bluuebunny/crossref_metadata_2025 huggingface dataset:
Interferon Alpha-2B (IFN-a) Is an Effective Agent for the Treatment of Primary Cutaneous T- and B-Cell Lymphoma.
Authors: Siakantaris Marina P. , Kyrtsonis Marie-Christine , Tzenou Tatiana , Dimitriadou Evangelia M. , Dimopoulou Maria N. , Angelopoulou Maria K. , Kokoris Styliani I. , Vassilakopoulos Theodoros P. , Korkolopoulou Penelope , Giannakakis Antonia , Plata Eleni , Patsouris Eustratios , Katsambas Andreas , Pangalis Gerassimos A.
Published: October 2019

Primary cutaneous lymphoma comprise a wide range of rare diseases of which the commonest T- cell subtypes include mycosis fungoides (MF), Sezary syndrome (SS) and peripheral T-cell lymphoma (PTCL) while follicle center cell lymphoma (FCL), diffuse large B-cell lymphoma (DLBL) and extranodal marginal zone B-cell lymphoma (MZL) represent the B-cell subtypes. The behavior of these diseases is different from nodal NHL and it has been shown that IFN-a can produce long-lasting remissions in early stages MF. On the contrary, there are no reports on the efficacy of IFN- a for primary cutaneous B-cell lymphomas (CBCL). The purpose of this study was to present our experience on IFN -a treatment for patients with both primary T-cell and B-cell cutaneous NHL. Forty-one patients, 25 with primary cutaneous T-cell NHL (CTCL) (16 MF, 4 SS, 5 PTCL) and 16 with CBCL (4 MZL, 3 FCL, 8 DLBL, 1 lymphoblastic NHL) were included in this study. Of the 25 CTCL patients, 20 were males (median age 48y); 16 received IFN-a as first line treatment, 5 after PUVA and 4 after failure of other treatment modalities. Of the 16 CBCL patients, 8 were males (median age 57y); 14 received IFN-a frontline and 2 after treatment failure. Three to 5 MU IFN-a were administered subcutaneously daily with paracetamol prophylactically 1 hour before injection and two hours after. 17 out of 24 CTCL patients responded (overall response rate 71%, 42% CR and 29% PR) (12/16 MF, 1/4 SS and 3/5 PTCL). Median time to response was 4 months (1–17). Median duration of response was 15 months (8–38). 11 patients relapsed, 1 responder rapidly discontinued treatment because of side effects (cataract), the others are still under treatment with IFN-a. Among patients with CBCL 12/14 responded (overall response rate 86%, 64% CR and 22% PR). Median time to response was 2 months (1–4) and median response duration 23 months (13–30); 4 patients relapsed, 1 discontinued treatment because of side effects (depression), another continued with Peg- IFN because of chronic fatigue and the others are still under IFN-a treatment. With respect to histologic subtype, all 4 MZL patients responded; 2/3 FCL and 4/8 DLBL also responded. Flu-like syndrome was observed in the majority of patients at treatment initiation and 60% of patients presented mild leukopenia. In conclusion IFN-a is an effective treatment in primary cutaneous NHL and is usually well tolerated. As already reported response rate is satisfactory in MF. The high response rate observed in CBCL, especially in low-grade subtypes, but also in half of DLBL patients has not been reported previously.
Intravenous Forodesine (BCX-1777), a Novel Purine Nucleoside Phosphorylase (PNP) Inhibitor, Demonstrates Clinical Activity in Phase I/II Studies in Patients with B-Cell Acute Lymphoblastic Leukemia.
Authors: Furman Richard R. , Gandhi Varsha V. , Bennett J. Claude , Bantia Shanta , Kilpatrick J. Michael
Published: October 2019

Forodesine is a potent, specific transition-state analog inhibitor of PNP with clinical activity in T-cell malignancies. Pharmacodynamic studies support that this anti-leukemic effect is mediated through the accumulation of plasma 2′-deoxyguanosine (dGuo) and intracellular Dgtp. In vitro studies indicated that B-cell acute lymphoblastic leukemia (B-ALL) cells can also accumulate Dgtp. These preclinical data led to a phase I/II multicenter dose-escalation study to evaluate forodesine in pts with various hematologic malignancies. 15 pts were treated with forodesine, including 6 with B-ALL. Forodesine was given via a 30-min IV infusion followed 24 hrs later by doses every 12 hrs for a total of 9 doses. A second course could be repeated after a 2-week break. Doses were escalated by 50% to pts grouped in cohorts of 3 (starting dose: 40 mg/m2). There was a rapid rise in plasma dGuo and a maximum PNP inhibition was achieved at 40 mg/m2 (Fig 1). 7/15 treated pts (2 T-cell malignancies and 5 B-ALL), including 5/6 treated B-ALL pts, demonstrated a hematologic benefit, defined as a decrease in tumor burden. 3 of the responding B-ALL pts were further treated with 6 courses of forodesine on a compassionate use protocol. One pt treated at 135 mg/m2 demonstrated a complete response with a decrease in bone marrow blast cells from 22% to 5% at the end of therapy. The other 2 pts, treated with 90 mg/m2 and 135 mg/m2 forodesine respectively, showed dramatic improvement in their CBC despite the persistence of bone marrow blasts. The dramatic fall in WBC count was accompanied in each pt by a rise in plasma dGuo (Cmax: 1.9–7.1 μM) and intracellular Dgtp (380–800 pmoles/107 cells vs 25–40 pmoles/107 cells pre-treatment), The response of the pt treated with 90 mg/m2 forodesine is shown in Fig 2. The absolute neutrophil count (ANC) improved from ~0 at pre-treatment to 1800 cells/mm3 at Day 18.

