bluuebunny/crossref_metadata_2025 huggingface dataset:
Ligand Mediated Targeting of FVIII Inhibitor Specific Primary B Cells Via Surface Immunoglobulin.
Authors: Kessel Christoph , Kreuz Wolfhart , Brassat Katharina , Klingebiel Thomas , Königs Christoph
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. In hemophilia A recombinant (rFVIII) or plasma derived (pdFVIII) preparations are substituted to restore coagulation activity. However, a major problem of current treatment in hemophilia A is the development of an antibody response (inhibitors) to FVIIII. Various Immune Tolerance Therapies (ITTs) are being applied to overcome the FVIII specific immune response. In this study, phage displayed random peptide libraries were screened with various patients’ plasma samples. We recently reported about the identification of a conformational epitope in the A2 subunit of FVIII. Phages as well as corresponding synthetic peptides specifically block inhibitor binding and crossreact with heterologous plasma. Peptide ligands specifically stained primary B cells (CD19+/CD27+ double positive cells) of the inhibitor patient, but not of a healthy control by FACS analysis. To increase the potential of small synthetic peptide ligands as tools for the development of novel inhibitor elimination strategies, the peptide was expressed in a multimeric form in an eukaryotic expression system. In multimeric form the peptide ligand is highly stable and reveals better inhibitor blocking capacities than the synthetic peptide we sowed before. The multimer now serves as basis for the construction of LECs (Ligand Effector Conjugates) for the specific targeting and destruction of inhibitor specific B cells.
Rituximab for the Treatment of High Titer Acquired Factor VIII Inhibitors Refractory to Conventional Chemotherapy.
Authors: Field Joshua J. , Fenske Timothy S. , Blinder Morey A.
Published: April 2020

Acquired factor VIII inhibitors are a rare cause of serious bleeding which are associated with a high mortality rate. Individuals with high titer antibodies (>100 Bethesda units) often have difficulty achieving a complete sustained remission and although different treatment strategies have been advocated for these patients, no approach has been well defined. Rituximab is a monoclonal antibody targeting the CD20 antigen on B lymphocytes which has demonstrated efficacy in the treatment of a variety of immune-mediated hematologic diseases including patients with acquired factor VIII inhibitors. However, there is limited data in patients with high titer inhibitor antibodies. In this study, we prospectively studied 4 patients with acquired high titer factor VIII inhibitors who have had long-term follow up. All patients were resistant to initial therapy with cyclophosphamide, vincristine, and prednisone. The patients ranged from 40 to 71 years of age and three of the four were women. The male patient had an associated autoimmune disease but the other patients were thought to have an idiopathic inhibitor. The patient’s inhibitor titers ranged from 249 BU to 836 BU at the time of treatment with rituximab and all received 4 weekly infusions of rituximab at 375 mg/kg. No significant treatment-related complications were noted. Each patient initially responded to rituximab therapy with a decline in inhibitor titer. Subsequently, three of the four patients relapsed. The relapses occurred 4, 8 and 15 months following rituximab therapy and were associated with an elevation in inhibitor titers and clinically significant bleeding in all 3 patients. The individual who did not relapse achieved a partial response with a decline in her inhibitor titer from 836 BU to 5 BU over 13 months and then died of causes unrelated to her coagulopathy. After control of the clinical bleeding, the three remaining patients received an additional course of rituximab with responses observed in all patients. One patient has been in a sustained remission for 19 months following the second dose of rituximab. A second patient had a notable improvement in his inhibitor titers from 418 BU to 10 BU over 9 months after the second dose. The third patient continues to have high inhibitor titers with intermittent bleeding following the additional dose. We conclude that in patients with acquired high titer acquired factor VIII inhibitors, treatment with rituximab alone results in improvement of inhibitor titers but is not sufficient to achieve a sustained response. Rituximab in combination with other therapies may provide a better result in this high risk population.
Inhibitor Haemophilia A - Cases with Reduced Haemostatic Response to Wildtype rFVIIa Who Achieved a Normal Clotting Profile with a Novel Potent Analogue of rFVIIa.
Authors: Sørensen Benny , Persson Egon , Ingerslev Jørgen
Published: April 2020

Development of inhibitory antibodies to coagulation factor VIII represents a serious complication in patients with congenital or acquired haemophilia A. Recombinant factor VIIa (rFVIIa, NovoSeven®, Novo Nordisk, Bagsvaerd, Denmark) has proven efficacious in management of bleeding episodes in patients with inhibitors. Recently, a novel analogue of rFVIIa (NN1731, Novo Nordisk) has been developed. NN1731 has increased potency to generate factor Xa on the surface of activated platelets. We have developed a thrombelastographic (TEG) whole blood (WB) coagulation model, employing activation with minimal amounts of tissue factor. This global clotting assay reflects the clinical severity of haemophilia A and can be used to ex vivo assess the haemostatic capacity of rFVIIa and NN1731. Adopting the TEG model, we report on two cases where ex vivo addition of rFVIIa only induced minor improvements of the WB coagulation profile. However, ex vivo addition of NN1731 revealed a normalization of the WB coagulation signature. The first case was a 74 years old female with acquired haemophilia A (APTT=51 sec, FVIII:C = 0.08 IU/mL, Inhibitor = 3.6 BU/mL. The second case was a patient with congenital haemophilia A and inhibitors (APTT=101 sec, FVIII:C<0.01 IU/mL, Inhibitor=9 BU/mL). In both cases, the WB thrombelastographic analysis demonstrated a severely compromised haemostatic capacity. Ex vivo dose titration studies with rFVIIa induced minor improvements only of the suppressed WB clot formation profile. However, ex vivo addition of NN1731 at 2 μg/mL normalized the coagulation signature. The observations that the analogue NN1731 normalized the haemostatic WB clotting profiles in cases where rFVIIa induced minor changes stimulates further pre-clinical investigations.
Central Venous Access Device-Related Infections in Hemophilia.
Authors: Journeycake Janna M. , Brambilla Don , Ragni Margaret V.
Published: April 2020

