bluuebunny/crossref_metadata_2025 huggingface dataset:
WT1 Overexpression after Induction Therapy in Children AML Is Associated with Higher Risk of Relapse.
Authors: Blaise Annick , Leroy Hugues , Auvrignon Anne , Ballerini Paola , Lapillonne Helene , Perot Christine , Mazingue Francoise , Fasola Sylvie , Flamant Cyril , Douay Luc , Leverger Guy , Preudhomme Claude , Landman-Parker Judith
Published: April 2020

The Wilms’ s tumor gene (WT1) is a tumor suppressor gene highly expressed in most acute leukemias. To determine whether WT1 gene expression is a valuable and informative marker for minimal residual disease in pediatric AML, we quantified WT1 transcript amount by RQ-PCR in 92 de novo AML and 20 normal controls. The WT1 transcripts obtained were normalized with respect to the number of TBP transcripts and expressed as WT1 copy numbers by the ratio WT1/TBPx1000. The WT1 levels were extremely low in normal controls, and the median number of WT1 copies was 10 (range 4–30 ). A level above 50 copies was considered as significant. All the patients (aged 2 months-18 years, median age:5.9 years, male/female ratio 0.87) were treated for de novo AML between 1995 and 2003 in two French institutions and enrolled in LAM91, LAM01 and APLs French collaborative protocols. According to the FAB classification the repartition was: MO 5.4%, M1 4.3%, M2 18.5%, M3 14.1%, M4 and M4Eo 12%, M5 33.6 %, M7 10.9% and unclassified 1%. Cytogenetics features according to the MRC classification were favourable, intermediate or poor in 27% (23/83), 59% (49/83) and 13% (11/83) respectively. With a median follow up of 24 months (range 8–97months) OS was 75± 8% and EFS 60 ± 6%. At diagnosis WT1 overexpression was detected in 78.3% (72/92) with a median copy number of 2231 (range 50-429200). The WT1 values were significantly higher (p=0.02) in M2-FAB subtype and lower (p=0.01) in M5-FAB subtype while no correlation was found with WBC count or cytogenetic abnormalities. WT1 quantification for MRD was evaluable in 41/72 pts and positive in 9/32 at D40-50, 5/25 at M3-M5, 5/7at M6-M8. At least one analysis above 50 copies after induction therapy is associated with a significant risk of subsequent relapse 8/11 vs 8/30 ( p=0.007) RR=22 (IC 95%:46–118) and death 10/14 vs 3/30 (p=0.001) RR=7.3 (IC95%: 1–34). Although retrospective analysis may include bias, we conclude that WT1 is a useful and informative molecular marker for MRD in pediatric AML and performed as prospective analysis in ELAM02 protocol.
NUP98 Is Fused to PRRX2, a New Homeobox Partner Gene in t(9;11)(q34;p15), in a Therapy-Related Acute Myeloid Leukemia.
Authors: Gervais Carine , Mauvieux Laurent , Perrusson Nathalie , Helias Catherine , Struski Stephanie , Leymarie Vincent , Lioure Bruno , Lessard Michel
Published: April 2020

The nucleoporin gene NUP98 is known to be rearranged in several recurrent translocations occurring in de novo and therapy-related myelodysplastic syndrome and acute leukemia, in children or adults. All these abnormalities seem to have bad prognosis, thus, it is important to identify NUP98 rearrangements. As other genes (MLL, ETV6, RUNX1, ...), NUP98 may fuse to different partners, often homeobox genes such as HOXA, HOXC, HOXD, PMX1, but also non homeobox genes such as RAP1GSD1, NSD1, NSD3, LEDGF, ADD3, DDX10, and TOP1. The fusion transcript always juxtapose the N-terminal FG repeats of NUP98, required for its docking function, to the C-terminus of the partner gene, which contains the homeodomain in the case of a homeobox partner gene. We identified a t(9;11)(q34.1;p15.5) by conventional cytogenetic analysis, in a 65 years old Caucasian female who developed a t-AML four years after lymphoma treatment. The involvement of NUP98 gene was confirmed by FISH analysis using BAC RP11-120E20 and PAC RP11-1173K1. 3′RACE analysis allowed to identified a novel partner gene, the class II homeobox gene PRRX2 (Paired Related homeobox 2), located on 9q34. The breakpoint occurred in an infrequent breaking region in exon 11 of NUP98, and in exon 2 of PRRX2. The NUP98-PRRX2 fusion transcript was cloned and sequenced, and confirmed by RT-PCR. As its homologue PRRX1 (PMX1), PRRX2 is a DNA-binding transcription factor that is essential for fetal development. PRRX1 has been described to be fused with NUP98 in t(1;11)(q23;p15), but it is the first time PRRX2 is implicated in leukemia, and even in malignancy. Further studies are necessary to confirm the recurrence of this translocation and its prognosis. Furthermore, transformation assays in cells lines and transgenic mice studies would be interesting to understand the leukemogenicity induced by the NUP98-PRRX2 fusion transcript.
Identification of a Second Acute Promyelocytic Leukemia (APL) Patient with the STAT5b-RARα Fusion Gene among PML-RARα-Negative Eastern Cooperative Oncology Group (ECOG) APL Protocol Registrants.
Authors: Gallagher Robert E. , Mak Serena , Paietta Elisabeth , Cooper Brenda , Ehmann W. Christopher , Tallman Martin S.
Published: April 2020

