with genetic diversity index (Hd) being 0.932 and 98.61% of the genetic variation occurring among populations, indicating that this complex has undergone strong intraspecific differentiation. The cytotype II had the highest haplotype diversity (Hd = 0.885), while cytotype IV harbored the highest nucleotide diversity (π = 4.09 × 10–3). We detected significant leaf morphological differences not only between cytotype II and IV but also between west lineage and east lineage within cytotype IV. These results illustrated that aneuploidy contributed to extensive morphological and genetic differentiation in L. aurea complex. It was suggested that L. aurea complex should comprise multiple independent evolutionary lineages, and accurate species delimitation needs to be established further in an integrative taxonomic approach.

Misregulation of the IgH Locus in Thymocytes.

Functional antigen receptor genes are assembled by somatic rearrangements that are largely lymphocyte lineage specific. The immunoglobulin heavy chain (IgH) gene locus is unique amongst the seven antigen receptor loci in undergoing partial gene rearrangements in the wrong lineage. Here we demonstrate that breakdown of lineage-specificity is associated with inappropriate activation of the Eμ enhancer during T cell development by a different constellation of transcription factors than those used in developing B cells. This is reflected in reduced enhancer-induced epigenetic changes, eRNAs, formation of the RAG1/2-rich recombination center, attenuated chromatin looping and markedly different utilization of DH gene segments in CD4+CD8+ (DP) thymocytes. Additionally, CTCF-dependent VH locus compaction is disrupted in DP cells despite comparable transcription factor binding in both lineages. These observations identify multiple mechanisms that contribute to lineage-specific antigen receptor gene assembly.

Spontaneous refolding of the large multidomain protein malate synthase G proceeds through misfolding traps

Most protein folding studies until now focus on single domain or truncated proteins. Although great insights in the folding of such systems has been accumulated, very little is known regarding the proteins containing multiple domains. It has been shown that the high stability of domains, in conjunction with inter-domain interactions, manifests as a frustrated energy landscape, causing complexity in the global folding pathway. However, multidomain proteins despite containing independently foldable, loosely cooperative sections can fold into native states with amazing speed and accuracy. To understand the complexity in mechanism, studies were conducted previously on the multidomain protein malate synthase G (MSG), an enzyme of the glyoxylate pathway with four distinct and adjacent domains. It was shown that the protein refolds to a functionally active intermediate state at a fast rate, which slowly produces the native state. Although experiments decoded the nature of the intermediate, a full description of the folding pathway was not elucidated. In this study, we use a battery of biophysical techniques to examine the protein's folding pathway. By using multiprobe kinetics studies and comparison with the equilibrium behavior of protein against urea, we demonstrate that the unfolded polypeptide undergoes conformational compaction to a misfolded intermediate within milliseconds of refolding. The misfolded product appears to be stabilized under moderate denaturant concentrations. Further folding of the protein produces a stable intermediate, which undergoes partial unfolding-assisted large segmental rearrangements to achieve the native state. This study reveals an evolved folding pathway of the multidomain protein MSG, which involves surpassing the multiple misfolding traps during refolding.

Genome sequence comparison between Chinese hamster ovary (CHO) DG44 cells and mouse using end sequences of CHO BAC clones based on BAC-FISH results.

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology as a mammalian host cell platform for expressing genes of interest. Previously, we constructed a detailed physical chromosomal map of the CHO DG44 cell line by fluorescence in situ hybridization (FISH) imaging using 303 bacterial artificial chromosome (BAC) clones as hybridization probes (BAC-FISH). BAC-FISH results revealed that the two longest chromosomes were completely paired. However, other chromosomes featured partial deletions or rearrangements. In this study, we determined the end sequences of 303 BAC clones (BAC end sequences), which were used for BAC-FISH probes. Among 606 BAC-end sequences (BESs) (forward and reverse ends), 558 could be determined. We performed a comparison between all determined BESs and mouse genome sequences using NCBI BLAST. Among these 558 BESs, 465 showed high homology to mouse chromosomal sequences. We analyzed the locations of these BACs in chromosomes of the CHO DG44 cell line using a physical chromosomal map. From the obtained results, we investigated the regional similarities among CHO chromosomes (A-T) and mouse chromosomes (1-19 and sex) about 217 BESs (46.7% of 465 high homologous BESs). Twenty-three specific narrow regions in 13 chromosomes of the CHO DG44 cell line showed high homology to mouse chromosomes, but most of other regions did not show significant correlations with the mouse genome. These results contribute to accurate alignments of chromosomes of Chinese hamster and its genome sequence, analysis of chromosomal instability in CHO cells, and the development of target locations for gene and/or genome editing techniques.

