bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the Transcription Factor PAX5/BSAP in Childhood Acute Leukemia Cells and Haematological Tumor Cell Lines.
Authors: Zhang Bei , Tie Li-Jun , Ye Qidong , Zhao Yanxia , Gu Long-Jun , Tang JingYan , Yuan Xiangliang , Shen Li-song
Published: April 2020

PAX5 gene is a paired-box PAX gene family member,and encodes the transcription factor BSAP(B-cell specific activator protein) which is a key regulator of B-cell development and differentiation.Dysregulation of PAX5 gene function may contribute to tumorigenesis in lymphoid malignancies.But up to now,a detailed examination of PAX5/BSAP expression in acute leukemia(mainly acute B-lineage lymphoblastic leukemia) has not been reported.In this study,a real-time RT-PCR assay for the relative quantitation of PAX5 and CD19 mRNA expression was developed.It was applied on 6 haematological tumor cell lines and bone marrow cells of 6 normal children,58 previously untreated and 4 relapse acute leukemic children,including 39 cases of B-ALL,10 cases of T-ALL,and 13 cases of AML.PAX5 and CD19 mRNA expression were detected in B-cell lines tested,but almost not in other T- and myeloid cell lines.Among clinical samples,expression of PAX5 mRNA in B-ALL was significantly higher than that in T-ALL and AML(P=0.029 and P=0.013,respectively).PAX5 expression was significantly lower in T-ALL and AML than normal controls.The mRNA levels of PAX5 between T-ALL and AML had not any difference.Individual difference of PAX5 mRNA expression levels in children with B-ALL was great.Because binding sites for BSAP have been identified in the promoters of CD19,the study found that in B-ALL,there was clear correlation between the level of PAX5 expression and that of CD19,which was also studied by real-time RT-PCR.BSAP expression by Western Blotting analysis was also performed in haematological tumor cells,including 6 haematological tumor cell lines and 4 clinical samples(2 cases of B-ALL,1 case of T-ALL,and 1 case of AML).The results of Western Blotting analysis showed a 52-KD BSAP band in B lineage cells,but not in T- and myeloid lineage cells.The intensity of BSAP bands was in accordance with PAX5 mRNA expression level detected by real-time RT-PCR.It was concluded that PAX5 transcripts are readily detectable and quantified in clinical materials with B-ALL by real-time RT-PCR.The strong PAX5 mRNA expression in some B-ALL can be considered to be particularly interesting for further analysis.
Proteomic Analysis of Targeted Therapeutic Agents in Multiple Myeloma (MM).
Authors: Ghobrial Irene M. , Eckel Jeanette E. , Hoering Antje , Timm Michael , Leontovich Alexey A. , Fonseca Rafael , Greipp Philip R. , Witzig Thomas E.
Published: April 2020

The objective of this study was to analyze protein changes that occur a the molecular level with novel therapeutic agents used in MM in order to identify potential combinations for clinical trials. We employed antibody protein microarrays (BD Clonetech, CA) to measure changes in the patterns of protein expression in Kas 6/1 MM cell line after treatment with bortezomib, dexamethasone, CC-5013, and 17-AAG. The antibody array is a new technique that assays protein differences directly by hybridizing fluorescently labeled protein mixtures from cell extracts onto glass slides spotted with 512 different monoclonal antibodies specific for human proteins. Induction of apoptosis was assessed by Annexin/Propidium iodide FACS analysis at 24 and 48 hours using different concentrations of the inhibitors. Protein arrays were performed at concentrations and treatment times that did not induce more than 25% apoptosis to ensure adequate analysis of early changes in signaling pathways. Bortezomib 5nM at 10 hours, dexamethasone 100mM, CC-5013 250nM, and 17-AAG 1mM at 24 hours were used. Control cells were treated with vehicles ETOH or DMSO at the same concentrations as the inhibitors. To assess differential protein expression, the mean of the Cy5/Cy3 ratios for each sample and its control were analyzed using the Clontech software and an internally normalized ratio (INR) was calculated. INR values >1.3 or <0.77 were considered as valid changes in protein abundance for this study. All inhibitors induced the upregulation of apoptosis proteins including BAK, BIK, SMAC/DIABLO, and caspase 14. Several proteins were inhibited by all 4 inhibitors indicating common pathways affected by these agents. These included members of the PI3K pathway (PI3K-p85a, p70S6kinase, and cyclin dependent kinases), MAPKinases (ERK1 and 2), antiapoptotic protein Bclx, heat shock proteins Hsp70 and HSP60, and ubiqutin/NFkB pathway (Ubch6, IKKalpha, and IKBe). These results indicate that inhibitors of the PI3K pathway such as mTOR inhibitors, IKK inhibitors or MAPkinase inhibitors may be used in the future in combination with bortezomib, dexamethasone, CC-5013 or 17-AAG. Fatty acid synthetase was downregulated by bortezomib and dexamethasone indicating potential use of fatty acid synthase inhibitors in combination with bortezomib or dexamethasone in future clinical trials. These results suggest that antibody protein arrays may be used in the rational design of combinations of targeted therapies in MM.
T Cell Activation by t(9;22) Acute Lymphoblastic Leukemia-Derived Dendritic-Like Cells Is Associated with Increased Expression of Antigen Processing Machinery Components.
Authors: Claus Jonathan A. , Brady Michael A. , Lee Jaewoo , Donohue Kathleen A. , Ferrone Soldano , Wetzler Meir
Published: April 2020