In addition to the PML-RARα fusion gene, which accounts for >98% of APL cases, a common segment of 5′-truncated RARα has been found to fuse with the following 4 alternative genes: promyelocytic leukemia zinc finger (PLZF-RARα), nucleophosmin (NPM-RARα), nuclear mitotic apparatus protein (NUMA-RARα) and signal transducer and activator of transcription 5b (STAT5b-RARα). While recurrent cases of PLZF-RARα and NPM-RARα have been documented, there are only single case reports of NUMA-RARα and STAT5b-RARα. In two Phase III clinical trials, E2491 and C9710, the ECOG registered 10 and 12 presumptive APL patients, respectively, based on initial clinical and cytological assessment, which were negative for PML-RARα by reverse transcriptase-polymerase chain reaction (RT-PCR) testing. In this study, we tested 20/22 of these PML-RARα-negative protocol patients for the presence of the 4 alternative fusion genes by RT-PCR, using the corresponding normal transcripts for the rearranged genes as RNA transcription controls. All specimens were positive for the control gene, while 1 case each was positive for PLZF-RARα or STAT5b-RARα. The karyotypes for these 2 cases, respectively, were 46, XY, t(11:17)(q23;q21) and 46, XY, t(10;11)(q22:q25), i(17)(q10). None of the X-RARα-negative cases had abnormalities of chromosome 17. Immunophenotypically, the PLZF-RARα case was consistent with APL; the STAT5b-RARα case had all features of APL (negative for CD11a, CD18, CD34, HLA-DR) except for weak CD133 expression. These findings imply that it is the RARα rearrangement that prompts the APL immunophenotype. Sequence analysis of the STAT5b-RARα mRNA disclosed the same break/fusion point of the STAT5b gene as the only previously-reported case of STAT5b-RARα-positive APL (Arnould, et al, Hum Mol Genet8, 1741, 1999). Also like the first case (67 years), our STAT5b-RARα patient was an elderly male (57 years). These results suggest that X-RARα fusion genes will rarely be found in the absence of cytogenetic evidence of chromosome 17 abnormalities and establish that STAT5b-RARα, although rare, is a recurrent pathogenetic factor in APL.
The Presence of FLT3/ITD Mutations Is an Independent Prognostic Factor in Acute Myeloid Leukemia Patients with Normal Karyotype.
Authors: Kim Yeo-Kyeoung , Lee Je-Jung , Lee Yu-Ra , Lee Il-Kwon , Kim Hee-Nam , Kim Nan-Young , Park Kyeong-Soo , Yang Deok-Hwan , Park Moo-Rim , Kim Hyeoung-Joon
Published: April 2020

An internal tandem duplication of the FLT3 gene (FLT3/ITD) has been detected in approximately 20–30% of patients with acute myeloid leukemia (AML). These are frequently associated with poor outcome in AML patients, but it is still a matter of debate whether the FLT3/ITD mutations play a role in the prognosis of AML patients independently or not. We investigated the presence of FLT3/ITD mutation in 165 patients with de novo AML, except acute promyelocytic leukemia (APL), to evaluate its clinical and prognostic significance. The FLT3/ITD mutations were studied on bone marrow samples at diagnosis using PCR assay. Of the patients, 58 patients (35.2%) demonstrated the aberrant FLT3/ITD mutations. The patients with FLT3/ITD had significantly higher WBC counts at presentation compared with patients without FLT3/ITD (52.9 ± 66.9 ×109/L vs. 32.4 ± 41.8 ×109/L, p < 0.05). However, there was no statistically significant difference in age, gender, hemoglobin level, platelet count, percentage of peripheral or bone marrow blasts, or the presence of molecular abnormalities between the patients with FLT3/ITD and the patients without FLT3/ITD. To analyze the response to or outcome of therapy, we evaluated 118 patients who received intensive induction chemotherapy. In univariate analysis, there was no significant difference in complete response rate (p = 0.21), in median duration of overall survival (13.9±3.8 ms. vs. 16.5±0 ms., p = 0.07), or in median duration of leukemic-free survival (LFS) (9.8±3.5 ms. vs. 34.6±17.9 ms., p = 0.09) between the patients with FLT3/ITD and the patients without FLT3/ITD. However, the presence of FLT3/ITD was associated with lower LFS in the patients with a cytogenetically intermediate-risk group (p < 0.05). Furthermore, in multivariate analysis, FLT3/ITD mutations were an independent prognostic factor in LFS in AML patients with normal karyotype (p < 0.05). In conclusion, this study demonstrates that the presence of FLT3/ITD mutations is a significantly poor prognostic factor for leukemic free survival in non-APL patients with a cytogenetically intermediate-risk group, especially normal karyotype.
FAS Promoter Polymorphism and Etiology and Outcome of Childhood Acute Myeloid Leukemia.
Authors: Mehta Parinda A. , Alonzo Todd A. , Elliot James S. , Gerbing Robert B. , Wilke Tiffany A. , Perentesis John P. , Lange Beverly J. , Davies Stella M.
Published: April 2020

FAS (TNFRSF6/CD95/APO-1) is a cell surface receptor involved in apoptotic signal transmission. Dysregulation of this pathway is believed to result in down regulation of apoptosis and subsequent persistence of a malignant clone. A single nucleotide polymorphism resulting in guanine-to-adenine transition in the FAS promoter region occurs at position −1377, affecting an SP1 transcription factor binding site; an adenine residue at position −1377 of FAS promoter significantly reduces SP1 binding compared to guanine residue causing a decrease in FAS expression. Recent data in adult leukemia indicate that a variant allele at this site increases susceptibility to AML (Morgan et al; Cancer Research 63, 4327–4330). We hypothesized that FAS genotype would also increase risk of childhood AML and, by altering susceptibility to apoptosis might impact outcome of AML therapy. Methods: 442 children treated for AML on CCG studies 2941 and 2961 (intensive induction with daunorubicin, idarubicin, Ara-C, 6-thioguanine, etoposide, dexamethasone, +/− fludarabine, Ara-C, idarubicin; consolidation with high-dose Ara-C and L-asparaginase or HLA-matched sibling BMT) were genotyped for the −1377 FAS promoter polymorphism using mismatch amplification coupled with real time PCR (MAMA assay). Genotype frequencies were compared with published normal control frequencies. In addition patient outcomes were analyzed according to genotype.