Abstract 2721: Prospective whole exome and whole transcriptome sequencing of hematologic cancers for biomarker-driven treatment : Preliminary results of a randomized pragmatic clinical trial

Abstract Introduction: Biomarker-driven early clinical trials of molecularly targeted agents have reported improvement of the response rate in various malignancies. In this pragmatic clinical trial, we aimed to assess whether biomarker-driven treatment strategy could improve outcomes of incurable hematologic cancer patients. Methods: This is an open-label, randomized, pragmatic trial in adult patients with hematologic cancer. Patients with any kind of hematologic cancer refractory to standard-of-care are eligible. We randomly assigned these patients to receive a matched molecularly targeted agent (experimental group) or treatment at physician’s choice (control group). Randomizations are determined by central block randomization in 2:1 ratio. Clinicians and patients are not masked to allocation. Whole exome and whole transcriptome sequencing (WES/WTS) are applied for detecting all classes of genomic alterations: single nucleotide variation (SNV), insertions and deletions (INDEL), copy number alterations, Fms-Related Tyrosine Kinase 3 (FLT3) internal tandem duplication (ITD), Mixed-lineage leukemia partial tandem duplication and gene rearrangements. The primary endpoint is response rate in the intention-to-treat population. Results: Twenty-three patients with median age of 53 years and male to female ratio of 11:12 were enrolled between December 2015 and October 2016; 15 patients in experimental group, 8 patients in control group. Actionable mutations were identified in four patients in experimental arm. A 78-year-old female with acute myelogenous leukemia (AML) had FLT3 ITD at exon 14, which was identified as a long insertion mutation in SNV/INDEL calling. She received ASP2215 (FLT3 inhibitor), and showed a dramatic reduction in blast counts of peripheral blood. A 75-year-old female with myelodysplastic syndrome had TSC1P39A mutation. Because mTOR exists in downstream signaling pathway of TSC1, everolimus was administered and regression of disease activity, reflected by reduction of C-reactive protein level, was observed. NOTCH2 c.17′ and KITD815V mutation were identified in two patient; 69-year-old female with multiple myeloma and 42-year-old male with AML, respectively. Among 8 patients in control arm, seven patients received cytotoxic chemotherapy. One undergone another comprehensive genomic profiling called FoundationOne Heme, however no actionable mutations was identified. Conclusions: Among patients with incurable hematologic malignancy, actionable mutations could be identified in one fourth of patients for whom WES/WTS were performed. They showed favorable initial response to molecularly targeted agents. This randomized pragmatic clinical trial is currently ongoing(NCT02758080). Citation Format: Sanga Kim, Ryul Kim, Youngil Koh, Choong Hyun Sun, Hogune Im, Hong Seok Yun, Dong-Yeop Shin, Inho Kim, Sung-soo Yoon. Prospective whole exome and whole transcriptome sequencing of hematologic cancers for biomarker-driven treatment : Preliminary results of a randomized pragmatic clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2721. doi:10.1158/1538-7445.AM2017-2721

An unusual case of acute leukemia

We report the case of a 31 year-old man diagnosed with an atypical acute leukemia difficult to characterize cytologically. The immunophenotyping identified a blastic population co-expressing myeloid, lymphoid B and lymphoid T markers suggesting the diagnosis of either a mixed phenotype acute leukemia (MPAL) or an early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Because of the poor prognosis linked to these leukemias, the patient benefited from chemotherapy targeting both myeloid and lymphoid components, followed by allogeneic hematopoietic stem cell transplantation. DNA-based techniques analyzing B and T-cell clonality identified partial rearrangements in immunoglobulin and TCR genes, allowing the monitoring of minimal residual disease. This observation highlights the difficulty to classify some atypical cases of acute leukemias. It emphasizes on the complementarity of cytomorphology, immunophenotyping by flow cytometry and molecular techniques in order to promptly characterize and treat these leukemias.

Spermatogenic failure and the Y chromosome.