To-date forodesine has been safe and well-tolerated at all dose levels tested with no dose-limiting toxicities. Clinical activity has been seen in T- and B-ALL. It is interesting to note the restoration of normal hematopoiesis, indicating the specificity of forodesine for leukemic cell populations and supporting that forodesine represents an important breakthrough in the development of less toxic ALL therapy. This encouraging clinical activity has led to the initiation of a phase II trial in pts with B-ALL. Additional clinical and pharmacodynamic results will be presented.
Long-Term Follow-Up of 5,930 Unrelated Volunteers Donating Bone Marrow or Peripheral Blood Stem Cells.
Authors: Ehninger Gerhard , Rall Gabi , Kroschinsky Frank , Schuler Ulrich S. , Schmidt Alexander , Hoelig Kristina , Bornhaeuser Martin , Rutt Claudia
Published: October 2019

More than 1,1 million donors are registered in the DKMS Deutsche Knochenmarkspenderdatei donor center. Since the start of the program, 5,930 have donated bone marrow or peripheral blood stem cells. These donors were included in a follow up program with questionnaires and blood cell counts 1, 6, and 12 months, and thereafter annually. Non responding donors were interviewed via a telephone call which enables us to have a follow-up of all donors. The age range at harvest was 18 to 60 years (median 32.4 years). The cumulative observation years were 13,350 years for 2,644 bone marrow donors and 7,980 years for 3,286 peripheral blood stem cell donors. No severe immediate complications occurred during the harvests. Seven out of 2,644 bone marrow donors (0.3%) suffered from long lasting severe local pain. Three of them were hospitalized within 6 weeks after donation with hematoma, local infection or severe local pain. One donor of the peripheral blood stem cell group suffered from severe hyperthyroidism 14 days after donation. In this donor, euthyroidism was documented at the day of leukapheresis. Antibodies to thyroid peroxidase were already detectable in retrospectively analyzed cryopreserved samples from these time points. TSH receptor stimulating immunoglobulin appeared only after harvest accompanied by symptoms of hyperthyroidism. Rheumatoid arthritis was diagnosed in 2 peripheral blood stem cell donors (interval from harvest: 0 days and 1 year) and joint complaints were addressed by additional 4 donors. Vascular events were seen in 4 peripheral blood stem cell donors (cerebral ischemia in 3 donors 3 months, 1 year and 2.5 years after donation; myocardial infarction in a 58 years old male donor 2 years after donation) and in one bone marrow donor (Brown-Séquard-syndrome 4 years after harvest). Four donors of the bone marrow group and 5 donors of the peripheral blood stem cell group developed malignancies during the follow-up observation. Four donors of the bone marrow group died 1 year to 8 years after donation (sudden death, car accident, suicide, and unknown). In unrelated donors, bone marrow harvests and peripheral blood stem cell donations are safe procedures in specialized collection centers. Even in a large donor center as DKMS, the numbers of adverse events in the follow up period are small. The documentation of diseases early after harvest (autoimmune diseases, thromboembolic events etc.) needs a careful follow-up. A case-controlled study with sex-, age- and HLA-matched volunteers within the donor pool will therefore be initiated by DKMS.
Is a Younger 10-Allele-Matched Unrelated Donor (MUD) Better Than an Older 6-Antigen-Matched Sibling Donor (MSD) for Allogeneic HSCT?.
Authors: Mehta Jayesh , Gordon L. , Tallman M. , Winter J. , Williams S. , Evens A. , Grinblatt D. , Kaminer L. , Singhal S.
Published: October 2019

56 adults with hematologic malignancies underwent blood cell allografts from A/B-DR-identical MSDs or A/B/C/DRB1/DQB1-allele-identical MUDs after 100 mg/m2 melphalan with (n=39; no prior autograft) or without (n=17; prior autograft) 50 mg/kg cyclophosphamide. GVHD prophylaxis comprised mycophenolate with cyclosporine (MSD) or tacrolimus (MUD). No growth factors were used. Supportive care was uniform. There were significant differences between groups (Table 1); most strikingly with respect to donor age.

MSD (n=37) MUD (n=19) P Pt age 53 (38–66) 47 (27–62) 0.085 Pt age >55 32% 32% 0.95 Donor age 51 (31–69) 33 (24–50) <0.0001 Donor age >45 76% 16% <0.0001 Refractory disease 49% 68% 0.16 PS 2–3 22% 32% 0.42 LDH 172 (83–1298) 237 (105–1919) 0.082 Abnormal LDH 43% 63% 0.16 CD34+ cell dose 5.0 (3.0–7.6) 6.0 (1.4–11.8) 0.12 CD34+ cell dose >6 11% 47% 0.002

10 patients experienced transplant-related mortality (TRM), and 29 relapsed (23 dead). The following factors were analyzed in a Cox model for effect on outcome: chemosensitive (n=25) vs refractory disease (n=31), patient age ≤55 (n=38) vs >55 (n=18), performance status (PS) 0–1 (n=42) vs 2–3 (n=14), normal (n=28) vs abnormal (n=28) LDH, prior autograft or not, CD34+ cell dose ≤6 (n=43) vs 6 (n=13) x 106/kg, MSD vs MUD, male (n=38) vs female (n=18) donor, and donor age ≤45 (n=25) vs >45 (n=31). Donor age was also analyzed as a continuous variable. Patient age >55 and PS 2–3 resulted in higher TRM. Refractory disease and abnormal LDH resulted in higher relapse, and lower disease-free (DFS) and overall (OS) survival. PS 2–3 resulted in lower DFS and OS. Patient age >55 and MSD resulted in lower OS. OS was reanalyzed after excluding donor type as a variable (*) because of its correlation with donor age. Table 2 shows the favorable effect of donor age ≤45 on relapse, DFS and OS.

Outcome RR (95% CI) P Relapse 0.28 (0.12–0.64) 0.003 DFS 0.27 (0.13–0.57) 0.001 OS* 0.42 (0.19–0.94) 0.035

When analyzed as a continuous variable, lower age resulted in lower relapse and higher OS with donor type in the model, and with higher DFS when donor type was excluded. Figures 1 and 2 show the effect of donor age on relapse and DFS in plots generated from the Cox model at the means of the covariates.

