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Goncharova, Anna S., Evgeniy N. Kolesnikov, Ekaterina V. Zaikina, A. V. Volkova, M. V. Mindar, D. V. Khodakova, Ekaterina A. Lukbanova, et al. "Correlation of SOX-2 and NOTCH1 copy numbers with vimentin expression in orthotopic patient-derived xenografts of cardioesophageal cancer in immunodeficient mice." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e15034-e15034. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e15034.
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e15034 Background: Cardioesophageal cancer is one of the most common tumors affecting the mucosa of the cardiac stomach and distal esophagus. Despite the variety of treatment strategies and chemotherapy agents, the prognosis for the patients remains poor. The purpose of the study was to analyze the relative copy numbers of SOX-2 and NOTCH1 and vimentin expression in orthotopic patient-derived xenografts of cardioesophageal cancer. Methods: The model of cardioesophageal cancer was created in Balb/c Nude mice with surgical bioptates of moderately differentiated adenocarcinoma obtained from a donor with infiltrative ulcerative cancer of the lower third of the esophagus with a transition to the cardiac stomach into the distal esophagus of Balb/c Nude mice. The index of proliferative activity in the bioptates was assessed by IHC. The relative copy numbers of SOX-2 and NOTCH1 and vimentin expression were determined by Real-Time qPCR. Results: Expression of vimentin was absent in tissues of the donor tumor. The levels of vimentin expression statistically significantly increased in xenografts (1+ and 3+). The SOX-2 and NOTCH1 relative copy numbers were statistically significantly increased in tissues of the donor tumor (0.9 and 0.7), compared to xenografts (1.5±0.03 and 1.7±0.03). Molecular genetic analysis demonstrated an association between an increased vimentin expression and changes in the relative copy numbers of SOX-2 and NOTCH1 (p = 0.013 and p = 0.0001). Conclusions: The relative copy numbers of SOX-2 and NOTCH1 genetic loci was associated with increased expression of the epithelial-mesenchymal transition marker vimentin in tumor tissue samples and changed with each new generation of orthotopic xenografts.
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Ghassemzadeh, Mitra, Klaus Harms, Kurt Dehnicke, and Jörg Magull. "μ2-Chlorokomplexe von Succinimid und N-Chlorsuccinimid. Die Kristallstrukturen von PPh4[Cl(Succinimid)2], PPh4[[Cl(N-Cl-Succinimid)2] und N-Chlorphthalimid / μ2-Chloro Complexes of Succinimide and N-Chlorosuccinimide. The Crystal Structures of PPh4[Cl(Succinimide)2], PPh4[Cl(N-Cl-Succinimide)2] and N-Chlorophthalimide." Zeitschrift für Naturforschung B 49, no. 4 (April 1, 1994): 506–12. http://dx.doi.org/10.1515/znb-1994-0413.
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Abstract The donor-acceptor complexes PPh4[Cl(succinimide)2] and PPh4[Cl(N-chlorosuccinimide)2] have been prepared by reactions of PPh4Cl with succinimide and N-chlorosuccinimide, re spectively. in acetonitrile solutions. The complexes have been characterized by IR spectros copy and by crystal structure determinations. The crystal structure of N-chlorophthalimide has also been solved.
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Kaneda, M., S. Watanabe, Y. Hirao, S. Akagi, S. Haraguchi, T. Somfai, K. Takeda, M. Hirako, M. Geshi, and T. Nagai. "47 DIFFERENCES IN MITOCHONDRIAL DNA COPY NUMBER AND EPIGENETIC PATTERNS OF MITOCHONDRIA-RELATED GENES IN CLONED COWS FROM THE SAME DONOR CELLS." Reproduction, Fertility and Development 25, no. 1 (2013): 171. http://dx.doi.org/10.1071/rdv25n1ab47.
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The genetic codes of cloned animals and the donor are identical; however, incomplete reprogramming of donor nuclei during NT causes epigenetic abnormalities in cloned animals. Due to the genetic identity and epigenetic differences among clones, we can study epigenetic effects on the phenotypes by analyzing genetically identical clones. During the NT process, donor cell mitochondria (mt) are transferred into the recipient oocytes and mtDNA heteroplasmy is observed. Previous studies have reported various mtDNA transmission patterns not only in the cloned animal itself but also in the offspring of clones. However, differences in mtDNA copy number in cloned animals have not been reported, especially genetically identical ones. To analyze the genetic effects on mtDNA copy number in cattle, we compared actual mtDNA copy number per diploid genome in various tissues of clones derived from the same donor cells. From 5 genetically identical cloned cows (Japanese Black cattle, ages 68 to 82 months) and 6 non-cloned cows (Japanese Black cattle, ages 52 to 129 months), we isolated DNA from 8 kinds of tissues (heart, lung, liver, kidney, spleen, small intestine, muscle, and spinal cord) and measured mtDNA copy number by using real-time PCR. The absolute copy numbers of 2 mtDNA-encoded genes (COX1 and CytB) and 2 nuclear-encoded genes (H19 and IGF2) were measured and analyzed. To examine the epigenetic effects on mitochondria-related genes, we also analyzed DNA methylation patterns of mitochondria-related gene ANT4 (mitochondrial ADP-ATP translocase) in these tissues by the combined bisulfite restriction analysis (COBRA) method. The actual mtDNA copy number per diploid genome varied in tissues and individuals both in clones and non-clones (average in clones v. non-clones: heart: 11 839 ± 6210 v. 9569 ± 2555; lung: 2027 ± 1153 v. 1383 ± 173; liver: 5644 ± 2278 v. 4799 ± 1848; spleen: 1080 ± 844 v. 393 ± 265; kidney: 7034 ± 4448 v. 2939 ± 784; small intestine: 1330 ± 573 v. 437 ± 171; muscle: 9861 ± 3640 v. 7907 ± 3229; spinal cord: 3961 ± 1819 v. 2756 ± 496). The variability of mtDNA copy number in clones was significantly higher in the lung, spleen, kidney, small intestine, and spinal cord (P = 0.001, 0.026, 0.005, 0.021, and 0.014, respectively; F-test), but not in other tissues. Methylation of the ANT4 gene is quite tissue dependent: hypomethylated in the liver, muscle and spinal cord; moderately methylated in the heart, lung, and kidney; and highly methylated in the spleen and small intestine. The methylation patters of ANT4 were not different between clones and non-clones. These results suggest that mtDNA copy number is more influenced by nongenetic factors than genetic background.
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Guo, Wenjun, Sunil Kumar, Frederik Görlitz, Edwin Garcia, Yuriy Alexandrov, Ian Munro, Douglas J. Kelly, et al. "Automated Fluorescence Lifetime Imaging High-Content Analysis of Förster Resonance Energy Transfer between Endogenously Labeled Kinetochore Proteins in Live Budding Yeast Cells." SLAS TECHNOLOGY: Translating Life Sciences Innovation 24, no. 3 (January 10, 2019): 308–20. http://dx.doi.org/10.1177/2472630318819240.
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We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by µManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence. For data analysis, we used custom MATLAB-based software tools to perform kinetochore foci segmentation and local cellular background subtraction and fitted the fluorescence lifetime data using the open-source FLIMfit software. We validated the methodology using endogenous KPs labeled with mTurquoise2 FP and/or yellow FP and measured the donor fluorescence lifetimes for foci comprising 32 kinetochores with KP copy numbers as low as ~2 per kinetochore under an average labeling efficiency of 50%. We observed changes of median donor lifetime ≥250 ps for KPs known to form dimers. Thus, this FLIM high-content analysis platform enables the screening of relatively low-copy-number endogenous protein–protein interactions at spatially confined macromolecular complexes.
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Daher, May, Kai Cao, Rafet Basar, David Marin, Takuye Sekine, Gabriela Rondon, Weicheng Zhao, et al. "Donor NKG2C Copy Number: An Independent Predictor for CMV Reactivation after Double Cord Blood Transplantation." Blood 132, Supplement 1 (November 29, 2018): 2077. http://dx.doi.org/10.1182/blood-2018-99-114407.
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Abstract Cytomegalovirus (CMV) is a member of the herpesviridae family and remains latent in the host cells after primary infection. The virus reactivates in the immunosuppressed host and is a leading cause of morbidity following allogeneic hematopoietic stem cell transplant (allo-HSCT). Natural killer (NK) cells provide the first-line defence against viruses and a subset of NK cells expressing NKG2C have been shown to play a role in the immune surveillance of human CMV. Deletion of the NKG2C gene has been reported as a risk factor for viral infections including CMV and HIV, but current data on the influence of NKG2C gene-copy number variations in the donor graft on the risk of CMV reactivation after allo-HSCT is limited. Following cord blood transplantation (CBT), where the T-cell compartment is functionally naïve, the NKG2C genotype may have an even more pronounced impact on the risk of CMV reactivation. Therefore, we studied NKG2C copy number in the donor graft and the risk of CMV reactivation after double umbilical cord blood transplantation (DUCBT) in 100 CMV seropositive DUCBT recipients and their corresponding cord blood grafts (n=200). The gene encoding NKG2C is present at different copy numbers in the genomes of different individuals, with possible genotypes including 2 copies (wt/wt), 1 copy due to heterozygous deletion (wt/del) or 0 copies due to homozygous deletion (del/del). Among the donor CB units, approximately 2/3 had both copies of the gene (wt/wt), 1/3 had only one copy (wt/del) and only a minority of units had 0 copies with both alleles deleted (del/del). In the setting of DUCBT, the combined graft may contain 0 to 4 functional copies of NKG2C gene. In our cohort, patients whose combined grafts contained only 1 or 2 NKG2C gene copies had a significantly higher probability of CMV reactivation than patients whose combined grafts had 3 or 4 NKG2C copies. The 6-month cumulative incidence of CMV reactivation for the 4 groups was 100%, 92.9%, 60.5% and 55.9% respectively (p=0.005). No patient received a graft with zero gene copies. For the rest of the analysis we divided the patients into two groups, namely the 84 patients who received a combined graft with 3 or 4 NKG2C copies and the 16 patients who received two units with 1 or 2 NKG2C copies. The 6-month cumulative incidence of CMV reactivation for the two groups was 58.4% and 93.7% respectively (p=0.0003). In a multivariate analysis, receiving a combined graft with low NKG2C copy number (1 or 2 copies) (HR=2.72, CI=1.59-4.64; p<0.0001) and reduced intensity conditioning (RIC) (HR=0.59, CI=0.35-0.99, p=0.046) were the only independent predictors for CMV reactivation. Interestingly, the NKG2C copy number of the dominant cord was not predictive for CMV reactivation, suggesting that both cord blood units contribute to the antiviral response early post-DUCBT. Our study points to an important role for donor NKG2C in protection against CMV reactivation after DUCBT. If confirmed in larger numbers of CBT recipients, NKG2C genotyping of the CB graft may be a useful biomarker for predicting the risk of CMV infection after CBT, thus guiding the intensity of CMV prophylaxis for individual patients. Moreover these novel findings may provide a compelling rationale for considering NKG2C genotype as a criterion in the algorithm of CB selection for DUCBT. Disclosures Oran: ASTEX: Research Funding; AROG pharmaceuticals: Research Funding; Celgene: Consultancy, Research Funding. Champlin:Sanofi: Research Funding; Otsuka: Research Funding. Shpall:Affirmed GmbH: Research Funding.
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Jan, Max, Matthew J. Leventhal, Elizabeth A. Morgan, Jordan C. Wengrod, Anwesha Nag, Samantha D. Drinan, Bruce M. Wollison, et al. "Recurrent genetic HLA loss in AML relapsed after matched unrelated allogeneic hematopoietic cell transplantation." Blood Advances 3, no. 14 (July 19, 2019): 2199–204. http://dx.doi.org/10.1182/bloodadvances.2019000445.
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Abstract Immune evasion is a hallmark of cancer and a central mechanism underlying acquired resistance to immune therapy. In allogeneic hematopoietic cell transplantation (alloHCT), late relapses can arise after prolonged alloreactive T-cell control, but the molecular mechanisms of immune escape remain unclear. To identify mechanisms of immune evasion, we performed a genetic analysis of serial samples from 25 patients with myeloid malignancies who relapsed ≥1 year after alloHCT. Using targeted sequencing and microarray analysis to determine HLA allele-specific copy number, we identified copy-neutral loss of heterozygosity events and focal deletions spanning class 1 HLA genes in 2 of 12 recipients of matched unrelated-donor HCT and in 1 of 4 recipients of mismatched unrelated-donor HCT. Relapsed clones, although highly related to their antecedent pretransplantation malignancies, frequently acquired additional mutations in transcription factors and mitogenic signaling genes. Previously, the study of relapse after haploidentical HCT established the paradigm of immune evasion via loss of mismatched HLA. Here, in the context of matched unrelated-donor HCT, HLA loss provides genetic evidence that allogeneic immune recognition may be mediated by minor histocompatibility antigens and suggests opportunities for novel immunologic approaches for relapse prevention.
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Bétermier, Mireille, Sandra Duharcourt, Hervé Seitz, and Eric Meyer. "Timing of Developmentally Programmed Excision and Circularization of Paramecium Internal Eliminated Sequences." Molecular and Cellular Biology 20, no. 5 (March 1, 2000): 1553–61. http://dx.doi.org/10.1128/mcb.20.5.1553-1561.2000.
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ABSTRACT Paramecium internal eliminated sequences (IESs) are short AT-rich DNA elements that are precisely eliminated from the germ line genome during development of the somatic macronucleus. They are flanked by one 5′-TA-3′ dinucleotide on each side, a single copy of which remains at the donor site after excision. The timing of their excision was examined in synchronized conjugating cells by quantitative PCR. Significant amplification of the germ line genome was observed prior to IES excision, which starts 12 to 14 h after initiation of conjugation and extends over a 2- to 4-h period. Following excision, two IESs were shown to form extrachromosomal circles that can be readily detected on Southern blots of genomic DNA from cells undergoing macronuclear development. On these circular molecules, covalently joined IES ends are separated by one copy of the flanking 5′-TA-3′ repeat. The similar structures of the junctions formed on the excised and donor molecules point to a central role for this dinucleotide in IES excision.
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Faridi, Rehan Mujeeb, Taylor J. Kemp, Poonam Dharmani, Victor A. Lewis, Noureddine Berka, Jan Storek, and Faisal M. Khan. "Copies of Donor Killer Immunoglobulin-like Receptor Genes and Motifs Titrate Natural Killer (NK) Cells' Functional Response to Epstein - Barr Virus Infections and Influence the Risk of Developing Post-Transplant Lymphoproliferative Disease (PTLD) after Allogeneic Hematopoietic Cell Transplantation." Blood 126, no. 23 (December 3, 2015): 741. http://dx.doi.org/10.1182/blood.v126.23.741.741.
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Abstract BACKGROUND: Recipientsof allogeneic HCT remain vulnerable to a heightened risk of reactivation of otherwise latent viral infections owing to a compromised immune system early after transplantation. Uncontrolled reactivation of Epstein-Barr virus (EBV) leading to post-transplant lymphoproliferative disorder (PTLD) is one of such major complications after T-cell depleted HCT. Recovering within weeks after transplantation and being first in line of defense against viral infections, natural killer (NK) cells are deemed important in the immunopathogenesis of EBV complications. Their role however remains elusive. NK cell responses are regulated by a series of activating and inhibitory cell surface receptors, central to which are the Killer Immunoglobulin-like Receptors (KIR). Through these receptors NK cells discriminate healthy cells from 'altered' self-cells by scaling the perturbations in HLA expression after viral transformation of the target cell. Here, we set out to determine whether and how KIR gene and motifs' content of HCT donors and/or recipients influences the development of PTLD after allo-HCT. STUDY DESIGN: Hypothesizing that diverse NK cell receptor repertoires can titrate NK cell functional responses to EBV infections/reactivation and can potentially modify the risk of developing PTLD, we determined the KIR gene repertoires of 356 HLA-matched donor-recipient pairs of first allo-HCT and 50 healthy donors through Next Generation Sequencing of the KIR locus on the Illumina MiSeq platform. Based on the presence/absence and number of copies of individual genes, the KIR genotypes were determined and classified into four common centromeric (cA01, cB01, cB02 and cB03) and two telomeric (tA01 and tB01) motifs along with their variants. PBMNCs from KIR typed healthy volunteers were stimulated with EBV-transformed target cells to enumerate NK cell response to EBV (degranulation and/or IFNγ production) as a function of KIR gene content and motifs' distribution using a multicolor flow cytometry-based assay. Effect of KIR gene profile on development of PTLD was analyzed using binomial competing risks regression statistics. Distribution of NK cell functional response across various KIR characterized groups was analyzed using Mann-Whitney U statistics. RESULTS: Donor telomeric A motifs (tA01, KIR3DL1+ve KIR2DS4+ve; KIR3DS1/2DS1+/-ve), strongly protected against PTLD (p=0.0001, SHR=0.17; Figure 1). An increased protection against PTLD with increasing number of tA01 was noted with at least one copy required for a significant protective effect (Figure 1B). Copy number analysis of tA01 gene contents yielded similar associations. Further, the number of EBV induced functional NK cell subsets were significantly higher in individuals with than without KIR genotypes containing tA01 motifs (Figure 2 A-C) and was found to be increasing with an increasing number of tA01 copies (Figure 2 A'-C'). There was no influence of recipients' KIR repertoire on the risk of developing PTLD CONCLUSIONS: NK cell responsiveness, a function of KIR gene repertoire has a profound effect on the development of PTLD. Appropriately characterized KIR gene profile based identification of HCT recipients at high risk of developing PTLD will enable closer monitoring of EBV DNAemia and facilitate prompt therapy. Figure 1. Donor KIR telomeric A motif (tA01) protects against the risk of developing PTLD (A). Presence of at least one copy of donor KIR tA01 motif confers significant protection from PTLD (B) Figure 1. Donor KIR telomeric A motif (tA01) protects against the risk of developing PTLD (A). Presence of at least one copy of donor KIR tA01 motif confers significant protection from PTLD (B) Figure 2. KIR telomeric A motifs (tA01) titrate NK cells' functional response to Epstein-Barr virus infected cells (A-C), with and increasing %functional NK cells and subsets (measures as expressing CD107a, IFN-γ, or both) are observed with increasing tA01 motifs' copies (A'-C') Figure 2. KIR telomeric A motifs (tA01) titrate NK cells' functional response to Epstein-Barr virus infected cells (A-C), with and increasing %functional NK cells and subsets (measures as expressing CD107a, IFN-γ, or both) are observed with increasing tA01 motifs' copies (A'-C') Disclosures No relevant conflicts of interest to declare.