We have recently shown that dendritic-like cells derived from t(9;22) acute lymphoblastic leukemia (ALL) cells can activate T cells, while the original unmodified leukemic blasts cannot. To define the molecular basis of this functional difference, we have compared the level of antigen processing machinery components in unmodified blasts and ALL-derived dendritic-like cells, since they play a major role in antigen presentation. Six patient samples and one t(9;22) ALL cell line (Z119) were studied.

Dendritic-like cells were generated by in vitro culture with CD40 ligand, interleukin (IL)-1β, IL-3, IL-7, stem cell factor and tumor necrosis factor-α for six days. Maturation was verified by morphology and CD80/CD86 expression. Cells were studied for HLA class I and class II antigen expression. Furthermore, antigen processing machinery component expression was analyzed by intracellular staining with a unique panel of monoclonal antibodies which recognize the constitutive proteasome (Z, MB1, delta) and immunoproteasome (LMP2, LMP7, LMP10) subunits, the transporter subunits TAP 1 and 2 and the chaperones calnexin, ERp57, calreticulin and tapasin. HLA Class I antigen and HLA-DR/DQ/DP antigen expression was significantly up-regulated on the dendritic-like cells. LMP2, LMP7 and tapasin expression was significantly up-regulated in all t(9;22) ALL-derived dendritic-like cells, in comparison to the unmodified blasts. No significant difference was detected in the expression of the other antigen processing machinery components. These results suggest that T cell activation by t(9;22) ALL-derived dendritic-like cells is associated with increased expression of some, but not all, of the antigen processing machinery components. Whether t(9;22) ALL-derived dendritic-like cells can be used as a cellular vaccine for adoptive immunotherapy of ALL is being investigated.
Amphiregulin Is Produced by Myeloma Cells and Is Involved in Tumor-Environment Interactions in Multiple Myeloma.
Authors: Mahtouk Karène , Hose Dirk , Reme Thierry , De Vos John , Jourdan Michel , Moreaux Jérome , Fiol Geneviève , Raab Marc , Jourdan Eric , Moos Marion , Goldschmidt Hartmut , Rossi Jean-François , Cremer Friedrich W. , Klein Bernard
Published: April 2020

Multiple myeloma (MM) is characterized by the accumulation of clonal malignant plasma cells in the bone marrow. One of the hallmarks of plasma cells is the expression of the heparan-sulfate proteoglycan syndecan-1. In epithelial cells, syndecan-1 plays a major role as a coreceptor for heparin-binding growth factors and chemokines. This stresses that heparin-binding growth factors may play a major role in the biology of MM cells. Recently we have demonstrated that heparin-binding EGF-like growth factor (HB-EGF), one of the ten members of the Epidermal Growth Factor (EGF) family, is produced by the tumor microenvironment and is able to trigger myeloma cell growth. As amphiregulin (AREG) is another member of the EGF family that also binds heparan-sulphate chains, we investigated its role in MM.