Central venous access devices (CVAD) are commonly used in young children with hemophilia who need frequent factor infusions, including prophylactic infusions given every other day to prevent joint disease and immune tolerance therapy given every day to neutralize inhibitors, both of which result in better health outcomes. CVAD use, however, may be complicated by infection or sepsis, catheter replacement, and rarely, subacute bacterial endocarditis (SBE) or death. Infection rates have varied widely among institutions, possibly in part because of variable diagnostic and reporting methods. In order to identify risk factors for CVAD-related infection in children with hemophilia, we distributed an electronic survey to 14 U.S. hemophilia treatment centers participating in the TMH network regarding children with hemophilia whose initial CVAD was placed between January 1, 2002 and December 31, 2004. Thirteen (94%) sites responded, providing data on 94 patients with CVAD. All but two (98%) were ports. CVADs were placed for prophylaxis in 66 (70%) patients and for immune tolerance therapy in 28 (30%) patients. Thirty-one (33%) patients developed CVAD-related infections, all in the first year following insertion. One subject developed SBE, and one died of CVAD-related sepsis. Infection was significantly more common in subjects receiving immune tolerance, 19 of 28 (68%), than on prophylaxis 12 of 66 (18%) (odds ratio = 9.5, p<0.001). The time from CVAD insertion to initial post-operative CVAD access did not influence the risk for infection (p = 0.13), but the duration of factor replacement following CVAD insertion did. Factor replacement for fewer than 7 days following insertion was associated with an increased incidence of CVAD-related infection (odds ratio = 2.2, p= 0.04). We conclude that individuals using CVAD for immune tolerance and/or receiving fewer than 7 days of factor replacement following CVAD placement are at greater risk for first CVAD-associated infection. The reasons for these findings are not known. However, the more frequent access of CVAD in patients on immune tolerance (daily vs. every other day) increases exposure to skin organisms, which could account for the higher rate of infection in these patients. Those receiving fewer than 7 days of factor replacement after CVAD placement may have insufficient wound healing or suffer local hematoma formation, which is a potential culture medium for local skin organisms. The high rate of CVAD infection in hemophilic children, >30% overall, constitutes a major public health problem for which prevention trials are in development.
High Efficacy of Combination Therapy with Pegylated Interferon and Ribavirin in Hemophiliacs with Chronic Hepatitis C.
Authors: Mancuso Maria E. , Santagostino Elena , Rumi Maria G. , Linari Silvia , Coppola Antonio , Rocino Angiola , Iorio Alfonso , Dragani Alfredo , Barillari Giovanni , Tagariello Giuseppe , Nicolin Roberto , Colombo Massimo , Mannucci Pier M.
Published: April 2020

Pegylated interferon (Peg-IFN) plus ribavirin is the standard treatment for patients with chronic hepatitis C. In a multicenter open trial, we assessed the efficacy and tolerability of this treatment in 78 HIV negative hemophiliacs (age: 20–64 years) with persistently high transaminase values. Sixty-four were naïve and 14 were relapsers to IFN monotherapy. Cirrhosis was clinically detected in 12 patients (15%). HCV genotype was 1 in 69%, 2a/c in 14%, 3a in 14% and 4 in 3%. Peg-IFN alpha-2b was given subcutaneously at doses of 1.5 mcg/Kg/week for 48 weeks in genotypes 1 and 4 and for 24 weeks in genotypes 2 and 3; oral ribavirin at 800–1200 mg/day based on body weight. Treatment was stopped in patients with polymerase chain reaction positive HCV-RNA at month 6.

Results: 11 patients (14%) withdrew for side effects (4) or non-compliance (7). Neutropenia (<500 cells/mmc), decompensated diabetes, ALT flares, and vomiting not responding to antiemetic drugs were reasons for treatment discontinuation. The median fall in hemoglobin levels was 3.1 g/dL. Weight loss (38%), fatigue (33%) and cephalalgia (15%) were frequent side effects. Thirty-two patients (41%) required dose reduction of ribavirin (23, 29%) or Peg-IFN (20, 26%). At the end of the 6-month follow-up, sustained virological response (SVR) was achieved in 43 patients (55%): 40/64 naïve (63%) and 3/14 relapsers (21%, p = 0.007). Five patients (6%), all relapsers to IFN monotherapy, had a virological breakthrough during treatment and 4 (5%) relapsed during the post-treatment follow-up period. SVR was obtained in 86% genotypes 2/3 and 43% genotypes 1/4 (p = 0.001). Predictors of SVR were evaluated by univariate analysis in the 64 naïve patients. SVR was significantly associated with HCV infection type 2 and 3 (86% vs 50% in HCV type 1 and 4; p = 0.008), absence of cirrhosis (97.5% vs 75% in non-responders; p = 0.02) and higher pre-treatment serum ALT levels (111 vs 75 IU/L in non-responders; p = 0.02). SVR rates did not differ in relation to patient’s age, pretreatment HCV viremia, median disease duration and compliance to full-dose treatment.