Results: Comparison of gene frequencies in AML patients and reported normal controls showed similar frequencies (78.7% GG, 18.6% GA, 2.7% AA in patients vs. 78% GG, 20% GA and 2% AA in controls; p=0.6). Stratification of cases by age at diagnosis, white blood count at diagnosis, AML subtype, or cytogenetics revealed no difference in genotype frequencies. Outcome data showed no significant difference in OS (p=0.22), EFS (p=0.31), TRM (p=0.42) or relapse rate (p=0.57) between patients with 1377GG genotype vs. 1377GA/1377AA genotypes. However, there was trend towards improved survival (OS at 5 years 75% AA, 55% GA, 49% GG; p=0.16) and EFS (66% AA, 43% GA, 39% GG; p=0.19) in AA cases, but small numbers (n=12 AA) may have contributed to the results not being statistically significant. Conclusion: These data, representing the only data in pediatric AML, suggest that FAS genotype does not affect the etiology of childhood AML and that children with AML differ from adults in terms of biology of their disease. However, further studies are needed to evaluate a potentially important role of FAS genotype in outcome of AML therapy.
Heparin-Induced Thrombocytopenia Is a Thymus (T cell) Dependent Disorder.
Authors: Suvarna Shayela , McCracken Emily K.E. , Arepally Gowthami M.
Published: April 2020

Heparin-Induced Thrombocytopenia (HIT) is a drug-dependent immune disorder caused by autoantibodies to Platelet Factor 4 (PF4) and heparin. The immune basis of HIT is poorly understood. Recent studies describing transient antibody responses and absence of immunologic memory in HIT suggest that PF4/heparin autoantibodies may develop independently of T cell help. To investigate the cellular basis of the HIT immune response, we have developed a murine autoimmune model in which anti-murine PF4/heparin (anti-mP+H) arise de novo. Cohorts of BALB/c mice were immunized daily either intravenously (IV, n=10) or intraperitoneally (IP, n=10) for five days with complexes of murine (m) PF4/heparin (IV, n=5 or IP, n=5), heparin alone (IV, n= 2) or buffer (IV, n=3 or IP, n=5). Mice were screened for anti-mP+H for four weeks after immunization using a murine PF4/heparin ELISA. Peak antibody responses to antigen were seen at 11–15 days in 2/5 mice injected with IV mP+H (Day 11, mouse IV P+H #0 peak A450nm =0.34±0.01; Day15 mouse IV P+H #2 peak A450nm =0.69±0.01), and at days 22–25 in 2/5 mice injected by IP route (Day 22, mouse IP P+H #0 peak A450nm =0.37±0.01; Day 25 mouse IP P+H #2 peak A450nm =0.78±0.02). Anti-mP+H were not detected in mice injected with heparin alone or buffer alone at any time point (peak maximum IV A450nm= 0.1±0.001, Control #2; mouse IP P+H Control #2 A450nm =0.04±0.002). Serologically, murine autoantibodies were similar to anti-human (h) PF4/heparin. Anti-mP+H reactivity was specific for murine antigen (mouse IV P+H #2 A450nm=0.65±0.06), and was reduced with antigen in the presence of excess heparin (A450nm=0.38±0.01). Minimal reactivity was seen with wells coated with hP+H (A450nm=0.09±0.005), albumin (mouse IV P+H #2 A450nm=0.15±0.03), or PBS alone (mouse IV P+H #2 A450nm =0.16±0.01). Similar to human HIT antibodies, anti-mP+H were of IgG1 subclass. To determine if T cells are required for development of anti-mP+H, mice lacking T cell function (BIG:BALB/c-Nu, n=10) were injected IV with mP+H daily for five days. Unlike euthymic mice, nude mice did not manifest any antibody responses to IV injections of mP+H. In summary, we have developed a novel murine autoimmune model of anti-PF4/heparin that recapitulates many salient features of the human immune syndrome. Using this murine model, we demonstrate that T cells are essential for development PF4/heparin autoantibodies. Studies are currently underway to delineate mechanisms of T cell regulation and peripheral tolerance in HIT.
Comparison of Notoginsenoside R1 and TPO on Thrombopoiesis in Irradiated Mice.
Authors: Yang Mo , Lam T.L. , Li Karen Kwai Har , Leung L.K. , Lau KM , Cheng S.W. , Pong N.H. , Lau C.P. , Fung K.P. , Fok Tai Fai
Published: April 2020