Figure Figure

These data appear to suggest that a young MUD may be preferable to an older MSD. However, small numbers preclude obtaining an answer to the question of the age at which a sibling donor should be considered too old. Additionally, logistic issues involved in obtaining a MUD may reduce any benefit from having a young donor. We conclude that a younger donor should be chosen whenever possible. Further work is required to address the intrguing possibility of superiority of MUD over MSD based on age.
Clinical and Biological Relevance of NF-κB Activation in B-CLL.
Authors: Rodriguez Antonia , Martinez Nerea , Camacho Francisca I. , Ruiz-Ballesteros Elena , Algara Patrocinio , Artiga Maria J. , Garcia Juan-Fernando , Piris Miguel A.
Published: October 2019

Based on previous observations, we postulate that the molecular and clinical variability of Chronic Lymphocytic Leukemia (CLL) could reflect differences in the degree of NF-κB activation. To test this hypothesis, the expression profile (mRNA and protein expression) of a series of 41 B-CLL cases with lymph node involvement was analyzed at the same time, with a cDNA microarray (OncoChip, 7K clones) and an oligo microarray (Human 1A Agilent, 22K sequences). Results were correlated with the IgVH mutational status, ZAP-70 expression, clinical outcome and NF-κB activation status, as determined by EMSA and phosphorylated IκBα (p-IκBα) detection by Western Blotting. The study showed a correlation between the presence of a surrogate marker of NF-κB activation, p-IκBα, and changes in the degree of NF-κB activation, as determined by EMSA. NF-κB activation was associated with the expression of 2200 and 894 genes using the oligo and cDNA microarray platforms respectively. These sets of genes comprise key genes involved in the control of B-cell receptor signaling, signal transduction and apoptosis, including SYK, LYN, BCL2, CCR7, BTK, PIK3CD, BAFF. Cases with higher levels of NF-κB activation showed longer overall survival than cases with lower activation. A Cox regression model was derived to analyze variables of potential prognostic interest in this series: IgVH mutational status, ZAP-70 and p-IκBα expression. The multivariate analysis disclosed p-IκBα and ZAP-70 expression as independent prognostic factors related with overall survival. In conclusion, a variable degree of activation of NF-κB, as determined by the expression of p-IκBα and EMSA, is a relevant event in CLL, associated with changes in the survival probability, and correlated with the expression of a large set of genes identified by two different microarray platforms.
Treatment with AMD3100 in Multiple Myeloma or Non-Hodgkin’s Lymphoma Patients to Increase the Number of Peripheral Blood Stem Cells When Given with a Mobilizing Regimen of Chemotherapy and G-CSF.
Authors: Dugan Michael J. , Akard Luke P. , Thompson James M. , Nademanee Auayporn , Maziarz Richard T. , Bensinger William I. , Liesveld Jane , Badel Karin , Dehner Christine , Gibney Colleen , Calandra Gary , Jansen Jan
Published: October 2019

AMD3100 is a reversible inhibitor of the binding of SDF-1α to its cognate receptor CXCR4. Patients with MM and NHL given AMD3100 alone have increased circulating C D34+ cells. The purpose of the present study is to determine if AMD3100 added to a chemotherapy and G-CSF mobilization regimen can increase the circulating levels of PBSC, and the removal of such cells by leukapheresis. The only change to the standard care is the addition of AMD3100 to a cyclophosphamide (2.5 g/m2) mobilization for MM and either (R)ESHAP or (R)ICE in the case of NHL. In both arms G-CSF is given at 10 mcg/kg/d. AMD3100 is given after the first apheresis collection. To date, 10 patients (8 MM, 2 NHL) have completed the mobilization phase of the protocol. During recovery the mean number of PB CD34+ cells/μL was 191. This rose to 304 [2.0 fold increase, p=0.008] following the administration of AMD3100. The baseline collection resulted in a mean of 16.7 x 106 CD34+ cells/kg. This rose to 24.0 [1.8 fold increase, p=0.007] following the administration of AMD3100. Historic controls with MM receiving cyclophosphamide (4 g/m2), etoposide (400 mg/m2) and G-CSF had an inferior collection on day 2 [37.9 x 106 CD34+ cells/kg day 1 vs. 29.5 day 2, p=0.009]. Untoward events related to the combination of chemotherapy, G-CSF, and AMD3100 have not been noted. The 9 patients transplanted following this mobilization strategy have achieved an ANC > 500 at a median of 10 days, and platelet independence (>20k) at a median of 11 days. Validation of this strategy continues in additional patient cohorts to assess alternative AMD3100 administration schedules and to define CD34+ cell recruitment/release during recovery from mobilizing chemotherapy.
Activation of Classical and Alternative Nuclear Factor-kappaB (NF-kB) Pathways in Diffuse Large B-Cell Lymphomas.
Authors: Timar Botond , Chadburn Amy , Knowles Daniel , Cesarman Ethel
Published: October 2019

Activation of the NF-kB pathway is involved in many human neoplasms. In this study we examined the status of the NF-kB canonical (IkB, p50/p65) and non-canonical (p52, RelB) pathways in diffuse large B-cell lymphomas (DLBCL), which are a common and heterogeneous group of lymphoid malignancies. DLBCL have been divided into activated B-cell (ABC) like, and germinal center B-cell (GCB) like subgroups, which have been reported to have high and low NF-kB activity, respectively. However, the nature of the NF-kB complexes in this lymphoma entity has not been previously evaluated. Therefore we performed Western blotting, electrophoretic mobility shift assays (EMSA) and real time quantitative RT-PCRs on nuclear and cytoplasmic protein and total RNA extracts in 20 primary tumor samples and 9 different DLBCL cell lines. In the cell lines, presence of NF-kB proteins in nuclear extracts correlated with expression of NF-kB target genes (CCR7, IkBa, CCND2, BCL-2, IRF4) as determined by RT-PCR. Therefore, these could be assigned into GCB and ABC-like categories. In primary DLBCLs, EMSA showed NF-kB binding in all but one case. The same cases (19/20) had high p52 in the nucleus indicating activation of the alternative pathway. TNF family ligand BAFF was also found to be expressed in all primary samples and most cell lines. The classical pathway, as determined by nuclear p50, was also present in these cases. Levels of p65 and RelB expression were variable, but did not correlate with the mRNA expression of NF-kB target genes. In conclusion, while DLBCL cell lines may be divided into two distinct categories, the primary samples represented a spectrum of NF-kB target gene activity, while levels of NF-kB expression were high. BAFF expression and activation of the alternative pathway may be important in the pathogenesis of DLBCL.
Once Per Cycle Pegfilgrastim Versus Daily Filgrastim in Pediatric Patients with Ewing Sarcoma.
Authors: Wendelin Gerald , Lackner Herwig , Schwinger Wolfgang , Sovinz Petra , Urban Christian
Published: October 2019