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Lee, TH, E. Donegan, S. Slichter, and MP Busch. "Transient increase in circulating donor leukocytes after allogeneic transfusions in immunocompetent recipients compatible with donor cell proliferation." Blood 85, no. 5 (March 1, 1995): 1207–14. http://dx.doi.org/10.1182/blood.v85.5.1207.bloodjournal8551207.
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Donor leukocytes in therapeutic blood components are implicated in transfusion-related complications ranging from alloimmunization to graft-versus-host disease (GVHD) to viral transmission and reactivation. To further characterize the kinetics of donor leukocyte clearance after allogeneic transfusion, we developed allele-specific polymerase chain reaction (PCR) assays directed at a single-copy Y chromosome gene and HLA class II alleles. These assays enable sensitive detection and quantitation of donor leukocytes at concentrations ranging from one cell to greater than 1,000 cells per 125 microL of recipient blood. When applied to serial samples from five consecutive orthopedic surgery patients who met study criteria, we observed 99.9% clearance of donor leukocytes over the initial 2 days posttransfusion, followed by a transient, 1-log increase in circulating donor leukocytes on days 3 to 5. This phenomenon was reproduced in a canine transfusion model, where the transient donor leukocyte expansion phase was prevented by gamma irradiation of donor blood, and was not observed after transfusions into alloimmunized dogs. We hypothesize that this transient increase in circulating allogeneic donor cells represents one arm of an in vivo mixed lymphocyte reaction, with activated donor T lymphocytes proliferating in an abortive GVHD reaction to HLA- incompatible recipient cells. Further investigation of this phenomenon should provide insight into the mechanisms involved in donor-recipient leukocyte interactions posttransfusion and the relationship of these interactions to leukocyte-induced complications.
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Flesch, Brigitte Katharina, Vanessa Scherer, Burkhard Just, Andreas Opitz, Oswin Ochmann, Anne Janson, Monika Steitz, and Thomas Zeiler. "Molecular Blood Group Screening in Donors from Arabian Countries and Iran Using High-Throughput MALDI-TOF Mass Spectrometry and PCR-SSP." Transfusion Medicine and Hemotherapy 47, no. 5 (2020): 396–408. http://dx.doi.org/10.1159/000505495.
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Background and Aims: Only little is known about blood groups other than ABO blood groups and Rhesus factors in Arabian countries and Iran. During the last years, increased migration to Central Europe has put a focus on the question how to guarantee blood supply for patients from these countries, particularly because hemoglobinopathies with the need of regular blood support are more frequent in patients from that region. Therefore, blood group allele frequencies should be determined in individuals from Arabian countries and Iran by molecular typing and compared to a German rare donor panel. Methods: 1,111 samples including 800 individuals from Syria, 147 from Iran, 123 from the Arabian Peninsula, and 41 from Northern African countries were included in a MALDI-TOF MS assay to detect polymorphisms coding for Kk, Fy(a/b), Fynull, Cw, Jk(a/b), Jo(a+/a–), Lu(a/b), Lu(8/14), Ss, Do(a/b), Co(a/b), In(a/b), Js(a/b), Kp(a/b), and variant alleles RHCE*c.697C>G and RHCE*c.733C>G. Yt(a/b), S–s–U–, Velnull, Conull, and RHCE*c.667G>T were tested by PCR-SSP. Results: Of the Arabian donors, 2% were homozygous for the FY*02.01N allele (Fynull), and 15.7% carried the heterozygous mutation. However, 0.8% of the German donors also carried 1 copy of the allele. 3.6% of all and 29.3% of Northern African donors were heterozygous for the RHCE*c.733C>G substitution, 0.4% of the Syrian probands were heterozygous for DO*01/DO*01.-05, a genotype that was lacking in German donors. Whereas the KEL*02.06 allele coding for the Js(a) phenotype was missing in Germans; 0.8% of the Syrian donors carried 1 copy of this allele. 1.8% of the Syrian but only 0.3% of the German donors were negative for YT*01. One donor from Northern Africa homozygously carried the GYPB*270+5g>tmutation, inducing the S–s–U+w phenotype, and in 2 German donors a GYPB*c.161G>A exchange, which induces the Mit+ phenotype, caused a GYPB*03 allele dropout in the MALDI assay. The overall failure rate of the Arabian panel was 0.4%. Conclusions: Some blood group alleles that are largely lacking in Europeans but had been described in African individuals are present in Arabian populations at a somewhat lower frequency. In single cases, it could be challenging to provide immunized Arabian patients with compatible blood.
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Wang, Jingbo, Xiaojun Huang, Ying Hu, Song Xue, Haoyu Cheng, Yuming Yin, Weijie Zhang, Jiangying Gu, Junbao He, and Fan Yang. "Safety and Efficacy of Donor-Derived Virus Specific Cytotoxic T Lymphocytes(CTL) Administered in Treatment for CMV/EBV Infections after Allogenetic Stem Cell Transplantation." Blood 128, no. 22 (December 2, 2016): 5744. http://dx.doi.org/10.1182/blood.v128.22.5744.5744.
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Abstract Objective: To investigate the safety and efficacy of donor-derived cytomegalovirus(CMV) specific cytotoxic T lymphocytes(CTL) and Epstein-Barr virus(EBV) specific cytotoxic T lymphocytes(CTL) as immune therapy in CMV viremia and EBV viremia postallogeneic transplant. Methods: A total of fifty-three patients receiving haplo-identical allogeneic HSCT for hematological malignancy were eligible for recruitment to the study from February 2013 to December 2015. Thirty-eitht patients of CMV viremia postallogeneic transplant received CMV CTL. Fifteen patients of EBV viremia postallogeneic transplant received EBV CTL. The criteria for inclusion were: (1) plasma CMV/EBV DNA >1×103/ml. (2) Antiviral therapy (ganciclovir and foscaret) was not effective.(3) Antiviral therapy was not tolerated for pancytopenia. The criteria for exclusion were: (1) plasma CMV/EBV DNA <1×103/ml. (2) Prophylactically administered CMV-specific T cells and EBV-specific T cell. T-cell infusion: Patients were infused with a single dose of 2-3×105/kg weekly. Median number of infusion was 8(2-15). Criterion of therapeutical effect: Virus DNA copy number decreased by a log rank or above (Valid). Virus DNA copy number to zero(negative). Virus DNA copy number increased or not reduced ( Invalid). Results: 38 cases of CMV viremia were effective in 28 cases, the effective rate was (73.7±7.1)%, the median onset time was 5 days (2-10). Plasma CMV was negative in 28 cases, the negative rate was (73.7±7.1)%, the median negative time was 11 days (2-24). 15 cases of EBV viremia were effective in 13 cases, the effective rate was (86.7±8.8)%, the median onset time was 5 days (2-11). Plasma EBV was negative in 13 cases, the negative rate was (86.7±8.8)%, the median negative time was 10 days (3-24). Adverse events: fever(n=1), skin rash(n=3), hypoxemia(n=2), diarrhea (n=1 uncertain), Tachycardia of sinus( n=1) Conclusion: Safety and efficacy of virus specific cytotoxic T lymphocytes(CTL) administered postallogenetic HSCT are exact. CTL can be used as second-line therapy of viral infections postallogenetic HSCT. Disclosures No relevant conflicts of interest to declare.
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Sucheston-Campbell, Lara E., Leah Preus, Marcelo C. Pasquini, Philip L. McCarthy, Kenan Onel, Xiaochun Zhu, Stephen R. Spellman, et al. "Combined Donor and Recipient Non-HLA Genotypes Show Evidence of Genome Wide Association with Transplant Related Mortality (TRM) after HLA-Matched Unrelated Donor Blood and Marrow Transplantation (URD-BMT) (DISCOVeRY-BMT study)." Blood 126, no. 23 (December 3, 2015): 61. http://dx.doi.org/10.1182/blood.v126.23.61.61.
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Abstract Transplant-related mortality (TRM) is the largest limiting factor to successful URD-BMT as curative therapy. Identification of non-HLA genetic factors in either recipients or donors could improve BMT outcomes through better matching at these loci. We performed a genome-wide association study (GWAS), named DISCOVeRY-BMT (Determining the Influence of Susceptibility COnveying Variants Related to one-Year mortality after BMT) of 1-year TRM in 3,532 patients treated for AML, ALL or MDS (recipients) reported to CIBMTR from 2000-2011 and their HLA-matched URD (donors); donor DNA was available for 98% of HCT recipients. Cohort 1 includes 2,240 donor-recipient pairs; patients received a 10/10 HLA URD-BMT from 2000-08. Cohort 2 includes 823 donor-recipient pairs; patients received either a 10/10 HLA URD-BMT from 2009-11 or 8/8 (but <10/10) HLA URD-BMT. Genotyping of recipient and donor DNA was performed using the Illumina HumanOmniExpress-24 BeadChip containing 729,293 single nucleotide polymorphisms (SNPs) at University of Southern California. Due to the small number of non-European individuals, we report analyses of European American recipients only. After quality control, Cohort 1 includes 2,052 donor-recipient pairs and Cohort 2 includes 763 donor-recipient pairs typed at 637,655 SNPs. For each SNP, a shared genotype variable was created to capture donor-recipient allele sharing. The shared genotype was assigned a value of 0 if the recipient and donor have the same genotype at a given SNP and a value of 1 if they differ by 1 or 2 alleles. This idea, that a "matched" genotype can impact risk of TRM, is similar to the idea behind HLA matching. For all survival analyses we accommodated the competing risk of death due to disease; covariates including age, body mass index, and graft type (blood or marrow) were included in all analyses. Analyses of the shared genotype variable with TRM were run for all diseases together and excluding ALL (AML+MDS). P-values for each cohort were combined using METAL software with weights proportional to the square root of the number of cases. We report on results for combined P-value (Pmeta) <5 x 10-8. In analyses of AML+MDS donor-recipient pairs four typed SNPs, rs9884653, rs16850885, rs1246576 and rs10014791, spanning 865,040 base pairs in and near MTHFD2L and EPGN on chromosome 4q13.3 were significant at Pmeta <5 x 10-8; several SNPs in this region approach genome wide significance level. The most significant SNP, rs16850885 (P=7.6 x 10-7 in Cohort 1, P=6 x10-3 in Cohort 2, Pmeta =1.8 x 10-8) at 74,304,423 bp, places recipients who differ from their donors by at least one allele (5.2% of patients in Cohort 1 and 3.9% in Cohort 2) at a 2.6 fold increased risk of TRM (Tables 1 and 2). It is in perfect linkage disequilibrium (r2=1) with rs9884653 at 74,280,426 and rs10014791 at 75,145,466; rs1246576 is in strong linkage disequilibrium with rs16850885 (r2 =.91). All four SNPs shows hazard ratios (HRs) of similar magnitude across both cohorts for patients who differ from their donors by at least 1 allele. These SNPs are not associated with death due to disease in either cohort. The difference was not allele specific; of those patients who died of TRM with at least one allele difference approximately 50% of the donors provided the less common A allele (donor A/G or A/A with recipient G/G) and 50% of the recipients had the A allele (recipient A/G with a G/G donor). Analyses of the donors and recipients separately each show some evidence of association with the minor allele A at rs16850885 (Table 2), but it is patients who differ from their donors at this locus who have greatest risk of TRM. A previous GWAS showed that individuals with at least one copy of the A allele at rs16850885 have lower levels of secreted IL-1β following small pox vaccination compared with those who are homozygous GG (P=7.3 x 10-9). IL-1β, a pro-inflammatory cytokine secreted early in the inflammatory response, may be contributing to the risk of TRM through the combination of initiation and maintenance of host tissue inßammation and donor cell inßammatory response. Our study, DISCOVeRY-BMT, is the largest GWAS of TRM HLA-matched URD-BMT. Further confirmation of these findings in a third cohort may aid in donor selection. Disclosures Sucheston-Campbell: NIH/NHLBI: Research Funding. McCarthy:The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hahn:NIH/NHLBI: Research Funding; Novartis: Equity Ownership.
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Srirattana, K., and J. C. St. John. "6 THE EFFECTS OF DEPLETING DONOR CELL MITOCHONDRIAL DNA ON CATTLE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER." Reproduction, Fertility and Development 28, no. 2 (2016): 132. http://dx.doi.org/10.1071/rdv28n2ab6.
Full textAbstract:
Although somatic cell nuclear transfer (SCNT) is a valuable tool for producing animals for agricultural and research purposes, the resultant mixing of mitochondrial DNA (mtDNA) from the donor cell and recipient oocyte (heteroplasmy) affects embryo development and offspring survival and health. The aim of this study was to determine the effects of depleting donor cells of their mtDNA before SCNT on embryo development. mtDNA was depleted from cattle fibroblasts using 2′,3′-dideoxycytidine. mtDNA copy number in cells depleted for 30 days (0.85 ± 0.05) was significantly decreased when compared with nondepleted cells (150.12 ± 29.90; P < 0.0001, ANOVA). Moreover, mtDNA copy number in depleted cells could not be replenished after depletion for 30 days. Depleted cells and nondepleted cells were used as donor cells for SCNT. Somatic cell nuclear transfer embryos were produced by electrofusion of a single donor cell with an enucleated cow oocyte. Reconstructed oocytes were chemically activated and cultured for 7 days (nontreated embryos). Another cohort of embryos was treated with Trichostatin A (TSA), to enhance reprogramming, by activating reconstructed oocytes and culturing them in the presence of 50 nM TSA for up to 10 h. The embryos were then cultured in the absence of TSA. In nontreated groups, the fusion rates of depleted cells (78.0 ± 0.8%) were significantly lower than those of nondepleted cells (92.1 ± 1.4%; P < 0.05). No positive effect on fusion rates was found after TSA treatment. The blastocyst rate for SCNT embryos derived from depleted cells (18.7 ± 4.9%) was significantly lower than the nondepleted group (32.5 ± 3.1%; P < 0.05). Trichostatin A treatment increased blastocyst rates for SCNT embryos derived from depleted cells (32.5 ± 5.3%) to levels equivalent to those of nondepleted cells but did not have any beneficial effect on SCNT embryos derived from nondepleted cells. We have analysed blastocysts for the presence of donor cell mtDNA by high resolution melting analysis. Four out of 10 SCNT blastocysts derived from nondepleted cells were heteroplasmic, whereas others had no donor cell mtDNA. However, all 10 analysed SCNT blastocysts derived from depleted cells were homoplasmic as they harboured only oocyte mtDNA. From RNA sequencing results, TSA treatment of SCNT blastocysts derived from depleted cells increased the expression of key developmental transcription regulators and decreased expression of the mtDNA-specific replication factors, which is essential for embryo development. In conclusion, homoplasmic SCNT embryos were successfully produced by using mtDNA depleted donor cells. Trichostatin A treatment enhanced nuclear reprogramming efficiency in SCNT embryos derived from depleted cells. This work was supported by MitoStock Pty. Ltd., Australia.
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Rezvani, Katayoun, Abdul Tawab, Yasemin Kilical, Giuseppe Sconocchia, Jonming Li, Nancy Hensel, Roger Kurlander, and John Barrett. "PRAME-Specific CD8+ T Cells Are Spontaneously Present in a Large Proportion of Healthy Donors and Leukemic HLA-A0201 Patients." Blood 104, no. 11 (November 16, 2004): 2535. http://dx.doi.org/10.1182/blood.v104.11.2535.2535.
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Abstract The tumor antigen PRAME (preferentially expressed antigen of melanoma) is expressed in up to 50% of patients with AML. Four HLA-A*0201-restricted epitopes in the PRAME protein have been identified: P100-108 (P100), P142-151 (P142), P300-309 (P300) and P425-433 (P425). To detect very low frequencies of PRAME-specific CD8+ T cells, we used quantitative real-time reverse transcription polymerase chain reaction (qPCR) to measure interferon-g mRNA (IFN-g) production by PRAME peptide pulsed CD8+ T cells from 11 healthy donors and 10 HLA-A*0201+ patients with AML, one of whom had received an allogeneic stem cell transplant (SCT). After isolation, 1 x106 CD8+ T cells were stimulated in vitro with C1R-A2 cells (an MHC class I-defective LCL expressing HLA-A*0201) loaded with test peptides at concentrations of 0.1, 1 and 10 mM, to determine functional avidity. CD8+ T cells were also stimulated with CMV pp65 (positive control) and gp100 (209-2M) (negative control) peptides. After 3h coculture, cells were harvested for RNA extraction and cDNA synthesis. qPCR was performed for IFN-g mRNA and normalized to copies of CD8 mRNA from the same sample. Parallel assays using tetramers demonstrated the IFN-g copy number to be linearly related to the frequency of tetramer-binding T cells, sensitive to frequencies of 1 responding CD8+ T cell/100 000 CD8+ T cells. A positive response was defined as a threshold of 100 or more IFN-g mRNA copies/104 CD8 copies and a stimulation index (SI) of 2 or more, where SI = IFN-g mRNA copies/104 CD8 copies in peptide pulsed cultures/unpulsed cultures. Using this sensitive technique, we found responses in 8/11 HLA-A2-positive healthy donors and 7/10 AML patients. Four of eight healthy donors and 5/7 AML responders recognized 2 or 3 peptide epitopes. The mean response against each of the four epitopes was greater in leukemic patients compared with healthy donors (3597–9371 versus 172–1288 IFN-g mRNA copies/104 CD8 copies). PRAME peptide mediated responses, particularly to P300, were also detected using an optimized ELISPOT assay in 2/4 AML patients and 5/8 normal donors tested. In cross-comparison of 8 qPCR positive donors, 6 also generated IFN-g ELISPOTS confirming IFN-g mRNA transcription was also associated with protein secretion. Of note, the most immunogenic epitope in both donors and patients by both methods was P300. Six qPCR and ELISPOT assays were concordant, but there were 2 ELISPOT negative, qPCR positive patients, and 1 ELISPOT positive qPCR negative patient. In 2/5 samples tested (1 donor and 1 patient), peptide-specific ELISPOT responses expanded from 99 to 1627 and 280 to 758 spots per million plated PBMC respectively, after a single 7 day peptide pulse. Samples from a stem cell donor and the recipient pre and post SCT were also studied. The donor had CD8+ T-cell reactivity to P142, P300 and P425. The patient had no PRAME response prior to SCT but after SCT developed significant responses to P142 and P425 and P100, suggesting the transfer and expansion of PRAME-specific CD8+ T cells from donor to recipient. These results provide the first evidence for spontaneous T-cell reactivity against PRAME in healthy donors and AML patients. They support the immunogenicity of PRAME and its potential application in immunotherapy of leukemia.
