bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Expression Profiles as Prognostic Factors in Patients with Multiple Myeloma Treated with Conventional Versus Novel Agents in Correlation with Outcome.
Authors: Broyl Annemiek , Hose Dirk , de Knegt Yvonne , Lokhorst Henk , Goldschmidt Hartmut , van Duin Mark , Sonneveld Pieter
Published: October 2019

Background: Bortezomib (Bor), a proteasome inhibitor, and Thalidomide (Thal), an anti-angiogenic and immunomodulatory drug, have a remarkable effect in patients with relapsed or refractory MM with 30–40% response rates. In newly diagnosed patients the response rates vary from 70–85%. However, 15–30% of newly diagnosed patients do not respond to Bor or Thal. Secondly, 30% of the patients treated with these novel agents have to stop prematurely because of intolerable side effects, such as polyneuropathy, thrombocytopenia, thrombosis and gastro-intestinal symptoms.

Aim: To gain new insights regarding the mechanisms of drug response and toxicity associated with these agents, we have embarked on a prospective study to analyze gene expression profiles (GEP) of myeloma specific genes in plasma cells purified from bone marrow from myeloma patients at diagnosis who have been treated with these novel agents in order to learn which genes govern outcome upon treatment with Bor and Thal.

Methods: GEP of CD138 magnetic cell selected (MACS) myeloma plasma cells was performed using Affymetrix GeneChip Human Genome U133 plus 2.0 arrays. This program has been initiated in a large multicenter, prospective, randomized phase III trial, comparing Bor in combination with Adriamycin, Dex (PAD, arm A) followed by high dose therapy with stem cell rescue and maintenance therapy with Bor vs. Vincristine, Adriamycin and Dex (VAD, arm B) followed by high dose therapy with stem cell rescue and maintenance therapy with Thalidomide (HOVON65/GMMG-HD4). This cooperative trial in the Netherlands and Germany has started in April 2005 and has included 600 patients. Gene arrays were analyzed using correlation analysis in Omniviz. Differentially expressed genes in the different groups and in Bor responders were determined using a t-test with adjusted p-value (p<0.0001) and a 1000 permutation analysis (BRB tool).

Results: Based on correlated GEPs we divided myeloma patients into 10 clusters. We determined differentially expressed genes in the different clusters and correlated the clusters with chromosomal aberrations like 13q loss, 9 and 11 gain and translocations. Three clusters showed high expression of MMSET/FGFR3, MAF downstream targets and CCND1, respectively.

Conclusion: unsupervised cluster analysis led to the subdivision of myeloma patients into clusters, each with a specific gene expression signature. We determined differentially expressed genes in the different clusters and could confirm 3 subgroups (MMSET/FGFR3, MAF/MAFB, and a group with high CCND1 expression) as have been published before.
Laboratory Response to Intranasal Desmopressin in Women with Menorrhagia and Underlying Platelet Dysfunction.
Authors: Rose Shelonitda S. , Faiz Ambarina , Miller Connie H. , Saidi Parvin , Philipp Claire S.
Published: October 2019

Intranasal desmopressin (IN-DDAVP) is used for home treatment of menorrhagia in women with inherited bleeding disorders. The effect of IN-DDAVP on laboratory hemostatic parameters in women with menorrhagia and associated platelet dysfunction is unknown. We evaluated the effects of IN-DDAVP on hemostatic parameters in women with menorrhagia and platelet dysfunction and correlated them with menstrual blood flow. Eleven women (aged 18–45) with menorrhagia and abnormal platelet aggregation and/or platelet adenosine tri-phosphate (ATP) release had determination of factor VIII coagulant activity (FVIII: C), von Willebrand factor antigen (VWF: Ag), von Willebrand factor ristocetin cofactor (VWF: RCo) activity, platelet aggregation, and platelet ATP release pre and 60 minutes post IN-DDAVP. Eight of eleven women underwent Platelet Function Analyzer (PFA-100) closure time determination with collagen/epinephrine (CEPI) and collagen/adenosine diphosphate (CADP) cartridge pre and post treatment. IN-DDAVP was administered during two consecutive menstrual cycles. Menstrual blood flow was assessed during each cycle using a pictorial blood assessment chart (PBAC). Following administration of IN-DDAVP there was a 2–3 fold increase in FVIII: C, VWF: Ag, and VWF: RCo observed in most patients over baseline levels. Mean FVIII:C increased from 130% ± 34.8% to 218% ± 96.5% (P < 0.007), VWF: Ag increased from 86.9% ± 35.1% to 128.7% ± 64.3% (P < 0.011), and VWF: RCo increased from 89.8% ± 42.8% to 139.6% ± 86.5% (P < 0.02). The median baseline PBAC score of 235 decreased to 136 following IN-DDAVP. In addition, there were significant inverse correlations between changes in PBAC score and changes in VWF: Ag (rs = − 0.85, p = 0.02), VWF: RCo (rs = −0.81, p = 0.029), and FVIII:C (rs = 0.92, p = 0.003) with IN-DDAVP. Mean PFA-100 closure time with the CEPI cartridge shortened from 166.8sec ± 85sec to 90.9sec ± 49.3sec (p< 0.02), and the closure time with the CADP cartridge shortened from 104.5sec ± 34.3sec to 64.8sec ± 40.4sec (p <0.007). There were significant inverse correlations between post IN-DDAVP PFA-100 CADP closure time and post IN-DDAVP FVIII:C (rs = −0.86, p = 0.005), VWF: Ag (rs = −0.72, p = 0.04), and VWF: RCo (rs = −0.80, p = 0.01). In addition there were also significant inverse correlations between post IN-DDAVP PFA-100 CEPI closure time and post IN-DDAVP FVIII:C (rs = −0.73, p = 0.03), and VWF: RCo (rs = −0.74, p = 0.03), but not VWF: Ag (rs = −0.66, p = 0.07). In-vitro platelet aggregation and platelet ATP release response did not correct and did not correlate with changes in menstrual blood flow. Our results demonstrate a correlation between VWF parameters and menstrual blood flow following IN-DDAVP in women with menorrhagia and underlying platelet dysfunction.
Proteins of the Calnexin Cycle Regulate αIIbβ3 Surface Expression in Human Megakaryocyte-Lineage Cells.
Authors: Yazdani-Abyaneh Mahmoud , Mitchell W. Beau
Published: October 2019

