bluuebunny/crossref_metadata_2025 huggingface dataset:
Cost Effectiveness of Rituximab Maintenance (R-Maint) vs Autologous Stem Cell Transplant (ASCT) from a UK National Health Service (NHS) Perspective.
Authors: Millar Douglas R. , Lewis Gavin , Marcus Robert
Published: October 2019

Objectives: Both ASCT and R-Maint have been shown to be effective therapies for treating rituximab naïve patients with relapsed/refractory follicular lymphoma. The aim of this research was to assess the cost effectiveness of ASCT compared to R-Maint in this patient population (following a response to chemotherapy induction) from a UK perspective.

Methods: A literature review of randomised control trials (RCTs) and case series studies suitable for comparing the efficacy of the two treatments in relapsed / refractory follicular lymphoma patients was performed. Given the heterogeneity of relevant study outcomes and designs a naïve indirect comparison was performed. Equivalent efficacy between the two therapies was assumed based upon the indirect comparison; hence a cost minimization approach was utilized. The starting time point of the analysis was following 3 cycles of CHOP therapy. In the EORTC 20981 trial both RCHOP and CHOP induction therapies were used; to reduce heterogeneity between the trial populations being compared, only results from patients who received CHOP induction in this trial were considered. Drug, administration, adverse event, and monitoring costs were evaluated from the start of treatment to the point of relapse after which time-point cost equivalence was assumed between the two therapies. Resource use and unit costs were sourced from published NHS reference costs, the EORTC 20981 study, and clinical expert opinion. Uncertainty around the assumption of equal efficacy was explored using threshold analysis based on an assumed incremental cost effectiveness ratio (ICER) threshold of £30,000 and an average utility score of 0.71 for patients with follicular lymphoma (derived from the literature).

Results: One RCT and 6 case series for ASCT and one RCT for R-Maint were identified. The estimated cost of R-Maint and ASCT was £12,275 and £25,563 respectively, resulting in an estimated incremental cost of ASCT over R-Maint of £13,288 per patient. ASCT would have to offer an additional 7.5 months of overall survival to be considered cost effective compared to R-Maint.

Conclusions: There is a high degree of certainty that ASCT produces higher costs compared to R-Maint, however it is uncertain whether ASCT offers an increase in either progression-free or overall survival over R-Maint in rituximab naïve patients. Consequently R-Maint may be considered a cost-saving treatment option for such patients in the treatment of relapsed/refractory follicular lymphoma patients compared to ASCT.
Mouse Hematopoietic Stem Cells, Unlike Human and Mouse Embryonic Stem Cells, Exhibit Checkpoint-Apoptosis Coupling.
Authors: Rohrabaugh Sara , Mantel Charlie , Broxmeyer Hal E.
Published: October 2019

Cell cycle checkpoints guarantee that cells move through the events of the cell cycle in the appropriate manner. The mitotic spindle checkpoint, also known as the spindle assembly checkpoint (SAC), helps to ensure the proper segregation of chromosomes into daughter cells during mitosis. Our lab recently reported on the condition of the SAC in both mouse and human embryonic stem cells (ESCs). We found that ESCs do not initiate apoptosis when the SAC is activated, which allowed these cells to tolerate a polyploid state resulting from the aberrant mitosis (Mantel et al. Blood.109: 4518–4527. 2007). These results lead us to conclude that the spindle checkpoint is uncoupled from apoptosis in ESCs. Knowing whether adult tissue specific stem/progenitor cells, such as hematopoietic stem cells (HSCs), have checkpoints which are uncoupled from apoptosis is extremely important information. If HSCs were to manifest such checkpoint uncoupling as that which we defined for ESCs, this might present a problem for the ex-vivo expansion and transplantation of HSCs. Using multiparametric permeablized cell flow cytometric analysis, we found the mitotic spindle checkpoint to be functional in primary murine sca 1+/c-kit+/lin- cells (LSK cells), a population highly enriched in primitive hematopoietic stem/progenitor cells. Using nocodazole, which exerts its affect by depolymerizing microtubules, we were able to activate the spindle checkpoint in low density mononuclear cells collected from murine bone marrow. Through flow cytometric analysis of the LSK cells in the mononuclear fraction, we were able to determine that spindle checkpoint activation in LSK cells resulted in a cell cycle arrest in mitosis, which was determined by DNA content of the cells, and eventually this arrest lead to cell death via apoptosis, as indicated by caspase-3 activation. This behavior is unlike that of ESCs, which exit mitosis and become polyploidy after prolonged spindle checkpoint activation. Thus the mitotic spindle checkpoint appears to be coupled to apoptosis in this particular set of tissue specific stem/progenitor cells, which lessens the possibility that ex-vivo expansion of hematopoietic stem cells will result in abnormalities to these cells that may give rise to disease initiation or progression after their transplantation.
Proline-Rich Tyrosine Kinase-2 (PYK2): A New Target for the Treatment of Hematopoietic Neoplasms with TEL-FGFR3 Fusion or FGFR3 Active Mutation.
Authors: Okamura Daisuke , Yagasaki Fumiharu , Maeda Tomoya , Ishikawa Maho , Jinnai Itsuro , Bessho Masami
Published: October 2019