Conclusions: combination therapy with Peg-IFN plus ribavirin is the recommended therapeutic option for hemophiliacs with hepatitis C chronic infection. Relapsers to IFN monotherapy may benefit of re-treatment with Peg-IFN plus ribavirin achieving at least 20% SVR. Genotype 2 and 3 infection is the most significant predictor of SVR.
Increased Risk of Epstein-Barr Virus Associated Post-Transplant Lymphoproliferative Disorder (EBV-PTLD) after Anti-Thymocyte Globulin (ATG) Non-Myeloablative (NMA) Conditioning for Umbilical Cord Blood Transplantation (UCBT).
Authors: Brunstein Claudio G. , Weisdorf Daniel J. , DeFor Todd , Tolar Jakub , Wagner John E.
Published: April 2020

EBV-PTLD is a known complication of alternative donor hematopoietic stem cell transplantation (HSCT) particularly when used in combination with HSC T-cell depletion and ATG. Initial reports in the setting of UCBT suggested a low incidence of EBV-PTLD (2–5%). We reviewed 335 consecutive patients (pts) who underwent UCBT at our institution between 7/19/94 and 3/29/05. Median age, weight, and follow-up were 16 years (0.2–69), 53.7kg (3.8–134.0) and 1.2 years (77 days9.2 years), respectively. Equine ATG (ATGAM) was used as part of a myeloablative (MA, Cy 120 mg/kg and TBI 1320–1375 cGy ± Flu 75 mg/m2) and non-myeloablative (NMA, Cy 50 mg/kg, Flu 200 mg/m2 and TBI 200 cGy) preparative regimen in 175/240 and 30/95 patients, respectively. As previously reported the overall incidence of EBV viremia and EBV-PTLD remained low. Overall, use of ATG lead to an increase in the incidence of EBV viremia (defined as > 1,000 copies of EBV DNA per mL of whole blood) and EBV-PTLD (14/205 [7%] with vs. 1/130 [0.7%] without ATG [p=0.16]), but this was not statistically significant. In recipients of a MA preparative regimen, EBV viremia or EBV-PTLD was observed in 8/175 (5%) who received ATG and in 0/65 who did not (p=0.81). However, a higher risk of EBV viremia and PTLD was observed in recipients of ATG and a NMA preparative regimen. EBV viremia or EBV-PTLD after NMA preparative regimen was observed in 6/30 (20%) pts who received ATG and 1/65 (2%) who did not (p<0.01) For the whole group, median time to development of EBV-PTLD was 133 days (54–407) with no difference between the MA and NMA groups [132 days (92–407) vs. 133 days (54–247)]. Among 9 pts who developed EBV-PTLD, 5 are alive (2 MA and 3 NMA) 113-1668 days after UCBT. Therefore, we conclude that patients undergoing a NMA UCBT with ATG are at uniquely higher risk for the development of EBV reactivation or EBV primary infection and EBV-PTLD. Routine monitoring of EBV viral load and pre-emptive treatment of patients who develop a positive viral load must be considered for this group of patients.
Donor KIR Genotype Does Not Affect VZV Reactivation after Allogeneic Haematopoietic Stem Cell Transplantation.
Authors: Narayanan Sujaatha , Nagra Sandeep , Mahendra Premini , Clark Fiona J. , Craddock Charles F. , Osman Husam , Briggs David C. , Moss Paul A. , Cook Mark A.
Published: April 2020

Background. Varicella zoster virus (VZV) reactivation is a common occurrence after allogeneic haematopoietic stem cell transplant (HSCT) and a cause of significant morbidity. We have recently demonstrated the protective effect of donor KIR genotpye against cytomegalovirus (CMV) reactivation- specifically the protective effect of the broad ’B’ haplotype containing multiple activating KIR genes1. This retrospective study was designed to investigate whether donor KIR genotype confers an equivalent protective effect against VZV reactivation.

Method. 152 patients who underwent allogeneic HSCT at a single centre were identified. Those with pre-transplant serology consistent with previous exposure to VZV were defined as at risk of VZV reactivation. All patients received aciclovir 800mg daily as routine prophylaxis. The diagnosis of VZV reactivation was made clinically. Cases of VZV reactivation were identified by examination of the case records. KIR genotyping was performed on donor DNA using PCR-SSP. The individual KIR genes KIR2DL1, 2DL2, 2DL3, 2DL5, 3DL1, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5 and 3DS1, as well as the broad KIR haplotypes ’A’ and ’B’, were assessed by univariate and multivariate analysis for an effect on VZV reactivation. Also assessed was the impact of recipient HLA-C type, recipient HLA-Bw4/Bw6, donor type (sex-matched sibling, sex-mismatched sibling, volunteer unrelated), reduced intensity conditioning (RIC) regimen, the use of alemtuzemab as in vivo T cell depletion, CMV reactivation, and grade 2 or greater GVHD requiring steroid therapy.

Results. 128 (84.2%) patients had evidence of past infection and thus were deemed at risk of VZV reactivation. Of these, 47 (36.7%) had clinical evidence of reactivation. 60% of transplants were from a donor possessing the broad ’B’ haplotype. The rate of reactivation in the presence of donor ’B’ haplotype was 40% compared to 32% when no donor ’B’ haplotype was present (p=0.237). None of the individual donor KIRs were shown to significantly reduce the rate of VZV reactivation. Neither the use of a RIC regimen nor the presence of alemtuzemab in the conditioning regimen were shown to have an impact. None of the other factors analysed were associated with an increased rate of zoster reactivation.

Conclusion. Donor KIR genotype does not influence VZV reactivation after allogeneic HSCT. This contrasts with the findings for CMV reactivation. No influence of donor type, conditioning or GVHD could be demonstrated. This suggests 1) that there are differing mechanisms that control the reactivation of different Herpes viruses after transplantation and 2) that KIRs may have specificity for CMV.
The Peptide hLF1-11 as Broad Spectrum Antmicrobial Prophylaxis in HSCT Patients.
Authors: Wulferink Marty , Brouwer Carlo P. , Sluiter-van Dijk Astrid M.C. , Blijlevens Nicole N.A. , Donnelly J. Peter , Velders Markwin P.
Published: April 2020