The Y chromosome harbors a number of genes essential for testis development and function. Its highly repetitive structure predisposes this chromosome to deletion/duplication events and is responsible for Y-linked copy-number variations(CNVs) with clinical relevance. The AZF deletions remove genes with predicted spermatogenic function en block and are the most frequent known molecular causes of impaired spermatogenesis (5-10% of azoospermic and 2-5% of severe oligozoospermic men). Testing for this deletion has both diagnostic and prognostic value for testicular sperm retrieval in azoospermic men. The most dynamic region on the Yq is the AZFc region, presenting numerous NAHR hotspots leading to partial losses or gains of the AZFc genes. The gr/gr deletion (a partial AZFc deletion) negatively affects spermatogenic efficiency and it is a validated, population-dependent risk factor for oligozoospermia. In certain populations, the Y background may play a role in the phenotypic expression of partial AZFc rearrangements and similarly it may affect the predisposition to specific deletions/duplication events. Also, the Yp contains a gene array, TSPY1, with potential effect on germ cell proliferation. Despite intensive investigations during the last 20years on the role of this sex chromosome in spermatogenesis, a number of clinical and basic questions remain to be answered. This review is aimed at providing an overview of the role of Y chromosome-linked genes, CNVs, and Y background in spermatogenesis.

Acute Leukemia Trilineage Blasts Assignment and Epigenetic Mutations

Mixed Phenotype Acute Leukaemia (MPAL) is a heterogeneous group of rare leukemias in which assigning a single lineage of origin is not possible. We report a rare case of a MPAL without any clear cytological characterisation. Immuno-phenotyping identifies a blastic population co-expressing myeloid, B-lymphoid and T-lymphoid markers. The diagnosis of MPAL is considered and the patient receives chemotherapy targeting both myeloid and lymphoid components, followed by allogeneic hematopoietic stem cell transplantation. DNA-based techniques analyzing B and T-cell clonality identified the presence of partial rearrangements in immunoglobulin and TCR genes. Mutations in DNMT3A, IDH1/2, Flt3, NPM1 and ASXL1 genes were analysed. We did not observe any mutations in IDH2, Flt3 and ASXL1 genes. A several mutations were in DNMT3A, IDH1 and NPM1 genes, allowing the monitoring of minimal residual disease.

Control plane innovations to realize dynamic formulation of multicast sessions in inter-DC software-defined elastic optical networks

It is known that to support the applications such as datacenter backup and migration, multicast should be supported efficiently in inter-datacenter (inter-DC) networks to carry the corresponding point-to-multiple-point communications. Moreover, due to the traffic dynamics in inter-DC networks, we might have to consider the case that the multicast members can join or leave a multicast session dynamically. Therefore, in this work, we try to leverage control plane innovations to realize dynamic formulation of multicast sessions in inter-DC software-defined elastic optical networks (SD-EONs), which are equipped with multicast-incapable bandwidth-variable wavelength selective switches (MI-BV-WSS'). Here, one key issue to address is that the continuous changing of multicast group members can degrade the optimality of a multicast-tree. Hence, we propose to rearrange the multicast-trees adaptively to reduce their spectrum usage. Meanwhile, we try to minimize the frequency of rearrangements to avoid unnecessary operation complexity. Based on these considerations, we propose several multicast-tree rearrangement algorithms for updating multicast sessions dynamically with lightpath reroutings in inter-DC SD-EONs. Both partial and full multicast-tree rearrangements are studied. Simulation results indicate that the proposed algorithms can rearrange the multicast-trees intelligently such that the blocking probability can be reduced effectively with the least lightpath reroutings. Next, based on these theoretical investigations, we consider how to implement the proposed algorithms in the control plane of an inter-DC SD-EON. We extend the OpenFlow (OF) protocol to support the dynamic formulation of multicast sessions and also design the functional models in the control plane elements to realize multicast-tree rearrangements. Experiment results verify the effectiveness of our proposed algorithms and system design.