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Maruyama, Kana, Hiroyuki Maruyama, Yoshitaka Zaimoku, An T. T. Dao, Takamasa Katagiri, Hirohito Yamazaki, and Shinji Nakao. "Donor-Type Aplastic Anemia after Allogeneic Hematopoietic Stem Cell Transplantation: A Common Cause of Late Graft Failure in Patients with Complete Donor Chimerism." Blood 124, no. 21 (December 6, 2014): 2411. http://dx.doi.org/10.1182/blood.v124.21.2411.2411.
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Abstract 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. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.
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Ishiyama, Ken, Takumi Hoshino, Toru Sakura, Shuichi Miyawaki, Jun Ozaki, Toshiro Kurokawa, Takashi Yoshida, Go Aoki, Masaki Yamaguchi, and Shinji Nakao. "Preemptive Therapy of HHV-6 Encephalitis with Foscarnet Sodium for High Risk Patients After Hematopoietic Stem Cell Transplantation." Blood 114, no. 22 (November 20, 2009): 4655. http://dx.doi.org/10.1182/blood.v114.22.4655.4655.
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Abstract Abstract 4655 Umbilical cord blood from unrelated donors has been successfully used as an alternative hematopoietic stem cell source to treat hematologic malignancies in patients lacking HLA-matched donors. However, umbilical cord blood transplantation (UCBT) is associated with a higher risk of engraftment failure and more delayed immunological recovery than bone marrow transplantation and peripheral blood stem cell transplantation (PBSCT). Recently, human herpesvirus-6 (HHV-6) has been recognized as an important pathogen in allogeneic hematopoietic stem cell transplantation (HSCT). In particular, HHV-6 reactivation often causes limbic encephalitis with a dismal prognosis. We conducted a prospective, multicenter study to assess the safety and efficacy of preemptive therapy with foscarnet sodium (PFA) to prevent HHV-6 encephalitis after HSCT. Materials and methods Eligible patients were aged from 16 to 75 years with hematologic disorders refractory to conventional therapy and considered to require UCBT or HLA 1-haplotype mismatched HSCT (haplo HSCT) due to the unavailability of an HLA-identical relatives or a suitable unrelated donor. Informed consent was obtained from all subjects according to the Declaration of Helsinki, and this study protocol was approved by the institutional ethical committee. The amount of plasma HHV-6 DNA was measured 3 times per week between day 7 and day 36 after UCBT or PBSCT from HLA-haploidentical relative donors. PFA, 90 mg/kg/day, was given when the amount of HHV-6 DNA exceeded 5 ×102 copies/ml. Results Of 20 cases registered between September 2007 and January 2009, 12 of 15 UCBT recipients (80%) became positive for HHV-6 DNAemia, and 7 cases exceeded 5×102 copies/ml, while none of the 5 patients who received haplo HSCT became positive (UCBT vs. haplo HSCT; p<0.004). HHV-6 reactivation occurred earlier in patients who eventually required PFA due to an increase in the HHV-6 DNA copy number greater than 5×102 copies/ml than in patients who did not require PFA treatment (median date of developing HHV-6 DNAemia, day 17 vs. day 22 after HSCT, p<0.02). PFA was given to 7 patients whose HHV-6 DNA copy number exceeded 5×102 copies/ml on day 15 to day 20 (median, day 17) after UCBT. The amount of HHV-6 DNA in the plasma decreased the day after PFA administration in 4 of 7 patients, while the other 3 patients required 3-4 days until the copy number decreased. Two patients showed an increase in HHV-6 DNA copy number greater than 1×104 /ml prior to the initiation of PFA without accompanying symptoms suggestive of encephalitis. One patient developed limbic encephalitis with mild symptoms just after initial PFA administration, but the encephalitis resolved without any neurologic sequelae. Mild and transient adverse effects were associated with PFA in only 2 of 8 patients. Conclusion PFA administration guided by the HHV-6 copy number in the early posttransplant period is safe and may reduce the risk of severe limbic encephalitis. Disclosures: Nakao: Alexion: Research Funding.
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Volin, Liisa, Eeva Juvonen, Anne Nihtinen, Sanna Aalto, Klaus Hedman, and Tapani Ruutu. "EBV DNAemia after Allogeneic Stem Cell Transplantation (SCT): A Prospective Study in 131 High Risk Patients." Blood 104, no. 11 (November 16, 2004): 2242. http://dx.doi.org/10.1182/blood.v104.11.2242.2242.
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Abstract We have studied prospectively the incidence of EBV DNAemia and its early treatment in 131 adult allogeneic high risk SCT recipients transplanted during the years 2000 to 2003. Based on our retrospective experience, high risk patients for EBV infection and post-transplant lymphoroliferative disorder (PTLD) were matched unrelated donor (MUD) recipients (of whom a large majority had received anti-thymocyte globulin (ATG) in the conditioning), and patients receiving high dose corticosteroids (HDMP) +/− ATG for the treatment of acute GVHD (aGVHD). 130 patients had a malignant hematological disease, and 1 had aplastic anemia. 23 donors were siblings and 108 unrelated. All donors were A, B, DR matched. 56 grafts were bone marrow and 75 blood stem cells. The grafts were unmanipulated. 101 patients received a standard myeloablative and 30 patients a reduced toxicity conditioning. 101 MUD recipients were given ATG (ThymoglobulineR) as part of conditioning. GVHD prophylaxis consisted of cyclosporine and methotrexate, and 23 patients with a sibling donor received MP in addition. After reduced toxicity conditioning 15 patients received cyclosporine and mycophenolate mophetil. In MUD transplantation EBV was studied weekly for 4 months after SCT and then monthly up to one year after SCT. In sibling SCT EBV was studied from the HDMP treatment of aGVHD on. EBV DNAemia was diagnosed if two consecutive plasma samples had >1000 EBV genome copies/ml detected. The median follow-up of the patients was 12 months, range 2–12. None of the 23 sibling SCT recipients had EBV DNAemia detected during the follow-up despite that 26% of them received also ATG for the treatment of GVHD. Of the 108 MUD recipients 28/108 (26%) had EBV DNAemia detected and 80/108 (74%) remained EBV negative. The activation of EBV was significantly (p<0.001) predicted by the use of high dose (12 mg/kg) ATG during the conditioning treatment but not by the age of the patients, haematological diagnoses, conditioning, the source of the graft, or of the incidence of aGVHD. The median (range) day of the detection of EBV DNAemia was 53 (10–145). The median EBV copy number/ml at detection was 3300 (1200–553700). 20/28 (71%) of the patients had symptoms associated with the DNAemia, 12 fever, 7 enlarged tender lymphnodes, 3 fever and lymphnodes, and 1 had atypical blood lymphocytes. The median day of the first symptoms was 53 (8–102). 21 patients had early EBV treatment started on day 59 (12–139). The decision to initiate treatment was based on the copy number at the onset and its development, and the clinical signs and symptoms. The EBV copy number at treatment was 20150 (1700–2847000). The treatment consisted of Rituximab (R, 375 mg/m2) and decrease of immunosuppression (IS) in 9 patients, R alone in 5, decrease of IS in 7, irradiation of the lymphoma in 4, and donor lymphocyte infusion in 4 patients. The median number of R infusions given per patient was 1.5 (1–4). 12 months after SCT 18/28 (64%) of the patients were alive. Two patients who received EBV-treatment died of PTLD. Other causes of death were 1 rejection, 3 aGVHD, 4 relapses, and 1 epileptic seizure. In conclusion, of the present patients who had received unmanipulated grafts none of the sibling recipients developed EBV DNAemia even after intensive treatment of aGVHD. 26% of the present MUD recipients developed EBV DNAemia but due to the frequent monitoring and early intervention PTLD could in most cases be prevented or cured.
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Baker, M. D., L. R. Read, B. G. Beatty, and P. Ng. "Requirements for ectopic homologous recombination in mammalian somatic cells." Molecular and Cellular Biology 16, no. 12 (December 1996): 7122–32. http://dx.doi.org/10.1128/mcb.16.12.7122.
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Ectopic recombination occurs between DNA sequences that are not in equivalent positions on homologous chromosomes and has beneficial as well as potentially deleterious consequences for the eukaryotic genome. In the present study, we have examined ectopic recombination in mammalian somatic (murine hybridoma) cells in which a deletion in the mu gene constant (Cmu) region of the endogenous chromosomal immunoglobulin mu gene is corrected by using as a donor an ectopic wild-type Cmu region. Ectopic recombination restores normal immunoglobulin M production in hybridomas. We show that (i) chromosomal mu gene deletions of 600 bp and 4 kb are corrected less efficiently than a deletion of only 2 bp, (ii) the minimum amount of homology required to mediate ectopic recombination is between 1.9 and 4.3 kb, (iii) the frequency of ectopic recombination does not depend on donor copy number, and (iv) the frequency of ectopic recombination in hybridoma lines in which the donor and recipient Cmu regions are physically connected to each other on the same chromosome can be as much as 4 orders of magnitude higher than it is for the same sequences located on homologous or nonhomologous chromosomes. The results are discussed in terms of a model for ectopic recombination in mammalian somatic cells in which the scanning mechanism that is used to locate a homologous partner operates preferentially in cis.
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Jiang, Y., R. Kelly, A. Peters, H. Fulka, D. A. Mitchell, and J. C. St John. "108. INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER IS DEPENDENT ON COMPATIBLE CYTOPLASMIC FACTORS AND MITOCHONDRIAL DNA." Reproduction, Fertility and Development 22, no. 9 (2010): 26. http://dx.doi.org/10.1071/srb10abs108.
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Interspecies somatic cell nuclear transfer (iSCNT) offers significant opportunities to analyze and understand nuclear-cytoplasmic interactions. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility, specifically mitochondrial DNA (mtDNA), on successful development. Transfer of somatic murine fetal fibroblasts into enucleated porcine oocytes resulted in extremely low blastocyst rates (0.4%); increased DNA strand breaks; deficient nuclear pore complex arrangements and increased aberrant karyokinesis than observed in porcine-porcine SCNT embryos. Using allele specific-PCR analysis, murine mtDNA was detected at ever-decreasing levels to the blastocyst stage, with peak levels being 0.14 ± 0.055% in 2-cell embryos. Furthermore, these embryos reduced total mtDNA copy number during preimplantation development in a manner similar to porcine embryos. Injecting mouse embryonic stem cell extract and mitochondria along with the murine donor cell into a mitochondria depleted porcine oocyte, increased blastocyst zona pellucida thinning and blastocyst rates significantly (0.4% vs 3.42%) compared to the non-supplemented iSCNT group. They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout preimplantation development. At later stages of preimplantation development, they possessed 48.00% ± 17.38% murine mtDNA and exhibited a mtDNA copy number profile similar to murine embryos. Overall, these data demonstrate that the addition of species compatible cytoplasmic factors and mitochondrial DNA improve developmental competence of iSCNT embryos.
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Yang, Cai-Xia, Zhao-Hui Kou, Kai Wang, Yan Jiang, Wen-Wei Mao, Qing-Yuan Sun, Hui-Zhen Sheng, and Da-Yuan Chen. "Quantitative analysis of mitochondrial DNAs in macaque embryos reprogrammed by rabbit oocytes." Reproduction 127, no. 2 (February 2004): 201–5. http://dx.doi.org/10.1530/rep.1.00088.
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In cloned animals where somatic cell nuclei and oocytes are from the same or closely related species, the mitochondrial DNA (mtDNA) of the oocyte is dominantly inherited. However, in nuclear transfer (NT) embryos where nuclear donor and oocyte are from two distantly related species, the distribution of the mtDNA species is not known. Here we determined the levels of macaque and rabbit mtDNAs in macaque embryos reprogrammed by rabbit oocytes. Quantification using a real-time PCR method showed that both macaque and rabbit mtDNAs coexist in NT embryos at all preimplantation stages, with maternal mtDNA being dominant. Single NT embryos at the 1-cell stage immediately after fusion contained 2.6 × 104 copies of macaque mtDNA and 1.3 × 106 copies of rabbit mtDNA. Copy numbers of both mtDNA species did not change significantly from the 1-cell to the morula stages. In the single blastocyst, however, the number of rabbit mtDNA increased dramatically while macaque mtDNA decreased. The ratio of nuclear donor mtDNA to oocyte mtDNA dropped sharply from 2% at the 1-cell stage to 0.011% at the blastocyst stage. These results suggest that maternal mtDNA replicates after the morula stage.
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Kondo, Yukio, Takamasa Katagiri, Kohei Hosokawa, Kinya Ohata, Hirohito Yamazaki, Seishi Ogawa, and Shinji Nakao. "Loss of HLA Class-I Expression In Leukemic Cells That Relapsed After HLA-Matched and -Mismatched SCT." Blood 116, no. 21 (November 19, 2010): 2541. http://dx.doi.org/10.1182/blood.v116.21.2541.2541.
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Abstract Abstract 2541 Background: The loss or down-regulation of HLA class-I antigens often occurs in solid tumors, but it is rarely demonstrated in leukemic cells. This phenomenon may explain why leukemic cells are immunogenic to some degree with allogeneic hematopoietic stem cell transplantation (allo-SCT) or donor leukocyte infusion, because HLA class-I antigens on leukemic cells are thought to present minor histocompatibility antigens (mHAs) and leukemia-associated antigens (LAAs) to donor T cells to elicit anti-leukemic cytotoxic T-cell (CTL) responses. Recent analyses of leukemic cells that relapsed after HLA-haploidentical SCT revealed that the leukemic cells lose their unshared HLA haplotype expression as a result of acquired uniparental disomy (UPD) on the short arm of chromosome 6 (6p). The loss of HLA from leukemic cells may occur after transplantation from HLA-identical donors if CTLs specific to mHAs or LAAs play a substantial role in the eradication of leukemic cells. This study evaluated this hypothesis by investigating HLA class-I expression on leukemic cells from patients at both the time of diagnosis and relapse after allo-SCT from HLA matched and mismatched donors. Objectives/Methods: Leukemic cells were obtained from five patients with myeloid leukemia (1 CML, 4 AML) both at diagnosis and relapse after allo-SCT. HLA class-I expression on leukemic cells was determined by flow cytometry using monoclonal Abs specific for the HLA-A allele. SCT donors were an HLA-matched sibling, an HLA-A locus-mismatched mother, an HLA-B and C locus-mismatched father, and 2 HLA-matched unrelated donors. The copy number-neutral loss of heterozygosity on 6p in leukemic cells was analyzed by a single-nucleotide polymorphism (SNP) array in patients after haploidentical SCT. The origin of the patients' HLA alleles (paternal or maternal) was determined by family studies on HLA. The post-transplantation donor T-cell responses against the leukemic cells were analyzed with an IFN-γ secretion assay. Results: HLA-A expression of leukemic cells obtained at relapse after allo-SCT was down-regulated in all 5 patients compared to that of leukemic cell obtained at diagnosis. The patient possessing HLA-A2/A11 (Case 1) was the recipient of BM possessing HLA-A24/A11 showed that 66% of leukemic cells at relapse were deficient in HLA-A2 expression. Leukemic cells restored HLA-A2 expression when this patient relapsed after the 2nd SCT using HLA-A2-matched cord blood, but at lower level in comparison to the level at diagnosis (MFI at diagnosis 177 vs. MFI at relapse 101). In vitro, cell-surface HLA-A2 expression was completely restored when leukemic cells at the 2nd relapse were treated in culture with IFN-γ (200 U/mL) for 48 hr. HLA-A2 expression in the shared haplotype (A0206-B3902-Cw0702-DR0405) was unexpectedly missing in 12% of the relapsed leukemic cells in another patient (Case 2) with HLA-2 loci (HLA-B and -C)-mismatched BM (A3101-B5601-Cw0401-DR0901), and this proportion increased to 83% at relapse after 2nd allo-SCT from the same donor (Figure). IFN-γ failed to restore HLA-A2 expression (MFI without IFN-γ 32 vs. MFI with IFN-γ 10) in vitro, and the genomic DNA extracted from leukemic cells at relapse showed a UPD of 6p. Donor-derived T-cells stimulated with leukemia cells from case 2 at diagnosis in vitro secreted IFN-γ in response to leukemia cells at diagnosis better than leukemia cells at relapse (1.67% vs. 0.85%). Conclusion: Loss of HLA-class I antigen occurs frequently in myeloid leukemia cells at relapse after allo-SCT regardless of the HLA disparities. T-cell attacks specific to the major as well as the mHAs, and LAAs may favor proliferation of leukemic cells deficient in the expression of HLA class-I. Disclosures: No relevant conflicts of interest to declare.