hLF1-11, comprising the N-terminal 11 AA of the human lactoferrin protein, has previously been shown to have a broad antimicrobial spectrum. The efficacy towards several strains of bacteria and fungi was demonstrated in in vitro and in vivo models. At this moment, hLF1-11 is in clinical development for use as broad spectrum anti-fungal and anti-bacterial prophylaxis in hematopoetic stem cell transplantation (HSCT) patients. There is data to support that the hLF1-11 mechanism of action is not restricted to direct killing of the pathogen, but that the indirect, immune modulatory and immune activating properties are important for hLF1-11 efficacy in vivo at low doses. In earlier studies, efficacy of hLF1-11 was shown in neutropenic animals systemically infected with Candida albicans. In the present studies antimicrobial efficacy and safety of hLF1-11 was tested in animal models that resemble the immune compromised state of HSCT patients shortly after transplantation. In cyclophosphamid and cyclosporin treated animals, hLF1-11 significantly reduced intra muscular infection with methicillin resistant Staphylococcus aureus (MRSA) when injected 24 hours after infection. In the same model hLF1-11 showed prophylactic properties in that it was effective even when administered as a single injection 24 hours before local infection with MRSA. In order to assess drug-drug interactions of hLF1-11 with common antibiotics, hLF1-11 was co administered with either vancomycin or ciprofloxacin in the local MRSA infection. Data from these models show that hLF1-11 is effective in combination with standard antibiotic and immunosuppressive care in a HSCT setting. Moreover, the hLF1-11 peptide does not inhibit antibiotic activity of currently used antibiotics. Besides these preclinical safety data, clinical safety was evaluated in a single and multiple ascending dose study in human volunteers. During these studies pharmacokinetic and pharmacodynamic parameters were measured and no drug related adverse effects were observed. Now, preparations are made to evaluate safety and efficacy of hLF1-11 in autologous and allogenic HSCT patients as anti-fungal and anti-bacterial prophylaxis. In conclusion, hLF1-11 is a promising peptide to prevent and treat opportunistic infections in HSCT patients and other immune suppressed patient populations.
An Integrated Epigenomic Analysis Approach To Determine the Molecular Basis of Acute Leukemias.
Authors: Figueroa Maria E. , Ye Kenny M. , Paietta Elisabeth , Greally John M. , Melnick Ari M.
Published: April 2020

Acute leukemias are classified based on their immunologic, cytogenetic and morphologic characteristics. However, in most instances, response to treatment and survival probability cannot be accurately predicted, suggesting that the disease is even more complex and heterogeneous than can be shown with current techniques. Although prognostic value has been shown for certain gene expression profiles, expression profile studies are limited by the fact that only a snapshot of mRNA content is obtained in a basal state, failing to represent how genes will respond to different stresses, also failing to detect the roles of genes expressed at lower levels for which major changes in expression levels are often lost in the “noise” of expression arrays. In order to overcome these issues and to provide a more accurate molecular phenotype of acute leukemias, we established an integrated epigenomic and genomic high-throughput platform using novel techniques and custom high-density oligonucleotide arrays. We combine studies of i) genome-wide cytosine methylation using a novel technique we developed that provides accurate quantitative determination of DNA methylation levels, using genome-wide custom oligo arrays, ii) chromatin structure by ChIP on chip for histone code settings associated with active or repressed genes using 24 K promoter tiling arrays, iii) gene copy number by array-based comparative genomic hybridization (array CGH) at 6 kb resolution genome-wide and iv) 36 K gene expression arrays. Results are validated by single locus quantitative PCR techniques. Cross platform integration is facilitated by use of NimbleGen oligo arrays for all studies and analysis using novel bioinformatics and statistical models. We used this integrative analysis platform to perform an in-depth analysis of the epigenomic basis of AML and ALL using primary patient samples and cell lines. The data allowed us to generate a “gene activity index” which identified the ability of genes to be expressed to be characterized genome-wide in AML and ALL cells. This data also allowed a much more comprehensive analysis of pathways active in these cells to be identified in comparison to expression arrays alone. Our current studies apply integrative platform and gene activity indexing to large series of patients enrolled in multicenter clinical trials in order to provide high-resolution analysis of the molecular basis of acute leukemia.
CALM-AF10 and HOX11L2 Abnormalities Define Poor Prognostic Subgroups in Pediatric T-Cell Acute Lymphoblastic Leukemia.
Authors: van Grotel Martine , Meijerink Jules P.P. , van Noesel Max M. , Beverloo H. Berna , Langerak Anton W. , Poulsen Tim S. , van Wering Elisabeth R. , Janka-Schaub E. Gritta , Pieters Rob
Published: April 2020

Recognition of specific T-ALL subgroups based upon cytogenetic aberrations may have prognostic relevance. The prognostic relevance of recurrent molecular-cytogenetic abnormalities using RQ-PCR and FISH was established for T-ALL patients enrolled on the DCOG protocols, and assigned to SIL-TAL, HOX11, HOX11L2, or CALM-AF10 subgroups or a rest group lacking any of these abnormalities. RQ-PCR data almost completely matched with FISH data. CALM-AF10 was associated with a poor outcome (p=0.005), whereas HOX11L2 and TAL1 subgroups demonstrated a strong trend towards poor and good outcome, respectively. A significant association between HOX11L2 and poor outcome was validated in a second T-ALL cohort comprising 53 pediatric patients that were enrolled on the COALL97 protocol (p=0.01). The TAL1 subgroup was committed to αβ-lineage T-cell developmental stage. HOX11 T-ALLs were all CD1 positive without expression of Cytβ and/or TCR expression reflecting an early cortical developmental stage. The immunophenotype of the HOX11L2 subgroup was rather heterogeneous consisting of samples with TCRγδ expression as well as phenotypic immature cases. Only 41% of HOX11L2 positive cases expressed CD1, indicating that HOX11L2 and HOX11 represent different T-cell developmental stages. CALM-AF10 was associated with a very immature immunophenotype, frequently expressing CD34 and myeloid markers. The TAL1 subgroup was characterized by high expression levels of TAL1, but about half of the samples with no or other aberrations also highly expressed TAL1. LYL1 was highly expressed in the other subgroups, with the highest expression levels in the most immature subgroups. LMO2 gene expression levels closely matched with LYL1 levels. In conclusion, the presence of CALM-AF10 and HOX11L2 abnormalities define poor prognostic subgroups in pediatric T-ALL.
P-Loop an T315I BCR-ABL Mutations in Chronic Myelogenous Leukemia (CML) Patients Are of Poor Prognosis, but the Y253H + E255K P-Loop Mutations Are of Even More Severe Prognosis in Chronic Phase. On the Behalf of the French Intergroup of CML (Fi-LMC Group).
Authors: Nicolini Franck E. , Corm Sélim , Lê Quoc-Hung , Hayette Sandrine , Sorel Nathalie , Bories Dominique , Giraudier Stéphane , Tulliez Michel , Rousselot Philippe , Cayuela Jean-Michel , Mahon François-Xavier , Cony-Makhoul Pascale , Kiladjian Jean-Jacques , Tournilhac Olivier , Guilhot François , Roche-Lestienne Catherine , Preudhomme Claude , Michallet Mauricette
Published: April 2020