The use of combination of Chinese herbs as a treatment for thrombocytopenia has been reported to be effective and safe. We have reported that Angelica Polysaccharide (extracts of Angelica Sinensis) has promoting effect on blood stem cells and megakaryocytopoiesis (Yang et al, Blood, 2002, 100 (11); 53a). Sanqi, Radix Notoginseng, is the dried roots of Panax notoginseng (Burk.) F. H. Chen (Araliaceae). It has been used for treatment of trauma and bleeding due to internal and external injuries. Its main constituents are ginsenosides (a kind of saponins), as well as notoginsenosides (only rich in Notoginseng species). Although Sanqi is a well-known haemostatic drug, its effects and mechanisms on megakaryocyte/platelet production have not been studied. The objective of this study was to compare the effect of a purified notoginsenoside R1 (NR1) and thrombopoietin (TPO) on thrombopoiesis in irradiated mice. NR1 (2.5 mg) and TPO (0.25 ug) were dissolved in distilled water and given by intra-peritoneal injection daily for 14 days starting from the day after radiotherapy. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from NR1, TPO, and vehicle control groups on day 0, 7 and 14. On day 14, the mice were sacrificed and bone marrow cells were harvested for CFU-MK, CFU-GM, BFU-E and CFU-F (fibroblastoid) assays (n=5). Our results showed that NR1 enhanced the recovery of platelets, WBC, and RBC count. Moreover, NR1 also promoted the CFU-F (12 ± 0.7 vs 19 ± 0.38 colonies/2 x 106 cells, p=0.0034), CFU-MK (22 ± 1.9 vs 26 ± 3.8 colonies/2 x 105 cells, p=0.025), CFU-GM (26 ± 5.2 vs 37 ± 4.3 colonies/2 x 105 cells, p=0.002), and BFU-E (13 ± 2.9 vs 18 ± 1.9 colonies/2 x 105 cells, p=0.003) formation. Similar results were obtained in TPO-treated group. In in-vitro study, we further analyzed the effect of NR1 (0–50mM) on mouse CFU-MK formation using a plasma clot colony assay. The results showed that NR1 (20 mM) enhanced TPO (50 ng/ml)-induced CFU-MK formation (19 ± 2.2 vs 30 ± 6.8 colonies/2 x 105 cells, p=0.02, n=5). Furthermore, the effect of NR1 (5–50 mM) on the growth of bone marrow stromal cells was also investigated using CFU-F assay. NR1 (50mM) had a promoting effect on CFU-F growth (18 ± 3.7 vs 24 ± 1.8 colonies/2 x 106 cells, p=0.043, n=5). Our studies showed that NR1 enhances thrombopoiesis in vivo and the growth of bone marrow stromal cells as well as megakaryocytes in vitro. Therefore, we speculate that the thrombopoietic activity of NR1 may be mediated via promoting the progenitor of platelet, megakaryocytes, and bone marrow stromal cells. Although TPO has been used as an agent for the recovery of platelet production after the onset of thrombocytopenia, long-term clinical usage of TPO may induce potential side effects such as thrombosis. Here we reported that the effect of NR1 is comparable with TPO on the production of platelets in irradiated mice.
Fluorescence In Situ Hybridization (FISH) Using a Subtelomeric 11q Probe as a New Diagnostic Tool for Congenital Thrombocytopenia Caused by Deletions in 11q.
Authors: Gadzicki Dorothea , Michaelsen Gudrun , Rudolph Cornelia , Steinemann Doris , Happel Christoph M. , Welte Karl , Klein Christoph , Toennies Holger , Lichter Peter , Kontny Udo , Niemeyer Charlotte Marie , Schlegelberger Brigitte
Published: April 2020

Neonatal thrombocytopenia occurs in about 1% of all newborns. Inherited forms like 11q- or Jacobsen syndrome are rare. However, they may remain undetected with karyotyping because the deleted regions in 11q often involve small subtelomeric regions. Here we report on the detection of deletions in 11q in two newborns with normal routine karyotypes who were shown to carry subtelomeric deletions in 11q by means of fluorescence in situ hybridization (FISH) using a subtelomeric 11q probe (Abbott, Diagnostics, Wiesbaden, Germany). Both children showed thrombocytopenia (18.000/μl and 26.000/μl, respectively) and dysmegakaryopoiesis (absence of normal megakaryocytes and presence of micromegakaryocytes) associated with facial dysmorphism, cardiac defects and psychomotoric retardation. In the second case, the mother and the grandmother also showed mild thrombocytopenia. In both patients, FISH analyses on peripheral blood and bone marrow showed the loss of the telomere-associated region of 11q distal of the MLL gene. In the first patient, the deletion of 11q resulted from an unbalanced complex rearrangement with duplication of 11p. As the source of this chromosomal aberration, a paternal pericentric inversion of chromosome 11 was identified. The partial monosomy 11q and the partial trisomy 11p in the first patient were confirmed by comparative genomic hybridization (CGH) analysis. Array/matrix CGH assisted in determining the breakpoints at 11p15.1 and 11q24.1. No structural aberrations of 11q were found in the mother of the second patient, but further investigations are under way. These findings give further evidence that small subtelomeric deletions of 11q and probably mutations of genes located therein cause thrombocytopenia. Since it can be very difficult to detect these deletions by karyotyping, FISH using a subtelomeric 11q probe seems to be an extremely useful new diagnostic tool. This new method should be applied in children with congenital thrombocytopenia, in particular if they have additional complex dysmorphic features.
Are the Pfa-100® Closure Time and the Skin Bleeding Time Useful in the Diagnostic Work-Up of Patients with Bleeding Diathesis? Correlation with the Severity of Bleeding Symptoms and Sensitivity for Known Defects of Primary Hemostasis.
Authors: Podda GianMarco , Bucciarelli Paolo , Lussana Federico , Cattaneo Marco
Published: April 2020

We compared the performance of PFA-100® closure time (CT) and the skin bleeding time (BT) in the diagnostic work-up of 128 consecutive patients referred to our Centre from June 2002 through June 2003 to be screened for bleeding disorders, due to the presence of bleeding symptoms or the casual finding of abnormal screening tests of hemostasis. The correlation with the severity of the bleeding symptoms and the sensitivity of BT and PFA-100® CT for known defects of primary hemostasis were evaluated. All patients underwent a careful medical interview and were assigned a “bleeding score”, based on the number, type, frequency and severity (development of anemia, need for blood transfusion and/or surgical or medical intervention) of bleeding symptoms. In addition, all patients underwent a first-line screening, which included prothrombin time, activated partial thromboplastin time (APTT), BT and PFA-100® CT (with both the collagen-ADP and the collagen-epinephrine cartridges). The search for Von Willebrand disease (VWD), platelet function disorders (PFD), clotting factor defects and abnormalities of fibrinolysis was performed according to the results of the first line screening tests and the severity and type of bleeding history. Seven (6%) patients had type-1 VWD, 12 (9%) PFD, 29 (23%) defects of clotting factors, 18 (14%) had prolongations of the APTT due to abnormalities that are not associated with bleeding (factor XII deficiency and lupus anticoagulant), while in 63 (49%) all tests gave normal results. The sensitivity of PFA-100® for VWD was 86% (both cartridges), for PFD 75% (collagen-epinephrine) and 8% (collagen-ADP). The sensitivity of BT for VWD was 29%, for PFD 33%. The CT of the PFA-100® CT collagen-epinephrine cartridge was mildly prolonged in 38% of patients with clotting factor deficiency. After dividing the patient population in four quartiles of distribution, according to the severity of the bleeding score (null, low, intermediate and severe), only the CT of the PFA-100® CT collagen-epinephrine cartridge showed a progressive and significant prolongation from the first to the fourth quartile (p=0.04). No association of BT and collagen-ADP CT with the severity of bleeding history was found. In conclusion: 1) CT of the PFA-100® collagen-epinephrine cartridge was significantly associated with the severity of the bleeding history; 2) PFA-100® CT showed a better sensitivity than BT for VWD and PFD; 3) at variance with the BT, PFA-100® could help to discriminate between VWD (prolongation of CT of both cartridges) and PFD (prolongation of CT of the collagen-epinephrine cartridge only).
Ex Vivo Blockade of NF-[dappa]B Signaling in Alloreactive T Cells Prevents Murine Graft-Versus-Host Disease (GVHD).
Authors: O’Shaughnessy Matthew J. , Vogtenhuber Christine , Serody Jonathon S. , Sitcheran Raquel , Baldwin Albert S. , Dang Lenny , Jaffee Bruce , Blazar Bruce R.
Published: April 2020