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Selvakumar, Annamalai, Aisha Hasan, Vidhi Desai, Gianfranco Pittari, Lorna Barnett, Esperanza B. Papadopoulos, and Richard J. O'Reilly. "Quantitation of WT1 Transcripts by Copy Number Provides a Sensitive Indication of Minimal Residual Disease (MRD) and Response to Adoptive Cell Therapy in Patients Transplanted for Ph(+) CML." Blood 114, no. 22 (November 20, 2009): 4489. http://dx.doi.org/10.1182/blood.v114.22.4489.4489.
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Abstract Abstract 4489 The Wilm's tumor gene, WT-1, is markedly overexpressed in a high proportion of acute leukemias, advanced MDS and Ph(+) CML. Prior studies, employing semiquantitative PCR-based techniques, have suggested that elevated levels of WT-1 transcripts in bone marrow and PBMC are indicative of MRD, and predictive of relapse in AML and CML following chemotherapy. We have now developed a quantitative real time PCR-based assay for WT-1 transcripts and used it to evaluate the long term effects of donor lymphocyte infusions in a series of patients with Ph(+) CML treated for relapse or persistent MRD following allogeneic marrow transplants between 1993 and 2004 who are currently in molecular or cytogenetic remission. To quantitate WT-1 mRNA copy number in the marrow and PBMC, total RNA from patient samples received at different times that had been extracted with trizol reagent and stored at -20°C was transcribed into cDNA using MMLV reverse transcriptase. Real time PCR was performed with 1-2 μg of total RNA using the ABI 7300 instrument with a 25 base pair FAM labeled probe containing a sequence located at the boundary of exon 6 and 7 of the WT-1 gene in order to eliminate amplification of genomic DNA and insure measurement of all transcribed WT-1 isoforms. The forward and reverse primers were synthesized from sequences contained in exon 6 and 7 of the WT-1 gene respectively. Cloned plasmids containing defined copy numbers of WT-1 and human GAPDH were used to prepare standard curves using serial dilutions. Quantitation of WT-1 copy number expression was performed by calibration against these standard curves. The expression of WT-1 was normalized against the expression of the control GAPDH to adjust for variation in RNA and cDNA synthesis. Using this assay, we evaluated sequential samples from each patient for correlations between WT-1 copy number detected and the results of qualitative PCR assays for bcr/abl fusion gene transcripts previously performed on the same samples using nested primers as well as the results of clinical, hematologic and cytogenetic analyses of each patient obtained at each time of sampling. Blood samples from a group of 8 normal volunteers served as negative controls. The average WT-1 copy number in the blood of normal volunteers was 25 copies/ μg RNA (range 2-60). With one exception, blood and marrow samples from patients with CML in molecular remission (bcr/abl not detected) had WT-1 copy numbers < 50 copies/μg RNA. One sample of marrow from a patient who had been bcr/abl + until 6 months post transplant and had WT-1 copy numbers that had fallen from 12,932 one month post transplant to 1855 at that time, became bcr/abl negative at 11 months but still had a WT-1 copy number of 931/ μg RNA. In contrast, patients with molecular disease (+ bcr/abl) maintained WT-1 copy numbers ranging from 70-1000/ μg RNA. Samples from patients with cytogenetic relapse consistently had WT-1 copy numbers exceeding 1000/ μg RNA, with levels exceeding 10,000 in 4/6 such patients tested. In a subset of patients who have failed to achieve or sustain molecular remission following DLI, marrow and blood samples with detectable bcr/abl transcripts and WT-1 copy numbers ranging from 60-3000 have been repeatedly detected for periods of up to 6 years without additional treatment and without disease progression implying an ongoing mechanism of disease control. Therefore, quantitation of WT-1 copy number provides a useful technique for monitoring minimal residual disease and may be helpful in identification of patients at higher risk of cytogenetic relapse. Disclosures: No relevant conflicts of interest to declare.
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Bojesen, Stige, Mari Malkki, Ted Gooley, Lue Ping Zhao, Annamalai Selvakumar, Stephen R. Spellman, Effie W. Petersdorf, John A. Hansen, and Bo Dupont. "Genetic Allelic Variation in the p53 DNA Repair Pathway Constitute a Risk Factor for Long-Term Survival in Hematopoietic Stem Cell Transplantation." Blood 112, no. 11 (November 16, 2008): 337. http://dx.doi.org/10.1182/blood.v112.11.337.337.
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Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) from HLA-matched related and unrelated donors is well recognized as a potentially curative treatment for patients with high-risk leukemia and myelodysplastic syndrome (MDS), however, transplant-related morbidity and mortality limit the success of HSCT. Evidence from population-based studies suggests a role of genetic variation in the tumor suppressor gene p53 in health. P53 is a transcription factor that responds to cellular stress by regulating cell-cycle arrest, DNA repair and apoptosis. Studies in vitro indicate that the p53 codon 72Arg allele gene product predominantly increases apoptotic events, while 72Pro mediates cell-cycle arrest and DNA repair. We sought to define the role of germline allelic variations of the p53 Arg72Pro single nucleotide polymorphism (SNP) (rs1042522) in post-transplantation outcome. We determined the p53 Arg72Pro genotype in 1067 Caucasian unrelated donor-recipient pair samples obtained from the National Marrow Donor Program Research Repository and analyzed clinical outcome data from the Center for International Blood and Marrow Transplantation. Patients received myeloablative conditioning and marrow (n =1025 ) or peripheral blood stem cells (n = 42 ) from HLA-A, B, C, DRB1 and DQB1 10 of 10 allele-matched unrelated donors between 1985 and 2002 (50.9% were transplanted during the years 1992–1997) for the treatment of acute lymphoblastic leukemia (ALL) (15.9%), acute myelogenous leukemia (AML) (25.9%), chronic myelogenous leukemia (CML) (57.6%) or MDS (0.6%). 457 (42.8%) of the recipients were female. The allele frequency for the minor p53 72Pro allele was 0.255 (donors) and 0.249 (recipients) corresponding to the expected allele frequency for Caucasian individuals of mixed European origin. Presence of 1 or 2 minor p53 codon 72Pro alleles in patients was associated with an increased hazard of mortality: HR=1.12 (1.00–1.26, p=0.05). There was no statistically significant effect of donor genotype for SNP Arg72Pro on mortality, and no such effect of donor or recipient genotype on severity of acute graft-versus-host disease (GVHD) or disease recurrence. We further investigated the role of genetic variation in the MDM2 oncogene, a well-recognized central node in the p53 pathway that binds and inhibits p53 by regulating location, stability and activity. The minor G allele at position -309 (rs2279444) of the MDM2 promoter increases transcription of MDM2. We determined the genotype of the -309 SNP in the MDM2 promoter in the donors and recipients. Patients with 2 p53 72Proalleles and at least one copy of the -309 G allele had a 1.41-fold increase in mortality risk (95% CI, 1.00–1.98, p=0.05) compared to patients with 0–1 p53 72Pro alleles and zero G alleles at -309 (reference group), while patients with 2 p53 72Pro alleles and zero -309 G alleles had a similar outcome to that in the reference group (hazard ratio 1.00 (95% CI 0.65–1.56, p=.99)). In summary, our results are consistent with the hypothesis that p53 codon 72Pro is associated with increased post-transplant mortality, but with the increase due primarily to recipients that also have at least one G allele in MDM2 SNP -309. Inspection of Kaplan-Meier curves for patients with different genotypes of SNP p53 codon 72 demonstrates the increase in mortality for recipients with the 72Pro/Progenotype 6–10 years post-transplantation. These results suggest that genotyping of HSCT recipients for SNP in p53 codon 72 and MDM2 -309 may have predictive value for late transplant outcome. If confirmed in additional studies, prospective genotyping of SNPs in p53 codon 72 and MDM2 -309 and possibly other significant genomic positions could be used to plan transplant regimens and aid in risk assessment.
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Wang, Yipeng, Stephanie Greene, Angel Rodriguez, Jerry Lee, Mark Landers, Ryan Vance Dittamore, and Dena C. Marrinucci. "The use of whole genome copy number variation (CNV) to measure genomic instability in mCRPC CTCs." Journal of Clinical Oncology 34, no. 2_suppl (January 10, 2016): 307. http://dx.doi.org/10.1200/jco.2016.34.2_suppl.307.
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307 Background: AR targeted therapies in combination with PARP inhibitors have recently shown efficacy in mCRPC patients with specific DNA repair gene mutations in metastatic tissue biopsies. Homologous recombination DNA repair deficiencies (HRD), associated with response to PARP inhibitors, can also be assessed by genomic instability/scarring. Accurate genomic scarring measurement in tumors can be confounded by intra-tumor heterogeneity and/or non-tumor genome contamination. To better identify PARPi sensitivity in mCRPC patients, we developed a genomic instability and scarring assay starting from single CTCs. Methods: VCaP, LnCaP or PC3 cell lines were spiked into healthy donor blood. Individual spiked cells were identified and recovered for genomic analysis using the standard Epic CTC assay. Post recovery, cells were lysed, whole genome amplified, constructed into shotgun libraries and sequenced to ~2M 2x150bp PE reads. Following alignment, whole genome copy number and instability analysis was performed to identify large scale transitions (LST, n of chromosomal breaks between adjacent regions of at least 10 Mb), % of genome altered (percentage of 1Mbp bins with copy number alterations), as well as specific tumor suppressor/oncogene copy number alterations. The association of PTEN loss with increases in genomic instability and scaring was performed. Results: Loss of PTEN function was previously shown to be associated with genomic instability. Our assessment of PTEN deletion was confirmed in PC3, while at least one genomic copy was observed in LnCaP and VCaP. The number of LSTs and % of genome altered was higher in PC3 (n = 19 +/- 3; 9.3% +/- 2.6%) than both VCaP and LnCap (n = 8 +/- 2; 6.1% +/- 0.4%). Conclusions: The association of higher genomic instability in a PTEN null cell line (PC3) vs. those with either heterozygous (LnCaP) or wild type (VCaP) PTEN status, matches published reports associating PTEN loss with increased genomic instability. Although, PC3 only demonstrates mild PARPi sensitivity in vitro, the detection of increased genomic scarring vs. cells with at least 1 functional PTEN allele confirms the assays ability to quantify genome instability at the single cell level from CTCs in a liquid biopsy.
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McDermott, David H., Paejonette Jacobs, Qian Liu, Jiliang Gao, and Philip M. Murphy. "CXCR4 Gene Dosage Is Critical for HSC Engraftment." Blood 126, no. 23 (December 3, 2015): 3066. http://dx.doi.org/10.1182/blood.v126.23.3066.3066.
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Abstract Introduction: Warts, Hypogammaglobulinemia, Infections and Myelokathexis Syndrome (WHIMS) is an autosomal dominant immunodeficiency resulting from gain-of-function mutations in the chemokine receptor CXCR4. We recently described a unique WHIMS patient who underwent spontaneous genetic and phenotypic reversion at approximately age 30 after being severely affected as a child. Her reversion was due to a single catastrophic genetic event known as chromothripsis (chromosome shattering) resulting in the deletion of one copy of 163 genes in addition to her mutant copy of CXCR4 on chromosome 2. This event was traced to a hematopoietic stem cell (HSC) that had spontaneously repopulated her bone marrow; however, which of the genes was responsible and the mechanism required further investigation. Methods: Mouse models of CXCR4 haploinsufficiency (Cxcr4+/o) and WHIMS (Cxcr4+/S338X) were used in competitive bone marrow repopulation experiments transplanting whole bone marrow cells or purified HSC. Recipient mice were treated with / without lethal irradiation prior to transplant. Genome editing with TALENs and CRISPR-Cas9 technology was used to target CXCR4 for deletion in human cell lines. Results: Cxcr4 haploinsufficiency markedly enhanced HSC engraftment potential in recipient WHIM mice whether the donor HSC were purified from whole bone marrow cells or not, and whether the recipient was conditioned by lethal irradiation or not. Enhanced engraftment by Cxcr4 haploinsufficient donor HSC also occurred in wild-type mouse recipients, but to a lesser extent, and was also HSC intrinsic. Genome editing experiments have been successful at deleting one or both copies of CXCR4 in human cell lines in up to 40% of treated cells, and in reducing CXCR4 surface expression. Conclusion: While CXCR4 was already understood to be important in HSC biology, this patient and subsequent murine experiments have proven that the gene dosage of CXCR4 is a critical factor affecting HSC engraftment. Genome editing is a promising technology for deleting one copy of CXCR4, ideally the WHIM allele,in autologous HSC as a strategy to cure WHIM syndrome. Disclosures McDermott: US National Institutes of Health: Employment, Patents & Royalties: pending. Jacobs:US National Institutes of Health: Employment, Patents & Royalties: pending. Liu:US National Institutes of Health: Employment, Patents & Royalties: pending. Gao:US National Institutes of Health: Employment, Patents & Royalties: pending. Murphy:US National Institutes of Health: Employment, Patents & Royalties: pending.
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Tew, Ben Yi, Christophe Legendre, Mark A. Schroeder, Tim Triche, Gerald C. Gooden, Yizhou Huang, Loren Butry, et al. "Patient-derived xenografts of central nervous system metastasis reveal expansion of aggressive minor clones." Neuro-Oncology 22, no. 1 (August 21, 2019): 70–83. http://dx.doi.org/10.1093/neuonc/noz137.
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Abstract Background The dearth of relevant tumor models reflecting the heterogeneity of human central nervous system metastasis (CM) has hindered development of novel therapies. Methods We established 39 CM patient-derived xenograft (PDX) models representing the histological spectrum, and performed phenotypic and multi-omic characterization of PDXs and their original patient tumors. PDX clonal evolution was also reconstructed using allele-specific copy number and somatic variants. Results PDXs retained their metastatic potential, with flank-implanted PDXs forming spontaneous metastases in multiple organs, including brain, and CM subsequent to intracardiac injection. PDXs also retained the histological and molecular profiles of the original patient tumors, including retention of genomic aberrations and signaling pathways. Novel modes of clonal evolution involving rapid expansion by a minor clone were identified in 2 PDXs, including CM13, which was highly aggressive in vivo forming multiple spontaneous metastases, including to brain. These PDXs had little molecular resemblance to the patient donor tumor, including reversion to a copy number neutral genome, no shared nonsynonymous mutations, and no correlation by gene expression. Conclusions We generated a diverse and novel repertoire of PDXs that provides a new set of tools to enhance our knowledge of CM biology and improve preclinical testing. Furthermore, our study suggests that minor clone succession may confer tumor aggressiveness and potentiate brain metastasis.
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Faridi, Rehan M., Taylor J. Kemp, Victor A. Lewis, Noureddine Berka, Jan Storek, and Faisal Khan. "Donor-Recipient Matching For Killer Immunoglobulin-Like Receptor Genotypes Confers Protection Against Graft Versus Host Disease Without Affecting Disease Relapse After Allogeneic Hematopoietic Cell Transplantation." Blood 122, no. 21 (November 15, 2013): 4628. http://dx.doi.org/10.1182/blood.v122.21.4628.4628.
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Background Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for malignant and non-malignant hematological disorders. Unfortunately, complications of HCT, primarily graft versus host disease (GVHD) and relapse of the underlying disease are substantial. In Alberta, all HCTs are performed with antithymocyte globulin conditioning and >95% of these use HLA matched donors. However, in spite of that approximately 35% Albertan HCT recipients die or suffer long-term from GVHD while another 20% die due to disease relapse. Unfortunately, strategies/treatments aiming to control GVHD in most cases lead to increase in the rate of disease relapse and infections. In the recent years, the natural killer (NK) cell genetic system has garnered substantial research interest as an immunogenetic system that significantly influences HCT outcomes. The complexity of NK cell function is modulated by a series of activating and inhibitory cell surface receptors known as the Killer Immunoglobulin-like Receptors (KIR). Here we set out to determine the influence of KIR matching between HCT donor and recipient pairs on allogenic HCT complications. Study design Hypothesizing that donor-recipient KIR gene/profile mismatch would affect the allo-HCT outcomes, we genotyped 92 (discovery cohort) and 111 (validation cohort) allo-HCT pairs by Luminex-based rSSO method. The KIR genotypes were classified into AA and Bx genotypes on the basis of KIR gene constitution of the individual. Donor (D) and recipients (R) were ‘matched' for KIR genotypes when both carried either AA (two copies of group A haplotypes) or B/x (at least one copy of group B haplotype) genotypes. Effect of KIR matching on significant GVHD (described as grade 2-4 acute GVHD or chronic GVHD needing systemic therapy) as well as disease relapse was analyzed using binomial regression and Kaplan-Meier statistics. Results As observed in both the discovery and validation cohorts, a significant protection against GVHD was conferred upon when both the donor and recipient were matched for the KIR genotypes (HR=2.224; p=0.01) without any effect on disease relapse (HR=1.098; p=0.934) (figure1). Conclusions In spite of D-R matching for major immunogenetic determinants like HLA, complications of allogeneic HCT are significant. Since GVHD represents a flip side to graft-vs-leukemia (GVL) reaction, strategies to decrease GVHD in most cases have resulted in increased relapse. Matching donor and recipients for KIR genotypes can offer a significant protection against GVHD without increasing the risk of disease relapse. Disclosures: No relevant conflicts of interest to declare.
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Zhang, QiGuo, Jian Ou-Yang, and Rong-Fu Zhou. "Clinical and Experimental Features of Natural Killer Cell Leukemias in a Chinese Population: A Single Center Experience of 10 Years." Blood 118, no. 21 (November 18, 2011): 4892. http://dx.doi.org/10.1182/blood.v118.21.4892.4892.