The αIIbβ3 integrin receptor mediates platelet aggregation, and individuals without functional αIIbβ3 manifest the mucocutaneous bleeding disorder Glanzmann thrombasthenia. Since platelets also participate in the pathophysiology of thrombosis, anti-αIIbβ3 agents have been successful in reducing mortality after percutaneous coronary interventions and from platelet-mediated coronary thrombosis. Accordingly, much research has focused on the structure, activation and signaling of the mature receptor on the cell surface. There is much to be gained medically and scientifically by understanding the intracellular processes by which integrins are assembled and expressed. Some forms of Glanzmann thrombasthenia result from mutations that disrupt normal integrin folding and assembly, causing the integrin subunits to be retained in the ER and degraded. These cases indicate the existence of a quality control mechanism for αIIbβ3 by which misfolded subunits are recognized, retained and degraded. Previous studies in HEK293 cells and murine megakaryocytes have identified the calnexin cycle as part of this quality control mechanism. We have expanded upon these findings and studied their functional implications by determining the effect of siRNA-induced knock down of proteins from six families of post-translational processing proteins on αIIbβ3 surface expression on human megakaryocyte-lineage cells derived from umbilical cord blood (UCB): lectins, glucosyltransferases, mannosidases, HSP90s, HSP70s, BiP, protein disulfide isomerases, and tetraspanins. We report that siRNA against two proteins in the calnexin cycle decreased surface expression of natively-folded αIIbβ3 on UCB-derived megakaryocytes as measured by binding of 10E5, an αIIbβ3 complex-dependent mAb. siRNA against UDP-glucose ceramide glucosyltransferase-like 1(UGGT, a glucosyltransferase) decreased binding by 36 +/− 9%, and siRNA against EDEM1 (a mannosidase) decreased binding by 15 +/− 14%, both p < 0.05. Additionally, siRNA against CD9 increased binding by 17 +/− 8%, p < 0.05, suggesting that CD9 may be a negative regulator of αIIbβ3 surface expression. UGGT is a glucosyltransferase protein and folding sensor of the calnexin cycle that recognizes and binds to glycoproteins that are in nearly-native folding conformations, but does not bind to fully folded glycoproteins. Glycoproteins that are glucosylated by UGGT may be bound by calnexin and retained in the ER, increasing their time for folding. Fully folded proteins cannot be reglucosylated by UGGT and escape the calnexin cycle, exiting the ER. EDEM1 is a mannosidase that interacts with calnexin to accept terminally misfolded proteins and direct them to degradation. Together, these data suggest a model in which the level of expression of native-folded αIIbβ3 on the cell surface is regulated in the ER by the calnexin cycle.
Cord Blood NK Cytotoxic Activity Can Be Selelctively Modulated by Accessory.
Authors: Xing Dongxia , Li Sufang , Simon Robinson N. , William Decker , Hong Yang , David Steiner , Michael Thomas , Champlin Richard E. , McMannis John D. , Vina Marcelo F. , Cooper Laurence J.N. , Simmons Paul J. , Zweidler-Mckay Patrick A. , Shpall Elizabeth J.
Published: October 2019

Introduction The expansion of cord blood (CB) derived natural killer (NK) cells is a promising cell therapy approach. CB NK cell expansion and activation has proven to be feasible and effective. However, the selectivity of NK killing in an allograft setting raises concern. Accessory cells, in particular dendritic cells and mesenchymal stem cells (MSCs), have been shown to interact with and modulate NK cell proliferation and function. We explored the ability of accessory cells to modulate NK activity in the setting of clinically-relevant CB NK expansion protocols.

Methods Human MSCs were isolated from normal bone marrow donors. 5 × 106 CD56-selected NK cells or 5 × 107 unselected or CD3-depleted CB mononuclear cells (MNC) (averaging 10% CD56+ NK cells) were layered on top of the MSCs in the presence of 200u/mL IL-2. CB NK cells were tested for cytolytic activity by 51Cr release assay using MSCs and K562 AML cells. IFN-g secretion was detected by Elispot assay.

Results Cord blood-derived NK cells activated by exposure to IL-2 efficiently kill allogeneic MSCs in vitro. A 4 hour chromium release assay revealed that CD56-selected CB NK cells lysed 60% of allogeneic MSCs at E:T ratio 10:1. MSC co-culture with CB NK induced IFN-g secretion as determined by IFN-γ Elispot assay. In contrast, unselected or CD3-depleted CB MNCs with equal numbers of CD56+ NK cells were unable to lyse MSCs despite IL-2 activation. This suggests that CB accessory cells can modulate the activation or cytolytic activity of CB-derived NK cells. To address these two possibilities, we tested the ability of these CB cultures to kill a classic NK target, K562 human myeloid leukemia cells. Surprisingly, in all three CB cultures NK efficiently lysed K562 cells, 95% of lysis at 10:1 E:T ratio by chromium release assay suggesting that these accessory cells did not prevent IL-2 activation of NK cells, but selectively-modulated the cytolytic activity. Thus, preserving anti-leukemia killing while protecting non-malignant MSCs.