We looked for AREG expression on a panel of 7 normal plasmablastic cells (PPCs), 7 normal bone marrow plasma cells (BMPCs), purified MM cells from 65 patients and 20 myeloma cell lines (HMCLs), with Affymetrix U133A+B microarrays. We showed that primary MM cells overexpress AREG compared to normal BMPCs and PPCs. We then investigated the expression of the ErbB receptors with real-time RT-PCR. Myeloma cells variably expressed the 4 ErbB receptors. Normal BMPCs also expressed ErbB1 and ErbB2 unlike PPCs that did not express any ErbB receptors. We demonstrated that the high AREG expression by primary myeloma cells may have a dual effect. On the one hand, AREG stimulated IL-6 production and growth of bone-marrow stromal cells that highly express the AREG ErbB1 receptor. On the other hand, AREG could promote HMCL proliferation, suggesting that a functional autocrine loop involving AREG and ErbB receptors is involved in MM cell growth. Finally, we looked for the effect of ErbB inhibitors on MM cells of 14 patients cultured for 6 days together with their bone marrow environment. A pan-ErbB inhibitor (PD-169540, Pfizer) and an ErbB1-inhibitor (IRESSA, Astrazeneca) induced strong MM cell apoptosis in respectively 71% of patients (10 of 14) and 29% of patients (4 of 14). Of major interest, when PD169540 or IRESSA were combined with dexamethasone, they induced a dramatic myeloma cell death (respectively 92% and 69% inhibition of MM cell survival), while non-myeloma cells were unaffected. Thus ErbB activation is critical to trigger MM-cell survival in short-term culture.

In conclusion, our findings provide evidence for a major role of AREG and HB-EGF in the biology of multiple myeloma and identify ErbB receptors as putative therapeutic targets. These data emphasize the interest of clinical evaluation of specific-ErbB-inhibitors in patients with MM, either used alone or in combination with dexamethasone.
BCR-ABL Activates STAT3 Via MEK Kinase 1 in Embryonic Stem Cells.
Authors: Nakamura Yukinori , Yujiri Toshiaki , Nawata Ryouhei , Tagami Kozo , Tanizawa Yukio
Published: April 2020

BCR-ABL oncogene, the molecular hallmark of chronic myelogenous leukemia, arises in a primitive hematopoietic stem cell that has the capacity for both differentiation and self-renewal. Its product, Bcr-Abl protein, has been shown to activate STAT3 and to promote self-renewal in ES cells, even in the absence of leukemia inhibitory factor (LIF). MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase involved in Bcr-Abl signal transduction (Oncogene22:7774, 2003). To investigate the role of MEKK1 in Bcr-Abl-induced transformation of ES cells, p210 Bcr-Abl was stably transfected into wild type (WT+p210) and MEKK1−/− (MEKK1−/−+p210) ES cells. Bcr-Abl enhanced both MEKK1 expression and activation in ES cells, as it does in other Bcr-Abl-transformed cells. In the absence of LIF, WT+p210 cells showed constitutive STAT3 activation and formed compact colonies having strong alkaline phosphatase activity, a characteristic phenotype of undifferentiated ES cells. MEKK1−/−+p210 cells, by contrast, showed less STAT3 activity than WT+p210 cells and formed large, flattened colonies having weak alkaline phosphatase activity, a phenotype of differentiated ES cells. These results indicate that MEKK1 plays an essential role in Bcr-Abl-induced STAT3 activation and in the capacity for LIF-independent self-renewal, and may thus be involved in Bcr-Abl-mediated leukemogenesis in stem cells.
All Trans-Retinoic Acid (ATRA) in Combination with PPAR-g Ligand Synergistically Enhances Inhibition of Cell Growth and Induction of Tumor Suppressor PTEN in Leukemic Cells.
Authors: Park Jinny , Lee Young-Rae , Shim Hyun-Jaung , Kwak Jae-Young , Yim Chang-Yeoul , Song Eun-Kyung , Han Myung-Kwan , Park Jin-Woo , Rho Hye-Won , Lee Sang-Young , Kim Jong-Suk
Published: April 2020