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Abstract Abstract 4892 Leukemia of natural killer (NK) cell lineage is rare which affects Asian population preferentially. Here 10 Chinese patients with NK cell leukemia were retrospectively evaluated. Among them 7 man and 3 women, the middle age was 47 years (range 22–57 years). Aggressive NK cell leukemias were finally diagnosed in 7 patients, myeloid/NK cell precursor acute leukemias in 2 patients and NK-CLPD in 1 patient. Pronounced extramedullary involvements (breast mass, multiple subcutaneous nodes and eye rectus muscle mass) were the main manifestation in 2 myeloid/NK cell precursor acute leukemias and 1 aggressive NK-cell leukemia. Clonal cytogenetic abnormality (46, XX, 16P-) was only found in 1 myeloid/NK cell precursor acute leukemia patient. Epstein-Barr virus test was performed in 8 patients and 3 were Positive (all were aggressive NK-cell leukemias). The overall survival of 4 cases of aggressive NK-cell leukemias combined with hematophagocytic syndrome were less than 1.3 months. During following up, 8 patients died of disease, 1 NK-CLPD patient and 1 aggressive NK-cell leukemia patient treated by allo-BMT were still alive. The former NK-CLPD, who was in indolent course, survived 60+ months. The later patient was first diagnosed as NK-CLPD but with lymphoadenopathy and splenomegaly, the metabolic activity of spleen by F-18 FDG uptake was in normal range, lymph node pathology showed reactive change and PB EBV-DNA copy was negative, later hematophagocytic syndrome occurred and EBV-DNA copy became positive. The lymph node pathology were reexamined by FISH method and scattered EBV positive cells were found, so the exact diagnosis from beginning should be occult aggressive NK cell leukemia, soon matched sibling donor BMT was performed and the patient got complete remission and the EBV-DNA copy became negative and lived well for 10+ months. Natural killer cell neoplasms are rare and most in aggressive clinical course, novel therapeutic regimens including allogeneic bone marrow transplantation should be investigated to improve outcome of this disease. Disclosures: No relevant conflicts of interest to declare.
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Tabayashi, Takayuki, Fumihiko Ishimaru, Koichi Nakase, Hiromi Nakashima, Itaru Kataoka, and Mitsune Tanimoto. "Minimal Residual Disease Detection by Real Time PCR of the Dominant-Negative Isoform of Ikaros in Patients with Acute Lymphoblastic Leukemia." Blood 108, no. 11 (November 16, 2006): 4416. http://dx.doi.org/10.1182/blood.v108.11.4416.4416.
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Abstract Introduction
 The transcription factor Ikaros plays an important role in lymphoid development and proliferation. We and other groups have demonstrated overexpression of the dominant-negative isoform of Ikaros, Ik-6, in patients with acute lymphoblastic leukemia (ALL). By alternative splicing Ik-6 lacks exon3-6, and the junction of exon2 and 7 is a specific sequence for Ik-6. Using this leukemia-specific sequence as a molecular marker, we monitored minimal residual disease (MRD) of ALL patients. Patients and methods
 We designed a Taqman probe on the junction of exon2 and 7 of Ik-6. Consecutive real time PCR was performed on Ik-6 positve ALL patients. Ik-6 copy numbers were expressed as a ratio to copy numbers of the control gene GAPDH. Results
 Patient 1
 This 27-year-old patient was found to have ALL with normal karyotype and treated with induction chemotherapy. Unfortunately, her disease was primary refractory and then she received peripheral blood stem cell transplantation (PBSCT) from HLA-identical sibling donor and discharged hospital at day 72 after PBSCT with CR. She was readmitted 10 months later because of recurrence of ALL. After repeating donor leukocyte infusion and chemotherapy, she could not reach CR and received second bone marrow transplantation (BMT) from unrelated donor. Her disease remained CR for one year, however real time PCR of Ik-6 could not remained negative and predicted later relapse. Patient 2
 The 49-year-old patient was diagnosed with Philadelphia chromosome-positive ALL and achieved CR with induction chemotherapy. She was treated with repeated consolidation chemotherapy and her disease remained CR for 7 months, however real time PCR of Ik-6 became positive and predicted later relapse. Patient 3
 ALL with normal karyotype was diagnosed in this 39-year-old patient. He received induction chemotherapy and achieved CR. Despite repeated consolidation chemotherapy, his disease recurred after 5 months. He was refered to our hospital for PBSCT from HLA-identical sibling donor, however, even after PBSCT, real time PCR of Ik-6 remained positive and predicted later relapse. Patient 4
 This 64-year-old patient was diagnosed with ALL with normal karyotype. She was treated with induction chemotherapy and achived CR. Her disease remained CR with consolidation and maintenance chemotherapy for one year, and real time PCR of Ik-6 is negative all the time. Patient
 5 ALL with Philadelphia chromosome was diagnosed in this 16-year-old patient. She achieved CR with induction chemotherapy and was refered to our hospital for BMT from HLA-identical sibling donor. She discharged our hospital at day 39 after BMT with CR and minor BCR/ABL transcript was not detectable. One year later thrombocytopenia recurred and she was maintained on Imatinib without reaching CR. After 6 months she received second BMT from unrelated donor and discharged our hospital at day 77 with CR. At discharge, real time PCR of Ik-6 was negative and minor BCR/ABL transcript was not detectable. Discussion
 Our results demonstrate that real time PCR of Ik-6 is useful to monitor MRD in patients with Ik-6 positive ALL.
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Jan, Max, Matt J. Leventhal, Elizabeth A. Morgan, Jordan Chase Wengrod, Anewsha Nag, Samantha D. Drinan, Bruce M. Wollison, et al. "Recurrent Genetic HLA Loss in Acute Myeloid Leukemia Relapsed after Matched Unrelated Allogeneic Hematopoietic Cell Transplant." Blood 132, Supplement 1 (November 29, 2018): 817. http://dx.doi.org/10.1182/blood-2018-99-113911.
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Abstract Introduction: Leukemic escape from the graft versus leukemia (GVL) effect of allogeneic hematopoietic cell transplantation (HCT) is poorly understood. During prolonged periods of post-transplant remission, immunologic selection pressure can favor relapse mediated by acquired resistance to GVL-mediated clearance, such as loss of mismatched HLA in haploidentical transplants. We hypothesized that genetic mechanisms of immune evasion can cause late relapses after matched unrelated donor transplants for myeloid malignancies. Methods: We evaluated 580 adult patients with myeloid malignancies who underwent allogeneic HCT at our institution between 2001 and 2014 and experienced subsequent disease relapse. In 19% of these patients (n=112) relapse occurred at least one year after transplantation. The 25 patients included in the study had specimens banked at each of three timepoints: 1) prior to transplantation, with AML or MDS involvement, 2) approximately 100 days after transplantation, during the period of remission 3) at the time of disease relapse. The indications for transplantation were AML (n = 20) and MDS (n = 5). Transplants were from matched unrelated (n=14), matched related (n=9), and mismatched unrelated (n=2) donors. 12 patients received myeloablative conditioning and 13 received reduced-intensity conditioning regimens. Targeted sequencing of 187 genes, selected based on pathogenic involvement in myeloid malignancies or suspected involvement in immune evasion, was performed on all three timepoints from each patient. Genome-wide microarray-based copy number assessment was performed onthe pre-transplant specimen and post-transplant relapse specimens. Results: We identified three recipients with relapse-specific HLA loss via 1-8 Mb deletions or chromosome 6p arm-level copy neutral uniparental disomy (UPD). One of the HLA alterations was a deletion spanning HLA-B and HLA-C in a donor/recipient pair mismatched at HLA-C. However, in two other cases, relapse-specific HLA losses were identified in donor/recipient pairs that were fully matched at A, B, C, and DRB1. These findings suggest that HLA loss may allow leukemic cells to escape allogeneic immune recognition of minor HLA discrepancies and/or the presentation of minor histocompatibility antigens in the context of matched MHC presentation. These three HLA losses were identified among 14 recipients of matched unrelated donor HCT. HLA losses were not identified among recipients of matched related (n = 9) or mismatched unrelated (n = 2) allogeneic HCT. To evaluate potential interactions between canonical myeloid driver mutations and immunologic alterations, we defined the genetic characteristics of paired pre-transplant MDS/AML samples and post-transplant relapsed samples. In 22 out of 25 cases, at least one driver mutation that was present in the pre-transplant sample was also detected in the relapse sample. Clonal genetic evolution was common at the time of relapse and predominantly involved the acquisition of new subclonal mutations affecting mitogenic signaling (n = 9) and myeloid transcription factors (n = 11). TP53 alterations, including point mutations and 17p deletions were identified in 6 out of 25 patients, including two that remained stable before and after transplantation, and four that were newly detected at the time of relapse. Conclusions: We identified recurrent HLA loss via 6p UPD and segmental deletions in 3 out of 14 patients with late relapse after matched unrelated HCT. Although HLA loss has been observed at relapse after haploidentical HCT, the role of HLA loss as a mechanism of relapse after non-haploidentical HCT has remained unclear. HLA loss in cases with late relapse indicates a prolonged period of immunologic equilibrium, where effective GVL serves as a selective pressure for clonal genetic mechanisms of alloimmune evasion. In the context of MURD HCT, HLA loss provides genetic evidence that allogeneic immune recognition may be mediated by minor histocompatibility antigens and suggests opportunities for novel immunologic approaches for relapse prevention. Disclosures Ho: Jazz Pharmaceuticals: Consultancy. Nikiforow:Kite Pharma: Consultancy. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Soiffer:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.
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Yin, Lihui, Rupa A. Udani, Mary Parlow, Daniel B. Bellissimo, and Joel A. Brochstein. "Assessment of Erythroid Chimerism in Sickle Cell Patients Undergoing HSCT By Digital Droplet PCR." Blood 124, no. 21 (December 6, 2014): 563. http://dx.doi.org/10.1182/blood.v124.21.563.563.
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Abstract Sickle cell disease (SCD) is an inherited autosomal recessive blood disorder associated with significant morbidity. Nonmyeloablative allogeneic hematopoietic stem cell transplantation (HSCT) is increasingly used in severely affected patients with SCD. These transplants result in mixed hematopoietic chimerism so accurate chimerism testing is important for monitoring the status of the transplant. Reconstitution of the erythroid compartment is essential. Since red cells lack a nucleus, DNA-based chimerism assays do not directly assess the chimerism in the erythroid compartment. Several studies have shown that the chimerism in the white cells and erythroid cells can be very different (W, C., etal. Exp. Hematol. 2003, 31:924; Andreanni, M., etal. Haematologica, 2011, 96:128). We developed a procedure for quantification of chimerism in the erythroid compartment of blood using RNA-based digital droplet PCR (ddPCR). The sensitivity and specificity of the assay was evaluated then tested post-transplant samples from sickle cell patients. Total RNA is reverse transcribed and amplified in a two-step RT-PCR approach. The PCR reaction containing allele-specific hydrolysis probes is partitioned into ~15,000 droplets then amplified. The copy number of HbA and HbS transcripts from cells of the erythroid lineage is determined with ddPCR (BioRad, QX-200). The %HbA and the %Donor can be calculated using the donor genotype (A/A or A/S). The assay is designed to have a sensitivity of 1% with donor genotype of A/A and 5% with donor genotype of A/S. Each post-transplant sample is tested in duplicate along with an AA control, SS control, AS control and 1% sensitivity controls (5 controls total). Thirty AA or SS samples were tested to assess assay specificity. The average %HbA detected in a SS sample was 0.03%; the average %HbS in a AA sample was as 0.02%. The background signal is significantly below the cutoff for 1% sensitivity. Using contrived samples with low, medium, and high %HbA, we demonstrate the assay is accurate and linear to 0-2%HbA across the reportable range of 0%HbA to 100%HbA. The % CV of the minor allele for samples in the range of 10%-90%HbA, were equal or below 15%. A total of 11 post-transplant samples from 7 transplanted sickle cell patients were tested, and the results were compared to DNA-based/FISH chimerism, if available. Our results were comparable with DNA-based chimerism (Table 1). Five of the samples had slightly higher %donor in the erythroid compartment compared to the white cell compartment. The erythroid chimerism reflected changes in chimerism status: the decrease then increase in chimerism (P2), stable chimerism (P5) and graft failure (P7). Abstract 563. Table 1: Erythroid chimerism status of post-transplant SCD patients Patient # Donor Genotype Sample # %HbA %Donor %Recipient %Donor by FISH/STR P1 AS 1 52 100 0 95 P2 AA 1 66 66 34 72 2 44 44 56 50 3 100 100 0 95 P3 AS 1 54 100 0 85 P4 AA 1 100 100 0 100 P5 AS 1 14 28 72 15 2 12 24 76 15 P6 AS 1 57 100 0 95 P7 AA 1 0 0 100 Rejecting graft 2 0 0 100 Rejecting graft Assessment of the chimerism in the erythroid lineage may be a better indicator of donor erythropoiesis. We describe an accurate and sensitive assay for monitoring erythroid chimerism and the effects of post-transplant therapies in sickle cell patients undergoing HSCT. This assay also demonstrates the feasibility measuring erythroid chimerism detection in other hematologic disorders, such as thalassemia. Disclosures No relevant conflicts of interest to declare.
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Kaeda, Jaspal, Simone Bonecker, Frauke Ringel, Michaela Schwarz, Bernd Dörken, Ilana Zalcberg, and Philipp Le Coutre. "Droplet Digital PCR Reliably Detects a Single Copy of BCR-ABL1." Blood 126, no. 23 (December 3, 2015): 2784. http://dx.doi.org/10.1182/blood.v126.23.2784.2784.
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Abstract Data show 40% of chronic myeloid leukemia (CML) patients maintain complete molecular remission (CMR), i.e. failure to detect BCR-ABL1, consenting to termination of Imatinib mesylate (IM) therapy, following undetectable disease for ≥2 years by quantitative PCR (Q-PCR). These findings suggest majority of the patients experience molecular relapse. Furthermore, majority relapse in the first 6 months, implying Q-PCR assay sensitivity is suboptimum, to confidently identify patients for discontinuation of IM. Droplet digital PCR (ddPCR) is suggested to have sensitivity that is one log greater than the Taqman (Q-PCR) assay. If verified, ddPCR would enhance safe withdrawal of IM therapy from CML patients. Here we present data comparing ddPCR with Q-PCR. In total we assayed 161 samples, of these 6 were serial dilutions of the International reference (IR) BCR-ABL1 plasmid, the remainder were cDNA samples. The 161 samples comprised of 4 sample groups; I: CML chronic phase samples (n=118); II: CML samples post stem cell transplant (SCT) (n=22); III: normal control (NC) samples (n=16); IV: Serially diluted BCR-ABL1 1.04x10e2 copies/µl, IR ERM-AD623e (n=6). Group I comprised of 121 samples from 21 CML patients in chronic phase treated with IM. Group II comprised of 21 samples from 19 CML patients (2 samples each for 2 of these 19 patients) who had undergone SCT. Group II comprised of samples from normal adult blood donor volunteers. Finally, Group IV included 6 serially diluted samples, ranging 100 to 0.001 copies of the IR (1.04x10e2 copies/µl) plasmid (Sigma, Munich, Germany). All the Group I, III and IV samples were subjected to Q-PCR Taqman assay and ddPCR (Biorad, California, USA). The Group III samples were in addition subjected to nested PCR. Only those samples with cycle threshold (Ct) <37 in 2 or more replicates by Q-PCR were recorded positive. For ddPCR those samples with sum of ≥3 positive droplets with a minimum 5500 droplets per well were reported positive. All the PCR reactions were performed in triplicate in final volume of 20µl, which included 5µl of cDNA or reference plasmid. Among Group I, Q-PCR detected BCR-ABL1 in 57 of the 118 patient samples assayed, with a median of 16.72 transcripts (range 1.38-47450). In only 2 (2.25 and 4.96 transcripts) of these 57 samples, ddPCR failed to detect BCR-ABL1 transcripts. Q-PCR and ddPCR failed to detect BCR-ABL1 in 45 (38.1%) samples. For 14 (11.8%) of the samples ddPCR was positive, median 3.4 copies (range 3-15) but negative by Q-PCR. Among Group II, one of the 21 samples was excluded from the analysis because <5500 droplets were generated. Of the 21 samples 7 were negative by ddPCR. Nested PCR was negative for all 7 samples. Three samples were positive by all 3 technologies, nested PCR, Q-PCR and ddPCR. Remarkably, 5 of the 21 samples were negative by nested, but positive by ddPCR; median 18.0 copies (6.2-22.0). These 5 samples were not subjected to Q-PCR. In Group III all 16 NC samples were negative by Q-PCR and ddPCR. In Group IV, ddPCR did detect BCR-ABL1 in the serially diluted IR sample calculated to have 1 copy (26 positive droplets of the 106095 total droplets), but Q-PCR failed. However, the lower dilutions, calculated to contain 0.1, 0.01 and 0.001 copies were negative by ddPCR and Q-PCR. We assayed 161 samples by ddPCR and Q-PCR, of these 139 were from CML patients. In addition 21 of the samples were also subjected to nested PCR. Our data support the notion ddPCR is at least one log more sensitive than Q-PCR. Of the 140 patient samples assayed, 19 (13.5%) were positive by ddPCR but negative by Q-PCR. Only 2 of the 118 samples in Group I were negative by ddPCR but positive by Q-PCR. There was in insufficient sample to repeat these 2 assays. The increased sensitivity of ddPCR as implied by the clinical samples was supported by assays performed using the IR. The serial dilution equivalent to 1.0 BCR-ABL1 copy was reliably detected by ddPCR, but was negative by Q-PCR. In summary, these data suggest ddPCR is more sensitive. However, the clinical significance of this must be assessed in context of long-term clinical outcome of patients with detectable BCR-ABL1 by ddPCR and negative by Q-PCR. But, clearly increased sensitivity is likely to enhance safe withdrawal of IM therapy for CML patients in CMR. Furthermore, regular monitoring of these patients by ddPCR would enable early detection of molecular relapse and thereby minimize the risk of disease progression. Disclosures Le Coutre: Novartis: Honoraria.