A failure of IL-2 transcription has been associated with tolerance induction. We hypothesized that inhibition of the NF-κB pathway in alloreactive T-cells, which is critical for IL-2 transcription, could lead to alloantigen-specific hyporesponsiveness and prevention of GVHD. PS1145, a potent inhibitor of IκB kinase, and hence NF-κB activation, was added to an MLR culture consisting of CD4+ T-cells and MHC class II-disparate stimulators. Inhibition of NF-κB activity was verified by EMSA and confocal microscopy. Global inhibition of cytokine production and T-cell hyporesponsiveness was observed which persisted after washing T-cells and re-exposure to alloantigen. Responses to non-specific mitogens remained largely intact and alloantigen hyporesponsiveness was reversed by exogenous IL-2. Treatment of T cells and stimulator cells with PS1145 was required for maximal effect. Depletion of CD4+CD25+ cells from the MLR indicated that these cells were not required for tolerance induction in this system. Using an MLR system containing alloreactive and non-alloreactive transgenic T cells indicated that PS1145 treatment increased the rate of T-cell apoptosis selectively in alloreactive cells. Data from each of 4 experiments showed that GVHD in recipients of ex vivo PS1145 treated cells was profoundly inhibited, whereas CD4+ T-cells recovered from a vehicle-treated 7-day MLR were uniformly fatal upon adoptive transfer into sublethally irradiated MHC class II-disparate recipients. Studies addressing non-alloreactive in vivo responses of PS1145 treated T cells will also be presented. Our studies indicate that the NF-κB pathway is a critical regulator of productive alloresponses and provide a novel ex vivo approach to induce alloantigen-specific tolerance as a means of preventing GVHD.
Epitope Mapping during FVIII Inhibitor Elimination with Rituximab Revealed Conformational Epitopes on FVIII and Identifies Potentially Small Molecules for Novel Therapies.
Authors: Königs Christoph , Kessel Christoph , Linde Richard , Klingebiel Thomas , Kreuz Wolfhart
Published: April 2020

Hemophilia A is an X-linked bleeding disorder. Mutations in the Factor VIII (FVIII) gene result in reduced or non-functional expression of FVIII. Patients are commonly treated by substitution of recombinant (rFVIII) or plasma derived (pdFVIII) FVIII preparations. However, a significant number of patients treated develop an antibody response (inhibitors) against substituted FVIII. Various Immune Tolerance Therapies (ITTs) are applied to overcome the FVIII specific immune response.

We recently reported on a novel approach to eliminate the FVIII specific immune response by several rounds of rituximab (Mabthera®) infusions after several failed ITT approaches.

In this study, phage displayed random peptide libraries were screened with sera from a patient before, under and after rituximab treatment. After several rounds of biopanning more than 100 phage clones specific for FVIII-inhibitors were identified and sequenced. One amino acid motif - EVPN - was most common in isolated clones. Computer based analysis identified a conformational epitope in the A2 domain of FVIII. The clones specifically inhibit binding of inhibitor to FVIII. Also sera from other inhibitor patients cross reacts with isolated phage clones. Inhibitor sera also bind to synthetic peptides, which sequences correspond to the peptide insert on isolated phage. Functional studies to target inhibitor specific B cells with isolated peptides are currently being performed.

Specific ligands for FVIII inhibitors were identified. The epitope was mapped to the A2 domain. When inhibitor relapsed after Rituximab treatment, the same epitope was recognized. Small peptides representing the epitope on the phage (mimotopes) bound to inhibitor positive sera and even cross-reactivity with heterologous sera was observed. The inhibitor specific peptide ligands may serve as useful tools to develop novel approaches for inhibitor elimination therapies.
Factor V G1691A and Factor II G20210A Mutations and Clinical Expression of Severe Hemophilia A (< 2%) in Children: Impact on Bleeding Episodes and Joint Damage.
Authors: Nowak-Gottl Ulrike , Escuriola Carmen , Schobess Rosemarie , Duering Christine , Bidlingmaier Christoph , Pollmann Hartmut , Horneff Sylvia , Kurnik Karin
Published: April 2020