Conclusions Our data provide evidence for selective modulation of cord blood-derived NK cell cytolytic activity by CB accessory cells. Indeed, activation of cord-derived NK cells in the presence on CD3-depleted accessory cells yields NK cells capable of killing K562 target cells but not allogeneic MSCs. Although the underlying mechanism is unknown, this selective modulation of NK cells may be exploited in the clinical expansion of cord blood NK cells.
Severe Donor Events after Allogeneic Hematopoietic Stem Cell Donation.
Authors: Halter Jörg , Kodera Yoshihisa , Ispizua Alvaro Urbano , Greinix Hildegard , Schmitz Norbert , Favre Geneviève , Baldomero Helen , Niederwieser Diegter , Apperley Jane , Gratwohl Alois
Published: October 2019

The risk for donors of allogeneic hematopoietic stem cells (HSC) by bone marrow (BM) or by peripheral blood (PB) harvest is generally considered negligible. Scattered reports of severe to life-threatening complications and a recent controversy on hematopoietic malignancies after GCSF administration for peripheral stem cell donation have challenged this opinion. Previous studies were limited by small numbers. In two consecutive retrospective surveys conducted in 2003 and 2006 amongst 338 allogeneic transplant centres from 38 European countries participating in the annual EBMT activity surveys, centres were asked to report all donor deaths, all severe adverse events (SAE’s), defined as occurring within 30 days and any hematological malignancy in a donor occurring after HSC donation. 262/338 teams (77.5%) responded to the first survey (1993–2002) and 169/262 (65%) centres replied to the second survey (2003–2005). The responding teams performed a total of 51’024 first allogeneic HSCT, 27’770 BM and 23’254 PB HSCT, which corresponds to 69% of all 73’947 first allogeneic HSCT reported during this time to EBMT. There were 5 donor deaths, 1 after BM and 4 after PB donation, an incidence of 0.98 per 10’000 donations (95% CI 0.32–2.29), 37 SAE’s (incidence 7.25/10’000 donations; 95% CI 5.11–9.99), 12 in BM (incidence 4.32/10000 donations; 95% CI 2.24–7.75) and 25 in PB donors (incidence 10.76/10’000 donations; 95% CI 6.97–15.85; p<0.02). In absolute numbers, there were 20 hematological malignancies occurring in donors (3.92/10’000 donations; 95% CI 2.39–6.05), 8 after BM (2.88/10’000 donations; 95% CI 1.24–5.68) and 12 after PB donation (5.16/10’000 donations; 95% CI 2.67–9.02; p = 0.3). Based on the different observation times, the incidence rates for developing hematological malignancies are 0.398 per 10’000 person-years for BM and 1.20 per 10’000 person-years for PB donation, resulting in a relative risk of 3.02 (95% CI 1.11–6.87, p=0.027). These data document a definitive risk for death, SAE’s and hematological malignancies with HSC donation. Deaths occur with similar frequency in both groups. SAE’s were more frequently reported after PB donation. The incidence rate for developing hematopoietic malignancies is higher after PB donation. These data clarify the recent controversy on HSC donation. They form a basis for donor counselling and underline the need for standardised donor follow up and international cooperation in order to define risk factors and to build up preventive measures.
Pegfilgrastim in Congenital Neutropenia: An Observational Survey from the French SCN Registry.
Authors: Beaupain B. , Leblanc T. , Reman O. , Vannier J.P. , Hermine O. , Suarez F. , Bertrand Y. , Bellannre-Chantelot C. , Donadieu Jean
Published: October 2019

PegFilgrastim is registered in France since 2002. Its pharmacokinetic characteristic allows the possibility to limit the number of injections in patients with congenital neutropenia receiving long term G-CSF therapy. To date, no report has been issued about the outcome of patients with congenital neutropenia receiving this drug. At the cut-off date of July 31th, 2007, 339 patients with congenital neutropenia had been included in the French Severe chronic neutropenia (Severe congenital neutropenia n=142, WHIM n=16, Cyclic n=75; GSDIb n=16 SDS n=90). Eight patients (SCN n=4, Cyclic n=3 and GSDIb n=1) had been identified to receive PegFilgrastim. Median age at PegFilgrastim onset was 18.2 years (min 6.1-max 37.6). In 7 cases, PegFilgrastim was given in the continuation of Filgrastim (n=3) or Lenograstim(4), with a median delay of 13 years after G-CSF onset and in one case, PegFilgrastim was the initial prescription of cytokine. Dose of PegFilgrastim was usually a vial per injection, except in one child (1/2vials) resulting in a dose per injection between 90 and 286 μg/kg per injection. The rhythm of PegFilgrastim injection varied from 1 injection every 7 days until 1 injection every 14 days, with discontinuation period. The median duration (between onset of PegFilgrastim and the last dose) was 0.7 year (min 0.3–2.7). The efficiency measured by ANC criteria appears to better than G-CSF (fig 1: baseline Median ANC 156/mm3, Under G-CSF 501/mm3, Under PegFilgrastim 1901/mm3/ Kruskall Wallis test p=0.02). Severe infection was never reported under PegFilgrastim, but neither in the previous G-CSF therapeutic periods for the 7 patients. More frequent ENT infection was observed in one patient who consider the PegFilgrastim as less efficient than G-CSF. The side effects appeared to be more frequent compare to the G-CSF therapy. Bone pains were reported in 7 patients, anemia and thrombocytopenia (WHO gr II) in one, urticarian chronic skin rash in 3. Finally, at the last update, 7 patients had withdrawn PegFilgrastim, and had resumed G-CSF therapy. Only one patient is still receiving PegFilgrastim. In conclusion, PegFilgrastim appeared efficient in Congenital neutropenia, but more frequent short term side effects were observed compare to standard G-CSF therapy, resulting in a limited quality of life improvement, leading to drug withdrawn in most of the patients.