The administration of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) Filgrastim for reducing the duration of severe neutropenia after cytotoxic chemotherapy has become an important part of oncologic supportive care. Due to the short serum half-life Filgrastim has to be administrated daily by subcutaneous injections. Frequent injections however mark a problem in pediatric patients. Studies in adult patients have shown a comparable effect of the new long lasting rhG-CSF Pegfilgrastim which has to be administrated only once per cycle. In the current study the effects of Pegfilgrastim in pediatric patients were analysed. Five patients (10–16 years) with Ewing sarcoma were treated in a cross over study design alternately with Pegfilgrastim and Filgrastim following the EURO E.W.I.N.G. 99 protocol. Starting on day 4 after chemotherapy patients received Filgrastim 10μg/kg daily by subcutaneous injection until an absolute neutrophil count (ANC) >1000/μl after the expected nadir. Pegfilgrastim 100μg/kg was administrated on day 4 once per cycle subcutaneously. In 3 patients the stimulation with rhG-CSF was performed after each of the 6 preoperative VIDE-(Vincristin, Ifosfamide, Doxorubicin, Etoposide) cycles, in 2 patients after 8 postoperative VAI-(Vincristin, Actinomycin D, Ifosfamide), and in 2 patients after 7 postoperative VAC-(Vincristin, Actinomycin D, Cyclophosphamide) cycles of the EURO-E.W.I.N.G. 99 protocol. The duration of grade 4 neutropenia after single administration of Pegfilgrastim was 2,8 ±3,1 (0–10) days, after daily administration of Filgrastim 3,1 ± 2,7 (0–8) days. The number of days with a body temperature over 38 degrees and grade 4 neutropenia at the same time was 0,9 ±1,5 (0–6) after Pegfilgrastim and 0,9± 1,4 (0–4) after Filgrastim. Filgrastim had to be injected 6,7 ± 1,8 (3–10) times per cycle. Bone pain associated with Pegfilgrastim was noted in only one patient. Costs for Pegfilgrastim were 16% lower than for Filgrastim. We conclude that in pediatric patients with Ewing sarcoma the duration of severe neutropenia and number of days with febrile neutropenia after once per cycle Pegfilgrastim and daily Filgrastim are comparable. By using Pegfilgrastim the number of subcutaneous injections can be reduced to one single injection and costs can be lowered.
Interferons and IRF8/ICSBP Stimulate Immune Protection Against BCR/ABL-Induced Leukemia.
Authors: Nardi Valentina , Mohammad Azam , Dikovskaia Eugenia , Daley George Q.
Published: October 2019

Chronic Myeloid Leukemia (CML) results from transformation of hematopoietic stem cells by the BCR/ABL oncoprotein. CML can be effectively treated with the targeted ABL kinase inhibitor Imatinib (STI571; Gleevec), but patients rarely achieve molecular remission by sensitive PCR methods, and drug resistance limits the durability of drug response. A significant minority of patients treated with Interferon (IFN) alpha achieve complete cytogenetic remission, and response is correlated with immune reactivity against leukemic clones. This suggests that IFN-induced immunotherapy might be an important adjunct to Gleevec, thus prompting us to investigate immunoprotective mechanisms. We have shown previously that ectopic expression of Interferon Regulatory factor 8 (IRF8, also called ICSBP) can stimulate a potent CD8+ cytotoxic anti-leukemia response in a murine model of BCR/ABL-induced leukemia (Deng and Daley, Blood 97;3491, 2001). In transcriptional profiling experiments with cells that overexpress IRF8/ICSBP, we detected increased expression of IFN alpha and beta. To test whether IFN alpha or beta can mediate the same protective immune-response as IRF8/ICSBP, we co-injected BCR/ABL-transformed BaF3 cells together with non-transformed BaF3 cells engineered to overexpress IFN alpha or beta. Expression of IFN alpha or beta in trans protected mice from developing leukemia. Control constructs in which IFN alpha or beta were cloned in the anti-sense orientation failed to confer protection, confirming the importance of IFN alpha or beta expression. Importantly, IRF8/ICSBP expression is protective against cells transformed by Gleevec-resistant mutant BCR/ABL proteins (T315I and L248R). Our data suggest that IFNs are functionally equivalent to IRF8/ICSBP in conferring a vaccine-like anti-leukemic immune effect in our model, and may act through an autoregulatory loop involving IRF8/ICSBP. Defining the precise epitopes targeted by the IFN/ICSBP-induced immune response may enable immunotherapy in human CML patients.
A New Functional Coagulation Assay for Measurement of Total Factor V Activity in Human Plasma and Its Application to Samples from Patients with Disseminated Intravascular Coagulation.
Authors: Samis John A. , Stewart Kenneth A. , Toh Cheng Hock , Downey Colin , Nesheim Michael E.
Published: October 2019