In CML, the most frequent mechanism responsible for IM resistance is the onset of BCR-ABL mutations. Mutations interfering with IM binding to the ABL-kinase domain that restore the tyrosine kinase activity of BCR-ABL (T315I and P-loop mutations) are of poor prognosis. In a retrospective analysis, we analysed the features and clinical outcomes of 50 CML patients and presenting IM resistance, and harbouring 33 P-loop (1 patient had mutations) [Group 1 (G1)] and 18 T315I mutations [Group 2 (G2)] detected by direct sequencing. Twenty-eight patients were in chronic phase (CP), 12 in accelerated phase and 9 in blastic phase when IM was started, with no difference between P-loop and T315I groups. There were 23 M and 9 F, with a median age of 53 (13–74) for G1; and 14 M and 4 F, with a median age of 52 (28–70) for G2. The median duration of IM was 25 Mo (2.4–50) for G1 and 20 (0.5–145) for G2 (p=ns). The duration of IFN prior to IM was equivalent for both groups (12 Mo (0–86) for G1 vs 10 Mo (4–131) for G2, p=ns). The median interval between diagnosis and day 1 of IM was 50 Mo(0.4–128) for G1 and 20 Mo (0.5–145) (p=ns) for G2. Multivariate analysis for gender, age, prior IFN, interval diagnosis-IM start, and major cytogenetic remission achievement with IM, did not show any significant impact of these variables. Overall and Progression free survival (PFS) Kaplan Meier curves for all phases did not show any difference, however, PFS curves for CP only showed a somewhat worse survival for Y253H+E255K P-loop mutations than for other P-loop mutations or T315I (p=0.05).

Figure Figure

Median survival for CP was 7 Mo for Y253H+E255K P-loop mutations, vs 24 for T315I vs 36 for other P-loop mutations. In conclusion, P-loop and T315I mutations are of equivalent poor prognosis, but for CP patients, the Y253H+E255K P-loop mutations are particularly severe within the P-loop group, as suggested by in vitro data.
Philadelphia Chromosome Positive Acute Myeloid Leukemia: An Aggressive Acute Leukemia with Clinicopathologic Features Distinct from Chronic Myeloid Leukemia in Blast Crisis.
Authors: Soupir Chad P. , Vergilio Jo-Anne , Dal Cin Paola , Muzikansky Alona , Kantarjian Hagop M. , Jones Dan , Hasserjian Robert P.
Published: April 2020

Philadelphia chromosome positive acute myeloid leukemia (Ph+ AML) is a rare entity reported to comprise 1% of tumors bearing the Philadelphia chromosome. Controversy exists over whether this represents a distinct entity or is merely presentation of patients in the blastic phase of chronic myeloid leukemia (CML). Our study sought to determine the clinicopathologic characteristics of Ph+ AML in comparison to CML in blast crisis. We searched the archival files of Massachusetts General Hospital, Brigham and Women’s Hospital, Dana Farber Cancer Institute, and MD Anderson Cancer Center from 1995 to 2005 for cases of apparent de novo Ph+ AML. After excluding biphenotypic leukemias and cases with any evidence of prior CML or other myeloid neoplasia, we retrieved 18 Ph+ AML cases. 20 cases of documented CML in myeloid blast crisis (CML-MBC) who had not received prior imatinib mesylate therapy or bone marrow transplant were used as a control group. The clinical and laboratory features, bone marrow morphology, immunophenotype, cytogenetics, and type of BCR/ABL transcript were analyzed. The Ph+ AML patients included 13 males and 5 females with a median age of 55 years, which was not significantly different from CML-MBC patients. The median WBC at presentation was 15.7 x 109/L. Compared to CML-MBC patients, the Ph+ AML patients were less likely to have splenomegaly (22% vs. 68%, p=0.008) or peripheral basophilia (median absolute basophil count 0.16 vs. 1.24 x 109/L, p=0.003). The bone marrow in Ph+ AML patients was less cellular (median cellularity 80% vs. 98%, p=0.002) with a lower myeloid:erythroid ratio (1.3 vs. 4.8, p=0.01), but more prominent erythroid and myeloid lineage dysplasia (p=0.04) than that of CML-MBC patients. While cytogenetic abnormalities in addition to t(9;22) occurred in 67% of Ph+ AML cases, the typical additional cytogenetic changes of CML-MBC (+8, +19, iso17q, and +Ph+) were uncommon (22% vs. 87% for CML-MBC, p=0.0004). In 5/6 Ph+ AML cases with multiple clones, the Ph+ was present in all clones, suggesting that this represents an early event in the evolution of these leukemias. RT-PCR revealed a predicted BCR/ABL product of p210 (10/13 cases), p190 (2/13 cases) or p230 (1/13 cases). 6/7 Ph+ AML patients treated with imatinib showed at least a partial hematologic response, but this was of a short duration (median 2.5 months, range 1–6 months) in comparison to the reported median response duration of 10 months in imatinib-responsive CML-MBC. The median survival of the Ph+ AML patients was 10.5 months, similar to that of CML-MBC and AML with adverse cytogenetics. In summary, Ph+ AML is a rare entity distinct from CML-MBC, with an aggressive clinical course and only transient response to imatinib.
Standardization of WT1 mRNA Quantification for Minimal Residual Disease (MRD) Monitoring in Acute Leukemia Patients: A European LeukemiaNet Concerted Action.
Authors: Gottardi Enrico , Cilloni Daniela , Daly Sarah , Green S. , Pallisgaard Niels , Hokland P. , Ostergaard M. , Nyvold C. , Nomdedeu J. , Pane Fabrizio , Schnittger Susanne , Van der Velden Vincent , Tobal Kalid , Jal Y. Yin , Hermitte Fabienne , De Witte Teo , Hehlmann R. , Saglio Giuseppe , Grimwade David
Published: April 2020

Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemia. In acute myeloid leukemia (AML), the reliability of real-time quantitative PCR (RQ-PCR) and its potential clinical value for MRD studies using fusion gene (FG) transcripts as PCR targets such as PML-RARa, AML1-ETO and CBFb- MYH11 has been demonstrated, but these markers are present only in a minority of cases. In order to overcome this problem several groups looked for alternative markers and growing evidence has suggested that the high expression of WT1 in a significant proportion of acute leukemia cases provides a suitable target for therapy as well as for monitoring of MRD. However, heterogeneity of molecular approaches resulted in a lack of comparability between different MRD studies. This has been solved using RQ-PCR in a network of 9 laboratories within the European LeukemiaNet. Overall 8 primer/probe sets were evaluated including published and “in-house” sets. The assays analyzed differed significantly in terms of efficiency and sensitivity. Three assays with superior performance were identified, achieving sensitivities of at least 1 in 10,000 in serial dilutions of HL60 cells (WT1 positive). Subsequent analysis of two of the primer/probe sets, which amplify ex. 6/7 and 7/8 of WT1 respectively, revealed the potential for false negative results, following documentation of deletions of the WT1 gene in this region in primary AML samples. In two of these cases, different deletions of sequences corresponding to part of WT1 ex.8 were documented by WT1 RNA sequencing. Therefore, a primer/probe set amplifying ex. 1/2 of WT1 has been subject to further analysis in a QC round involving 9 labs. The analysis of 33 normal BM, 32 normal PB and 12 CD34 enriched PBMNCs gave the following results: BM, mean 70,32 WT1 copies/104 ABL copies (range 8,99–209,82); PB, mean 3,30 WT1 copies/104 ABL copies (range 0–13,55); CD34 enriched PBMNCs, mean 8,16 WT1 copies/104 ABL copies (range 1,55–26,16). Overall, these analyses underline the importance of standardization in the development of RQ-PCR assays for MRD detection in leukemia. There is increasing interest in identification of genes that are over-expressed in leukemia as potential MRD targets. However, it is clear that incorporation of such MRD targets into risk-directed treatment protocols is critically dependent upon establishing thresholds of expression in normal blood and marrow on regeneration following myeloablative therapy.
Rac1 and Rac2 GTPases Play Redundant but Critical Role in T-Cell Development and Survival.
Authors: Guo Fukun , Hildeman David , Williams David A. , Zheng Yi
Published: April 2020

The Rac subfamily GTPases of the Rho family have been implicated in the control of actin dynamics, cell proliferation, apoptosis, adhesion and migration of many blood cell types including hematopoietic stem/progenitors, neutrophils and macrophages, but their role in T cell development remains poorly understood. T cells from the Rac2 deficient mice appear to mostly undergo normal development, whereas previous constitutively active mutant Rac2 or Rac1 overexpression studies suggest Rac GTPases are required for CD4+ and CD8+ T cell maturation. Using conditional gene targeting, we have achieved specific deletion of Rac1 or Rac1 together with Rac2 in the T cell lineage by cross-breeding the Lck-Cre transgenic mice with the Rac1flox/flox mice that contain a pair of loxP sites sandwiching the exon 1 sequences of Rac1 or the Rac1flox/flox;Rac2−/− mice. We show that similar to Rac2 deficiency, inactivation of Rac1 alone had little effect on various developmental stages of T cells in the animal. However, deletion of both Rac1 and Rac2 significantly affected both the immature CD4−CD8− (2.3 fold increase) and CD4+CD8+ (13% decrease) populations in the mouse thymus and the mature CD4+ and CD8+ populations in the thymus and spleen (Table). These developmental defects are associated with proliferation defects of thymocytes and splenocytes in response to ConA or PMA/ionomycin stimulation and deficient survival of various T cell populations at different developmental stages (Table). Together, these data show that Rac1 and Rac2 play overlapping and obligatory roles in T-cell development and serve as important cell survival regulators at various stages.