Figure 1: Median ANC by diagnosis Figure 1:. Median ANC by diagnosis
Reducing MCM Loading Causes Chromosomal Aneuploidy.
Authors: Orr Stephen J. , Gaymes Terry , Wang Rong , Czepulkowski Barbara , Ladon Darius , Chronis Constantinos , Mufti Ghulam J. , Marcotte Edward M. , Thomas N. Shaun B.
Published: October 2019

Normal DNA replication must be accurate and occur only once per cell cycle. Sites of DNA replication are specified by binding the origin recognition complex, that includes minichromosome maintenance (MCM) proteins. Paradoxically, in higher eukaryotes MCM proteins are present in >20 fold excess of that required for DNA replication. They are also downregulated by elevated expression of proteins such as cyclin E that occurs in cancers, including AML and breast cancer. We investigated why human cells need “excess” MCM proteins and whether the reduction of MCM protein levels might contribute to a malignant phenotype. We determined the consequences of reducing the levels of MCM proteins in primary human T cells in which cell cycle controls and DNA damage responses are normal. Mass spectrometry sequencing of chromatin/nuclear matrix-bound proteins and western blotting identified that Mcm7 is not present in quiescent, normal primary human T cells. Mcm7 is induced in mid G1after the G0→G1 commitment point, the point beyond which T cells are committed to entering the cell cycle. Reduction of Mcm7 with siRNA to <5% of normal during G0→G1→S-phase reduces chromatin-binding of each of the MCM proteins that form the DNA helicase. However, these cells still enter S-phase and replicate DNA. Reducing MCM levels by titrating siRNA causes dose-dependent DNA-damage responses involving activation of ATR & ATM and Chk1 & Chk2. However, cells depleted of Mcm7 do not undergo apoptosis, rather reducing MCM levels even by 50% causes gross non-clonal chromosomal abnormalities normally found in genomic instability syndromes. M-FISH identified chromosome translocations, as well as loss and gain of individual chromosomes, which can occur individually or together in the same cell. Reducing MCM levels also causes misrepair by non-homologous end joining (NHEJ), and both NHEJ and homologous recombination (HR) are necessary for chromosomal abnormalities to occur. Therefore, “excess” MCM proteins that are present in a normal, proliferating cell are necessary for maintaining genome stability and reduction of MCM loading onto DNA that occurs in cancers is sufficient to cause genomic instability.
Leukemic Promyelocytes Possess Self-Renewal Capacity and Leukemia Stem Cell Properties in a Mouse Model of Acute Promyelocytic Leukemia.
Authors: Wojiski Sarah A. , Lee Benjamin , Kindler Thomas , Gozo Maricel , Leeman Dena , McDowell Elizabeth , Fabian Attila , Guibal Florence , Tenen Daniel , Gilliland D. Gary
Published: October 2019

It has been determined that within the heterogeneous leukemic population, there is a rare subset of leukemia stem cells that are responsible for continued maintenance and propagation of the tumor. Recent evidence suggests that during disease pathogenesis, more mature progenitor cells are capable of acquiring stem cell-like characteristics including properties of self-renewal and the capacity to differentiate into progeny that lack self-renewing potential. Acute promyelocytic leukemia (APL) is associated with the t(15;17)(q22;q12) chromosomal translocation that produces the PML-RARa fusion protein, and is unusual among the various subtypes of AML in that it is characterized by hyperproliferation of differentiated promyelocyte progenitors. In a mouse model in which PML-RARa is expressed under the control of the endogenous murine cathepsin G promoter, we observed that whole bone marrow expressing PML-RARa derived from pre-leukemic mice has certain properties of self-renewal, such as serial replating potential in methylcellulose cultures, and serial transplantation of disease into recipient animals. Using multiparameter flow cytometry, we demonstrated that preleukemic PML-RARa animals have an expansion of mature Gr-1+c-kit-CD34− granulocytes that are not capable of self-renewal. However, in the leukemic state, the expanded population shifted to a more immature population with an immunophenotype of Gr-1+c-kit+CD34+. This population was capable of transplanting disease to secondary recipients that recapitulated the disease of the primary donor, and thus contained leukemia-initiating activity. Morphologically, these cells resembled promyelocytes, and their promyelocytic identity was confirmed by quantitative PCR detection of the expression of azurophilic primary granule components associated with the promyelocyte stage of development, including myeloperoxidase, neutrophil elastase, and cathepsin G. However, these cells lacked expression of the secondary granule protein gelatinase B associated with the more mature myelocyte stage of granulocytic differentiation. Additionally, these leukemia-initiating promyelocytes responded to differentiation and cell death signals mediated by administration of all-trans retinoic acid (ATRA). These data indicate that in APL, a highly differentiated promyelocyte compartment does in fact possess properties of leukemia stem cells. This suggests that an important step in disease pathogenesis mediated by PML-RARa is the acquisition of properties of self-renewal in a terminally differentiating progenitor that normally lacks this potential.
The Effects of Chronic Opiates Pain Therapy in Sickle Cell Anemia.
Authors: Jonassaint Charles R. , Jonassaint Jude C. , Ashley-Koch Allison , Adam Soheir S. , Telen Marilyn J. , De Castro Laura M.
Published: October 2019