Factor V (F.V) circulates as an inactive single chain procofactor. Only upon cleavage and activation with thrombin to F.Va does it function as a cofactor in the F.Xa-mediated conversion of prothrombin to thrombin. The conventional F.V coagulation assay measures the one-stage clotting activity, which most likely reflects the trace of F.V that is activated in the sample or becomes activated during the assay. Since F.V is activated by thrombin to F.Va, a second (two-stage) assay is crucial to fully assess the total F.V potential activity in the plasma sample. For this assay, normal human pooled plasma (diluted 100- to 500-fold) was treated with thrombin (1–5 NIH Units/ml, final concentration) for various times at 37°C to fully activate the F.V. The added thrombin was inhibited with a 2-fold molar excess of Phe-Pro-Arg chloromethylketone (PPACK) and the sample re-assayed. This second assay measures the total F.V activity of a sample. Maximal 10-fold activation of plasma F.V activity occurred with 2Units/ml thrombin after 1 to 2min at 37°C and this activity was stable for 30min prior to PPACK addition. Control experiments indicated that the levels of PPACK used did not affect the one-stage F.V activity of untreated plasma samples but were required to inhibit the added thrombin from clotting fibrinogen in the assay. Assay of timed plasma samples (n=48) from seven patients admitted to the Intensive Care Unit and subsequently diagnosed with disseminated intravascular coagulation (DIC), indicated that the total F.V activity, the specific activity (total F.V activity/antigen), and the activation quotient were decreased, on average, by 45%, 49%, and 31%, respectively, from that observed with normal human pooled plasma. The results indicate that the total amount of activatable F.V as well as its degree of activation decreases from normal as part of the pathogenesis of DIC in humans. Further, the decreased total F.V specific activity observed in DIC patient plasma samples indicates that the F.V that is present is less active per molecule than normal, possibly because of proteolytic inactivation. This total F.V clotting assay should provide new information about the biochemistry of F.V and its contribution to the pathogenesis of coagulation disorders.
Minimal Residual Disease (MRD) Analysis in Acute Myeloid Leukemia (AML) with Favorable Cytogenetics [t(8;21) and inv(16)] by Real Time PCR(RT-PCR) and Flow Cytometry (FC).
Authors: Perea Granada , Lasa Adriana , Aventin Anna , Domingo Alicia , Villamor Neus , de LLano Maria Paz Queipo , Llorente Andreu , Junca Jordi , Palacios Carlos , Fernandez Cristalina , Guallart Miquel , Font Llorens , Tormo Mar , Florensa Lourdes , Bargay Joan , Marti Josep M. , Vivancos Pilar , Torres Pio , Berlanga Juan , Brunet Salut , Sierra Jorge , Nomdedeu Josep F.
Published: October 2019

Objectives: To analyze MRD in 65 patients (pts) with good prognosis AML: 30 t(8;21) and 35 inv(16), using both FC and RT-PCR, and to investigate the prognostic value of MRD in the pts outcome.

Methods: MRD was monitored in CR pts (n=55) by FC in 101 follow-up samples obtained after various cycles of treatment, as follows: 40 post-induction (ind), 30 post-intensification (int) and 31 at the end of treatment (ttm), and by RT-PCR in 76 samples: 31, 23 and 22, respectively. In 35 pts the two techniques were applied at the same time of the ttm. MRD by FC was assessed using fixed combinations of three monoclonal antibodies. AML1/ETO and CBFb/MYH11 were analyzed following the BIOMED protocol.

Results: Twenty-seven percent (n=15) of CR pts relapsed: 6 with t(8;21) and 9 with inv(16). The mean MRD by FC was 1.1% after ind, 0.2% after int and 0.1% at the end of ttm. At the end of ttm, the MRD detected by FC in relapsed and not relapsed pts were significativaly different: 0.3% vs 0.08% (p=0.002). By RT-PCR, the mean of fusion transcript copies/ablx104 differed between relapsed and nonrelapsed pts: 2385 vs 122 (p=0.001) after ind, 56 vs 7.6 after int (p=0.0001) and 75 vs 3.3 (p=0.0001) at the end of ttm. Relapses were more commonly observed in those pts with FC MRD level >0.1% at the end of ttm than in pts with ≤0.1%: 50% vs 12% (p=ns); likewise, using RT-PCR, a cutoff level of >10 copies at the end of ttm correlated with high risk of relapse: 80% of pts with RT-PCR >10 relapsed compared to 12% of pts with levels <10 (p=0.009). The overall survival (OS) probability was 86% for pts with CF MRD ≤0.1 at the end of ttm and 0% for pts with MRD >0.1 (p=0.1) and the leukemia free survival (LFS) was 78% and 44%, respectively (p=0.05). For pts with RT-PCR ≤10 at the end of ttm, the OS was 100% and for pts with RT-PCR >10 it was 30% (p=0.007) and the LFS was 87% and 20%, respectively (p=0.001). MRD was identified after ind in 55% of relapsed pts and at the end of ttm in 83% of relapsed pts. Only 1 pt (1/13) with FC MRD <0.1 and RT-PCR <10 at the end of ttm relapsed. For patients in complete remission, the mean copy level of chimeric transcript was higher for pts with t(8;21) than for those with inv(16): 30.2 vs 17.4 (p=0.0001).

Comments: In tandem analysis of MRD by FC and RT-PCR could improve MRD detection in AML pts.
Identification of Natural Killer Cell Receptor Phenotypes Associated with Leukemia.
Authors: Verheyden Sonja J. , Bernier Michel , Demanet Christian J.
Published: October 2019

Introduction: Natural Killer (NK) cells play a key role in defense against tumor cells that have the capacity to downregulate Human Leukocyte Antigen (HLA) class I expression. It has been reported that leukemic cells can have down-regulated expression of HLA class I molecules. Apparently, the NK cells of these patients are not able to destroy these leukemic cells and may allow malignant cells to escape from innate immune control. This failure may be due to the fact that NK cells are part of the malignant clone and therefore might have a decreased function. An alternative hypothesis could be that these patients may display a NK cell Receptor (NKR) genotype incapable of destroying leukemic cells with aberrant expression of HLA class I molecules. The polymorphic nature of the NKR genes generates diverse repertoires in the human population, which display specificity in the innate immune response.