Constitutive activation of Fibroblast Growth Factor 3 (FGFR3) tyrosine kinase have been identified in various human cancers and have been reported to play an important role in some hematopoietic neoplasms. We have previously reported that TEL-FGFR3 in a patient with peripheral T-cell Lymphoma and AML conferred IL-3 independency to Ba/F3 cells and activates PLCγ, PK3K, STAT3, STAT5, MAPK through its constitutive tyrosine kinase activity in TEL-FGFR3 transfected Ba/F3 cells (TF-V5). In KMS-11, human multiple myeloma cell line which expresses constitutively active mutant FGFR3, activations of PI3K and STAT3 pathways have been reported. However, little is known about how FGFR3 tyrosine kinase (TK) activates these downstream molecules. Here, we show that PYK2, a member of focal adhesion kinases, plays a pivotal role for the activation of PI3K, STAT3 and STAT5 in FGFR3 oncogenic pathways, and is a candidate for therapeutic target. PP1/PP2, a kinase inhibitor of SRC and PYK2, inhibited the cell growth of TF-V5 and KMS-11 cells in a dose-dependent manner (IC50=15μM, 25μM respectively), not affecting the cell growth of IL-3 dependent Ba/F3 cells. Another specific SRC inhibitor did not affect the cell growth of TF-V5 and KMS-11 cells. TEL-FGFR3 transfection to Ba/F3 cells led to the overexpression of PYK2 but not FAK. Expression and phosphorylation of PYK2 were identified in KMS-11 cells. Immunoprecipitation analysis using FGFR3 TK inhibitor SU5402 showed that the activation of PYK2 which was recruited to FGFR3 was dependent on the kinase activity of FGFR3. The cell growth of TF-V5 was completely inhibited at the concentration of PP1/PP2(30μM), which inhibited auto-phosphorylation of PYK2. PP1/PP2 suppressed the activation of PI3K-ATK pathway and decreased expression of C-MYC, inducing G1-arrest of TF-V5. PP1/PP2 induced intrinsic apoptosis of TF-V5 and did not affect activation of BAX but decrease expression of BCL-2 and BCL-XL through inactivation of STAT3 and STAT5. PP1/PP2 also inhibited the activation of PI3K and STAT3 in KMS-11 cells, inducing G1-arrest and apoptosis. PP1/PP2 inhibited tyrosine kinase of PYK2 mesured by in vitro kinase assay (IC50=23μM, 13μM, respectively). Further PYK2 C-terminus Associated Protein (PAP) siRNA expression plasmid significantly decreased the proliferation of TF-V5 but not mock transfected Ba/F3 cells. Our data demonstrates that PYK2 is an attractive molecular target for FGFR3 associated hematopoietic neoplasm.
Pivotal Role of FoxO3 and mTORC1 in the Opposite Effects of SDF-1 and TGF-β on Cell Cycle Progression in CD34+ Stem/Progenitor Cells.
Authors: Chabanon Aurelie , Desterke Christophe , Rodenburger Emilie , Clay Denis , Guerton Bernadette , Boutin Laetitia , Pierre-Louis Olivier , Capron Claude , Bennaceur-Griscelli Annelise , Uzan Georges , Abecassis Lucile , Bourgeade Marie-Francoise , Lataillade Jean-Jacques , Le Bousse-Kerdiles Marie-Caroline
Published: October 2019

Control of cell cycling is a key process in stem cell fate. A balance between cell quiescence and proliferation within the stem cell niche in interaction with microenvironment is critical for sustaining long-term hematopoiesis and for protection against stress. Among growth factors regulating haematopoietic stem/progenitor cell (HSC/HP) cell cycling, TGF-β is known to be a major negative regulator by blocking cell cycle progression during the G1 phase. Besides its effect on HSC/PH and lymphocyte trafficking, the SDF-1/CXCL12 chemokine is another key regulator which helps maintaining hematopoiesis homeostasis. We have previous demonstrated that SDF-1 acts as a survival and cell cycle promoting factor for CD34+ HSC/HP by triggering the G0-G1 transition and then regulating early cell cycle phases suggesting that it may act as an antagonist of TGF-β on cell cycle progression (Lataillade et al., Blood 2000 and 2002). The general aim of this study was to investigate the mechanisms of this antagonist effect on cell cycle. This work was performed on CD34+ cells purified from the peripheral blood of healthy un-mobilized donors. Since they are mainly in G0, they allow dissecting the early cell cycle phases including the G0-G1 transition. We demonstrated that SDF-1 and TGF-β exert an opposite effect on the expression of cell cycle key regulators such as cyclins and CDKI. We showed that cross-talk between SDF-1 and TGF-β signaling pathways involving PI3K/Akt phosphorylation participates in the control of CD34+ cell cycling. We demonstrated a pivotal role of FoxO3 and mTOR in the TGF-β/SDF-1 control of the quiescence/cycling of hematopoietic progenitors. A model integrating a pivotal role for the activation of two Akt substrates, FoxO3 and mTORC1, in the control of CD34+ cells quiescence/cycling by TGF-β and SDF-1 is proposed. Altogether, our results shed new light on the intracellular signaling mechanisms of SDF-1 and of its implication, together with TGF-β, in cell cycle promotion in primary CD34+ cells. Regarding their opposite effect on cell cycle and survival, we suggest that SDF-1 and TGF-β can be considered as natural mutual competitors, both acting as organizers of the stem cell niche where a balance between HSC hibernation and activation is an essential part of the stemness control.
Caspase-3 Activation Is Involved in the Early Megakaryocyte Differentiation, but Not in Platelet Production.
Authors: Kozuma Yukinori , Yuki Satoshi , Suzuki Hidenori , Kojima Hiroshi
Published: October 2019