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Lamoureux, Didier, Daniel G. Peterson, Wanlong Li, John P. Fellers, and Bikram S. Gill. "The efficacy of Cot-based gene enrichment in wheat (Triticum aestivum L.)." Genome 48, no. 6 (December 1, 2005): 1120–26. http://dx.doi.org/10.1139/g05-080.
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We report the results of a study on the effectiveness of Cot filtration (CF) in the characterization of the gene space of bread wheat (Triticum aestivum L.), a large genome species (1C = 16 700 Mb) of tremendous agronomic importance. Using published Cot data as a guide, 2 genomic libraries for hexaploid wheat were constructed from the single-stranded DNA collected at Cot values > 1188 and 1639 M·s. Compared with sequences from a whole genome shotgun library from Aegilops tauschii (the D genome donor of bread wheat), the CF libraries exhibited 13.7-fold enrichment in genes, 5.8-fold enrichment in unknown low-copy sequences, and a 3-fold reduction in repetitive DNA. CF is twice as efficient as methylation filtration at enriching wheat genes. This research suggests that, with improvements, CF will be a highly useful tool in sequencing the gene space of wheat.Key words: gene enrichment, renaturation kinetics, gene-rich regions, bread wheat.
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Faraci, Frank M., Curt D. Sigmund, Edward G. Shesely, Nobuyo Maeda, and Donald D. Heistad. "Responses of carotid artery in mice deficient in expression of the gene for endothelial NO synthase." American Journal of Physiology-Heart and Circulatory Physiology 274, no. 2 (February 1, 1998): H564—H570. http://dx.doi.org/10.1152/ajpheart.1998.274.2.h564.
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We examined the hypotheses that responses to acetylcholine are impaired and responses to NO are enhanced in carotid artery from mice made deficient in endothelial nitric oxide synthase (eNOS) by gene targeting (eNOS-deficient mice). We also tested the hypothesis that deletion of one copy of the eNOS gene is sufficient to alter vascular responses. Vessels were studied in vitro from heterozygous (+/−) and homozygous (−/−) eNOS-deficient mice as well as wild-type [eNOS(+/+)] littermates. After precontraction with prostaglandin F2α, acetylcholine produced marked relaxation of carotid arteries in eNOS(+/+) mice, with impaired vasorelaxation in eNOS(+/−) mice. For example, 1 μM acetylcholine relaxed carotid arteries by 55 ± 5% (mean ± SE) in eNOS(+/−) mice ( n = 13) compared with 83 ± 3% in eNOS(+/+) mice ( n = 14, P < 0.001 vs. +/−). In contrast, acetylcholine caused no relaxation in carotid arteries from eNOS(−/−) mice ( P < 0.001 vs. +/+ and +/−). Relaxation of the carotid artery in response to nitroprusside [a nitric oxide (NO) donor] was enhanced ( P < 0.001) in eNOS-deficient mice. For example, in response to 10 nM nitroprusside, the carotid artery relaxed by 18 ± 2% in eNOS(+/+) mice ( n = 14), 33 ± 2% in eNOS(+/−) mice ( n = 13), and 47 ± 4% in eNOS(−/−) mice ( n = 5). Thus relaxation of the carotid artery is impaired with acetylcholine and enhanced with the NO donor nitroprusside in eNOS-deficient mice. Enhanced responses to NO may represent a compensatory response expressed in the absence of eNOS. The findings that vascular responses to acetylcholine and NO are altered in eNOS(+/−) mice compared with those observed in eNOS(+/+) mice suggest a “gene-dosing” effect.
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Schiffmann, R., JA Medin, JM Ward, S. Stahl, M. Cottler-Fox, and S. Karlsson. "Transfer of the human glucocerebrosidase gene into hematopoietic stem cells of nonablated recipients: successful engraftment and long-term expression of the transgene." Blood 86, no. 3 (August 1, 1995): 1218–27. http://dx.doi.org/10.1182/blood.v86.3.1218.1218.
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Abstract In trying to develop methods of gene therapy for Gaucher disease that will avoid the morbidity and mortality associated with bone marrow (BM) ablation, we transplanted BM stem cells transduced with a retroviral vector containing the human glucocerebrosidase cDNA into normal, nonablated, syngeneic mice. Donor BM from untreated male mice or treated with 5-fluorouracil (5-FU) was transduced ex vivo using a standard 4-day transduction protocol. Recipient female mice were injected one time only or once daily for 5 consecutive days or once a week for 5 consecutive weeks using 2 x 10(7) (untreated BM) or 2 x 10(6) (5-FU-treated BM) cells per injection. Initial transduction efficiency into colony-forming unit-spleen (CFU-S) was 80% to 100%. Recipient analysis was performed at least 6 months after the last transplantation. The best engraftment of donor stem cells, up to 5% by secondary CFU-S analysis, was obtained with multiple injections of transduced BM not previously treated with 5-FU. Polymerase chain reaction (PCR) amplification for both the transgene and the Y chromosome identified the progeny of transduced stem cells in various hematopoietic and non-hematopoietic organs. The copy number of the transgene in stem cells was 0.13 to 2.8. Transgene expression was shown by reverse transcriptase-PCR, in situ hybridization, and immunohistochemistry. No serious side effects of the procedure were noted. We conclude that multiple transplants of retrovirally transduced BM cells into nonablated recipients may be a safe and effective therapeutic modality for a number of genetic hematopoietic disorders.
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Schiffmann, R., JA Medin, JM Ward, S. Stahl, M. Cottler-Fox, and S. Karlsson. "Transfer of the human glucocerebrosidase gene into hematopoietic stem cells of nonablated recipients: successful engraftment and long-term expression of the transgene." Blood 86, no. 3 (August 1, 1995): 1218–27. http://dx.doi.org/10.1182/blood.v86.3.1218.bloodjournal8631218.
Full textAbstract:
In trying to develop methods of gene therapy for Gaucher disease that will avoid the morbidity and mortality associated with bone marrow (BM) ablation, we transplanted BM stem cells transduced with a retroviral vector containing the human glucocerebrosidase cDNA into normal, nonablated, syngeneic mice. Donor BM from untreated male mice or treated with 5-fluorouracil (5-FU) was transduced ex vivo using a standard 4-day transduction protocol. Recipient female mice were injected one time only or once daily for 5 consecutive days or once a week for 5 consecutive weeks using 2 x 10(7) (untreated BM) or 2 x 10(6) (5-FU-treated BM) cells per injection. Initial transduction efficiency into colony-forming unit-spleen (CFU-S) was 80% to 100%. Recipient analysis was performed at least 6 months after the last transplantation. The best engraftment of donor stem cells, up to 5% by secondary CFU-S analysis, was obtained with multiple injections of transduced BM not previously treated with 5-FU. Polymerase chain reaction (PCR) amplification for both the transgene and the Y chromosome identified the progeny of transduced stem cells in various hematopoietic and non-hematopoietic organs. The copy number of the transgene in stem cells was 0.13 to 2.8. Transgene expression was shown by reverse transcriptase-PCR, in situ hybridization, and immunohistochemistry. No serious side effects of the procedure were noted. We conclude that multiple transplants of retrovirally transduced BM cells into nonablated recipients may be a safe and effective therapeutic modality for a number of genetic hematopoietic disorders.
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Juvonen, Eeva, Sanna Aalto, Liisa Volin, Klaus Hedman, and Tapani Ruutu. "Impact of Serum EBV-qPCR Positivity on the Outcome of Allogeneic Stem Cell Transplantation: A Retrospective Study of 406 Patients." Blood 106, no. 11 (November 16, 2005): 3233. http://dx.doi.org/10.1182/blood.v106.11.3233.3233.
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Abstract The course of serum EBV-PCR positivity and its impact on the outcome of allogeneic stem cell transplantation (Tx) has not been known well until recently. We have studied retrospectively with quantitative real-time EBV-PCR (qPCR) 5445 serum samples (median 14 sera/patient, range 1–26) collected and frozen-stored according to a predefined schedule within 2 years after Tx from 406 consecutive allogeneic patients transplanted in 1988–1999. 398 patients had a malignant hematological disease, 8 had aplastic anemia. Of the donors 310 were sibling and 96 were unrelated. All donors were HLA A, B, DR matched. 374 grafts were bone marrow and 32 blood stem cells. The grafts were unmanipulated. Patients with an unrelated donor were given anti-thymocyte globulin (ATG). GVHD prophylaxis consisted of cyclosporin A and methotrexate, and in addition methylprednisolone (MP) in 270 patients. Second line treatment for MP-refractory GVHD was ATG. EBV-qPCR positivity (>500 copies/ml) occurred in 55 patients (13.5 %). The median time from the Tx to the first positive sample was 64 days (range day 10 pretransplant - day 537). In 13 patients EBV-qPCR positivity appeared after day 100 post Tx. In 17 patients the EBV-qPCR positivity converted back to negativity, in 25 patients the copy numbers progressively increased or the count was high in the first positive sample, and in 13 patients a positive EBV-qPCR with a low copy number was seen in the last available sample. Only 2 patients with post-transplant lymphoproliferative disorder (PTLD) received EBV-targeted treatment. 7 /55 (13 %) patients with positive EBV-qPCR were alive with a median survival of 3.6 (range 0.7–171) months, while 180 /351 (51%) EBV-qPCR negative patients were alive with a median survival of 51 (0.1–189) months (p<0.0001). In the EBV-qPCR positive group the main cause of death was GVHD in 26 patients, 9 patients relapsed, 6 patients died of infection, and 1 patient had VOD. In 6 patients the cause of death was PTLD. In addition, confirmed PTLD was diagnosed in 6 other patients (3 with GVHD, 2 with relapse and 1 with fungal infection). The cumulative survival was statistically significantly better in those with first positive serum after day 100 post Tx (0.31 vs 0.07, p<0.0001, Log Rank). In the EBV-qPCR negative group the cause of death was GVHD in 46, relapse in 84, infection in 21, other transplant related mortality in 17 patients, and other cause in 3 patients. 1 relapsed patient had also PTLD, and two patients with myeloid leukemia died of NHL. In univariant analysis significant risk factors for EBV-qPCR positivity were unrelated donor, acute GVHD, MP ≥ 2mg/kg/day, and the use of ATG (p<0.0001 each, Chi-Square). Positive EBV-qPCR decreased significantly also the survival of the patients without any of the given risk factor. When compared with the EBV-qPCR neg patients, the survival of EBV-qPCR+ patients with no acute GVHD (alive 6/21 vs 123/216, p= 0.02 Chi-Square), with no ATG given (3/12 vs 146/260, p = 0.04), or without the use of MP ≥ 2mg/kg/day (4/18 vs 125/211 p=0.003) was inferior. Conclusions: In allogeneic transplant patients the EBV-qPCR positivity had a negative impact on the outcome of the transplantation.
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Markiewicz, Miroslaw, Urszula Siekiera, Monika Dzierzak Mietla, Agnieszka Karolczyk, Tomasz Kruzel, Grzegorz Helbig, and Slawomira Kyrcz-Krzemien. "Monozygotic Twins Display Different Minor Histocompatibility Antigens." Blood 114, no. 22 (November 20, 2009): 4324. http://dx.doi.org/10.1182/blood.v114.22.4324.4324.
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Abstract Abstract 4324 Introduction Albeit it is generally presumed that monozygotic twins are genetically identical and that phenotypic differences between twins are mainly due to environmental factors, large-scale variation in copy number of DNA segments recently evidenced by Bruder et al. (AJHG, 2008) showed presence of genotypic diversity in monozygotic twins. The rationale of this study was to test whether monozygotic twins display disparities of minor Histocompatibility antigens (mHags) which may play role in syngenic HCT. We and others have previously shown that mHags constitute an important immunogenetic factor influencing immune responses following transplantation from HLA-matched allogeneic donors. Patients and Methods mHags HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, HwA-9, HwA-10, UGT2B17, HY genotypes were defined with use of Dynal AllSet kits by PCR-SSP method in secured DNA samples from 3 monozygotic twins pairs aged 34, 24 and 28, who underwent syngenic allo-HCTs due to different hematological malignancies (NHL, CML, AML) in the Department of Hematology and BMT in Katowice, Poland in years 2000-2004. Results In 2 out of 3 syngenic pairs we have found differences in genes encoding mHags: different allele of EB-1 was present in one pair (NHL) (recipient HH, donor HY), and two different alleles of HwA-9 (RR, RG) and HwA-10 (**, R*) were present in second pair (CML). No differences in mHags were observed in the third pair (AML). Conclusions Our results question the long-standing belief that monozygotic twins are genetically identical and open up a possibility to further study the role of disparate mHags in disease and transplantation. Disclosures: No relevant conflicts of interest to declare.
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Oneal, Patricia A., Joseph D. Schwartz, Nicole Gantt, Natarajan Bhanu, Y. Terry Lee, Robert Gherman, Alan N. Schechter, Naomi L. C. Luban, and Jeffery Miller. "Two Cellular Mechanisms for Gamma-Globin Gene and Protein Silencing in Humans." Blood 104, no. 11 (November 16, 2004): 374. http://dx.doi.org/10.1182/blood.v104.11.374.374.
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Abstract Although the genetic processes responsible for gamma-globin gene and protein silencing are not known, the prevailing model is that gamma-globin silencing results from a gradual change within a single hematopoietic cell lineage that is governed by intrinsic properties of the cells. In order to provide a more complete characterization of the silencing phenomenon, we studied globin expression patterns directly from clinical samples using single-cell, quantitative PCR, and globin protein phenotyping. We collected blood samples from untransfused donors: umbilical cords (n=3), infants (n=11; ages 1 day to 35 months), and adults (n=3). All samples were maintained at 4°C and analyzed within 72 hours. Flow cytometry (30,000 cells per donor) and HPLC analyses were used for globin protein phenotyping. For globin gene expression, we identified reticulocytes using a strategy that required no membrane permeabilization, and sorted them as single cells directly into lysis buffer. Oligo-dT reverse transcription of mRNA was followed by real-time PCR quantitation. Globin cDNA copy numbers were calculated using standard curves from serial dilutions of a plasmid DNA. We analyzed approximately 1000 single-cell quantitative PCR amplifications for gamma- and beta-globin gene expression. In cord blood, we detected both gamma- and beta-globin gene expression in 97.4% (112/115) of the reticulocytes. The average gamma-globin cDNA copy number was 1870±1390 copies, compared with an average beta-globin cDNA copy number of 2181±2138 copies per reticulocyte. HbF and HbA were also detected in >95% of the cord blood erythrocytes. In the adult samples, HbF was detected in <5% of the circulating erythrocytes and gamma-globin gene expression in only 1.5% (3/206) of the reticulocytes. The average gamma-globin cDNA copy number in the minor population of gamma(+) adult reticulocytes was 468±198 copies, and the average beta-globin cDNA copy number in the beta(+) adult reticulocytes was 3869±3733 copies. Compared with the relatively monotonous patterns of gamma-globin gene and protein expression in cord and adult blood, we clearly detected an age-based fluctuation between those patterns in the infant blood samples. During the first three years of life, a gradual loss in the level of gamma-globin gene and protein expression was identified among the gamma(+)beta(+) reticulocytes and the HbF(+)HbA(+) erythrocytes. In addition, discrete populations of gamma(−)beta(+)reticulocytes and HbF(−)HbA(+) erythrocytes were detected. Rapid expansion of those gamma-silenced populations became apparent soon after birth. Within four months, the proportion of gamma-silenced cells eclipsed that of the gamma(+)beta(+) cells to become the predominant population. By three years after birth, the two cell populations essentially merged to become a single, gamma-silenced population similar to that found in adults. These data suggest two cellular mechanisms for gamma-globin silencing in humans: 1) a gradual loss in gamma-globin expression in the gamma(+)beta(+) cells beginning prior to delivery and continuing during infancy, and 2) replacement of the gamma(+)beta(+) cells with a population of gamma-silenced cells that rapidly accumulate after birth, possibly in response to the dramatic increase in oxygenation or other environmental changes.
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Zhang, Rui, Juan Xiao, Zhouyang Liu, Yuan Sun, Sanfang Tu, Yuhua Li, Leping Zhang, et al. "CAR2.0 Therapy for the Management of Post-Transplantation Relapse of B-Cell Acute Lymphoblastic Leukemia." Blood 136, Supplement 1 (November 5, 2020): 7. http://dx.doi.org/10.1182/blood-2020-141531.