Materials and Methods: In the present study, 11 Killer cell Immunoglobulin-like Receptor (KIRs) and 2 CD94/NKG2 receptors were genotyped by PCR-SSP in 96 leukemic patients and 148 healthy Caucasians.

Results and Conclusion: We report a significant increased frequency of the more inhibitory AB KIR phenotype in leukemic patients compared to the controls (31.1% in healthy controls vs. 51.0% in leukemic patients, Pc = 0.002), which is related to the high prevalence of the inhibitory KIR2DL2 in this population (Pc = 0.007). Moreover, two specific KIR phenotypes AB1 and AB9, including all inhibitory KIRs, were significantly associated with leukemic patients. Our study suggests that an important percentage of leukemic patients express a KIR phenotype in favor of escape from NK cell immunity.
Clinical Outcome Correlations of Genomic Gains and Losses in Diffuse Large B-Cell Lymphoma by Array-CGH.
Authors: Chen Weiyi , Olsen Adam , Chaganti Seeta , Halaas Jeff , Zelenetz Andrew D. , Feldstein Julie , Houldsworth Jane , Chaganti Raju S.K.
Published: October 2019

Conventional karyotype and chromosomal comparative genomic hybridization (CGH) studies of diffuse large B-cell lymphoma (DLBCL) have revealed few chromosomal abnormalities associated with outcome. In order to evaluate the association between genomic copy number changes in DLBCL and clinical outcome at a higher resolution, we assayed a panel of 64 newly-diagnosed DLBCL specimens by array-CGH. For each specimen, the respective patient had a known response to anthracycline-based therapy, and the median follow-up was 5-years. Forward and reverse hybridizations were performed for all specimens to avoid dye-bias, using BAC/PAC arrays with 1-4 Mb resolution coverage of the genome (Spectral Genomics). After customized normalization, the circular binary segmentation (CBS) algorithm was used to identify regional copy number changes along each chromosome in each specimen. Of the 64 specimens, 59 (92%) displayed regional copy number changes. The frequency of gain/loss of each clone for all 64 specimens was then calculated based on the CBS results, and, additionally, based on singleton clone changes if they were outside of CBS-defined regions. Clones displaying change ≥ in 10% of specimens for at least two contiguous clones were further considered as recurrent sites for clinical correlations. 27 sites of recurrent gain and 25 sites of recurrent loss were identified. Association between loss or gain of sites and International Prognostic Index (IPI) were evaluated using Fisher’s exact test, and associations between loss or gain of sites and time to treatment failure (TTF) and overall survival (OS) were tested using the log-rank test. Table 1 lists the sites as independent markers that were significant at p < 0.05. Of these sites, five (with asterisk) were found to significantly predict outcome after stratifying by IPI using the stratified log-rank test. It is noted that 6 of the sites were 5 Mb or less in size, facilitating the identification of target genes. Losses of chromosome 7 (8–13 Mb) and chromosome 13 (56.7–63 Mb) were found to contribute to IPI prediction of outcome, but were not significantly associated with clinical features as independent markers. In summary, array-CGH has lead to the identification of several genetic markers with prognostic significance in DLBCL, which in some cases are of a size amenable for target gene identification.

Significant Genetic Markers Associated with Clinical Outcome

Chrom. Start (Mb) End (MB) Change # of cases IPI TTF OS 1 78.2 79.1 Loss 19 Better 2 2.4 4.1 Loss 8 Worse* 3 0.2 72.3 Gain 14 Better Better 3 101.3 136 Gain 18 Low Better 3 154.7 188.7 Gain 18 Low Better Better 6 62.2 170.5 Loss 32 Low Better Better 7 18.8 19.2 Loss 11 Better* 9 8.3 13 Loss 13 Better 9 83.9 96.3 Gain 9 Better 9 122.1 132.8 Gain 12 Better Better* 12 0 133.3 Gain 31 High 16 0.1 4.7 Gain 13 Better 16 33.8 35.6 Loss 7 Worse* Worse* 19 43.1 63.7 Gain 13 Better Better
Phenotype Correction of Hemophilia A Mice with Adeno-Associated Virus (AAV) Vectors Carrying the B Domain Deleted Canine Factor VIII Gene.
Authors: Mimuro Jun , Ishiwata Akira , Kashiwakura Yuji , Takano Katsuhiro , Ohmori Tsukasa , Madoiwa Seiji , Mizukami Hiroaki , Ozawa Keiya , Sakata Yoichi
Published: October 2019

Hemophilia A is a life-threatening bleeding disorder caused by mutations in the factor VIII (FVIII) gene that lead to deficiency of FVIII. Gene therapy is respected to provide an alternative to current FVIII supplemental therapy. Among a variety of vectors, AAV vectors are thought to be ideal for transfer of therapeutic genes since they are derived from non-pathogenic virus and have been demonstrated to provide sustained transgene expression in non-dividing cells with little toxicity, though, delivery of the FVIII gene using AAV vectors are limited by its small packaging capacity. To overcome the packaging capacity limit, we developed AAV1 vectors and AAV8 vectors carrying the B domain deleted canine FVIII gene (BDD cFVIII) utilizing a minimum promoter (150b) and expressed canine FVIII in hemophilia A mice. Previous reports suggested AAV1 serotype is suitable for transduction of skeletal muscles and AAV8 serotype is superior to other AAV serotypes for transduction of the liver, thus, AAV1 vectors carrying the BDD cFVIII gene (AAV1 cFVIII) were injected to the skeletal muscles of hemophilia A mice. Since the liver could be transduced with intravenously injected AAV8 vectors carrying the Lac Z gene as efficiently as intraportally injected vectors, AAV8 vectors carrying the BDD cFVIII gene (AAV8 cFVIII) were injected to hemophilia A mice intravenously. FVIII clotting activities measured by the APTT method increased in mice injected with AAV1cFVIII in a dose dependent manner. The FVIII activity level in peripheral blood increased to 2.9±1.0% in hemophilia A mice with the AAV1cFVIII dose at 1x1012 gc/body, suggesting partial correction of phenotype with AAV1cFVIII vectors. The FVIII clotting activity levels in hemophilia A mice injected with AAV8cFVIII increased also in a dose dependent manner, achieving supernormal FVIII levels (436±219 %) in hemophilia A mice with the AAV8 cFVIII dose at 1x1012 gc/body. It is apparent that transduction of the liver with AAV8cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding FVIII production, however, AAV1 vectors have advantages of removing the transgenes in case of inconvenience and of less systemic distribution of vectors. These data suggested that both AAV1 and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy.
Molecular Mechanism of Hepcidin Deficiency in a Patient with Juvenile Hemochromatosis.
Authors: Rideau Alexandra , Mangeat Bastien , Matthes Thomas , Trono Didier , Beris Photis
Published: October 2019