Megakaryocytes (Mgk) differentiate from hematopoietic stem cells through the complex process of endomitosis and cytoplasmic development, and finally form proplatelets in the terminal stage of the differentiation. To elucidate the role of a proapoptotic effecter protein, caspase-3, in megakaryopoiesis and thrombopiesis, we analyzed the phenotype of Mgk in vav-Bcl-2 transgenic (Tg) mice, which overexpress Bcl-2 exclusively in hematopoietic cells by the regulation of vav promotor. As was expected, Bcl-2-overexressing Mgk were remarkably resistant to caspase-3 activation induced by the depletion of thrombopoietin (TPO) from the culture. Percentages of proplatelet formation (PPF) assayed by culturing primary Mgk were slightly increased in the Tg mice. Platelet production from cultured Mgk, which was assayed by flowcytometry, was also slightly increased, indicating normal ability for Plt production from Bcl-2-overexpressing mature Mgk. To further analyze the ability for thrombopoiesis, we assayed the trends of Plt recovery following experimental thrombocytopenia. As Bcl-2-overexpressing mice had giant splenomegaly, we used splenectomized mice, which showed normal Plt counts. After the injection of 5-FU, we observed significant delay of the Plt recovery in the Tg mice, as well as the lack of the overshoot of Plt counts during the recovery phase. To gain insights about the mechanism underlying the delayed Plt recovery, we analyzeded the ploidy of DNA content. In the steady state, peak ploidy of Mgk obtained from the bone marrow of the Tg mice shifted to 2–4N. In a sharp contrast, in Plt recovery phase after 5-FU injection, peak ploidy shifted to 16–32N, and the percentages of Mgk possessing less than 16N ploidy were dramatically decreased. Consistent with this finding, we observed the delay of PPF in Bcl-2-overexpressing cells when Mgk were derived from c-kit+/lineage- progenitor cells in the presence of TPO. These data indicate that differentiation of immature Mgk into mature Plt-producing stage is significantly delayed in the Tg mice, but once the maturation proceeds, Mgk normally produce Plt. Taken together, we conclude that caspase-3 activation is involved in the early stage of the differentiation of Mgk, but not in Plt production.
Selected Members of the Angiopoietin-Like Family of Human Proteins Enhance Survival and Replating Capacity of Immature Human Hematopoietic Progenitor Cells.
Authors: Broxmeyer Hal E. , Srour Edward F. , Cooper Scott , Wallace Carrie T. , Hangoc Giao , Youn Byung
Published: October 2019

Angiopoietin-like (ANGPTL) molecules are a family of secreted proteins which have characteristic structures of angiopoietins. This includes a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils (CC), a short linker peptide, and a globular fibrinogen-like domain (FLD). Zhang et. al. (Nat. Med., 12(2):240–245, 2006) reported that human ANGPTL-2, 3, 3CC, 5 and 7, but not ANGPTL4, enhanced ex-vivo expansion of highly enriched mouse bone marrow (BM) long term competitive repopulating hematopoietic stem cells in serum-free culture with SCF, TPO, IGF-2, and FGF-1. To the present, there have not been publications describing effects of human ANGPTL molecules on hematopoietic progenitor cells (HPC) or on human hematopoietic cells. Thus, we evaluated purified recombinant human ANGPTL-2CC, 3, 3CC, 3FLD, 4, 4CC, 5, 6 and 7 (AdipoGen, Inc, Seoul, Korea) for effects on proliferation and survival of HPC from human cord blood (CB). No endotoxin was detected in the ANGPTL molecule preparations (<0.1 EU/ug endotoxin per LAL method). None of the ANGPTL molecules at up to 500ng/ml stimulated HPC colony formation by themselves, or enhanced or inhibited HPC colony formation of low density (LD) or CD34+ human cord blood (CB) cells stimulated by GM-CSF, GM-CSF plus SCF, Epo plus SCF, or the combination of Epo, SCF, IL-3 and GM-CSF. However, ANGPTL-2CC, 3, and 3CC at 200 and 100, but not 10ng/ml significantly enhanced the survival of human LD and CD34+ HPC (CFU-GM, BFU-E, CFU-GEMM) subjected to delayed addition of growth factors (Epo, SCF, IL-3, GM-CSF). Survival is a measure of anti-apoptosis for the hematopoietic progenitor cells in this context. The other ANGPTL molecules were not active at up to 500ng/ml. The survival enhancing effects of ANGPTL-3 was neutralized by purified rabbit anti-ANGPTL-3 IgG, but not by anti-ANGPTL-4, -6, or -7. Replating of HPC colonies offers an estimate of the self-renewal capabilities of HPC. We found that ANGPTL-3, but not -4, -6, or -7 enhanced the replating capacity of single CFU-GEMM colonies by greater than 2 fold. Thus far, we have not detected significant effects of the ANGPTL molecules on ex-vivo expansion of human CB CD34+ cells, alone, or in combination with SCF, TPO, Flt3-ligand, with or without IL-3, after assessing output of HPC, % and numbers of CD34+ cells, or cell cycle status of produced cells. In summary, we have implicated a few members of the ANGPTL family of proteins in functional effects on human HPC survival and replating/”self-renewal” activity, effects requiring the CC domain of the ANGPTL molecules. This information may be of relevance to regulation of HPC, and of use for protocols to use these cells for transplantation.
Prospective Evaluation of the Prevalence of Elevated Tricuspid Regurgitant Jet Velocity and Associated Clinical and Echocardiographic Factors in Children and Adolescents with Sickle Cell Disease.
Authors: Campbell Andrew D. , Minniti Caterina , Sable Craig , Ensing Greg , Dham Niti , Rana Sohail , Darbari D. , Onyekwere Onyinye , Kato Greg J. , Gladwin Mark T. , Castro Oswaldo , Gordeuk Victor
Published: October 2019

Background: Echocardiography (ECHO)-determined tricuspid regurgitation jet velocity (TRV) ≥2.5 m/s occurs in about 30% of adults with sickle cell disease(SCD)and is associated with increased mortality. The frequency and significance of high TRV in children is not known. We determined the prevalence and risk factors of elevated estimated systolic pulmonary artery pressures in 228 patients with SCD and 39 controls.

Methods: Patients with SCD (Hb SS, SC, SB thalassemia or other major sickling phenotypes) aged 3 to 20 years were evaluated clinically and with ECHO and six minute walk tests at their steady state. Healthy, non-SCD controls, matched for ethnicity, sex and age, were also evaluated.