Peroxisome proliferator-activated receptor-γ (PPAR-γ), a transcriptional factor that plays an essential role in mediating the pharmacologic actions of PPAR-γ ligands, is highly expressed in normal monocytes, various leukemias, and epithelial malignancies. PPAR-γ ligands have been developed to induce differentiation, growth arrest, and apoptosis. PPAR-γ must form a heterodimer with the retinoic acid receptor (RAR) to bind DNA, and its transcriptional activity is thought to be maximal in the presence of both PPAR-γ ligand and RAR ligand. Recently, it has been shown that activated PPAR-γ and PPAR-γ ligand may influence tumor growth, directly and indirectly, through regulation of the tumor suppressor gene PTEN, a lipid phosphatase that plays a significant role in several cellular functions, including survival and proliferation, by antagonizing phosphatidylinositol 3-kinase (PI 3-kinase)-mediated signaling pathways. The human leukemia cell line HL60 displays controlled differentiation along the granulocyte or monocyte lineages in response to various inductive signals. Retinoic acid receptor ligands are inducers of HL60 granulocytic differentiation. PPAR-γ ligands and RAR ligands induced growth arrest and oxidative burst capacity in HL60 cells. Previously, we reported the induction of PTEN gene expression by the PPAR-γ ligand ciglitazone in HL60 cells in the presence of retinoic acid using northern and western blotting. Here, using RT-PCR and an AdenoVector system to examine the contribution of transcriptional control in PTEN regulation and for PTEN overexpression, we show that PPAR-g ligand and RAR ligand can synergistically upregulate PTEN in human promyeloid leukemia cells, resulting in inhibition of cell growth and cell cycle progression of acute leukemic cells. Adenovirus-delivered PTEN overexpression resulted in arrests of both cell growth and the G1 phase of the cell cycle in HL-60 cells, suggesting that the growth-suppressive effect of PTEN is associated with its ability to induce cell cycle arrest. The PPAR-γ ligand, ciglitazone, and the RAR ligand, retinoic acid, increased PTEN expression of HL-60 cells in a time- and dose-dependent manner, respectively. Upregulation of PTEN was significantly enhanced by a combination of both ciglitazone and retinoic acid. Moreover, these compounds synergistically induced arrests of both cell growth and the G1 phase of the cell cycle. These findings suggest that the activation of the PPAR-γ: RAR heterodimer represents a novel regulatory pathway for HL60 cells and there may be a possible role for PPAR-γ and RAR ligand in prophylactic and therapeutic approaches for controlling leukemia through the upregulation of PTEN.
Mutations in the AML1 Gene Define an Unfavorable Subgroup in Acute Myeloid Leukemia with FAB M0.
Authors: Dicker Frank , Klaus Mirjam , Haferlach Torsten , Kern Wolfgang , Hiddemann Wolfgang , Schoch Claudia , Schnittger Susanne
Published: April 2020

The AML1/RUNX1 gene is the most frequent target for chromosomal translocations in leukemia. Recently point mutations in the AML1 gene have been demonstrated as another mode of genetic aberration. AML1 mutations have been reported in de novo MDS and AML, as well as in therapy related MDS and AML. The AML M0 subtype has been found to be most frequently affected by sporadic AML1 gene mutations. We analysed AML1 gene mutations in a cohort of 49 M0 patients. Mutation screening was performed either with SSCP (n=21) and/or denaturating High Performance Liquid Chromatography (dHPLC) (n=33), 5 cases were analyzed by both methods. SSCP screening of exons 3–5 of the AML1 gene was carried out at the genomic level. These exons cover the socalled Runt domain, which is most frequently mutated. Fragments with aberrant mobility were sequenced. With this method 5 cases were found to be mutated. Subsequently, to improve the screening efficiency an assay using dHPLC was established. Hereby, we screened the cDNA of patient samples for mutations in amino acid codons 1–277 of the AML1b transcript, where the Runt domain is located between codons 49 and 178. All 5 cases detected by SSCP were confirmed by dHPLC. Nine mutations were detected in the cohort of 28 cases (32%) which had not been analyzed by SSCP. In total, 14 of the 49 samples (29%) tested were identified to be mutated, which is a slightly higher frequency than previously reported. In the cohort of 35 AML1 non-mutated cases 20 (57%) had a normal karyotype and 15 (43%) an aberrant karyotypes, whereas only 6 of the 14 AML1 mutated cases (43%) had a normal karyotype (p=0.001). Three of the AML1 mutated cases (21%) also had FLT3 mutations. One had an FLT3-LM, one an FLT3-TKD mutation, and one case both LM and TKD mutations. Clinical follow up data were available for 33 patients (22 AML1 non- mutated, 11 AML1 mutated). The median OS and EFS of the AML1 non-mutated versus the mutated group was 276 days versus 63 days (p = 0.0679) and 276 vs. 63 days (p=0.0630) respectively. Thus the AML1 mutated cases tend to have a worse clinical outcome. When other AML subtypes were screened for AML1 mutations, i.e. M1 (n=26), M2 (n=21) and M4 (n=3), only 1 additional AML1 mutation was detected, confirming the highest prevalence of AML1 mutations in M0. In conclusion, 1) we established a new assay to screen for AML1 mutations. 2) We confirmed the high incidence of AML1 gene mutations in AML M0, both in cases with normal and aberrant karyotype. 3) For the first time we demonstrated that AML1 mutations define an unfavorable subentity in AML M0.
A New Polycythemia Syndrome: Congenital Polycythemia with High Erythropoietin and Propensity for Malignant Hypertension Due to Paraganglioma.
Authors: Guan Yongli , Wu John K. , Jastaniah Wasil , Moss Larry G. , Digman Colleen , Mostacci Salvatore , Prchal Jaroslav F. , Prchal Josef T.
Published: April 2020