In eukaryotes protein translation is initiated at an AUG codon typically located within a so-called Kozak sequence (GCCRCC AUG G), a motif that presumably promotes the correct positioning of the mRNA in the ribosome. Recently, we described a patient suffering from juvenile hemochromatosis resulting from a lack of hepcidin production (Blood;15 June 2004; Epub ahead of print). We further revealed that this defect coincided with a G-to-A point mutation at position +14 of the 5′untranslated region (5′UTR) of the HAMP gene. This nucleotide change led to the creation of an ATG codon, out of frame with the physiological pre-hepcidin initiator located at positions +39-41. We speculated that this new ATG, which was embedded within a Kozak consensus sequence, acted as an aberrant translation initiation site that precluded hepcidin synthesis. In order to verify this hypothesis, we cloned in a mammalian expression vector the wild-type and mutant HAMP 5′UTR upstream of a modified EGFP cDNA, replacing the EGFP ATG by the HAMP physiological initiation codon. The resulting vectors were transfected into 293T cells. Presence of the additional ATG in the HAMP 5′UTR led to a 93 % decrease in GFP expression compared with wild-type. To confirm that this effect resulted from aberrant translation initiation at this site, a one-nucleotide deletion was introduced at the very 5′ end of the EGFP coding sequence, in order to place it in frame with the mutant HAMP 5′UTR proximal ATG (GTG in the wild-type sequence). This restored GFP expression to levels obtained when its coding sequence was in frame with the physiological hepcidin ATG of a wild-type HAMP 5′UTR. Our results very strongly suggest that the G to A mutation at position +14 of the HAMP gene 5′UTR blocks hepcidin production by inducing aberrant translational initiation of the pre-hepcidin mRNA. Whether a stable protein is synthesized from the mutated HAMP mRNA, which does not contain a stop codon in frame with the +14 AUG, remains to be determined. It is however unlikely that such protein contributes to the pathology observed in our patient, since the two other cases of hepcidin deficiency reported so far exhibit the same phenotype as our patient in spite of harbouring different mutations in the HAMP gene.
Hemojuvelin Mutations in Whites, Blacks and Asians with Primary Iron Overload and in Control Subjects.
Authors: Lee Pauline L. , Barton James C. , Brandhagen David J. , Niyongere Sandrine , Bohannon Sean , Rivers Charles A. , Acton Ronald A. , Beutler Ernest
Published: October 2019

Mutations in the hemojuvelin gene (HJV) are a cause of juvenile hemochromatosis (JH), but do they influence the phenotype of patients with adult-onset hemochromatosis with or without mutations of the HFE gene? We sequenced the coding region of the HJV gene of 133 subjects with primary iron overload (75 whites: 50 HFE, 25 non-HFE associated; 51 blacks: 1 HFE-associated; 7 Asians). A 63 year old white woman with non-HFE iron overload was found to be a compound heterozygote for HJV mutations: one previously identified (G320V); the other novel (C321W). The patient was diagnosed at age 30 (SI 223 μg/100 mL, TIBC 293 μg/100 mL, TS 76%, hepatic iron 31,154 μg/g, hepatic iron index 19.2). She had 2 healthy children, developed amenorrhea at age 23, had a history of hypothyroidism and developed Type 2 diabetes at age 59. She was iron depleted after 66 phlebotomies. Among 74 other white subjects with iron overload we found 3 polymorphisms: IVS2-6 C>G (3 subjects); nt 165 T>A (G55G), and IVS4-50 C>T (1 subject, each). Among 51 black iron overloaded and 132 black control subjects, the most notable polymorphism was a DNA triplet insert predicting an insertion of glycine. This was found in 2 subjects, one with and one without iron storage disease. Among the iron overloaded black subjects, we also found IVS2-6 C>G (1 subject), nt -140 G>C (3 subjects), nt 792 G>C (S264S) (1 subject), nt 828 C>A (I275I) (1 subject), and nt 929 C>G (A310G) (2 subjects). These polymorphisms, excluding S264S and I275I, were found in control subjects as well. No coding region mutations or polymorphisms were found in the 7 Asian iron overloaded subjects. We also determined the gene frequency of the HJV I222N and G320V mutations in adult control subjects from central Alabama, where a JH index case had been found with these mutations. Allele frequencies of I222N and G320V were 0.002 (n=240) and 0 (n=241) respectively in control whites. A white control female heterozygous for the I222N was also heterozygous for HFE C282Y but had normal iron measures. No I222N and G320V mutations were found in 124 and 118 black subjects respectively. We conclude that: 1) iron overloaded patients without HFE mutations, with or without a JH phenotype should be screened for HJV; 2) HJV mutations do not account for most cases of increased penetrance of HFE-associated hemochromatosis; 3) double heterozygosity for HFE C282Y and HJV I222N does not necessarily cause iron overload.
Geographic and Racial/Ethnic Differences in HFE Mutation Frequencies and Iron Phenotypes in the Hemochromatosis and Iron Overload Screening (HEIRS) Study.
Authors: Acton Ronald T. , Snively Beverly M. , Barton James C. , Adams Paul C. , Eckfeldt John H. , Harris Emily L. , Dawkins Fitzroy W. , McLaren Christine E. , Gordeuk Victor R. , McLaren Gordon D. , Reboussin David M. , Sholinsky Phyliss , Speechley Mark R. , Holup Joan , Leiendecker-Foster Cathy
Published: October 2019