Sickle cell disease (SCD) patients experience high rates of morbidity and early mortality. Hydroxyurea (HU) therapy is associated with decreased morbidity and mortality as well as improved patient health outcomes (Charache et al, 1995 and Steinberg et al, 2003). However, the effect of chronic opiate pain management on HU treatment outcomes is unknown. In the current study, we evaluated the effects of HU and chronic long acting opiate therapy-OxyContin, methadone, fentanyl and MS Contin-on the following laboratory parameters: white blood cell count (WBC), hemoglobin (Hb), and mean corpuscular volume (MCV). Changes in these variables are known to be linked with HU effects on morbidity and mortality in SCD. 142 homozygous sickle cell anemia patients (75 females and 67 males) from the outpatient clinic at Duke University Medical Center were included in this study. Clinical data were collected by patient interview as well as review of medical records after informed consent. Patients were classified in 4 groups based on report of chronic daily use of long acting opiates and/or HU (current therapy with HU) as follows: No HU or opiates (None); opiates only; HU only; and HU and opiates. ANOVAs were used to identify combined effects of opiate therapy, HU therapy and the 3 laboratory parameters. Overall, we found that patients on HU (n=76, 54%), had lower WBC and higher Hb and MCV than the no HU patients (n=66, 46%), with p<.01 for all associations, as could be expected. In contrast to the positive effects of HU, patients on opiates had higher WBC than non opiate patients (p<.01). Surprisingly, when adjusting for gender and age, opiate therapy appeared to moderate the effect of HU treatment on WBC (p=.05) and MCV (p=.04). Patients who were on HU and opiates concurrently exhibited 17% higher WBC and 6% lower MCV than patients who were on HU alone, suggesting that opiate therapy may be attenuating the positive effects of HU. The findings from the current study confirm that HU treatment is associated with better patient health indices, i.e. lower WBC, higher Hb and MCV. In contrast, chronic opiate pain management, often a concomitant therapy with HU, was associated with poorer patient laboratory indices, both with or without concurrent HU treatment. Overall, these data indicate that the chronic use of opiates may decrease the efficacy of HU treatment in this patient population. Further studies are needed to study the mechanism of drug interaction between opiate and HU in SCD patients. The previously suggested inhibitory effect of HU on the cytochrome P450 CYP3A, an important cytochrome in the metabolism of some opiates (Reardon et al, 2005), should be considered for future studies of drug-drug interaction in SCD.

Figure Figure
Asthma Is Associated with Acute Chest Syndrome, but Not an Increase Rate of Hospitalization for Pain among Children with Sickle Cell Anemia: A Retrospective Cohort Study.
Authors: Bernaudin Francoise , Strunk Robert C. , Kamdem Annie , Arnaud Cecile , An Ping , Torres Martine , Delacourt Christophe , DeBaun Michael R.
Published: October 2019

Introduction In this study, we sought to validate the previous findings that asthma increases the incidence of acute chest syndrome (ACS) and pain in children with sickle cell anemia (SCA).

Methods A retrospective cohort was comprised of children with SCA evaluated for at least six months from a single medical center. Asthma was defined as being present when the first wheezing episode was heard by a physician after the age of 2 years or after 3 episodes of bronchiolitis before the age of 2 years. ACS was defined as a recent abnormal X-ray of the lungs associated with fever, respiratory signs or thoracic pain. A painful episode was defined as pain that resulted in hospitalization. Therapeutic intervention (hydroxyurea, blood transfusion therapy or transplant) was uniformly applied to all children with three or more episodes of pain that required hospitalization within a 12 month period. Patient years were accumulated from birth until death, lost to follow up, last visit to the center or a therapeutic intervention, whichever came first.

Results A total of 297 children with SCA were evaluable for a doctor diagnosis of asthma for a total of 1,805 patient-years. The mean length of follow-up was 6.1 patient-years. A doctor diagnosis of asthma was present in 8.4% (25 of 297). Among the children with asthma 75% (19 of 25) were consistently prescribed a beta 2 agonist or inhaled corticosteroids. After adjustment for the effect of age, asthma was significantly associated with ACS event (p = 0.03) but pain was not (>0.05).

Conclusion Among children with SCA, asthma is associated with an increased incidence of ACS, but not pain that required hositalization. The absence of an association between asthma and pain may be related to uniform therapeutic intervention for children with repetitive painful episodes that require hospitalizations coupled with active treatment for asthma in most of the children.
A Phase I Trial of Subcutaneous (SQ) Dose-Escalated Alemtuzumab in Patients with T-Cell Lymphoproliferative Disorders (T-LPD).
Authors: Porcu Pierluigi , Baiocchi Robert A. , Lin Thomas S. , Blum Kristie A. , Curtis Patricia , Michael Grever R. , Byrd John C.
Published: October 2019

BACKGROUND: T-cell lymphoproliferative disorders (T-LPD) are heterogeneous and highly chemoresistant malignancies without standard therapy. With few exceptions, cure rates with combination chemotherapy do not exceed 25–30%. We have shown that alemtuzumab (A), a humanized IgG1 targeting the CD52 antigen expressed on most human leukocytes, is cytotoxic for malignant T-cells in vitro and in vivo, regardless of p53 mutational status (Blood106: 3380–3382, 2005). We initiated a Phase I study with A and CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) in T-LPD.

METHODS: Accrual goal: 18–21 pts with untreated (u) or relapsed (r)T-LPD, excluding untreated ALK-1-positive or primary cutaneous anaplastic large cell lymphoma. CD52 expression was not assessed. Other exclusion criteria: pregnancy, HIV+, HCV+, or HBV+. Primary objective: A maximal tolerated dose (MTD). DLT defined as grade (G) ≥ 3 (or non-reversible grade 2) non-hematologic toxicity or G4 neutropenia or thrombocytopenia requiring >7 day delays in therapy for > 3 times. All pts received single agent SQ A loading (3, 10, 30 mg) over 5 days followed by one SQ A dose with each cycle of CHOP every 21 days for a total of 8 cycles. A dose levels: 3, 10, 20, 30 mg. All pts received valacyclovir, trimethoprim-sulfamethoxasole prophylaxis and G-CSF. Erythropoietin was given according to published guidelines.