We report three patients, two females and one male, with congenital polycythemia and high erythropoietin. All were extensively investigated in childhood; no known polycythemic conditions were diagnosed nor were any tumors known to be associated with polycythemia uncovered. At the ages of 14, 16, and 32 years the patients developed malignant hypertension secondary to multiple paragangliomas. In addition, one of patients has also developed a malignant pancreatic carcinoid tumor that expressed somatostatin. After the surgical resection of paragangliomas, hypertension subsided, but polycythemia persisted in all three subjects.

Comprehensive screening for germ-line mutations of von Hippel-Lindau (VHL) gene as well as genes known to be causing familial paragangliomas - catalytic succinate dehydrogenase subunits B (SDHB), and catalytic succinate dehydrogenase subunit D (SDHD), has not revealed any mutations. In one of these patients studied so far there was no abnormality of the any of the three hypoxia control associated proline hydroxylase genes expressions found. Unlike the erythroid progenitors of Chuvash Polycythemia, these patients had normal sensitivity of erythroid progenitors to erythropoietin. The search for somatic mutations of VHL, SDHB, and SDHD genes, as well as quantitative analysis of the expressions of hypoxia inducible factor 1 alpha subunit (HIF-1a), vascular endothelial growth factor (VEGF), VHL, SDHB, and SDHD mRNAs by real-time PCR is ongoing.

In summary, we report a new polycythemic syndrome of congenital polycythemia with high erythropoietin and the normal sensitivity of erythroid progenitors to erythropoietin, which is not associated with VHL mutation. This is in contrast to our previous experience with about 200 patients, homozygous for Chuvash Polycythemia mutation or compound heterozygous for Chuvash Polycythemia R200W and other VHL mutations, who have not developed paraganglioma or any VHL syndrome tumors. This new polycythemic syndrome shares some clinical features with both von Hippel Lindau syndrome and with the Chuvash polycythemia. While its molecular defect is yet to be elucidated, it is a distinct clinical and genetic entity.
Analysis of Common Single Nucleotide Polymorphisms in MDR1 Gene in Patients with Multiple Myeloma.
Authors: Jamroziak Krzysztof , Balcerczak Ewa , Smolewski Piotr , Piaskowski Sylwester , Cebula Barbara , Panczyk Mariusz , Janus Agnieszka , Mirowski Marek , Robak Tadeusz
Published: April 2020