Table. Frequency and apoptosis rate of different T-cell subsets in thymocytes and splenocytes T cell subsets WT (n=10) Rac1−/− (n=6) Rac1−/−Rac2−/−(n=6) Total thymocyte number (×106) 101.3±30.0 98.0±25.0 44.7±25.5 CD4−CD8− thymocyte frequency (%) 5.5±1.9 4.5±0.7 12.7±4.3 apoptosis rate (%) 20.1±2.2 15.0±1.3 CD4+CD8+ thymocyte frequency (%) 76.2±3.2 77.3±4.1 66.2±5.4 apoptosis rate (%) 18.8±4.3 27.9±2.8 CD4+ thymocyte frequency (%) 14.5±3.4 14.4±2.4 7.9±2.3 apoptosis rate (%) 13.3±2.3 21.5±4.5 CD8+ thymocyte frequency (%) 3.9±1.2 3.8±1.0 13.2±2.2 apoptosis rate (%) 12.5±2.2 8.8±1.1 Total splenocyte number (×106) 60.4±21.8 62.0±13.0 51.1±28.9 CD4+TCRβ+ splenocyte frequency (%) 9.7±2.2 8.0±2.3 3.2±1.1 apoptosis rate (%) 15.1±3.1 27.5±6.9 CD8+TCRβ+ splenocyte frequency (%) 3.2±0.8 2.4±0.9 0.6±0.4 apoptosis rate (%) 13.1±3.0 24.5±6.4
Maintenance of Telomere Length in Peripheral Blood CD4+CD25+ Regulatory T-Cells of Cancer Patients Despite Active Proliferation.
Authors: Wolf Dominik , Rumpold Holger , Koppelstaetter Christian , Gastl Guenther , Gunsilius Eberhard , Tilg Herbert , Wolf Anna M.
Published: April 2020

CD4+CD25+ regulatory T-cells (Treg) are increased in the peripheral blood of cancer patients. It remains unclear whether this is due to redistribution or active proliferation. The latter would require the up-regulation of telomerase activity, whose regulation also remains unknown for Treg. We therefore isolated Treg and the respective CD4+CD25− control T-cell population from peripheral blood of cancer patients (n=23) and healthy age-matched controls (n=17). Analysis of their content of T-cell receptor excision circles (TREC) revealed that the observed increase of Treg frequencies in peripheral blood is due to active cycling rather than to redistribution from other compartments (i.e. secondary lymphoid organs or bone-marrow), as Treg from cancer patients are characterized by a significant decrease of TREC content when compared to TREC content of Treg isolated from healthy age-matched controls. Surprisingly, despite their proven in vivo proliferation, telomere length is not further shortened in Treg from peripheral blood of cancer patients as shown by Flow-Fish, Real-Time PCR and Southern Blotting. Accodingly, telomerase activity of Treg was readily inducible in vitro by OKT3 together with IL-2. Notably, sorting of in vitro proliferating Treg using dilution of CFSE revealed a significant telomere shortening in Treg with high proliferative capacity (i.e. CFSElow fraction) under conditions of strong in vitro stimulatory growth conditions despite a high telomerase activity. Thus, under conditions of strong in vitro stimulation induction of telomerase seems to be insufficient to avoid progressive telomere shortening. In contrast, in actively proliferating peripheral blood Treg from patients with epithelial malignancies induction of telomerase activity is likely to compensate for further telomere erosion.
Transcription Factor ICSBP/IRF8 Regulates B Cell Development at Multiple Checkpoints.
Authors: Wang Hongsheng , Lee Chang Hoon , Qi Chen-Feng , Kerrikatte Nishant , Tailor Prafullakumar , Ozato Keiko , Morse Herbert C.
Published: April 2020

The generation of lymphoid and myeloid lineage cells from hematopoietic stem cells is controlled by multiple transcription factors regulating distinct developmental and functional aspects. Interferon consensus sequence binding protein (ICSBP)/interferon regulatory factor 8 (IRF8) is a transcription factor known to regulate the differentiation of macrophages, granulocytes, and dendritic cells. Our recent findings that IRF8 transcripts and protein are highly expressed in germinal center (GC) B cells suggest that IFR8 may also play a role in normal B cell development. In IRF8 deficient mice, the number of early B lineage cells (pre-pro-B) was reduced by 5-fold, indicating a defect in early B lineage commitment. While the numbers of late pre-B and immature B cells were moderately reduced (~2-fold), recirculating mature B cells were almost undetectable in the bone marrow of mutant mice. This deficiency in early stage B cells is correlated with increased expression of PU.1, a crucial transcription factor for myeloid and lymphoid lineage specification. Interestingly, the number of splenic transitional 1 (T1) cells was slightly increased but the numbers of T2 and follicular (FO) B-2 cells were moderately decreased in mutant mice. This indicates that positive selection of T2 cells into the mature B-2 pool is regulated by IRF8. The marginal zone (MZ) B cell and peritoneal CD11b+ B-1b cell compartments were also slightly expanded in IRF8 knockout mice. Overall, these results provide compelling evidence that IRF8 regulates B cell differentiation and function at multiple stages.
Murine Embryonic Liver Differentiates Human Stem Cells into a Spectrum of NK Precursors and Polyclonal KIR Expressing NK Cells.
Authors: Miller Jeffrey S. , Brungaard Karen , Oostendorp Robert A.J. , McCullar Valarie
Published: April 2020