It has been recently shown that the first bleeding onset in children with severe hemophilia A (HA) carrying prothrombotic risk factors is significantly later in life than in non-carriers.1 The present multicenter study was performed to determine whether the factor (F) V G1691A or the F II G20210A are associated with decreased annual bleeding episodes (ABE) in 106 pediatric PUP patients with severe HA (Intron 22 58.6%) consecutively admitted to German pediatric hemophilia treatment centers. Treatment was initiated according to the frequency of bleedings, and most patients received on demand therapy with a switch over to prophylactic therapy 3x/week (40–60 IU/kgKG factor VIIII concentrate) when more than three bleedings (range 2–6) had occurred into the same joint (n=49). Prospective median(range) patient follow-up was 14(4–35) years. Heterozygosity of the FV mutation was found in 8 subjects, homozygosity in one, and 5 children carried the FII mutation once combined with protein C-deficiency. Carriers of the FV and FII mutations had significantly fewer ABE than non-carriers (p=0.004). 66 of 106 PUP patients developed at least one target joint with a median(range) Pettersson score of 1(0–12) available in 57 patients clearly dependent on age (p=0.039) as well as ABE (p=0.037). The “Nuss” joint score available in 33 subjects highly correlated with the Pettersson score (p=0.007). Data presented here give evidence that the clinical expression of severe HA in children is influenced by the co-expression of the FV and FII mutation.
Slug Plays an Essential Role in the Radioprotection of Hematopoietic Progenitors In Vivo by Antagonizing p53-Mediated Apoptotic Pathways.
Authors: Wu Wen-Shu , Xu Dong , Heinrichs Stefan , Look A. Thomas
Published: April 2020

An antiapoptotic role for Slug/Snail in mammals was suggested by studies in C. elegans, where CES-1/Scratch, a member of the Slug/Snail superfamily, was found to control the apoptotic death of NSM sister neurons by acting as a transcriptional repressor of EGL-1, a BH3-only proapoptotic protein. Identification of Slug as the target gene of the E2A-HLF oncoprotein in human pro-B leukemia cells led us to demonstrate its antiapoptotic function in IL-3-dependent murine pro-B cells. In contrast to its aberrant expression in pro-B leukemia cells, endogenous Slug is normally expressed in both LT-HSC and ST-HSC, as well as committed progenitors of the myeloid series, but not in pro-B and pro-T cells, implying its function in myelopoiesis. Using Slug−/− mice produced in our laboratory, we showed that these knockouts are much more radiosensitive than Slug+/− and wild-type mice, and that apoptotic cells increase significantly in the hematopoietic progenitor cells of Slug−/− mice as compared to wild-type mice following γ-irradiation, indicating a radioprotective function in vivo. We showed here that although the development of myeloid progenitors is not impaired under steady-state conditions, their repopulation is incomplete γ-irradiated in in Slug−/− mice. We demonstrate further the radiation-induced death of Slug−/− mice is exclusively a result of bone marrow failure with no apparent contribution from systemic injures to other tissues. By two-way bone marrow transplantation, we provide firm evidence that Slug protects mice from γ-irradiation-induced death in a cell-autonomous manner. Interestingly, regenerative capacity of hematopoietic stem cells (HSC) was retained in irradiated Slug−/− mice, which could be rescued by wild-type bone marrow cells after irradiation, indicating that Slug exerts its radioprotective function in myeloid progenitors rather than HSCs. Furthermore, we establish that Slug radioprotects mice by antagonizing downstream of the p53-mediated apoptotic signaling through inhibition of the p53-resposive proapoptotic gene Puma, leading in turn to inhibition of the mitochondria-dependent apoptotic pathway activated by γ-irradiation in myeloid progenitors. More interestingly, we observed that Slug is inducible by γ-irradiation in a p53-dependent manner. Together, our findings implicate a novel Slug-mediated feedback mechanism by which p53 control programmed cell death in myeloid progenitor cells in vivo in response to γ-irradiation.
Treatment of Anemia with Aranesp® (Darbepoetin-alfa) Given Once Every Two Weeks in HIV Seropositive Patients.
Authors: Tulpule Anil , Dharmapala Dharshika , Burian Pamela , Espina Byron M. , Jayne Gerald , George Shelly , Levine Alexandra M.
Published: April 2020

Background: Anemia is common in patients (pts) infected with the Human Immunodeficiency Virus (HIV). In these pts, anemia is associated with diminished quality of life (QoL) and is an independent prognostic factor for mortality. Aranesp® (darbepoetin-alfa) is a recombinant erythropoiesis-stimulating protein with extended serum half-life allowing for less frequent dosing than conventional recombinant erythropoietin. We studied Aranesp® in anemic HIV-positive pts to evaluate safety, hemoglobin (Hgb) response, and quality of life (QoL).

Methods: Entry criteria included HIV infection, Hgb < 11.5 g/dL, and adequate renal and liver function. Patients with anemia due to bleeding, malignancy, B12, folate, or iron deficiency were excluded. Aranesp® 3.0 mg/kg was given sc every 2 weeks (Q2W) for 24 weeks. Treatment response was defined as Hgb increase of ≥ 1.5 g/dL from baseline sustained for ≥ 4 weeks duration at any time during the study. If no response by wk 8, Aranesp® was escalated to 5.0 μg/kg.

Results: Thirteen female and 10 male pts have been enrolled to date. Median age was 43 years (range 27–67), median entry Hgb was 10.6 g/dL (range 8.6–11.5), and median CD4 count was 197 cells/mm3 (range 29–821). Nine (39%) pts had a prior clinical AIDS-defining illness or opportunistic infection, 19 (87%) pts received concurrent HAART therapy; 11 (58%) were receiving a zidovudine- containing regimen with either combivir (n=6) or trizivir (n=5). Aranesp® was well tolerated. The only treatment-associated side effect was mild pain at the injection site. Pts who received ≥ 8 wks of Aranesp® were analyzed (n=20). 17 of the 20 pts (85%) had a Hgb response (median duration 12+ weeks [range 4+ to 22+]). One of these pts required a dose escalation. Evaluation of change in QoL using a Linear Analog Scale in 20 evaluable patients showed a mean improvement in energy level (+23.2 mm), activity level (+19.8 mm) and overall QoL (+15.5 mm) over the first 8 wks of treatment.