P-glycoprotein (P-gp) encoded by MDR1 gene is a xenobiotic transporter involved in protection of normal tissues against environmental toxicants and multi-drug resistance of cancer cells. Recently, a number of authors, including our group, have indicated that phenomenon of single nucleotide polymorphism (SNP) in MDR1 may influence susceptibility or prognosis in various diseases as well as pharmacokinetics of P-gp substrates. Although environmental chemicals are suspected carcinogens in multiple myeloma (MM) and P-gp transported drugs are used in the treatment of this malignancy, little data is known on the significance of MDR1 SNPs in MM. In this study, three common MDR1 SNPs: C1236T, G2677T/A and C3435T were assessed in 89 MM patients using automated sequencing and RFLP method. Moreover, in the control group of 215 healthy individuals MDR1 C3435T SNP was genotyped in all subjects and P-gp expression and activity were examined in a proportion of subjects. The haplotypes were inferred using Partition-Ligation algorithm showing highest frequency of the haplotype with wild-type alleles at all sites studied (1236C-2677G-3435C, 30%), followed by haplotype with all mutant alleles (1236T-2677T-3435T; 27%) and 1236C-2677G-3435T haplotype (12%). Carriers of a mutant allele at MDR1 1236 site showed a trend to longer overall survival as compared to wild-type homozygotes MDR1 1236CC (p=0.08), while no correlations with survival were observed regarding other polymorphisms. As compared to the control group, the MDR1 C3435T SNP allele frequency did not differ in MM patients, however the carriers of at least one T-allele (CT and TT genotypes) were significantly more frequent in the MM cohort (79% vs. 65%, p=0.02). The flow cytometry results of anti-P-gp MRK16 antibody staining and functional rhodamin123/PSC833 assay performed on peripheral blood mononuclear cells, CD19+ B-lymphocytes and CD3+ T-lymphocytes were similar among healthy individuals having MDR1 3435CC, CT and TT genotypes. In conclusion, we observed that MDR1 gene SNPs may exert some effect in genetic predisposition and prognosis in MM, which should be further examined in larger cohorts of uniformly treated patients.
Serum Levels of Vascular Endothelial Growth Factor (VEGF) and Basic Fibroblast Growth Factor (bFGF) in Children with Acute Lymphoblastic Leukemia.
Authors: Palgan Izabela , Wysocki Mariusz , Palgan Krzysztof , Debski Robert , Odrowaz-Sypniewska Grazyna , Styczynski Jan
Published: April 2020

Angiogenesis is a process of blood vessel formation from the preexisting capillaries. It plays an essential role of growth and development of cancer. The aim of this study was to evaluate serum levels of the most important angiogenic stimulators, Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF) in children with acute lymphoblastic leukemia (ALL) and their relation to clinical manifestations of the disease, and in healthy controls. Although VEGF and bFGF have been suggested to be reliable prognostic indicators and important tools for treatment approach in hematopoietic malignancies and solid tumors, experience in childhood ALL has been limited. Patients and methods: 46 children with ALL (24 male, 22 female) aged 2-18 years (median 8 years) at the time of diagnosis and in remission and 70 healthy children (31 male, 39 female). For the quantitative determination of human VEGF and bFGF, the Quantikine (R&D Systems, Minneapolis, MN, USA), a solid phase enzyme-linked immunosorbent assay method was used. Elevated VEGF and bFGF were defined as being higher than the 95 percentile value in control group. Results: The range of VEGF serum levels in healthy controls was 24.73–467.7 pg/ml (median 168.9 pg/ml), 95 percentile was 431.85 pg/ml and the range of bFGF serum levels was 0–10.8 pg/ml (median 2.9 pg/ml), 95 percentile was 6.95 pg/ml. In children with ALL, the range of VEGF serum levels at the time of diagnosis was 0–532.3 pg/ml (median 91.18 pg/ml), and bFGF 0–64.48 pg/ml (median 5.11 pg/ml). In children in remission, the range of serum levels of VEGF was 53.15–962.67 pg/ml (median 209.73 pg/ml), and bFGF 0–32.5 pg/ml (median 5.98 pg/ml). The median level of VEGF at diagnosis was lower than those of the control group (p=0.006), and higher in remission, when compared to values obtained in children on diagnosis and in the control group (p=0.0005; p=0.01). Elevated level of VEGF was observed in 8.7% children at the time of diagnosis and in 24.4% of patients in remission. The median levels of bFGF at diagnosis and in remission were significantly higher than those in control group with 40% of children having elevated levels. A positive correlation between VEGF serum concentration and platelet number, and negative correlation between VEGF serum levels and WBC were observed, with no other correlations between growth factors (VEGF, bFGF) and age, type of lymphoblasts (FAB), risk group, and drug resistance. Conclusion: These results suggest that bFGF more than VEGF can play an important role in childhood ALL. The serum levels of angiogenic factors may be related to the activity of the disease, while both growth factors can possibly be a target of anti-angiogenic therapy.
Microarray Gene Expression Studies Reveal Potential Classifier Genes That Allow Prediction of Response to Interferon in Chronic Myeloid Leukemia Patients.
Authors: Barbany Gisela , Hagberg Anette , Strömberg Ulla Olsson , Isaksson Anders , Simonsson Bengt
Published: April 2020