We have shown that a murine fetal liver cell line (AFT024) and human cytokines (IL-15, IL-7, IL-3, Flt3-ligand and c-kit ligand) are needed to induce NK cell differentiation and KIR acquisition. To understand the level of maturation where these factors orchestrate NK cell development, a switch culture was designed to separate early and late events. Cord blood CD34+/Lin−/CD38− stem cells were cultured on AFT024 for 28 days. Use of IL-3 or Flt3-L alone resulted in minimal growth. In contrast, we show that NK cell differentiation can occur, albeit at low frequency, with a combination of IL-3 and Flt3-L, in the absence of IL-15. These early NK cells were negative for both CD94 and KIR. These conditions also allowed accumulation of CD56− NK cell precursors. CD34+CD7−, CD34+CD7+ and CD34−CD7+ cells were detected in cultures lacking IL-15. Each precursor was tested in secondary cultures containing AFT024 with IL-15 alone, IL-15+IL-3, or IL15+IL-3+Flt3-L. After an additional 2–4 weeks, NK cells differentiated from each distinct cell population. A few predominantly KIR negative NK cells resulted from IL-15 alone. Addition of IL-3 or IL-3+Flt3-L significantly increased the absolute number of NK cells as well as the acquisition of CD94 heterodimers and KIR. We next explored other stromal cell lines in attempt to identify novel factors important in early NK cell maturation. A novel cell line derived from murine embryonic liver (EL08-1D2), identified for its ability to support expansion of mouse stem cells, was compared to AFT024. To test the differential capacity of these microenvironments, single cord blood stem cells were plated on the two feeders supplemented with all cytokines. After 4 weeks, EL08-1D2 induced 125,852±1400 NK cells from a single stem cell, significantly more than with AFT024 (23,143±8117). KIR+ NK cells were also significantly more frequent with EL08-1D2 (3689±801 vs. 799±491), always in a polyclonal pattern. NK cell development and KIR acquisition were dependent on direct contact with EL08-1D2. Increased development could be from greater differentiation, proliferation or both. Cord blood stem cells were cultured in direct contact with EL08-1D2 under primary culture conditions with IL-3 and Flt3-L but in the absence of IL-15. All CD56− NK cell precursors developed with greater frequency on EL08-1D2 than AFT024. In conclusion, EL08-1D2, derived from a primitive microenvironment during mouse ontogeny, efficiently recapitulates NK cell development by inducing NK cell differentiation and proliferation. IL-3 and Flt3-L, but not IL-15, facilitate the isolation and study of distinct NK cell precursors. Direct contact with EL08-1D2 induces KIR acquisition, suggesting that unique environmental factors conserved between mouse and man contribute to the extrinsic signals which lead to KIR acquisition.
Functional and Molecular Analysis of Maturation of Thymocytes in Bcl10 or Malt1 Deficient Mice.
Authors: Jost Philipp J. , Ferch Uta , Weiss Stephanie , Leeder Stephanie , Gross Olaf , Mak Tak W. , Peschel Christian , Ruland Juergen
Published: April 2020

Development of immature T cells in the thymus requires signals through the clonotypic T cell receptor (TCR). Thymocytes expressing a functionally inactive or autoreactive TCR are deleted via apoptosis (negative selection). Thymocytes expressing a functionally active but not autoreactive TCR are selected through inhibition of cell death (positive selection). Deregulation of this process is likely to result in autoimmunity or lymphomagenesis of T cells. The intracellular mechanisms by which the balance between TCR-dependent survival and apoptosis are regulated are largely unknown. A central regulator of survival and apoptosis in the immune system is the transcription factor NF-κB. Activation of NF-κB in mature T-cells requires the adaptor proteins Bcl10 and Malt1. Using gene-targeted mice deficient for Bcl10 or Malt1, we show that Bcl10 and Malt1 are also required for TCR-induced NF-κB activation in immature T cells. Furthermore, to elucidate the process of T cell selection within the thymus, we have crossed Bcl10 or Malt1 deficient mice into mice with genetic backgrounds expressing defined TCR transgenes. Using specific peptide agonists of these TCR transgenes, we show that neither in vivo nor in vitro development into single positive (SP) CD4 or CD8 positive T cells is altered in Bcl10 or Malt1 deficient mice. Absolute numbers and ratio of SP T cells found within the thymus or in peripheral lymphnodes of transgenic animals are normal. These findings indicate that Bcl10 and Malt1 activate NF-κB in thymocytes but are dispensable for maturation of immature T cells in this model system.
A Prospective Risk Model for Neutropenic Complications in Patients with Malignant Lymphoma.
Authors: Lyman Gary H. , Crawford Jeffrey , Wolff Debra , Culakova Eva , Poniewierski Marek S. , Dale David C.
Published: April 2020

Background: Myelosuppression including severe and febrile neutropenia continues to represent a major cause of dose-limiting toxicity of cancer chemotherapy. Neutropenic complications in cycle 1 have been shown to frequently lead to reduced dose intensity or addition of a myeloid growth factor. A prospective, nationwide study was undertaken to develop and validate risk models for first cycle neutropenic events associated with cancer chemotherapy.

Methods: Patients with malignant lymphoma initiating a new chemotherapy regimen have been prospectively registered at 115 randomly selected practice sites. Data on at least one cycle of chemotherapy were available on 357 patients including 56 with Hodgkin’s disease and 301 with non-Hodgkin’s lymphoma. A logistic regression model for first cycle neutropenic events based on pretreatment characteristics was developed and predictive test performance characteristics examined.

Results: Severe or febrile neutropenia occurred in 81 (22.7%) of patients in cycle 1. Two-thirds of patients with one or more neutropenic events experienced their initial event in cycle 1. Significant independent pretreatment predictive factors for first cycle neutropenic complications were: history of renal disease (OR=33.15, P=.011) or recent infection (OR=12.41, P=.035), Caucasian (OR=4.13, P=.053), use of an anthracycline-based chemotherapy regimen (OR=6.9, P<.001), baseline absolute neutrophil count (OR=0.95, P<.001) and lymphocyte count (OR=0.95, P<.001), anemia (OR=2.14, P=.043), elevated lactate dehydrogenase (OR=2.0, P=.040) and elevated bilirubin (OR=2.25, P=.051). Model fit was excellent (P<.001), R2= 0.503 and c-statistic = 0.769 [95% CL: .71–.83, P<.0001]. Individual predicted risk of cycle 1 events based on the model ranged from 0 to 96% with mean and median probabilities of 0.23 and 0.21, respectively. Two-thirds of patients were classified as high risk with mean risk scores in high and low risk subjects of 32.0% and 5.6%, respectively. Model test performance characteristics [±95% CL] included: sensitivity: 92% [84–96]; specificity: 40% [34–46]; likelihood ratio positive: 1.53 [1.36–1.73]; likelihood ratio negative: 0.19 [0.09–0.42]; positive predictive value: 32% [26–38]; negative predictive value: 94% [88–97] and diagnostic odds ratio: 7.92 [3.32–18.91].

Discussion: The risk model identified lymphoma patients at increased risk for first cycle neutropenic complications using common clinical parameters. Validation of the model in a separate population of patients is in progress.