RESULTS: 18 pts were enrolled (uPTCL=11, uT-PLL=2, rCTCL=3, uSezary Syndrome=2). Enrollment according to dose level: 3mg = 6 pts; 10mg = 3 pts; 20 mg = 6 pts; 30 mg = 3 pts. Median age: 62 years. All pts completed single agent SQ A loading on time with minimal local reactions. Ten pts completed all planned therapy. Six pts did not complete therapy: 2 rCTCL=stable disease, 1 uPTCL=poor compliance, 2 PTCL=pt witdrew, 1 uT-PLL=progression. Two pts (30 mg dose level) are receiving therapy.

Toxicity: G4 febrile neutropenia=1; G3 fatigue=2, G3 anemia=2, G3 dyspnea=1, G3 emesis=2, G3 CMV infection=1. Cohort 1 was expanded due to asymptomatic CMV reactivation requiring hospitalization for thrice daily foscarnet. Subsequent asymptomatic CMV reactivations (N=4) were treated with oral valganciclovir until clearance. No symptomatic CMV reactivation or other viral or fungal infections were seen. Out of 107 cycles of A/CHOP given, only 3 had to be delayed. Four pts (1 PTCL, 1 T-PLL and 2 Sezary) are in continuous clinical and molecular (PCR) complete response at 36, 28, 18 and 12 months respectively.

CONCLUSIONS: SQ A was safely administerd up to 20 mg with each cycle of CHOP chemotherapy and growth factor support without excessive myelosuppression or infectious AEs. Treatment at 30 mg dose level is in progress. Asymptomatic CMV reactivation can be managed with oral valganciclovir without discontinuation of therapy. Durable responses have been seen, including molecular clearance of the malignant T-cell clone.
Continued Late Conversion to Complete Remission (CR/CRu) and Durability of Remission (DUR) in Pts with B-Cell Follicular Lymphoma (FL) Treated with Rituximab Followed by Mitumprotimut-T (Id-KLH, FavId®) Active Immunotherapy.
Authors: Koc Omer N. , Redfern Charles , Wiernik Peter H. , Rosenfelt Fred , Winter Jane N. , Guthrie Troy H. , Kaplan Lawrence , Holman Peter , Densmore John , Hainsworth John , Lin Thomas S. , Castillo Rene A. , Janakiraman Nalini , Ghalie Richard G.
Published: October 2019

Background: We have previously reported results from a Phase 2 trial in which both treatment naïve (TN) and relapsed/refractory (R/R) pts with stable disease (SD) or a clinical response (PR or CR/CRu) following 4 weekly doses of rituximab received mitumprotimut-T active immunotherapy (Koc et al, Blood2006; 108: #691). We report here 4-year follow up of conversion of pts into complete remission (CR/CRu) and an assessment of the DUR in this population.

Treatment: Pts received rituximab (375mg/m2 iv weekly x 4) and those with ≥SD assessed at Week 11 received mitumprotimut-T, 1 mg sq every month (mo) x 6 starting on Week 12 along with Leukine (sargramostim, GM-CSF), 250 mcg, sq on Days 1–4. Pts continued to receive booster injections on a reduced schedule (every 2 mos x 6, then quarterly) until disease progression. Radiological scans were performed every 3 mos for the first 2 years then every 6 mos, and reviewed centrally. Objective response and progression were assessed using modified IWG criteria.

Results: 89 pts (54 RR, 35 TN) had ≥SD following rituximab received mitumprotimut-T + Leukine. Two pts (RR) achieved a CR following rituximab and prior to receiving mitumprotimut-T. As shown in Fig. 1, an additional 16 pts (9 TN, 7 RR) converted to CR/CRu over the next 31 mos. At a median follow-up of 42 mos, only 6 (3 TN, 3 RR) of the 18 CR pts have relapsed (Fig. 2).

Discussion: Long-term follow-up has shown a continuing conversion of pts into CR occurring as late as 31 mos. These data suggest clinical activity of mitumprotimut-T in these pts as determined by the increasing CR rate and the durability of this response. These data are of particular interest since a blinded interim analysis of best response in an ongoing controlled Phase 3 study of single agent rituximab vs. rituximab followed by mitumprotimut-T showed 47% of pts in this study to be in CR/CRu with 12–18 mos of follow-up (Freedman et al, Blood2006; 108: #2756).

Figure Figure Figure Figure
Relapse from Complete Remission More Than 5 Years after Therapy for Diffuse Large B-Cell Lymphoma (DLBCL): Relapse Histology Most Commonly DLBCL with a Germinal Center B-Cell (GCB) Phenotype.
Authors: Vose Julie M. , Chan John C. , Bierman Philip J. , Arevalo Alejandro , Loberiza Fausto , Fu Kai , Weisenburger Dennis , Bociek Robert G. , Bast Martin , Armitage James O.
Published: October 2019