Results: A comparison of patients and controls is summarized in Table 1. 184 patients (81%) had hemoglobin SS phenotype and 28 (12%) had hemoglobin SC. TRV was measurable in 214 (94%) of the patients and 34 (87%) of the controls. In a multivariate logistic regression model, each 10 mm Hg increase in systolic blood pressure was associated with an estimated 2.0-fold increase in the odds of TRV≥2.5 m/sec (P = 0.002) and a 10-fold increase in LDH with a 25-fold increase in the odds (P = 0.015).

Conclusion: This study of children with SCD who have limited iron overload and limited renal dysfunction compared with the aging SCD population provides a unique window into the early pathogenesis of PH in SCD. TRV and LVMI are significantly higher in children and adolescents with SCD at steady state than age-matched control participants. Among children with SCD, TRV elevation is independently associated with systolic BP and LDH concentration. In this analysis we find a strong association with hemolysis and systemic hypertension and surprisingly no associations with vaso-occlusive events or biomarkers (VOC, ACS, HU use, WBC and platelet counts).

Table I. Comparison of SCD and control subjects. Results in mean (SD) unless otherwise stated. SCD(n=228) Control (n=39) p Age in years 12 (5) 12 (5) 0.7 Female (%) 48% 46% 0.8 Systolic BP (mm Hg) 112 (11) 116 (15) 0.1 Diastolic BP (mm Hg) 64 (9) 68 (10) 0.040 Six minute walk in meters 454 (76) 498 (84) 0.006 TRV in m/sec 2.3 (0.3) 2.1 (0.3) 0.003 TRV 2.5 m/sec (%) 21% 3% 0.012 LV intern. diastolic diam Z-score 1.2(1.4) −0.3(1.2) <0.001 LV mass index 88 (25) 59 (13) <0.001 Table II. Comparison of SCD patients according to TRV category. Results in mean (SD) unless otherwise stated. Variables TRV <2.5 (n= 169) TRV ≥2.5 (n=45) p History of acute chest syndrome (%) 20% 40% 0.034 Hydroxyurea therapy 42% 44% 0.8 Hemoglobin in g/dl 9.3 (1.8) 8.9 (1.7) 0.071 LDH in U/L 393 (163) 474 (170) 0.009 Total Bilirubin in mg/dL 2.7 (1.9) 3.8 (2.2) 0.004
Variation in the Small Guanosine Triphosphate (GTP)-Binding Protein, Secretion-Associated and RAS-Related (SAR1A) Gene and Response to Hydroxyurea Treatment in Sickle Cell Disease.
Authors: Kumkhaek Chutima , Zhu Jianqiong , Taylor James G. , Hoppe Carolyn , Kato Gregory J. , Rodgers Griffin P.
Published: October 2019

Hydroxyurea (HU) is a well-known chemotherapeutic agent and a potent ribonuclease reductase inhibitor that induces Hb F synthesis in sickle cell disease and some thalassemia syndromes. Our group has shown that the HU-inducible small guanosine triphosphate (GTP)-binding protein, secretion-associated and RAS-related (SAR) protein plays a role in g-globin gene induction and erythroid cell maturation by causing cell apoptosis and G1/S-phase arrest through the reduction of PI3 kinase and ERK phosphorylation and increased p21 and GATA2 expression. Our preliminary analysis indicates that HU inducibility is mediated at the transcriptional level, and is localized to elements in the SAR1A promoter. The aim of this study was to assess whether polymorphisms in the SAR1A gene promoter are associated with Hb F levels or HU therapeutic responses. We studied 386 sickle cell disease patients consisting of 269 adults treated with or without HU and 117 newborns with sickle cell disease identified from a newborn screening program. Twenty point mutations, including one nonsynonymous variant, were identified in SAR1A, including nine previously reported in SNP databases. A difference in genotype frequencies was observed between adults and newborns for rs2310991 in the 5′UTR (odd ratio [OR] = 1.9, 95% confidence interval [CI] = 1.1–3.2, P=0.009) and +31 T>C in 5′UTR (odd ratio [OR] 9.8 [ 1.3–73.9]; confidence interval [CI] 95%; P<0.001). Three previously unknown SNPs in the upstream 5′UTR (−809 C>T, −502 G>T and −385 C>A) were significantly associated with the HbF response in Hb SS patients treated with HU (P<0.05). Our data suggest that the SAR1A polymorphism might contribute to the regulation of HbF expression and modulate patient responses to HU in sickle cell disease.
Efficacy of High-Dose Sequential Chemotherapy and Autograft for Transformed Non Hodgkin’s B Cell Lymphoma: Results of a Retrospective Analysis from GITIL (Gruppo Italiano Terapie Innovative nei Linfomi).
Authors: Di Nicola Massimo , Devizzi Liliana , Rambaldi Alessandro , Zanni Manuela , Benedetti Fabio , Patti Caterina , Barbui Tiziano , Tarella Corrado , Gianni Alessandro M.
Published: October 2019

Background - Follicular lymphoma (FL) transforms in a more aggressive lymphoma in 25–60% of patients representing the outgrowth of a more malignant subclone. Transformation is usually associated to a rapidly progressive clinical course, refractoriness to treatment, and short survival. To define the impact of high dose sequential (HDS) therapy and peripheral blood stem cell (PBSC) autograft on outcome of transformed FL (TFL), we analyzed a consecutive series of 66 pts with a confirmed diagnosis of TFL registered at the GITIL centers from March 1988 to September 2004 and treated with the HDS regimen.