Pattern of immunoglobulin and T-cell receptor-δ/γ gene rearrangements in Iranian children with B-precursor acute lymphoblastic leukemia

IntroductionAcute lymphoblastic leukemia (ALL) cells have unique rearranged immunoglobulin heavy chain (IgH), immunoglobulin light chain (IgK), and T-cell receptor (TCR) genes, which can be used as markers for clonality assay and evaluation of minimal residual disease. In this study, we have evaluated the pattern of IgH, IgK chains, and TCRG/D gene rearrangements in precursor-B ALL.Materials and methodsIn our prospective study, hyper-variable regions (CDRI and III) of IgH, TCRD (Vδ2–Dδ3 and Dδ2–Dδ3), TCRG (Vγ, VγI, and VγII), and IgK (Vκ-Kde) were studied in 126 cases with diagnosis of B-precursor ALL.ResultsOne hundred and fourteen (90.5%) out of 126 patients had clonal rearrangements of IgH using consensus primers for CDRI and/or CDRIII regions. Monoclonal, biclonal, and oligoclonal patterns were observed in 63 (57.8%), 38 (34.9%), and 6 (5.5%) patients with IgH (CDRIII) rearrangements, respectively. Clonal rearrangements of TCRG (Vγ) and VγI/II were present in 79.3 and 64.9% of patients, respectively, and only 5% of cases showed biclonal pattern. The VγII rearrangement was the most common (46.8%) type in TCRG. Vδ2–Dδ3 and Dδ2–Dδ3 partial gene rearrangements were observed in 47 (45.2%; n = 104) and 11 (16.6%; n = 66) patients, respectively. Biclonal/oligoclonal patterns were present in 13 (27.7%) and 2 (4.3%) cases with Vδ2–Dδ3 rearrangement, respectively. Only one patient had biclonal Dδ2–Dδ3 rearrangement. Clonal pattern of IgK-Kde was detected in 59 cases (67%; n = 88).ConclusionOur findings showed that clonal rearrangements of IgH and TCRD (Vδ2–Dδ3 and Dδ2–Dδ3) genes had similar patterns to other studies. Frequency of TCRG (VγI and VγII) and IgK rearrangements was found to be slightly higher than previous reports. Among the IgK rearrangements, VKI (25%) was the most common.

Clinical relevance of Y-linked CNV screening in male infertility: new insights based on the 8-year experience of a diagnostic genetic laboratory

AZF microdeletion screening is routinely performed in the diagnostic work-up for male infertility; however, some issues remain debated. In this study, we provide insights into the sperm concentration cutoff value for routine testing, the predictive value of AZFc deletion for testicular sperm retrieval and the Y-background contribution to the interpopulation variability of deletion frequencies. In the Spanish population, partial AZFc rearrangements have been poorly explored and no data exist on partial duplications. In our study, 27/806 (3.3%) patients carried complete AZF deletions. All were azoo/cryptozoospermic, except for one whose sperm concentration was 2 × 10(6)/ml. In AZFc-deleted men, we observed a lower sperm recovery rate upon conventional TESE (9.1%) compared with the literature (60-80% with microTESE). Haplogroup E was the most represented among non-Spanish and hgr P among Spanish AZF deletion carriers. The analysis of AZFc partial rearrangements included 330 idiopathic infertile patients and 385 controls of Spanish origin. Gr/gr deletion, but not AZFc partial duplications, was significantly associated with spermatogenic impairment. Our data integrated with the literature suggest that: (1) routine AZF microdeletion testing could eventually include only men with ≤2 × 10(6)/ml; (2) classical TESE is associated with low sperm recovery rate in azoospermic AZFc-deleted men, and therefore microTESE should be preferred; (3) Y background could partially explain the differences in deletion frequencies among populations. Finally, our data on gr/gr deletion further support the inclusion of this genetic test in the work-up of infertile men, whereas partial AZFc duplications do not represent a risk for spermatogenic failure in the Spanish population.

Minimal Residual Disease Quantification in Acute Lymphoblastic Leukemia Using High-Throughput Sequencing of Multiple Immunoreceptor Genes Predicts Relapse and Survival After Allogeneic Hematopoietic Cell Transplantation.