Although many patients are successfully treated for DLBCL, relapses can occur especially in the higher risk patients. Relapses in successfully treated DLBCL patients most frequently occur within the first 2–3 years. However in a small number of patients, relapses occurring after 5 years do happen and have been frequently reported to be a relapse of follicular lymphoma. We evaluated 805 patients with DLBCL treated with an anthracycline based chemotherapy through the Nebraska Lymphoma Study Group from 1983–1998. The patients were treated prior to the use of rituximab in this patient population. Two hundred and three patients relapsed from a documented complete remission. Of these, 30 (15%) relapsed more than 5 years after treatment (range 5.3 – 14.5 years, median 6.8 years). The median age at relapse was 71 years (range 32 – 85) and 58% were male. Patients with late relapses had relatively good prognostic features at the time of diagnosis. None had a low performance status, 71% were stage I/II, 78% had no systemic symptoms, 78% had a normal LDH, and 67% had an IPI of 0/1. All patients had biopsies to document relapse. Eighty one percent of the relapses were documented DLBCL, 11% had composite lymphoma with DLBCL and follicular grade 3 (FL3) lymphoma, 4% had FL3 alone, and 4% had follicular grade 1 (FL1). Using immunohistochemistry to predict GCB vs. non-GCB origin, 72% of the patients had a GCB phenotype at diagnosis and 90% had a GCB phenotype at relapse. Four patients had no therapy at relapse and 2 patients had radiotherapy. The remaining patients received a variety of combination chemotherapy regimens with or without rituximab and 2 patients underwent autologous stem cell transplantation. Seven patients had durable complete remissions to their second line chemotherapy regimens (4 with chemo + rituximab and 2 autologous stem cell transplantation). Three of these survivors died of unrelated causes and 4 are alive in remission 19–119 months after the salvage therapy or autologous transplantation.

Conclusions: Most patients with late relapses of DLBCL initially present with good prognostic characteristics at the time of the origninal diagnosis. Unlike previous studies, the most common type of histology in this study at the time of late relapse was DLBCL and not follicular lymphoma. However, the vast majority of the late relapse DLBCL patients had a GCB phenotype both at diagnosis and at relapse. Patients with late relapses usually do respond to salvage therapy and can have second prolonged remissions with chemotherapy and/or autologous stem cell transplantation.
C-MYC Correlates with Expression of Multidrug Transporters ABCC1 and ABCC4.
Authors: Ho Lye Lin , Chong Li , Ling Silvia , Lau Diana , Lau Edwin , Iland Harry , Joshua Douglas E. , Allen John
Published: October 2019

C-MYC is an oncogene with protean effects and there is some speculation that aberrant overexpression in cancer may be associated with drug resistance by increased expression of the Multi-Drug Resistance Transporters, for example ABCC4 (MRP4) and ABCC1 (MRP1). A relationship between N-MYC and these transporters has been established in the limited context of childhood neuroblastoma and it would be tantalising to extrapolate this to the wider scope of adult tumours. The corollory would be that modulating the levels of C-MYC might have a beneficial secondary effect of making a tumour more sensitive to chemotherapeutic treatments. We have determined that the ABCC4 promoter directly responds in vitro to C-MYC and N-MYC contransfection and that this effect can be abolished by the progressive disruption of the C-MYC binding sites i.e. mutation of the E-boxes. Transient or stable overexpression of C-MYC or N-MYC did not increase multidrug transporter protein expression in 5 tumour cell lines, possibly due to the methylation status of the E-boxes or that the transporters present are already maximally expressed. However, knocking-down C-MYC by shRNA results in a concomitant reduction in ABCC1 and ABCC4. Together, these results suggest both direct and indirect regulation may be important in the functional control of these transporters. A survey was performed of Chronic Myeloid Leukaemias (CML, n=26) and Myelomas (n=23), assessing the expression of C-MYC, ABCC4 and ABCC1 by quantitative RT-PCR. The genes of interest were normalised to housekeeping genes and linear regression applied to examine the relationship of C-MYC levels to transporter expression. In CML, a direct relationship was seen between C-MYC and both ABCC4 and ABCC1. In Myeloma, a correlation was noted between C-MYC and ABCC1 but was not seen with ABCC4, as the levels detected were very low. The inference is that high levels of C-MYC may be associated with elevated multidrug transporter levels in other tumours and may be of use in predicting transporter-mediated drug resistance.
Pharmacogenetic Study on Multiple Myeloma Patients Treated with DAV Regimen and Autologous Stem Cell Transplantation.
Authors: Buda Gabriele , Maggini Valentina , Orciuolo Enrico , Galimberti Sara , Martino Alessandro , Morabito Fortunato , Barale Roberto , Rossi Anna Maria , Petrini Mario
Published: October 2019

As previously reported, genetic variability in xenobiotic metabolism (GSTP1 and NQO1), multidrug resistance (MDR1), folate transport (SLC19A1) and DNA synthesis (TYMS) can influence the outcome after chemotherapy and autologous transplantation in patients with multiple myeloma (MM) (Maggini et., Leuk Res 2007 in press; Buda et al., Leuk Res 2007, 31(8):1029–30; Buda et al., BJH 2007, 137(5):454–6; Maggini et al., Leukemia 2007, 21(1):176–8). To test the reciprocal influence of the examined polymorphisms on the outcome, multivariate analyses were performed on 98 patients receiving DAV regimen, followed by conditioning regimen with melphalan and autologous stem cell transplantation (ASCT) in 84 cases. As shown in table I, a worse outcome following DAV therapy was significantly associated with GSTP1 G/G, SLC19A1 T/T and TYMS A/A genotypes. Only a borderline effect was seen for NQO1 T/T carriers. After ASCT, no significant influence of the analysed polymorphisms was observed, but a higher percentage of responders presented the SLC19A1 T/T genotype. A significantly improved overall survival (OS) was found in patients carrying SLC19A1 T/T genotype or MDR1 T allele (Cox regression analysis, p=0.01). Multivariate analyses confirmed the previously reported findings: the estimated effects are comparable, even though the significance resulted slightly reduced as expected for multiple comparisons. These preliminary results, if sustained by extended analyses, suggest that these polymorphisms could be regarded as pharmacogenetic markers and as prognostic factor in order to predict the outcome of MM patients.