Methods - Biopsy-proven histological transformation (HT) in diffuse large B cell lymphoma (DLBCL) was observed at diagnosis (n=24; 36%) or at relapse after a treatment for FL (n= 42; 64%). Main patient characteristics were as follows: male/female 36/30; median age 51 yrs (range 33–66), stage I-II/III-IV 8/58, IPI score 0–1/≥2 28/38. HDS regimen included: i. 3 APO or 3 DHAP courses (for patients relapsed after anthracycline-containing regimens); ii. sequential administration of hd-CTX (7g/mq), hd-Ara-C, (2g/mq q12h for 6 days) hd-Etoposide (2.4 g/mq), with PBSC harvests following hd-CY and hd-Ara-C; iii. myeloablative regimen with hd-Mitoxantrone/L-Pam (n=28) or BEAM (n=28 pts who could not receive additional anthracycline), or TBI-PAM (n=3); iv. PBSC autograft; v. consolidation radiotherapy on bulky disease. From January 1999, hd-CTX and hd-Ara-C has been supplemented with Rituximab (RHDS; n=34) with in-vivo purging intent.

Results - Overall 59 patients achieved a complete remission (CR; 89%), 1 patient responded partially and underwent allogeniec bone marrow transplantation, 6 patients died for progressive disease while on therapy (PD; 9%). With a median follow-up of 67 months (range 23–170), 42 patients are alive (63.6%), 24 patients relapsed and died for progressive disease (n= 23) or toxicity (n= 1). Five-year event free survival (EFS) and overall survival (OS) are 53.0% and 63.6%, respectively. No significant differences in OS and EFS were observed between patients with HT at diagnosis or at relapse, with IPI O-1 vs IPI >2. Of note, pts treated with R-HDS showed an improved clinical outcome (OS: 76% vs. 50%; EFS: 67.6 vs. 37.5 respectively), with a large difference that did not reach statistical significance because of the limited number of patients.

Conclusion - Our data strongly suggest that HDS regimen, in particular when supplemented with rituximab (R-HDS), is a very effective regimen in transformed B cell lymphoma.
Effects of Steroid Treatment on Childhood ALL Stem Cells.
Authors: Cox Charlotte V. , Diamanti Paraskevi , Kearns Pamela R. , Blair Allison
Published: October 2019

Several lines of evidence indicate a central role for stem cells in the pathogenesis of human leukaemias and exemplify the need to develop strategies that target this sub-population of cells. It is proposed that these cells may exhibit different chemo-sensitivity and consequently may be resistant to drug regimens designed to kill the bulk leukaemia population. Inherently resistant leukaemia stem cells may contribute to subsequent disease relapse. Clearly, there is a need to assess the relative efficacy of therapeutic agents on the sub-populations of cells in addition to the bulk leukaemia. We have previously demonstrated that the sub-population of childhood acute lymphoblastic leukaemia (ALL) cells, capable of serially engrafting NOD/SCID mice, have a CD34+/CD19− or CD34+/CD7− phenotype in B cell precursor (BCP) ALL and T-ALL, respectively. In this investigation we have compared the efficacy of a current glucocorticoid therapeutic agent on these putative ALL stem cells with their effects on the bulk leukaemia population. The effect of dexamethasone (dex), a key component of the treatment of childhood ALL, on primary ALL cells from 13 paediatric cases was examined. Unsorted ALL cells were co-cultured with and without dex for 48 hours. Subsequently, cell viability and apoptosis were evaluated by flow cytometry using propidium iodide and annexin V staining, with Flow-Count fluorospheres to directly determine absolute cell counts. Primary cells from 11 patients with BCP ALL were sorted for expression of CD34/CD19 and cells from 2 T-ALL cases were sorted for expression of CD34/CD7. The unsorted cells and the sorted sub-fractions were co-cultured with increasing concentrations of dex (0.05 to 500 μM) to compare the relative chemosensitivity of the bulk and putative leukaemia stem cell populations. The unsorted leukaemia populations were completely refractory to dex with no significant difference in the levels of apoptosis observed or the absolute number of viable cells in the treated samples and in the untreated controls. Interestingly, when the sorted populations were assessed, an increase in the absolute numbers of viable CD34+/CD19− (1.2–9.7 fold, P<0.02) and CD34+/CD7− (2.6–5 fold, P<0.04) leukaemia cells were observed even at the highest steroid dose, compared to the respective untreated sub-fractions. The other leukaemic sub-fractions did not show a significant increase in the number of viable cells following dex exposure. These data show that 10 out of 11 drug treated primary leukaemia cells were resistant to dex. The putative CD34+/CD19− BCP ALL cells and CD34+/CD7− T-ALL cells showed a significantly enhanced proliferative potential when exposed to the drug, suggesting that it is these cells that may be responsible for disease relapse.
Effect of FMS-Like Tyrosine Kinase-3 Mutations on Prognosis in Acute Myeloid Leukemia: A Single Institution Experience between 2000 and 2007.
Authors: Ghanem Hady A. , Jones Dan , Kantarjian Hagop M. , Faderl Stefan , Estey Elihu H. , Pierce Sherry , O’Brien Susan , Cortes Jorge , Ravandi Farhad
Published: October 2019