AbstractAbstract 2512 Background:Tremendous progress has been made in the management of acute lymphoblastic leukemia (ALL) in children, in part, through the wide use of minimal residual disease (MRD) monitoring to guide therapeutic intensification before and after allotransplantation. Unfortunately, poor accessibility and the high costs of MRD testing have limited its use in the management of adult ALL. A universally applicable MRD quantification method has the potential to revolutionize the management of ALL in adults. Most B- and T-cell ALL patients exhibit clonal rearrangements of one or more immunoglobulin (heavy chain, IGH; light chain, IGK/IGL) or T-cell receptor (beta, B; delta, D; gamma, G) genes. Such rearrangements may be quantified in a mixture of polyclonal B or T cells by massively parallel high-throughput sequencing (HTS), enabling highly sensitive MRD quantification. Methods:Thirty-six allografted ALL patients were selected for this retrospective study based on availability of a diagnostic sample containing leukemic cells, which was necessary for validating the amplification and sequencing method with each disease clonotype. Using Sequenta’s LymphoSIGHT platform, we amplified and sequenced rearranged immunoreceptor (IR) loci from genomic DNA extracted from peripheral blood (PB) or bone marrow aspirates (BM) using V and J segment consensus primers for each IR gene (IGH, TCRB, TCRD, and TCRG) and, in some cases, D segment primers for incomplete IGH-DJ rearrangements. Sequences were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high prevalence in a PB or BM sample at a time of high disease burden. MRD levels were then determined in serial samples of PB or BM and quantified using spiked-in reference sequences. A total of 227 samples with a median 442,672 input genomes (range 8,038 – 7,162,715) were evaluated by IR-HTS. Results:A clonal IGH sequence was identified in 17/36 (47%) ALL patients. Amongst patients who did not have a detectable IGH clonotype, 14/19 (74%) had clonal sequences at one or more other loci, including partial IGH-DJ rearrangements (5/14; 36%), TCRB (4/14; 29%), TCRD (3/14; 21%), or TCRG (6/14; 43%). In total, 31 of 36 (86%) ALL patients had a clonal IR sequence suitable for MRD quantification. In 4 of 5 cases without an identified clonal sequence, only PB, but no BM samples, were available. Twenty patients achieved MRD negativity following HCT and 11 did not. In the MRD negative group, 12/20 patients (60%) ultimately relapsed with a median time to clinical progression of 231 days (range 77–889), whereas 11/11 patients (100%) in the MRD positive group relapsed with a median time to clinical progression of 139 days (range 60–304). The 12 patients who relapsed after achieving MRD negativity showed a median time to molecular disease progression of 93 days (range 59–689 days). Of the 8 patients who maintained MRD negativity following HCT, 7 remain alive at a median 1542 days (range 1133–2557 days). All 22 patients with MRD detected following HCT relapsed and 21 (95%) died (median survival 387 days; range 85–1991 days) (Figure 1). The lead time between molecular disease detection by IR-HTS and clinical relapse was a median 69 days (range 0–207 days) with significant likelihood of MRD detection in a PB sample at least one, three, and six months (each p<0.0001) prior to clinical relapse. Amongst 50 contemporaneous PB and BM samples, 25 (50%) were concordantly MRD negative, 7 (14%) were MRD positive in BM but negative in PB, and 15 (30%) exhibited significantly higher MRD in BM versus PB (median 41-fold higher, range 1.1 – 411-fold). Conclusions:We demonstrate a method for quantifying acute lymphoblastic leukemia MRD in the majority of patients using high-throughput sequencing of multiple immunoreceptor genes. IR-HTS MRD quantification provides valuable lead-time prior to clinical relapse that could provide an opportunity to apply additional therapeutic maneuvers while disease burden is low. Because MRD sensitivity in BM is significantly higher than in PB, marrow assessment may further improve the lead-time for predicting disease recurrence using IR-HTS. The dismal outcome of patients with ALL, particularly when disease burden persists after allogeneic transplant, demands further clinical study of both post-transplant MRD monitoring and novel methods for treating ALL relapse in evolution. [Display omitted] Disclosures:Faham:Sequentia Inc.: Employment. Carlton:Sequenta, Inc.: Employment. Zheng:Sequenta: Employment. Moorhead:Sequenta: Employment. Klinger:Sequenta, Inc.: Employment. Willis:Sequenta, Inc.: Employment.