Designing an optimal screening approach for hemochromatosis and iron overload requires knowledge of racial/ethnic subpopulations and frequencies of HFE mutations and phenotypes in geographic areas. In the HEIRS Study, HFE C282Y and H63D genotypes and prevalences of participants who met biochemical criteria for further evaluation (transferrin saturation >50% and ferritin >300 ng/mL, men; >45% and >200 ng/mL, women) were compared in a primary care-based sample of ~100,000 adults ≥25 years from 5 Field Centers. There were different respective HFE C282Y and H63D genotype frequencies in Whites, Blacks, Asians, and Hispanics across geographic areas (all p<0.0001), but not in Native Americans or Pacific Islanders. In Whites, the C282Y/C282Y proportion was significantly higher in AL than in CA or OR/HI. AL also had a significantly higher proportion of C282Y/wt than CA or OR/HI. In Blacks, D.C. had a significantly higher proportion of C282Y and H63D genotypes than AL. ONT had a significantly higher H63D genotype proportion than AL. In Asians, CA had a significantly higher proportion of H63D genotypes than OR/HI or ONT. In Hispanics, there were significant differences between: AL and CA; AL and D.C.; CA and D.C.; CA and OR/HI; D.C. and OR/HI; and D.C. and ONT. We also observed:

Prevalences of Participants Who Met Biochemical Criteria for Further Evaluation

Race/Ethnicity Geographic Area Prevalence (%) 95% CI Asians California 5.54 4.98, 6.16 Oregon/Hawaii 5.06 4.29, 5.95 Ontario 4.25 3.69, 4.88 Alabama 1.41 0.25, 7.56 D.C. 0.96 0.26, 3.42 Pacific Islanders California 3.66 1.25, 10.21 Oregon/Hawaii 3.47 2.23, 5.36 Native Americans Alabama 2.65 0.91, 7.52 Oregon/Hawaii 2.50 0.69, 8.66 D.C. 0.99 0.17, 5.40 Ontario 0.75 0.21, 2.71 Whites Ontario 2.31 2.08, 2.56 California 1.93 1.56, 2.37 Oregon/Hawaii 1.52 1.34, 1.73 Alabama 1.48 1.26, 1.74 D.C. 1.40 0.80, 2.43 Hispanics Ontario 1.92 0.82, 4.41 California 1.52 1.28, 1.79 Oregon/Hawaii 1.34 0.73, 2.4 Alabama 0.84 0.23, 3.01 D.C. 0.74 0.48, 1.16 Blacks D.C. 1.75 1.56, 1.96 Ontario 1.55 0.52, 4.45 Alabama 1.09 0.90, 1.32 Oregon/Hawaii 0.92 0.31, 2.66 California 0.86 0.29, 2.51

The percentage of men who met biochemical criteria for further evaluation was greater than that of women. We conclude that 1) C282Y and H63D genotype frequencies vary significantly within a single racial/ethnic group across geographic regions; and 2) the percentage of participants who met biochemical criteria for further evaluation varies by racial/ethnic group.
Identification and Characterization of Hemoangiogenic Progenitors during Cynomolgus Monkey ES Cell Differentiation.
Authors: Umeda Katsutsugu , Heike Toshio , Yoshimoto Momoko , Shiota Mitsutaka , Ma Feng , Suemori Hirofumi , Luo Hong Yuan , Chui David H.K. , Torii Ryuzo , Shibuya Masabumi , Nakatsuji Norio , Nakahata Tatsutoshi
Published: October 2019

Previous reports about the close association of development of hematopoietic (HCs) and endothelial cells (ECs) have suggested the presence of the hemangioblast, a putative common precursor of both lineage cells in mouse. The presence of putative ancestors is suggested by the fact that both HCs and ECs share common markers such as vascular endothelial growth factor receptor (VEGFR)-2, CD34, and CD31. The precise analysis about the relationship of HCs and ECs in human embryogenesis, however, remains unclear because of obstacle including the ethical restrictions on experiments using their embryo. To further understand the mechanisms that regulate the differentiation of HCs and ECs in humans, it is currently necessary to employ primate (human and monkey) ES cells. In this study, we identified the intermediate-stage cells with HC and EC differentiation potentials from nonhuman primate ES cells by coculture with OP9 stromal cells. Sequential FACS analysis demonstrated that during in vitro HC and EC differentiation, the expression of common surface markers such as CD34 and CD31 could first be detected, followed by the expression of lineage-specific markers (CD45, CD41 and VE-cadherin) in order. Sequential FACS and immunostaining analyses showed that both lineage developments concomitantly occurred, which were preceded by emergence of two hemoangiogenic progenitors, VEGFR-2high CD34− and VEGFR-2high CD34+ cells. While both VEGFR-2 high cells could differentiate into primitive and definitive HCs as well as ECs, VEGFR-2high CD34+ cells possessed higher hemoangiogenic potential than VEGFR-2high CD34− cells. In contrast, HC and EC production cluster was rare in the VEGFR-2low or − fractions. During HC and EC differentiation, most of VEGFR-2high cells first expressed CD34, and then diverged into both lineage cells at the VEGFR-2high CD34+ stage. We found that the VEGFR-2high cells can generate both HCs and ECs at the single cell level, which strongly supports the existence of hemangioblasts in primates. To our knowledge, this is the first time that hemangioblasts have been shown to be present during primate ES cell differentiation. Furthermore, differentiation pathway into HC and EC lineages can be defined by surface markers, which facilitates further investigations in vitro and in vivo.