Mutations of the FMS-like tyrosine kinase-3 gene (FLT3), including internal tandem duplication (ITD) of the juxtamembrane domain or activating point mutations of the second tyrosine kinase domain involving codon 835 or 836 are among the most common molecular abnormalities in acute myeloid leukemia (AML). A clear negative impact of FLT3 mutation on survival has been demonstrated in AML patients younger than 60 years; however, this negative effect on prognosis or survival has not been confirmed in older patients. We evaluated 661 patients with AML treated at MD Anderson Cancer center between 2000 and 2007 for FLT3 mutations including 281 patients ≤ 60 years and 380 patients > 60 years. Median age for the entire group was 65 years (range, 17 to 86). A FLT3 mutation was present in 124 patients (18.7%). Patients with FLT3 mutations had a significantly higher WBC (median, 16.5 vs. 4.2, p<0.001) and a higher percentage of bone marrow blasts (median, 62% V/S 40%, p<0.001) at initial presentation. These differences were equally seen in both sexes. Patients with FLT3 mutations were younger than those without mutations (median age 58 vs. 64, p=0.005). Patients with FLT3 mutations were less likely to have an antecedent hematological disease compared to those without mutations (p<0.001). FLT3 mutations were less frequent in patients with core binding factor leukemia [t(8;21), inv(16)]. FLT3 mutations were more common in patients with acute promyelocytic leukemia (APL) than other FAB subgroups (p<0.001). Induction death and complete remission rates were not affected by FLT3 status. When favorable prognosis subgroups [APL, t(8;21), inv(16)] were excluded, patients with FLT3 mutations had a significantly shorter overall survival; this difference was seen in patients younger than 60 (p=0.019), as well as in patients over 60 (p=0.015). We have further confirmed a clear negative impact of FLT3 mutations on survival in patients ≤ 60 years and have demonstrated a negative impact on survival even in patients older than 60 years. FLT3 inhibitors should be considered as a component of therapy in older patients with non-favorable AML and FLT3 mutation.
A JAK2-V617F Activating Mutation in Addition to C-KIT and FLT3 Mutations Is Associated with Clinical Outcome in Patients with t(8;21)(q22;q22;) Acute Myeloid Leukemia.
Authors: Nanri Tomoko , Iwanaga Eisaku , Matsuno Naofumi , Kawakita Toshiro , Suzushima Hitoshi , Kawano Fumio , Mitsuya Hiroaki , Asou Norio
Published: October 2019

AML1-MTG8 generated by t(8;21)(q22:q22) contributes to leukemic transformation but additional events are required for full leukemogenesis. Mutations in the receptor tyrosine kinase (RTK) including C-KIT and FLT3 genes appear to be the genetic events that cause acute myeloid leukemia (AML) harboring t(8;21) and are associated with unfavorable prognosis (Nanri et al. Leukemia19:1361;2005). On the other hand, the activating V617F mutation in the JAK2 cytoplasmic tyrosine kinase has been detected in a significant proportion of patients with myeloproliferative disorders. Although the same mutation has been identified in a small number of AML patients, it is likely that a relatively high incidence of JAK2-V617F mutation has been seen in t(8;21) AML patients. However, whether they are associated with other biological parameters and whether they influence clinical prognosis in patients with t(8;21) AML have not been determined. To clarify the biological and prognostic impact of the JAK2 mutation, we examined the clinical and prognostic relevance of the mutation. Of 45 patients with t(8;21) AML, mutations in the C-KIT and internal tandem duplications (ITD) in the FLT3 were observed in 18 (40%) and 3 (6.7%), respectively. We detected the JAK2-V617F mutation in 3 patients with t(8;21) AML (6.7%), which was consistent with previous studies. Although the occurrence of C-KIT and FLT3 mutations was mutually exclusive, one patient harboring JAK2 mutation also had a C-KIT second tyrosine kinase mutation and another one patient had a K-RAS mutation. Collectively, a total of 23 (51%) patients showed mutations in the RTK pathway. One patient carrying both C-KIT and JAK2 mutations did not respond to multiple induction chemotherapies. Another patient with the JAK2 and K-RAS mutations achieved a complete remission (CR) but later relapsed. Remaining one patient received allogeneic stem cell transplantation during first CR and continued CR. As we were limited to study a small number of mutated cases for a comparison of clinical and genetic features, we examined the clinical significance of C-KIT, FLT3 and JAK2 mutations as a collective group. A cumulative incidence of relapse (CIR) in 21 patients with RTK mutations was 77%, whereas CIR in 19 patients lacking mutations was 26% (P=0.0083). The 6-year relapse-free survival in patients with mutations was 20% compared to 54% in those without mutations (P=0.0595). These results suggest that the mutations in the JAK2, C-KIT and FLT3 could be an additional genetic events leading to the development of AML and are associated with the clinical outcome in patients with t(8;21) AML.
Reguatory T-Cell Subsets Demonstrate Quantitative Differences but Not Functional Abnormalities in Patients with Multiple Myeloma (MM) Compared with Healthy Controls.
Authors: Feyler Sylvia , von Lilienfeld-Toal Marie , Marles Lee , Rawstron Andy , Owen Roger G. , Ashcroft John , Cook Gordon
Published: October 2019