PAX5 Is a Frequently Altered Transcription Factor In Genetically Unclassified Childhood Common/B-Cell Precursor Acute Lymphoblastic Leukemia

Abstract 1458 Introduction:Genetic lesions define treatment and prognosis in childhood common/B cell-precursor Acute Lymphoblastic Leukemia (c/pre-B ALL) (Pui CH et al., J Clin Oncol, 2011). However, up to 40% of childhood pre-B ALL cases, lack the presence of prognostically significant abnormalities; and relapses are common in this group (Mejerink J et al., Semin Hem, 2009). Molecular genetic characterization of genetically unclassified ALL is critical for a better understanding of the biology of this disease, to determine new prognostic markers and to optimize treatment for this group of patients. Several transcription factors, including EBF1, IKZF1 and PAX5, which regulate B-cell development, have recently been shown often to be altered in B-ALL (Mullighan CG et al., Nature, 2007; den Boer ML et al., Lancet, 2009). We decided to investigate the incidence of abnormalities involving B-cell transcription factors, in a cohort of patients with childhood c/pre-B ALL lacking significant prognostic genetic aberrations. Materials and methods:60 patients diagnosed with c/pre-B ALL in Great Ormond Street Hospital (London, UK) from 1994 to 2011 were included in the study. The median age was 3.43 (range: 1.14–12.24), with a median white blood cell count of 14.0 (range: 1.36–213.2). All patients showed an absence of high-hyperploidy/hypodiploidy, BCR-ABL1, MLL, ETV6-RUNX1 and E2A rearrangements, and also i(amp)21 by karyotype, fluorescence in-situ hybridization (FISH) and/or reverse transcriptase-polymerase chain reaction (RT-PCR) investigations. We performed FISH using commercially available probes and BAC clones, in parallel with RT-PCR, to detect deletions/rearrangements of the EBF1, IKZF1 and PAX5 gene regions. Direct sequence analysis was performed for all exons of the PAX5 gene. Real-time PCR was used to determine the level of expression of PAX5 and its transcriptional targets, mostly components of the pre-B cell receptor (pre-BCR) complex including CD19, CD79α, VpreB, BLNK and BLK genes. Western blotting was further performed for the identification of an abnormal PAX5 protein. Results:25/60 patients (42%) showed abnormalities involving PAX5, 2/60 patients (3%) showed deletions of IKZF1. No deletions/rearrangements of EBF1 were detected. Several forms of PAX5 deletions were observed: the entire loss of one PAX5 allele (32%), loss of 5’PAX5 (20%), loss of 3’PAX5 (12%) and loss of 3’PAX5 due to unbalanced rearrangements (8%). Balanced PAX5 rearrangements comprise 12% of the PAX5 alterations. Sequence analysis revealed 9 somatic mutations of PAX5 in 8/60 patients (13%): 6 frameshifts, 2 splice-site and 1 non-stop mutations. The mutations were located in functional exons of PAX5 and resulted in the initiation of premature stop-codons, exon skips, and the formation of abnormal transcripts. Interestingly, 2 patients with an identical splice site mutation, showed the same karyotypic abnormality of an isochromosome of the long arm of chromosome 9 leading to loss of the second PAX5 allele. Moreover, further investigation DNA and mRNA from remission samples demonstrated that in one case the mutation was acquired, and that in the second case the mutation was a germ-line variant. In total, 20% of patients with PAX5 abnormalities had bi-allelic alterations of the PAX 5 gene. PAX5, BLNK and BLK gene-expression levels were lower (p<0.05) in the group of patients with loss of one entire PAX5 allele. There were no significant changes in expression levels of the tested genes in patients with partial PAX5 deletions, rearrangements or mutations. Expression of an abnormal PAX5 protein was confirmed by western blot analysis. Conclusion:The high incidence of PAX5 alterations in childhood c/pre-B ALL, without prognostically significant genetic abnormalities, suggests a pathogenetic role for PAX5 in these acute lymphoblastic leukemia cases. Down-regulation of expression of the BLNK and BLK genes could be one of the mechanisms by which abnormal PAX5 blocks B-cell differentiation and contributes to the initiation of leukemia. Disclosures:No relevant conflicts of interest to declare.