Table I Multiple genotype testing of MM patients are responders (R) versus non-reponders (NR) to DAV therapy (CT) or ASCT by unconditional logistic regression analysis.
Myeloperoxidase Is Required for the Combined Apoptotic Effects of Arsenic Trioxide with Ascorbic Acid or Epigallocatechin-3-Gallate in Myeloid Leukemia Cells.
Authors: Xia Lijuan , Lu Min , Chen Duo , Waxman Samuel , Jing Yongkui
Published: October 2019

Arsenic trioxide (As2O3) is a drug used world-wide that selectively causes the death of acute promyelocytic leukemia (APL) cells by novel mechanisms and induces complete clinical remission in 90% of patients without significant toxicity. The outstanding success of As2O3 therapy in relapsed APL patients has not been replicated by an equivalent success in other types of acute myeloid leukemia (AML). We have studied As2O3 mechanism(s) of action and identified agents to use in combination to improve the use of As2O3 as a treatment for other types of AML. As2O3 produces higher levels of H2O2 and apoptosis in APL NB4 cells than in other AML cells at therapeutic concentrations of 1-2 uM. As2O3 does not induce apoptosis in HL-60 cells but is synergistic with ascorbic acid (AA) or epigallocatechin-3-gallate (EGCG). Both AA and EGCG produce H2O2 which is augmented by the addition of As2O3. Apoptosis induction by As2O3 in combination with AA or EGCG is inhibited by catalase and the antioxidant N-acetylcysteine. Myeloperoxidase (MPO), a major neutrophil enzyme, augments H2O2-induced apoptosis by converting it into more potent reactive oxygen species. HL-60, NB4, SKNO-1 and PLB985 cells which express high level of MPO, but not U937 and K562 cells without expression of MPO, are responsive to As2O3 plus AA or EGCG-induced apoptosis. HP-100 cells, a subclone of HL-60 cells without MPO expression, are resistant to both combination treatments. MPO stable transfection sensitizes K562 cells to apoptosis following treatment with As2O3 and AA or EGCG. NADPH oxidase is an enzyme complex which generates O2− and H2O2 in neutrophils. In APL NB4 cells, As2O3 induces expression of NADPH oxidase members and is thought to participate in H2O2 production required for As2O3 response. X-CGD cells, a subclone of PLB-985 with targeted disruption of the gp91phox gene (Zhen et al, PNAS90:9832, 1993) and loss of NADPH oxidase activity but with high levels of MPO activity, are responsive to treatment with As2O3 and AA or EGCG. These results suggest that AA and EGCG enhance As2O3-induced apoptosis through a synergistic production of H2O2 which is independent NADPH oxidase activation in non-APL AML cells. MPO augments the apoptotic effect of H2O2 produced by As2O3 plus AA or EGCG and it could be used as a marker to predict the sensitivity of AML cells to both combination treatments.
Prognostic Value of Minimal Residual Disease by Real-Time Quantitative PCR in AML with CBFB-MYH11 Rearrangement: The French Experience.
Authors: Guièze Romain , Renneville Aline , Cayuela Jean-Michel , Abdelali Raouf Ben , Boissel Nicolas , Rubio Marie-Thérèse , Mazingue Françoise , Macintyre Elizabeth A. , Sigaux François , Fenaux Pierre , Dombret Hervé , Preudhomme Claude
Published: October 2019

Background. Despite the favorable prognosis of AML patients with inv(16)/t(16;16) leading to CBFB-MYH11 rearrangement, relapses still occur in about 40% of the cases. With the possible exception of receptor tyrosine kinase mutations, no pretreatment-factor can strongly predict risk of relapse.

Methods. To explore minimal residual disease (MRD) prognostic impact, we prospectively monitored CBFB-MYH11 rearrangement by real-time quantitative PCR (RQ-PCR) in patients with inv(16)/t(16;16) treated in French cooperative trials; most patients were treated in Acute Leukemia French Association (ALFA) trials and Leucémies Aiguës Myéloblastiques de l’Enfant (LAME) Cooperative Groups trials. RQ-PCR was performed in three molecular French labs (CHU of Lille, Necker Hospital and St-Louis Hospital) according to the EAC procedure and results are expressed as CBFB-MYH11/100 ABL copies(%).

Results. 61 AML patients with inv(16)/t(16;16) who had reached CR with one of the anthracyclin-aracytin regimens were analyzed. Median age was 37.5 years (range 2.1–76.5) and median baseline WBC 46 G/L (range 1.8–246). With a median follow-up of 23.3 months, median overall DFS was 23 months and median OS not reached (93.5% of patients alive). No initial clinical or hematological characteristic including age, gender and initial WBC was predictive of relapse in adults, but children had poorer DFS than adults (17.6 months vs not reached, p=0.03). Regarding chromosomal abnormalities in addition to inv(16)/t(16;16), trisomy 22 was the most frequent but no significant prognostic impact was observed. Of the 61 patients, MRD monitoring could be performed sequentially in 45. Pretreatment CBFB-MYH11 transcript level had no impact on DFS. However, after induction therapy, transcript levels >0.5% were significant predictors of poorer DFS (median 16.4 months vs not reached if <0.5%, p=0.002). Likewise, after first consolidation therapy course, transcript level >0.001% was also predictive of poorer DFS (median 22 months when >0.001% vs not reached when <0.001%; p=0.03). 34 paired analyses of bone marrow (BM) and peripheral blood (PB) samples revealed only moderate correlation (R2=0.86); especially for low MRD levels, CBFB-MYH11 transcript expression being generally higher (up to 78-fold) in BM samples than in PB samples.

Conclusion. MRD monitoring by RQ-PCR appears to be a major prognostic factor of DFS in AML with CBFB-MYH11 rearrangement and could be a powerful tool to early identify good and bad responders which could have an impact on therapeutic decisions for consolidation therapy.

DFS according to MRD level after first consolidation treatment DFS according to MRD level after first consolidation treatment