The immunologically hostile microenvironment of MM contributes to the limited success of immunotherapy strategies. In addition to direct tumour-induced immunosuppression, tumour cells may generate suppressor cells such as Treg cells which can profoundly suppress immune responses and induce tolerance. This study aimed to determine if Treg subsets are increased in the peripheral blood (PB) and bone marrow (BM) of patients with MGUS/MM and how this correlates with increasing disease burden. PB samples from 166 patients with MGUS/MM (Newly diagnosed (ND), n=34; Plateau/low disease burden (LD), n=63; Relapsed/refractory (R/R), n=27 and MGUS, n=42) with a median age of 69 years (range 39–89 yrs) were analysed by FACS and compared to PB from 32 age/sex matched controls. Using a sequential gating strategy, naturally occurring Treg cells (nTregs) were identified as CD4+/CD25+/FoxP3+ T-cells and expressed as a percentage of the CD4+ T-cells. Double negative T cells (DN Tregs) were identified as CD3+/CD4−/CD8−/alphabeta-TCR+/gammadelta−TCR− and expressed as a percentage of the CD3+ cells. Sera were analysed by ELISA for IL10 and TGF-beta. nTReg cells were significantly increased in patients with MGUS/MM compared with controls (Controls 1.6% ± 0.2, MGUS 2.3% ± 0.3, ND 2.4% ± 0.2, LD 4.2% ± 0.7 & R/R 3.8% ± 0.5; p=0.003). There was a positive correlation of nTRegs number with paraprotein level (R=0.3, p=0.005). Patients on Thalidomide at the time of sample collection had significantly higher numbers of nTRegs of 6% in comparison to patients who never received Thalidomide (3%) and patients who previously received the drug (3.5%, p=0.005). Analysis of BM (n=12) demonstrated a significantly reduced number of nTRegs in comparison to PB (1.6% vs 3.0%, p=0.025), which is higher than the number of nTRegs in the BM of controls (0.7%). Functionally, nTRegs from patients demonstrated suppressive activity of both autologous and allogeneic T-cells similar to nTRegs cells from control PB (p=NS). In contrast, DN TRegs were significantly reduced in patients with myeloma (ND 1.0% ± 0.2, LD 1.1% ± 0.1, R/R 1.3% ± 0.2) compared with MGUS and controls (2.1% ± 0.9 & 3.3% ± 0.7, respectively; p=0.02). Serum IL10 levels were significantly lower in ND (17 ± 38 pg/ml) and LD (29 ± 36 pg/ml) than in Controls (55 ± 89 pg/ml), MGUS (72 ± 151 pg/ml) and R/R (90 ±182 pg/ml, p=0.02). TGF-b levels differed significantly between groups (Controls 3765 ± 1593 pg/ml, MGUS 3506 ± 1504 pg/ml, ND 4774 ± 9879 pg/ml, LD 1915 ± 1324 pg/ml, R/R 2802 ± 2011 pg/ml, p=0.025). These results provide further evidence of immune dysregulation in MM. The association with advanced disease stage suggests a causal association.
Induction of UBC9 and the Sumoylation Pathway in Multiple Myeloma Promotes Plasma Cell Growth and Correlates with Decreased Patient Survival.
Authors: Driscoll James J. , Lefkimmiatis Konstantinos , Fulciniti Mariateresa , Prabhala Rao , Pelluru Dheeraj , Greipp Philip R. , Munshi Nikhil C.
Published: October 2019

The pursuit of rationale, targeted therapies relies on a detailed understanding of the mechanisms that subvert normal growth control and lead to development of Multiple Myeloma (MM). To further define the mechanistic steps that contribute to MM pathogenesis, we examined mRNA expression profiles of CD138+ plasma cells obtained from normal, Monoclonal Gammopathy of Unknown Significance (MGUS), and MM patient samples. Using genomic results in combination with molecular and cellular-based assays, we demonstrate a critical role for UBC9 and the sumoylation pathway in myeloma cell growth and survival. Notably, Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) demonstrated a ten-fold induction in UBC9; a gene that encodes the sole Sumo-conjugating enzyme in human cells. We demonstrated an elevation of UBC9 in both MM primary patient cells and in a number of patient-derived MM cell lines. In addition, a number of other sumoylation pathway components were induced in primary MM cells at the gene and protein level relative to normal plasma cells. We believe that induction of UBC9 is an early genetic event in the pathogenesis of MM since the induction was observed at the gene and protein level in plasma cells from patients with MGUS. Importantly, UBC9 induction was functionally significant since a different pattern of sumoylation was observed in total cell lysate from MM patient plasma cells relative to that of normal plasma cells. Furthermore, overexpresion of a mutant form of UBC9 deficient in Sumo-conjugating activity increased the sensitivity of plasma cells to apoptosis by chemotherapeutic agents and these cells were impaired in other essential functions that included cellular proliferation, DNA synthesis, resistance to apoptosis and adhesion to bone marrow stroma. Immunoblotting of MM patient cell lysates also demonstrated a similar induction of the UBC9 gene product (Ubc9) as well as induction of the Sumo ligases Nse2 and PIAS1. These studies identify UBC9 as a target upregulated early in the pathogenesis of MM and indicate a critical role for sumoylation in disrupting the controls that govern normal plasma cell growth. To further develop prognostically relevant molecular signatures and classifications of MM subtypes, we analyzed the survival outcome of patients that expressed induced levels of UBC9, as well as other sumoylation components, and demonstrate significantly reduced survival of such patients following current treatment modalities. The results provide evidence for critical role of UBC9 and sumoylation in MM pathogenesis. Furthermore, sumoylation pattern may serve as a therapeutic target in MM, help stratify clinical management and provide a framework for the identification of sumoylation pathway targets that govern MM cell growth and progression.
Kinetics of Thrombin Generation in Patients with Multiple Myeloma.
Authors: Petropoulou Anna D. , Gerotziafas Grigoris T. , Zervas Kostas , Mpanti A. , Samama Michel Meyer , Rendu Francine
Published: October 2019

Thalidomide has emerged as a promising treatment for multiple myeloma (MM). Thrombosis is the most serious complication of thalidomide therapy, essentially when it is combined with dexamethasone. The pathogenesis of thrombosis in MM patients (pts) treated with thalidomide is not clear and probably of multi factorial origin. We used the Thrombin Generation test (TGT) and measured the plasma levels of soluble thrombomodulin (sTM) to better clarify the MM-related and thalidomide-related thrombogenicity. TGT was performed in citrated frozen platelet poor plasma (PPP). Blood was obtained from 26 MM pts, Salmon and Durie stage II and III, 62.5 years old (42–77), 9 males and 17 females, 10 treated with thalidomide (100–200mg/d orally) and dexamethasone (40mg/d for 4 days) (TD group) and 16 receiving no treatment (MM group). 13 healthy volunteers formed the control group. Thrombin Generation (TG) was initiated by adding the PPP reagent (Thrombogram-Thrombinoscope®) and the triggering solution (CaCl2 and fluorogenic substrate). We analyzed the endogenous thrombin potential (ETP), the Cmax and the velocity index of TG. The plasma levels of sTM in PPP were measured by a specific ELISA (Diagnostica Stago, France). In the MM group we observed an increase of the ETP, though not significant compared to the controls. The Cmax was almost equal to the control group value, while the velocity index of TG was statistically lower in the MM group compared to controls. In the TD group, a statistically significant increase of ETP was observed as compared to the control group. The Cmax was higher, compared to controls, though not significantly, whereas the velocity index of TG was almost equal to the control group value. There was no significant difference in the TG parameters between MM and TD groups. sTM in the control group was 45±14ng/ml. Both groups of pts had significantly increased sTM plasma levels as compared to the control but the difference between the two groups did not reach significance. Results are shown in Table 1. In patients with MM coexists an increase of sTM, a marker of endothelial cell damage, together with an increased TG capacity. The addition of thalidomide treatment is associated with a slight but not significant increase of ETP and Cmax. The co-existence of endothelial cell damage with increased TG capacity could be associated to the increased thrombotic risk in MM patients treated with thalidomide. This hypothesis will be controlled in a prospective study.