Partial versus Productive Immunoglobulin Heavy Locus Rearrangements in Chronic Lymphocytic Leukemia: Implications for B-Cell Receptor Stereotypy

The frequent occurrence of stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences among unrelated cases with chronic lymphocytic leukemia (CLL) is widely taken as evidence for antigen selection. Stereotyped VH CDR3 sequences are often defined by the selective association of certain immunoglobulin heavy diversity (IGHD) genes in specific reading frames with certain immunoglobulin heavy joining (IGHJ ) genes. To gain insight into the mechanisms underlying VH CDR3 restrictions and also determine the developmental stage when restrictions in VH CDR3 are imposed, we analyzed partial IGHD-IGHJ rearrangements (D-J) in 829 CLL cases and compared the productively rearranged D-J joints (that is, in-frame junctions without junctional stop codons) to (a) the productive immunoglobulin heavy variable (IGHV )-IGHD-IGHJ rearrangements (V-D-J) from the same cases and (b) 174 D-J rearrangements from 160 precursor B-cell acute lymphoblastic leukemia cases (pre-B acute lymphoblastic leukemia [ALL]). Partial D-J rearrangements were detected in 272/829 CLL cases (32.8%). Sequence analysis was feasible in 238 of 272 D-J rearrangements; 198 of 238 (83.2%) were productively rearranged. The D-J joints in CLL did not differ significantly from those in pre-B ALL, except for higher frequency of the IGHD7-27 and IGHJ6 genes in the latter. Among CLL carrying productively rearranged D-J, comparison of the IGHD gene repertoire in productive V-D-J versus D-J revealed the following: (a) overuse of IGHD reading frames encoding hydrophilic peptides among V-D-J and (b) selection of the IGHD3-3 and IGHD6-19 genes in V-D-J junctions. These results document that the IGHD and IGHJ gene biases in the CLL expressed VH CDR3 repertoire are not stochastic but are directed by selection operating at the immunoglobulin protein level.

Then and now: Past Opitz winners discuss the influence of the award on their careers

Then and now: Past Opitz winners discuss the influence of the award on their careers

Significance of the pH-induced conformational changes in the structure of C-reactive protein measured by dual polarization interferometry

Emerging evidence indicates that the conformation of C-reactive protein (CRP) plays important roles in human inflammation and cardiovascular disease (CVD). The different conformations in the structure of CRP under different pH conditions remain an important issue to be investigated for explaining various functions of CRP under certain physiologic and pathologic conditions. We directly measured the pH-induced conformational changes in the structure of CRP by dual polarization interferometry (DPI). The CRP was attached to an aldehyde-functionalized DPI sensor chip at a concentration of 50 μg/ml, and attained 2.019 ng/mm 2 to form a surface coverage with a 1.71 × 10 −14 mol/mm 2 CRP monolayer. A pentagonal structure with an average monolayer thickness value of 5.70 ± 0.12 nm and a layer density of 0.374 ± 0.058 g/cm 2 was obtained at pH 7.0. Moreover, the DPI biosensor signals directly reflected the considerable structural parameters and phenomena of conformational changes of CRP in a pH range of 2.0–10.0. The results obtained showed that the pentameric structure of CRP might dissociated into monomers or monomer aggregates as the pH shifts toward both acidic and alkaline conditions, but only partial rearrangements of CRP subunits might occur at extremely acidic physiological conditions. Considering the proinflammatory effect and subclinical chronic inflammation, pH-induced conformational changes in the structure of CRP between monomeric and pentameric formations may strongly relate to vascular atherosclerosis and subsequent CVD.

Molecular dynamics study of the mechanical behavior of nickel nanowire: Strain rate effects

We present the analysis of uniaxial deformation of nickel nanowires using molecular dynamics simulations, and address the strain rate effects on mechanical responses and deformation behavior. The applied strain rate is ranging from 1 × 108 s−1 to 1.4 × 1011 s−1. The results show that two critical strain rates, i.e., 5 × 109 s−1 and 8 × 1010 s−1, are observed to play a pivotal role in switching between plastic deformation modes. At strain rate below 5 × 109 s−1, Ni nanowire maintains its crystalline structure with neck occurring at the end of loading, and the plastic deformation is characterized by {1 1 1} slippages associated with Shockley partial dislocations and rearrangements of atoms close to necking region. At strain rate above 8 × 1010 s−1, Ni nanowire transforms from a fcc crystal into a completely amorphous state once beyond the yield point, and hereafter it deforms uniformly without obvious necking until the end of simulation. For strain rate between 5 × 109 s−1 and 8 × 1010 s−1, only part of the nanowire exhibits amorphous state after yielding while the other part remains crystalline state. Both the {1 1 1} slippages in ordered region and homogenous deformation in amorphous region contribute to the plastic deformation.