Conclusions: In HIV-infected pts with anemia, Aranesp® at a dose of 3.0 mg/kg Q2W is well tolerated, is associated with a Hgb response in the majority of pts, effectively maintains Hgb levels, and improves QoL. Further work is on-going.
Akt Regulates the Activation-Induced Apoptosis of T Cells Mediated by HIV-1 gp120.
Authors: Anand Appakkudal R. , Ganju Ramesh K.
Published: April 2020

Multiple mechanisms contribute to the loss of CD4+ T cells in HIV-1 infected individuals. Activation-induced apoptosis of bystander T cells mediated by HIV-1 gp120 is one of the critical mechanisms leading to T cell loss in AIDS. Clinical studies have shown that T cells in the lymph nodes of HIV-1 infected individuals undergo activation-induced apoptosis. In the present study, we used a model where T cells undergo apoptosis after HIV-1 gp120/CD4 cross-linking in conjunction with CD3/T cell antigen receptor activation. We have shown that treatment with HIV-1 gp120 (10 nM) and anti-gp120 MoAb induces approximately 20–25% apoptosis in Jurkat T cells in the presence of immobilized anti-CD3 antibody. However, the molecular mechanism by which HIV-1 gp120 mediates the apoptosis of T cells is still unclear. We have also examined the role of Akt/Protein kinase B in HIV-1 gp120-induced apoptosis. Akt is a cell survival molecule that has been shown to block cell death. We observed a decrease in Akt phosphorylation upon gp120 treatment of Jurkat T cells and peripheral blood mononuclear cells (PBMCs). In contrast, only CD3 stimulation was shown to increase the phosphorylation of Akt. To further confirm the role of Akt in gp120-induced apoptosis, Jurkat T cells were transfected with HA epitope-tagged wild type Akt, dominant-negative Akt that lacks kinase activity, or with a control vector. The transfected cells were treated with gp120 and apoptosis was evaluated by Annexin-PI staining. The T cells expressing wild type Akt showed reduced gp120 apoptosis as compared to the vector control-expressing cells. Conversely, expression of a dominant-negative mutant of Akt accelerated cell death as compared to the vector control. We then further assessed the role of upstream regulators of Akt, such as PI-3 kinase. In this regard, we have shown that inhibition of PI-3 kinase leads to enhanced gp120-induced apoptosis. At present, we are elucidating downstream effectors of the Akt pathway. Taken together, these studies suggest that Akt plays a key role in HIV-1 gp120-induced apoptosis, and that identification of Akt-mediated signaling pathways may provide novel therapeutic targets to combat immune deficiency in AIDS.
Stanford V (SV) Regimen Versus ABVD for the Treatment of Advanced Hodgkin Lymphoma (HL): Results of a UK NCRI/LTO Randomised Phase II Trial.
Authors: Johnson Peter , Hoskin Peter , Horwich Alan , Hancock Barry , Smith Paul , Stevens Lindsey , Qian Wendy , Sweetenham John William
Published: April 2020

Background The ABVD regimen (doxorubicin, bleomycin, vinblastine, dacarbazine) is widely accepted as standard treatment for patients with advanced HL. There are potential long term cardiac and pulmonary toxicites, particularly in combination with mediastinal radiotherapy. The SV regimen comprises brief duration combination chemotherapy, with relatively low cumulative doses of alkylating agents, doxorubicin and bleomycin, followed by consolidation radiotherapy to sites of bulk disease. High response and disease free survival rates are reported in single institution and multi-centre phase II studies. This randomized phase II trial comparing SV with ABVD was designed to determine 1) the feasibility of the SV regimen; 2) response rate compared with ABVD; and 3) acute toxicity of SV.

Methods Patients with previously untreated advanced HL were randomised to receive SV chemotherapy for 12 weeks or 6 to 8 cycles of ABVD. All patients with bulky disease (mediastinal disease > 1/3 of CTR, nodal masses >5cm, macroscopic splenic nodules on CT scan) received involved field radiotherapy (IFRT). ABVD (days 1 & 15: doxorubicin 25mg/m2, bleomycin 10units/m2, vinblastine 6mg/m2, dacarbazine 375mg/m2), q28. SV (mustine 6mg/m2, wks 1,5,9, doxorubicin 25mg/m2, wks 1,3,5,7,9,11, vinblastine 6mg/m2 wks 1,3,5,7,9,11, prednisone 40mg/m2 alternate days, vincristine 1.4mg/m2 wks 2,4,6,8,10,12, bleomycin 5iu/m2 wks 2,4,6,8,10, 12, etoposide 60 mg/m2 wks 3,7,11).

Results From Mar 1998 to Jan 2002, 150 (74 SV, 76 ABVD) patients were randomised from 15 UK centers. Pre-treatment characteristics were balanced between the two groups: median (range) age 33 (18–67), male 57%, stage II 43%, III 27%, IV 29%, 76% with B symptoms. 97% SV patients completed 12 weeks treatment, 88% patients received 6 or more cycles of ABVD. In both groups, 75% patients received IFRT. Grade 3 or 4 toxicity was rare. 49% SV and 79% ABVD patients required G-CSF. Overall response rate was 86% in SV and 88% in ABVD. With median follow-up of 42 months, 2-year overall survival was 95%, (95% CI=92%–98%). No treatment related death occurred.

Conclusion Stanford V is a potentially less toxic alternative to ABVD. Phase III trial evaluation is underway in the UK.
Activating Transcription Factor 3 (ATF3) Is Overexpressed in Classical Hodgkin Lymphoma and Is Required for Survival of Hodgkin and Reed-Sternberg Cells in Response to Cellular Stress.
Authors: Janz M. , Hummel M. , Truss M. , Wollert-Wulf B. , Mathas S. , Hagemeier C. , Bommert K. , Stein H. , Dorken B. , Bargou R. C.
Published: April 2020