Table 1: Thrombogram parameters and sTM plasma levels of studied pts. Control MM group TD group * Results significantly different between the MM and TD groups and the control group (p<0.05 vs the control group) ETP (nM×min) 1399±297 1651±478 1747±448* Cmax (nM) 366±54 342±52 402±99 Velocity Index (nM/min) 198±45 160±19* 184±65 STM (ng/ml) 45±14 84±42* 73±30*
Use of Palifermin for the Prevention of High-Dose Methotrexate-Induced Oral Mucositis.
Authors: Schmidt Eva , Thoennissen Nils H. , Rudat Annika , Bieker Ralf , Schliemann Christoph , Mesters Rolf M. , Zühlsdorf Michael , Müller-Tidow Carsten , Berdel Wolfgang E.
Published: October 2019

Oral mucositis is a frequent problem in high-dose methotrexate (HD-MTX) based chemotherapy, impairing the patient’s quality of life, leading to higher rates of infections and delaying subsequent chemotherapy. Numerous substances have been used to prevent or treat oral mucositis, but none of them has proven clinical benefit. Palifermin, a recombinant human keratinocyte growth factor, can lead to a prevention of oral mucositis by different effects. Clinical activity of palifermin has been proven in a controlled randomized study with patients undergoing high-dose therapy with autologous stem cell rescue. The purpose of this report is to describe the effect of palifermin in patients treated within the GMALL-B-ALL 2002 protocol containing HD-MTX who developed a severe mucositis in cycles A1 or B1. Ten patients who were treated within this protocol developed a severe WHO grade III–IV oral mucositis in cycles A1 or B1. Before and after the subsequent similar or identical cycles A2 or B2 palifermin was given to reduce the risk of mucositis. Thus, patients serve as their own control for efficacy of palifermin. All ten patients developed a grade III–IV mucositis in cycles A1 or B1 without palifermin, whereas only 2/10 developed a grade III–IV mucositis in corresponding cycles A2 or B2 with palifermin (Mann-Whitney-Wilcoxon-test p < 0.05). Five patients were treated with a lower palifermin dose than recommended. Also these patients did not develop a higher grade mucositis. Only 4/10 patients showed infections in the cycles with palifermin compared to 10/10 patients without palifermin. The duration of mucositits in patients who aquired a higher-grade (III, IV) mucositis despite treatment with palifermin could be reduced from 12.9 days (median) without to 10.4 days with palifermin. In conclusion, palifermin can significantly reduce the incidence and severeness of oral mucositis and may influence clinical sequelae such as infection and quality of life.
Rituximab-EPOCH, an Efective Front-Line Therapy for Mantle Cell Lymphoma.
Authors: Palacios Andres , Lopez Andres , Salar Antonio , Cervera Marta , Gironella Merche
Published: October 2019

Introduction: Mantle-cell lymphoma accounts for 3–10% of non-Hodgkin’s lymphomas, with a median survival not exceeding of 3–4 years and its remains incurable with conventional therapy. CHOP plus Rituximab can induce a molecular complete response in 36% of patients. More aggressive combinations, as Hyper CVAD achieved an overall response rate of 97% with 87 of complete response, data no further confirmed in other studies in which Hyper-CVAD together with Rituximab achieve an overall response rate of 62.5%, with 33% of complete responses (CR). Being toxicity high mainly in elderly patients. Infusional chemotherapy combinations have shown efficacy in mantle-cell lymphoma (as VAD). Based this premise and in the efficacy of infusional R-EPOCH in aggressive lymphomas (DLBCL and PMBCL) we have conducted a compassionate prospective study of non-adjusted infusional EPOCH-R in patients with mantle -cell lymphoma as first-line therapy.

Aim: To evaluate the clinical activity and toxicity of non-escalated infusional EPOCH-R as upfront therapy in patients newly diagnosed of mantle-cell lymphoma.

Patients and methods: Herein, 12 patients of an ongoing compassionate prospective study in newly diagnosed patients with mantle-cell lymphoma are reported. EPOCH-R consisted on Rituximab 375 mg/m2 day 1, vincristine 0.4 mg plus doxorubicine 40 mg/m2 plus etoposide 50 mg/m2 days 1 to 4 in four day continuous infusion, cyclophsphamide 750 mg/m2 day 5, and prednisone 60 mg/m2 for 5 days, repeated every 21 days if feasible for 6 cycles. The median age of 65 yrs (range, 49–76). 50% of patients were males. 91% of patients presented with an Ann Arbor stage III–IV, high LDH in 50% of cases, leukemic status in 66.6%, Bone Marrow involvement in 66.6% and ECOG <2 was present in 91% of the cases.

Results: The response rate to EPOCH-R was 100% with 91% complete response (11 out of 12 patients). Neutropenia grade III–IV was observed in 16% of cases and anemia grade III–IV in 16% of cases. One case of neutropenic fever and two cases of grade III diarrhoea.

Conclusions: These preliminary results suggest that EPOCH-R is an effective as other more aggressive combinations and probably with less toxicity profile. More experience and longer follow-up is warranted to confirm this initial appealing experience.