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BACKGROUND: Allogeneic haematopoietic stem cell transplantation (allo-HCT) is a standard treatment for relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL). However ~30-40% of patients (pts) still relapse after HCT. We report a cohort of 20 r/rB-ALL pts, who relapsed after HCT, and enrolled in the CAR2.0 study receiving one or two types of CAR-T cells targeting various B-ALL antigens. METHOD: Pts with r/r B-ALL who relapsed after allo-HCT and did not have significant active comorbiditeis, were enrolled in the study. The target antigens were determined based on immunostaining of each pt's leukemia cells, and CAR-T infusions included a single, or a combination of CAR-Ts targeting the following antigens: CD19, CD22, CD123 and CD38. T cells were collected from pts (N=4) or their allogeneic donors (N=16) and transduced with an apoptosis-inducible, safety-engineered lentiviral CAR with the following intracellular signaling domains: CD28/CD27/CD3ζ-iCasp9 (4SCAR). Pts received cyclophosphamide/fludarabine lymphodepleting therapy before infusion of 0.2-5.8x106 CAR-T/kg per infusion. In addition to disease response, we carefully monitored the quality of apheresis cells, efficiency of gene transfer, T cell proliferation rate, CAR-T infusion dose, and the CAR-T copy number in peripheral blood. RESULTS: Among the 20 enrolled pts, 11 were <18 years of age, and 7 were BCR- ABL (P190) positive. Before CAR-T treatment, 7 pts had ≤grade 2 active graft-versus-host disease (GVHD), and 13 pts received chemotherapy or targeted therapy after their relapse post HCT. Six pts had extramedullary relapse and 2 of them also had bone marrow relapse. The tumor burden in bone marrow ranged from minimal residual disease (MRD) negative to 66% of blasts, based on flow cytometry before CAR-T therapy. Five pts had >10% blasts in bone marrow, 8 pts had <3% blasts, and 7 pts had MRD negative bone marrow (summarized in the Table below). Based on the GVHD history, chimerism state and the available T-cell sources, 16 pts used allogeneic HCT donor T-cells for CAR-T preparation. All pts were full donor chimeras prior to CAR-T infusion, except one pt who had 41% donor cells in bone marrow. Eleven pts received a single CD19 CAR-T infusion, with a mean dose of 1.6x106 CAR-T/kg, and ten achieved an MRD remission and one had progressive disease (PD) within 60 days by flow cytometry. The remaining 9 pts received 2 CAR-Ts (CD19 plus CD22, CD123 or CD38 CAR-Ts) given on the same day, and resulted in 8 CR and 1 PD within 60 days. After CAR-T infusion, no cytokine release syndrome (CRS) was observed in 8 pts, and 12 pts experienced CRS of grade 1, which was consistent with the previously described low toxicity profile of the 4SCAR design. Acute GVHD ≤ grade 2 developed in 5 pts within one month following CAR-T cell infusion but all responded well to supportive care and/or cyclosporine infusion. The 2 pts who developed PD after CAR-T infusion included the one with 41% donor chimerism and had grade 2 GVHD and active infections before CAR-T infusion. The other pt with PD following CAR-T had severe bone marrow suppression, low leukocyte count, infections and was transfusion dependent before enrollment. This emphasizes the need for controlling comorbidities before infusion of CAR-T cells. In summary, total 18 patients (90%) achieved negative MRD remission within 2 months of therapy with acceptable CRS. Four pts relapsed (after being in remission for 3 months) and 14 pts are in continued remission, 6 of which for > 1 year. None of these 20 pts received a second HCT after CAR-T infusion. GVHD developed in 5/16 (31%) pts after donor source CAR-T cell infusion within one month, but all responded well to treatment. CONCLUSION: This study focuses on CAR-T cell therapy following relapse after HCT. While the expanded study is ongoing, we present results of the first 20 pts. Use of donor-derived or recipient-derived CAR-T products in pts who relapsed after allo-HCT is well tolerated and it may prolong life expectancy of these pts while maintaining good quality of life. Table Disclosures No relevant conflicts of interest to declare.
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Sadat, Mohammed, Cecilia Barese, Nancy Pech, W. Scott Goebel, Mervin C. Yoder, Sean Mooney, Kenneth Cornetta, Manuel Grez, and Mary C. Dinauer. "Reconstitution of Neutrophil NADPH Oxidase Activity in Murine X-CGD Following Transplantation of Retrovirus-Transduced Marrow: Potential Impact of Submyeloablative Conditioning." Blood 106, no. 11 (November 16, 2005): 3050. http://dx.doi.org/10.1182/blood.v106.11.3050.3050.
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Abstract X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of innate immunity in which neutrophils lack the superoxide-generating NADPH oxidase, resulting in recurrent pyogenic infections. Genetic blood diseases can potentially be treated by transplantation of autologous hematopoietic stem cells (HSC) transduced with the functional gene. However, in the absence of a selective advantage for HSC following genetic correction, marrow conditioning is required to enhance engraftment. The current study examined the efficacy of 300 cGy as submyeloablative conditioning using a murine model of X-CGD. We compared reconstitution of peripheral blood neutrophil NADPH oxidase activity in female recipients conditioned with either 300 cGy or ablative irradiation prior to transplantation of male bone marrow transduced with a monocistronic retroviral vector for expression of gp91phox, the product of the X-CGD gene, in the SFFV (spleen focus forming virus) backbone. Transduced cells were transplanted into each of 6 mice receiving 300 cGy (8 x 106 cells per recipient) and also into each of five 1100 cGy-irradiated mice (2 x 106 cells per recipient). Chimerism for donor cells in the 300 cGy group ranged from 40-75 % at 6 months (mean ± SD 63 ± 16%), as monitored by FISH for the Y chromosome. The fraction of oxidase-positive cells in the 1100 cGy group at 6 months was 41 ± 11%, and was stable in this range after 2 months post-transplant. Unexpectedly, the fraction of oxidase-positive donor neutrophils was higher in recipients conditioned with 300 cGy, which increased from 44 ± 17% at four months (N=6) to 100% (N=4) at six months, and was 61, 63, and 100% in 3 mice available for study at nine months. Two mice in each group were sacrificed at five months for analysis of vector copy number in spleen DNA, which was 2–3 in both the 300 and 1100 cGy-irradiated groups. In a second experiment, in 1 of 8 mice conditioned with 300 cGy prior to transplantation of 5 x 106 SFFV-91 transduced cells, the frequency of oxidase positive donor neutrophils was 75% at 5 months compared to 19 ± 6% oxidase-positive donor neutrophils in the other 7 mice and in the group conditioned with 950 cGy (18 ± 5%). Ongoing studies include analysis of provirus marking and insertion sites in secondary and tertiary CFU-S. There has been no evidence of abnormal hematopoiesis in primary and secondary transplant recipients. In summary, 300 cGy is effective conditioning for engraftment of transduced marrow in a murine model of X-CGD, and some recipients had an unexpectedly higher fraction of oxidase-corrected donor neutrophils compared to cohorts conditioned with ablative irradiation. Our previous studies suggest that recipients conditioned with submyeloablative irradiation provide a more competitive environment for engraftment of donor cells undergoing the retroviral transduction procedure (Goebel, et al Exp Hem30, 1432, 2002). We thus postulate that a higher frequency of gene-corrected donor neutrophils in 300 cGy-conditioned murine recipients might reflect a selective advantage for proviral integration at loci that are active in myeloid cells.
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Mullally, Ann, and Jerome Ritz. "Beyond HLA: the significance of genomic variation for allogeneic hematopoietic stem cell transplantation." Blood 109, no. 4 (September 28, 2006): 1355–62. http://dx.doi.org/10.1182/blood-2006-06-030858.
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Abstract The last 2 years have seen much excitement in the field of genetics with the identification of a formerly unappreciated level of “structural variation” within the normal human genome. Genetic structural variants include deletions, duplications, and inversions in addition to the recently discovered, copy number variants. Single nucleotide polymorphisms are the most extensively evaluated variant within the genome to date. Combining our knowledge from these studies with our rapidly accumulating understanding of structural variants, it is apparent that the extent of genetic dissimilarity between any 2 individuals is considerable and much greater than that which was previously recognized. Clearly, this more diverse view of the genome has significant implications for allogeneic hematopoietic stem cell transplantation, not least in the generation of transplant antigens but also in terms of individual susceptibility to transplant-related toxicities. With advances in DNA sequencing technology we now have the capacity to perform genome-wide analysis in a high throughput fashion, permitting a detailed genetic analysis of patient and donor prior to transplantation. Understanding the significance of this additional genetic information and applying it in a clinically meaningful way will be one of the challenges faced by transplant clinicians in the future.
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Afolayan, Adeleye J., Annie Eis, Maxwell Alexander, Teresa Michalkiewicz, Ru-Jeng Teng, Satyan Lakshminrusimha, and Girija G. Konduri. "Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension." American Journal of Physiology-Lung Cellular and Molecular Physiology 310, no. 1 (January 1, 2016): L40—L49. http://dx.doi.org/10.1152/ajplung.00392.2014.
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Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that a decreased endothelial nitric oxide synthase level leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAECs) from normal and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels, and ATP levels in PAECs and lung tissue of PPHN fetal lambs at baseline compared with gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) and sirtuin-1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O2 was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared with unventilated PPHN lambs. iNO administration, which facilitated weaning of FiO2, partly restored mtDNA copy number, ETC subunit levels, and ATP levels. DetaNONOate increased eNOS phosphorylation and its interaction with heat shock protein 90 (HSP90); increased levels of superoxide dismutase 2 (SOD2) mRNA, protein, and activity; and decreased the mitochondrial superoxide levels in PPHN-PAECs. Knockdown of eNOS decreased ETC protein levels in control PAECs. We conclude that ventilation with 100% O2 amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by iNO and weaning of oxygen.
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Weigert, Oliver, Nadja Kopp, Andrew A. Lane, Akinori Yoda, Suzanne E. Dahlberg, Donna Neuberg, Anita Y. Bahar, et al. "Molecular Ontogeny of Donor-Derived Lymphomas Occurring After Transplantation,." Blood 118, no. 21 (November 18, 2011): 3671. http://dx.doi.org/10.1182/blood.v118.21.3671.3671.
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Abstract Abstract 3671 Follicular lymphoma (FL) is characterized by the translocation t(14;18), which results in overexpression of the anti-apoptotic protein BCL2 through juxtaposition to the immunoglobulin heavy chain (IGH) locus. Additional genetic aberrations are required and recurrent mutations have been identified in FL, however their timing during lymphomagenesis remains unknown. We performed ultra-sensitive mutation detection to define in vivo clonal diversification in paired follicular lymphomas from a donor-recipient sibling pair that presented more than 9 years after hematopoietic cell transplantation. Briefly, a 41-year old woman with chronic myeloid leukemia (CML) underwent myeloablative bone marrow transplantation from her HLA-matched sister in 2000. She received three donor leukocyte infusions (DLI) for molecular relapse, with the last in June 2002. In November 2009, the donor was diagnosed with grade 2/3A FL. Six months later, the recipient was diagnosed with grade 2/3A FL. The FLs shared identical BCL2/IGH rearrangements, which was also recovered from the DLI at a frequency of 1-in-2000 cells. Both FLs also shared the same V(D)J rearrangement, with the exception of single base-pair mismatches and insertions/deletions (InDels) consistent with ongoing somatic hypermutation (SHM) during clonal divergence. Alignment with germline VH3-66 sequence indicated that the common ancestor had initiated SHM. Whole exome sequencing of both FLs identified 12 single nucleotide variants (SNVs) and 2 InDels in both lymphomas, 3 SNVs unique to the donor's FL, and 4 unique to the recipient's FL. All candidate mutations were validated and confirmed to be somatic by Sanger sequencing. Among the identical mutations identified in both FLs were two SNVs in BCL2, an in-frame deletion in EP300, and an in-frame insertion in KLHL6, which were recently found to be recurrently mutated in lymphoma. Among the SNVs unique to the recipient's FL was an ARID1A (adenine-thymine (AT)-rich interactive domain-containing protein 1A) R1276 premature stop. Loss-of-function mutations in ARID1A have been reported in solid cancers, but not yet in hematologic malignancies. On immunohistochemical staining both lymphomas had decreased ARID1A/BAF250 protein expression, suggesting that loss of ARID1A occurred through separate mechanisms in each FL (i.e. convergent evolution). In fact, the donor's lymphoma was found to have a copy number loss at this locus (1p35.3) by qPCR. To determine whether the somatic mutations that we identified were present at a low frequency within the DLI, we PCR amplified regions flanking each mutation site from the DLI and subjected the products to ultra-sensitive deep sequencing (average read coverage at mutation site, 361,723; range, 16,684–1,169,555). To correct for background frequencies of non-germline calls, we PCR amplified and deep sequenced the same positions from the donor's buccal swab (average read coverage at the mutation site, 418,499; range, 20,711–1,070,734). Eleven of the 12 SNVs and the 2 InDels that were identified in both lymphomas were ‘enriched' in the DLI, i.e., recovered at frequencies significantly above background, indicating that those mutations were present more than 7 years prior to presentation of either lymphoma. All 4 SNVs unique to the recipient's FL and a RAFTLIN V254M mutation identified only in the donor's FL were not enriched in the DLI, consistent with subsequent acquisition during clonal diversification. Of the final two mutations, one was detected only in the donor's FL and was enriched in the DLI. The other was initially detected only in the donor's FL, but deep sequencing recovered the mutation in 4.7% of reads from the recipient's FL and demonstrated enrichment in the DLI. The presence of a mutation in the donor's FL and DLI but not within the majority of recipient's FL cells is consistent with at least two scenarios: (i) the recipient's FL is derived from a clonally diversified population of ancestor cells transferred from the donor or (ii) the mutant allele was lost in a subset or in all cells of the recipient's FL during clonal evolution. In conclusion, we utilized ultra-sensitive mutation detection to elucidate the molecular ontogeny of follicular lymphoma during clonal evolution in separate hosts. This approach has broad applicability for identifying genetic variants within tumor populations that confer phenotypes like therapeutic resistance or metastatic potential. Disclosures: No relevant conflicts of interest to declare.
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Eldfors, Samuli, Mika Kontro, Kimmo Porkka, Olli Kallioniemi, and Caroline Heckman. "Landscape of Mutations in Relapsed Acute Myeloid Leukemia." Blood 124, no. 21 (December 6, 2014): 2367. http://dx.doi.org/10.1182/blood.v124.21.2367.2367.
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Abstract While the majority of acute myeloid leukemia (AML) patients respond to induction chemotherapy, disease recurrence and drug resistance is common. Recently, mutations underlying AML pathogenesis have been extensively characterized by sequencing large numbers of samples obtained at diagnosis. However, mutations driving disease progression and drug resistance in relapsed AML are not well characterized. In addition, understanding the clonal composition of relapsed AML is compounded by interference of donor cell variants present in those patients who have received an allogeneic hematopoietic stem cell transplant (alloHSCT). In this study we sought to identify mutations and copy number aberrations associated with development of drug resistant AML, and at the same time develop methods to identify and filter out donor variants. For the study we analyzed samples from patients who had relapsed after therapy (N=18) by exome sequencing. This included a set of patients where diagnosis and relapse samples were available (n=10), and one patient with diagnosis, remission and relapse samples. All patients had received prior chemotherapy and a subset had relapsed after receiving an allogeneic hematopoietic stem cell transplant (alloHSCT, n=6). Four patients had secondary AML that had developed after treatment for earlier hematologic malignancy. Tumor DNA was from bone marrow mononuclear cells and germline DNA from matched skin biopsies. Exome libraries were prepared then sequenced with the Illumina HiSeq instrument. Sequence data was processed and somatic variants identified as described previously (Koskela et al., NEJM, 2012). We identified relapse specific and relapse enriched somatic mutations by comparing mutation profiles of diagnosis and relapse samples. Donor derived germline variants in chimeric samples from patients relapsing after alloHSCT were identified with a bioinformatic methodology utilizing the dbSNP population variant database. Somatic mutations called from chimeric samples were filtered for common population variants present in the donor’s genome. Rare donor derived population variants that have not been previously described were identified as variants not present in the patient’s germline genome and which had similar tumor variant allele frequencies as the common donor derived variants. We estimated the level of chimerism based on the variant allele frequencies of all donor derived variants. In chimeric samples, the number of donor derived variants vastly exceeded the number of somatic mutations in AMLs (Fig 1). Donor cell content varied widely ranging from close to 100% in a post transplant remission sample to 10-40% in relapse samples. In post-transplant samples, we identified on average 6800 donor germline variants within the exome-capture regions, many of which occurred within cancer genes which could potentially be misinterpreted as driver mutations. Many recurrent driver mutations in cancer genes were identified in the relapse samples: FLT3 (n=6, 33%), DNMT3A (n=4, 22%), NPM1 (n=2, 11%), WT1 (n=2, 11%), TP53 (n=2, 11%), CBL (n=2, 11%), NRAS (n=1, 6%), KRAS (n=1, 6%), IDH1 (n=1, 6%), PHF6 (n=1, 6%) and PTPN11 (n=1, 6%). In several cases, we observed that relapse-specific driver mutations occurred in the same genes or pathways that already had initial mutations at diagnosis. For example, one patient’s AML had a FLT3-ITD at diagnosis; at relapse an activating mutation in CBL and a loss of function mutation in PTPN11 were acquired. Both CBL and PTPN11 act downstream of FLT3 (Fig 2). In two patients with a heterozygous WT1 mutation at diagnosis, we found additional WT1 mutations or deletion of the remaining wild type allele in the relapse sample, suggesting full loss of normal WT1 function contributes to disease progression. Our results suggest that AML progression and drug resistance may be caused by strengthening aberrant signaling through pathways already affected by a mutation present at diagnosis. Hence, the pattern of mutual exclusivity of mutations to genes affecting the same pathway, which has been observed in diagnostic samples, does not occur at relapse. On the contrary, in several cases the relapse specific mutations affected genes in pathways already affected at diagnosis. In addition, we show that donor derived germline variants can be identified and filtered from exome sequence data. Figure 1 Figure 1. Disclosures Porkka: BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Kallioniemi:Medisapiens: Consultancy, Membership on an entity's Board of Directors or advisory committees.
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Yu, Lian, Wei Hao Wu, WANG Shunqing, Ken H. Young, and Funeng Jiang. "CRISPR/dCas9 (D10A) -Mediated Hb CS Gene Editing and Identification of Genetically Modified Fibroblasts." Blood 134, Supplement_1 (November 13, 2019): 5754. http://dx.doi.org/10.1182/blood-2019-129931.