Hodgkin and Reed-Sternberg (HRS) cells display aberrant activity of several transcription factors, including nuclear factor kappa B (NF-kB) and members of the activator protein-1 (AP-1) family, that promote cytokine expression, cellular survival and proliferation. Using oligonucleotide microarrays, we set out to identify additional factors which are responsible for the malignant phenotype of HRS cells. Microarray analysis and subsequent Northern and Western blotting revealed that activating transcription factor 3 (ATF3), a member of the CREB/ATF family of transcription factors that is involved in the cellular response to stress signals, shows a strong constitutive expression in Hodgkin-derived cell lines. In contrast, ATF3 expression could not be detected in several Non-Hodgkin cell lines of distinct differentiation stages, ranging from pre-B cell lines to plasma B cell lines. To investigate the expression pattern of ATF3 in primary tumor cells, we performed immunohistochemistry on tissue microarrays. Immunohistochemistry confirmed a strong expression of ATF3 in primary HRS cells with a nuclear staining pattern. ATF3 expression was observed in almost all cases of classical Hodgkin lymphoma examined (69/71), whereas ATF3 expression was not detected in lymphocyte-predominant Hodgkin lymphoma. In addition, a significant number of anaplastic large cell lymphomas revealed a nuclear ATF3 staining pattern. In contrast, among all other B and T cell Non-Hodgkin lymphomas, ATF3 expression was observed only in rare cases (7/244), indicating that ATF3 expression is almost exclusively restricted to classical Hodgkin lymphoma and to a subset of anaplastic large cell lymphomas. To investigate the role of ATF3 for proliferation and survival of HRS cells, we generated vector-based siRNA constructs to downregulate ATF3 expression. Our vector system carries a puromycin-resistance gene thus allowing selection of siRNA expressing cells. Transfection of Hodgkin-derived cell lines with ATF3 siRNA constructs and subsequent selection of siRNA-expressing cells demonstrated a dramatic loss of viable cells. Our observations indicate that ATF3 is required for the survival of HRS cells exposed to the environmental stress imposed by antibiotic selection.
Proliferation Characteristics in Pediatric Hodgkin’s Disease Point to a Cell Cycle Arrest in G1 Phase.
Authors: Tiemann Markus , Claviez Alexander , Lueders Heike , Zimmermann Martin , Schellong Guenther , Doerffel Wolfgang , Parwaresch Reza
Published: April 2020

This study was undertaken to determine the prognostic value of proliferation rate in Hodgkin and Reed-Sternberg (HRS) cells in pediatric and adolescent patients with Hodgkin’s disease (HD).

231 paraffin-embedded biopsies were immunostained with proliferation associated monoclonal antibodies Ki-S5 (Ki-67 antigen) and Ki-S2 (repp86 antigen), which is characterized by an expression pattern restricted to G2, S and M phases. Results were compared with clinical data from 224 patients included in multicenter GPOH HD-90 and HD-95 trials.

High Ki-67 expression was a striking feature (median: 80%, range: 20–100%) in contrast to low Ki-S2 expression (median: 20%, range: 10–80%). Apoptosis was assessed in 133 patients by TUNEL method (median: 10%, range: <10–70%). Proliferation rate was independent of histological subtype, stage, presence of B symptoms and allocation to treatment group. Probability of event-free and overall survival at 5 years was 91% and 98%, respectively with no significant differences in patients with low or high proliferation rate.

The difference between Ki-67 and repp86 expression in HRS cells points to a possible cell cycle arrest in G1 phase, which may explain the obvious paradoxon of a highly proliferating but slowly growing paucicellular tumor. High proliferation rate does not seem to be an adverse prognostic factor in pediatric patients with HD treated by effective chemoradiotherapy regimens.
Greater Area under the Hemoglobin Change Curve Is Associated with Improved Outcomes in Elderly Patients Receiving Epoetin Alfa for Chemotherapy-Related Anemia.
Authors: McKenzie Scott R. , Duh Mei S. , Lefevbre Patrick , Fastenau John M. , Piech Catherine Tak
Published: April 2020

Background: Several measures have been used to assess the efficacy of erythropoietic agents in patients with chemotherapy-related anemia (CRA). Previous research has demonstrated the area under the 16-week hemoglobin (Hb) change curve (Hb AUC16) as a sensitive and comprehensive efficacy measure compared to traditional single time point or threshold-based measures. Higher Hb AUC16 values during epoetin alfa (EPO) therapy in patients with CRA are associated with reduced transfusion requirements, improved quality of life, increased hematologic response rates, and decreased EPO utilization. This subgroup analysis of elderly patients with CRA evaluates the clinical significance of higher Hb AUC16 values in this unique population.

Methods: Data from three, large, multicenter EPO clinical trials were evaluated. In EPO 1, patients received 10,000 Units TIW with potential escalation to 20,000 Units TIW. In EPO 2 and EPO 3 patients received 40,000 Units QW with potential escalation to 60,000 Units QW. Eligible patients for this analysis were >/=65 years, had a baseline Hb </=11g/dL, had a non myeloid malignancy and received chemotherapy with or without radiotherapy (EPO 1: N=935: EPO 2: N=1,270; EPO 3: N=269). Hb AUC16 was calculated using sequential trapezoidal methodology based on the Hb change and was stratified into quartiles to assess trends in outcome measures. Trend tests were performed to determine if the following outcomes had significant linear trends across the Hb AUC16 quartiles: proportion of patients transfused, hematopoietic response (Hb rise >/= 2 g/dL over baseline or Hb >/= 12 g/dL during study), EPO utilization, and quality of life [assessed by the Linear Analogue Scale Assessment (LASA, all studies) and the anemia subscale of the FACT-An (EPO 1 and EPO 2)].

Results: Greater Hb AUC16 values had a strong linear association with a decreasing proportion of patients transfused (p<.0001), increasing hematopoietic response rate (p<.0001), decreasing average weekly EPO dose (p<.0001), increasing LASA scores in all studies (p<.001) and FACT-An scores in EPO 1 and EPO 2 (p<.001).

Conclusions: In the elderly patient subset, greater Hb AUC16 values were associated with significant improvement in clinical outcomes including the percentage of patients transfused, hematopoietic response rates, QOL score improvements, and EPO utilization. Hb AUC16 is an objective and meaningful metric that summarizes hematologic response over the course of treatment and is associated with clinical benefits in elderly patients receiving erythropoietic therapy.