Full textAbstract:
Background: Alpha-thalassemia (α-thalassemia) is an inherited hemolytic disease caused by complete or absent synthesis of α-globin chains due to alpha-globin chain synthesis disorders and is one of the most common genetic diseases in the world. At present, the most commonly used strategies in the gene therapy research of thalassemia, the nonhomologous end joining (NHEJ) apparatus, is only suitable for the study of beta thalassemia and sickle-type anemia. The DNA double strand breaks have a high risk to induce frameshift mutations, leading to more serious clinical consequences. Therefore, it is still necessary to study a safer genetic repair program for alpha-thalassemia. Methods: The donor we chose to repair was from a patient with a genotype of -SEA/αCSα, whose mutation is one of the most common thalassemia alleles in Longyan city, and the HBA1 gene is normal. Due to the deletion of a copy of the HBA2 gene, only one copy of the mutation needs to be repaired, making it a suitable candidate for the study of alpha thalassemia gene therapy. To minimize the risk of non-specific mutations introduced into the locus of interest, we used the single-base editing apparatus, dCas9 (dead Cas9), which only cleaves one strand of DNA and precisely modifies one base, effectively improve the specificity of target cleavage and reduce the risk of off-target. The recognition sequence on the homologous arm in the template was lengthened to reduce the risk of off-target.(see Figure 1 ) Result We used the pmaxGFP plasmid as a positive control for optimizing electroporation conditions. More than 80% transfection efficiency was obtained (see Figure 2 ) Fig 2 pmaxGFP transfection to verify the transfection efficiency of fibroblasts ( 100x ) To investigate the effectiveness of sgRNA and donor plasmids for mutated gene repair, we analyzed the repair efficiency of fibroblasts. Primers were designed near the repair site and PCR sequences were amplified by PCR . After amplification of the genomic fragments, BamH1 digestion assay was performed. Fig 3 PCR and Bam HI digested results using primers for the gene of interest, showed the gene editing efficiency was 4~10%. The sequencing results showed that three clonals of α- thalassaemia mutations were successfully repaired. Fig 4 Hb CS gene mutation target sequence repair and sequencing results The sequencing results of Fig. 5 show that no mutation was detected at the six potential off-target sites. Fig 5 off-target effect detection The use of base editing is limited by off-target activity and DNA delivery efficiency to cells. Filtration into fibroblasts by plasmid electroporation, CRISPR/Cas9-mediated editing efficiency is usually 3~10%, and we achieve a gene editing efficiency of 4~10% (Figure 4). Conclusions Our data show that the gene transfer of normal HBA2 gene by electroporation the single-base editor CRISPR/dCas9 (D10A) and donor into cells can successfully repair Hb CS mutations. Disclosures No relevant conflicts of interest to declare.
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Greenfield, Hayley M., Maged I. Gharib, Andrew J. L. Turner, Mary L. Coussons, Andrew M. Will, Ruth F. Jarrett, Mario P. Macheta, and Robert F. Wynn. "The Impact of Monitoring Epstein Barr Virus PCR in Paediatric Bone Marrow Transplant Patients: Can It Successfully Predict Outcome and Guide Intervention." Blood 104, no. 11 (November 16, 2004): 5064. http://dx.doi.org/10.1182/blood.v104.11.5064.5064.
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Abstract Epstein Barr Virus (EBV) associated Lymphoproliferative Disease (LPD) is a complication of allogeneic haemopoietic stem cell tranplantation (HSCT). In certain groups (congenital immunodeficiency, unrelated and mismatched donor transplants, T cell depletion) the risk may be as high as 25% with significant morbidity and mortality. Strategies to predict such illlness and allow early intervention have therefore assumed importance. We have routinely screened peripheral blood of paediatric, transplanted patients by quantitiative PCR. We report the results of such analysis of 28 successive patients and the EBV serial quantitation in 4 patients with EBV LPD. The median age at time of transplant was 6.5 years. 17 patients received an unrelated donor transplant and one patient received a haplo-identical transplant. The remainder (n=9) received a matched family donor transplant. 23 patients received either Alemtuzumab (n=19) or ATG (n=4). 13 patients had leukaemia, 5 had mucopolysaccharide syndrome, 4 for congential immune deficiency and 6 for non malignant haeamtological conditions. 9 (32%) patients showed no evidence of EBV reactivation using serial PCR monitoring. 10 patients had low level EBV reactivation as defined with a PCR log[copy number] <4.5 copies/ml. 9 patients had a higher level of EBV reactivation. 2 patients had clinical EBV LPD and 2 additional LPD patients with LPD and with archived serial blood samples were also analysed. All patients with clinical LPD fell in the high level reactivation group. All patients with high level reactivation had received either Alemtuzumab or ATG. Patients within this high level group with LPD had a higher PCR log value again than those without LPD (all patients with EBV LPD had levels > 6, whilst the highest level without disease was 5.2). All 4 patients responded to therapy for EBV LPD, with a combination of rituximab, withdrawal of immune suppression or administration of donor lymphocytes - DLI). At higher EBV levels the quantitative PCR had increasing positive predictive value for clinical LPD. We therefore conclude that EBV serial quantitative PCR is useful in discriminating those who will develop LPD from those that will not. Our data suggest that it is possible to use EBV PCR quantitation further to discriminate asymptomatic EBV reactivation that will resolve without therapy from EBV reactivation that will require intervention. This prevents over exposure of patients to treatments (rituximab, DLI, immune suppression withdrawal) with significant associated toxicity (prolonged B cell aplasia, graft versus host disease).
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Takahashi, Yoshiyuki, Itzel Bustos, Yoshiki Akatsuka, Hideki Muramatsu, Nobuhiro Nishio, Asahito Hama, Hiroshi Yagasaki, et al. "Relapse of Leukemia with Loss of Mismatched HLA Due to Uniparentaldisomy Follwing Haploidentical Hematopoietic Transplantation." Blood 114, no. 22 (November 20, 2009): 2457. http://dx.doi.org/10.1182/blood.v114.22.2457.2457.
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Abstract Abstract 2457 Poster Board II-434 Introduction: Down-regulation or loss of human leukocyte antigen (HLA) expression can lead to impaired T-cell recognition and a blunted immune response to malignant tumors. We investigated HLA expression on leukemic cells derived from patients at the time of diagnosis and relapse after HLA haploidentical hematopoietic stem cell transplantation (HSCT) using flow cytometry with locus-specific antibodies. We hypothesized that the loss of HLA haplotype caused leukemic cells to escape immunosurveillance and consequently led to relapse of the disease. Materials and methods: The CD13+/34+ leukemic blasts were sorted by flow cytometry from bone marrow cells at the time of diagnosis and at the time of relapse. Genomic DNA was extracted from leukemic cells by fluorescence-activated cell-sorter as well as phytohemagglutinin-stimulated patient-derived T cells and subjected to single-nucleotide polymorphism (SNP) array analysis using GeneChip NspI arrays (Affymetrix, Tokyo, Japan). Allele-specific copy number was detected using Copy Number Analyser for GeneChip® software. The frequencies of cytotoxic T lymphocyte precursor (CTLp) specific for the recipient mismatched HLA molecules were analyzed using a standard limiting dilution assay. Allo-HLA-restricted CTL clones were isolated by standard limiting dilution and expanded for cytotoxicity assay against mismatched HLA transduced HLA class I-deficient 721.221 B-LCLs. Results: Two of three relapsed patients after HLA haploidentical HSCT demonstrated loss of HLA alleles on leukemic cells at the time of relapse and this loss was limited to the mismatched alleles in both patients. However, none of seven relapsed patients experienced haplotype loss following HLA matched HSCT. SNP array analyses of sorted leukemic cells at the time of diagnosis and at the time of relapse further revealed the copy number-neutral loss of heterozygosity, namely acquired uniparental disomy (UPD) on the short arm of chromosome 6, resulting in the total loss of the mismatched HLA haplotype. Recipient alloantigen-specific cytotoxic T-cell clones were generated from the donor that did not recognize the leukemic cells at the time of relapse, whereas those cells taken at diagnosis were recognized and efficiently killed. In one of the patients, we sought to determine if the number of CTLp had changed during the post-transplant course. A limiting dilution analysis with a split-well 51Cr-release assay was carried out to compare the CTLp frequencies specific for the mismatched antigens between the recipient and donor. Surprisingly, the CTLp frequencies reactive with recipient T cell blasts in CD8+ T cells obtained around Days 100, 180, and 300 (4 months before relapse) were undetectable, while the CTLp frequency obtained at Day 520 (1 month after the third DLI or 2 weeks after remission confirmed by bone marrow aspirate) was 8.6 × 10-5 [95% confidence interval (CI), 1.49 × 10-6 – 5.0 × 10-5]. The CTLp frequency in the donor CD8+ cells was 4.3 × 10-5 (95%CI, 7.2 × 10-5 – 2.5 × 10-5), which was close to that obtained after DLI in the recipient. Conclusions: These results suggest that the cytotoxic T lymphocyte response to mismatched HLA alleles can eradicate leukemic cells; however, escape from immunosurveillance by the loss of mismatched HLA alleles using UPD may be involved in relapse after haploidentical HSCT. Disclosures: No relevant conflicts of interest to declare.
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Yu, Xingxing, Xiangyu Zhao, Xunhong Cao, Xuefei Liu, Xuying Pei, Xiaosu Zhao, Ying-Jun Chang, et al. "Poor NKG2C+ Adaptive NK Cell Recovery at Early Stage after Allo-HSCT Increased the Occurrence of Refractory Cytomegalovirus Infection." Blood 132, Supplement 1 (November 29, 2018): 360. http://dx.doi.org/10.1182/blood-2018-99-115772.
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Abstract Backgroud: Refractory cytomegalovirus (CMV) infection remains important causes of morbidity and mortality after allogeneic hematopoietic stem cells transplantation (allo-HSCT). Previous researches reported that adaptive immunity, such as CD8+ CMV-CTL, plays an important role in the in the control of refractory CMV infection. In mouse, Lanier et al. found there existed subsets of adaptive NK cells with the features of expanding, contracting after control of mouse CMV, and generating long-lived "memory" NK cells. In human, these adaptive NK cells were initially identified based on the high expression of the NKG2C which against HCMV through their cytotoxic potential and the production of TNF-α and IFN-γ upon Ab-mediated stimuli in vitro. Meanwhile, the expression levels of the NKG2C+ adaptive NK cells has been positively correlated with the NKG2C copy number. Several researchers had found that NKG2C+ adaptive NK cells persistent expanded and were potent producers of IFN-γ during CMV reactivation after solid-organ transplant or allo-HSCT. However, the role of NKG2C+ adaptive NK cells on refractory CMV reactivation were still unknown. Whether the rapid reconstitution of NKG2C+ adaptive NK cells can reduce the refractory CMV reactivation merit to be investigated. Aims:In this research, we had investigated the impacts of the quantity and quality recovery of NKG2C+ adaptive NK cells on the occurrence of refractory CMV infection. Method: At first, continuous 364 patients underwent allo-HSCT since June 2012 to February 2016 were prospectively enrolled and we retrospectively analyzed the correlationship between their donor NKG2C genotype and refractory CMV infection occurrence post transplantation. Secondly, the second cohort comprising continuous 125 patients underwent allo-HSCT since May 2016 to April 2017 were prospectively enrolled to analyzed the effect of donor NKG2C genotype on NKG2C+ adaptive NK cell recovery as well as the effect of NKG2C+ adaptive NK cell recovery on refractory CMV infection. The cytotoxicity of reconstituting NKG2C+ adaptive NK cells were evaluated against K562 cells, AD169 CMV stain infected MRC-5 cells, and UL40 peptide pulsed 721.221 cells to detect the anti-tumor or anti-CMV function of NKG2C+ adaptive NK cells. Results: Firstly, from the first cohort, we found that donor NKG2C gene deletion was an independent prognostic factor for refractory CMV reactivation (P=0.010) through the multivariate analysis. Then, through in-depth investigation from the second cohort, we found that the absolute cell counts recovery and anti-tumor function of NKG2C+ adaptive NK cells were both significantly lower in patients accepting NKG2C+/del donor than those patients accepting NKG2C+/+ donors at day 30, 90, and180, respectively after transplantation. There was no NKG2C+ adaptive NK cell recovery post transplantation in the patients who accepted NKG2Cdel/del donors. Meanwhile, anti-CMV function recovery of NKG2C+ adaptive NK cells in patients with NKG2C+/del donors were significantly lower than those patients with NKG2C+/+ donors at day 30 post transplantation. Furthermore, we further analyzed the relationship between the early reconstitution of NKG2C+ adaptive NK cells and refractory CMV infection occurrence. The patients were divided into three groups: no CMV, CMV reactivation (persistent time of CMV infection <2 weeks), and refractory CMV infection (persistent time of CMV infection>2 weeks). We found that the absolute cell counts of NKG2C+ adaptive NK cells in refractory CMV group was significantly lower than that of other two groups at day 30 transplantation. When the patients were devided into high and low level groups based on the ROC cut-off percentage of NKG2C+ adaptive NK cells (1.42%), the result revealed that the patients with lower level of NKG2C+ adaptive NK cells at day 30 post-HSCT had an higher cumulative incidence rate of refractory CMV infection (81.1%) comparing with the higher one (40.5%) (P=0.0014). Moreover, Cox regression model further demonstrated that the lower level of NKG2C+ adaptive NK cells at day 30 post-HSCT was significantly associated with refractory CMV infection (HR=2.578, 95% CI 1.379-4.21, P=0.003). Summary/Conclusion: Our results indicated that donor NKG2C deletion damaged the reconstitution of NKG2C+ adaptive NK cells after allo-HSCT, therefore increased the occurrence of refractory CMV infection post transplantation. Disclosures No relevant conflicts of interest to declare.
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Purvey, Sneha, Enkhtsetseg Purev, Xin Tian, Jennifer Wilder, Lisa Cook, Catalina Ramos, Elena Cho, et al. "High Incidence of Late, Sustained and Clinically Inconsequential Epstein Barr Virus (EBV) Reactivation Following Combined Unrelated Cord Blood and Haploidentical CD34+ Cell Transplantation." Blood 124, no. 21 (December 6, 2014): 1180. http://dx.doi.org/10.1182/blood.v124.21.1180.1180.
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Abstract Introduction: Unrelated cord blood (UCB) transplantation is a useful alternative for patients (pts) with malignant or non-malignant hematological disorders who lack an HLA matched donor. However, because UCB is devoid of memory T-cells, viral infections are more problematic following this transplant approach. EBV is a latent γ-herpesvirus that infects more than 90% of the world's population. Following UCB transplantation, early (<100 days) reactivation of latent EBV is common and is associated with a high risk of post-transplant lymphoproliferative disorder (PTLD). The likelihood of PTLD following late EBV reactivation (>100 days) is less well characterized. We investigated EBV reactivation and PTLD development in early vs late post-transplant periods in severe aplastic anemia (SAA) pts undergoing combined UCB and haploidentical CD34+ cell transplantation. Methods: Pts with SAA and life-threatening neutropenia (ANC <500/uL) refractory to ≥2 immunosuppressive agents were eligible for treatment if they lacked an HLA-matched donor. Conditioning consisted of cyclophosphamide (120 mg/kg), fludarabine (125 mg/m2), equine ATG (160 mg/kg) and a single dose of 200 cGy of total body irradiation. Graft-vs-host disease (GVHD) prophylaxis consisted of tacrolimus and mycophenolate mofetil. Pts received a single ³4/6 HLA antigen matched UCB unit co-infused with a G-CSF mobilized, T-cell depleted CD34+ selected (Miltenyi CliniMacs system) allograft collected by apheresis of a haploidentical relative. EBV copy numbers were measured weekly for the first six months post-transplant and thereafter as clinically warranted. Results: 18 pts with treatment refractory SAA (median age 18 years; range 4-30), including 4 with SAA evolved to myelodysplastic syndrome (MDS) were transplanted. All pts were platelet and RBC transfusion-dependent. 11 pts received a ≥4/6 HLA matched UCB unit and 7 received a 5/6 HLA-matched unit. UCB units contained a median 2.8 x 107 TNCs/kg (range 1.7- 5.2) and 2.1 x 105CD34+cells/kg (range 0.5- 4.7) and haploidentical peripheral blood grafts contained a median 3.2 x106 CD34+cells/kg (range 3- 4.1) and 2.3 x 103 CD3+cells/kg (range 0.8- 5). All 18 pts were EBV IgG seropositive pretransplant. With a median follow-up of 689 days (range 160-2022), 16 pts survive, all disease free and transfusion independent. 16/18 (89%) pts developed EBV reactivation (EBV copy number >1000 copies/ml of whole blood or >150 genome equivalents/million PBMC) at a median 42 days post-transplant (range 2-1165). 12 pts (67%) had EBV reactivation before transplant day 100 (defined as early reactivation). Among those with early reactivation, 10/12 (83%) were treated preemptively with rituximab while 2/12 (17%) did not receive treatment and cleared the EBV viremia without intervention. Late EBV reactivation (defined as reactivation occurring after day 100) occurred in 15/18 (83%) pts. Among those with late reactivation, 12/15 (80%) were not treated while 3/15 (20%) received rituximab, which was given preemptively for PTLD in 2 pts and used to treat biopsy-confirmed PTLD on day 343 in one pt. The median time to first rituximab use was 37 days (range 8-343). Among the 12 pts with late EBV reactivation not given treatment, 9 (50% of those transplanted) had sustained viremia of >1000 copies lasting >30 days (defined as late and sustained EBV reactivation). All pts with late and sustained EBV reactivation remained asymptomatic despite high viral copy numbers, including 4 who had peak EBV copy numbers >30,000 copies/ml (Figure). There was no statistically significant difference in immune reconstitution, as determined by quantitative serum IgG levels and absolute numbers of CD4+ and CD8+ T-cells on day 100, 6 months, 1 year and > 1 year post-transplant in those with vs without late and sustained EBV reactivation. Conclusion: We observed that late and sustained EBV reactivation occurs commonly following combined UCB/haploidentical selected CD34+ cell transplantation, even in pts off immunosuppressive therapy and without CD4 and/or CD8 lymphopenia. In most cases, it was clinically inconsequential, and did not require rituximab treatment. The factors contributing to late sustained EBV reactivation including potential defects in viral-specific immunity remain under investigation. Figure: Figure: Late and sustained EBV reactivation in 4 cord/haplo transplant recipients Figure:. Late and sustained EBV reactivation in 4 cord/haplo transplant recipients Disclosures No relevant conflicts of interest to declare.