Interferon has been extensively used to treat CML patients over the past years. However, not all patients respond and the reasons for this heterogeneity are largely unknown. Microarray-based gene expression analysis enables the study of thousands of genes in one single experiment and has been shown to be a useful tool to assign samples to specific categories. The goal of the study was to identify gene/s whose expression will permit prediction of response to interferon. Blood samples (n=9) or leukapheresis products (n=11) were collected from 20 CML chronic phase patients at diagnosis and all patients were subsequently treated with a-interferon. Responders were defined as patients achieving a complete or major cytogenetic response at 12 months or earlier after initiation of interferon (n=9) and patients with no cytogenetic response 5 to 18 months after interferon start were assigned to the non-responder group (n=11).

Mononuclear cells from two healthy blood donors were pooled and the RNA used as a reference sample. RNA was extracted from mononuclear or leukapheresis cells and gene expression profiles were analyzed using cDNA microarrays with approximately 7500 genes. Genes were organized according to the absolute difference in the median expression levels between the two groups and the top 20 genes were then used in an “all pair” selection procedure to identify the gene/s with the highest prediction accuracy. Six genes were selected by this procedure: RNAse2, PRG2, NRGN, LTF, JARID1A and DEFA4. These genes had an estimated error rate in predicting interferon response of 0,13 with a Fisher linear discriminant test.

Our results show that gene expression analysis may permit the identification of genes whose expression levels may be useful in the prediction of response to interferon in CML diagnostic samples.
Posttransplant Immunotherapy with a GM-CSF-Based Tumor Vaccine (GVAX®) Following Autologous Stem Cell Transplant (ASCT) for Acute Myeloid Leukemia (AML).
Authors: DeAngelo Daniel J. , Alyea Edwin P. , Borrello Ivan M. , Levitsky Hyam I. , Stock Wendy , Sher Dorie , Damon Lloyd E. , Linker Charles A. , Donnelly Amy , Hege Kristen M.
Published: April 2020

Background: Preclinical models have demonstrated the efficacy of GM-CSF secreting cancer vaccines accompanied by vaccine-primed lymphocyte infusion following ASCT.

Methods: Patients ≤ 60 years old with de novo AML (excluding M3) were enrolled. Leukemia cells were harvested at diagnosis followed by induction and consolidation chemotherapy and ASCT. A pretransplant vaccine composed of irradiated autologous leukemia cells mixed with GM-CSF gene-modified K562 cells (CG9962) was given followed by collection of vaccine-primed lymphocytes that were reinfused with the stem cell graft. Posttransplant vaccinations were initiated at week 6 (or upon platelet engraftment) and given every 3 weeks (10x107 tumor cells + 4x107 CG9962 cells) x 8 vaccinations.

Results: Leukemia cell harvest was successful in 51/54 patients (blood draw − 22, leukapheresis − 28, bone marrow aspirate − 4). To date, 44/54 patients (81%) have achieved a complete remission (CR) following induction chemotherapy, 27 have received the pretransplant vaccination, and 15 have initiated posttransplant vaccinations. Local vaccine injection site reactions developed in all patients. Delayed-type hypersensitivity (DTH) reactions to irradiated, autologous tumor were induced post vaccination in 4/11. Antibodies reactive against autologous tumor were induced in 5/11 and against CG9962 cells in 10/10. Minimal residual disease (MRD) is being monitored by quantitative analysis of peripheral blood (PB) and bone marrow (BM) for WT1, a leukemia-associated gene, by RT-PCR. All patients had detectable WT1 transcripts in PB and BM at diagnosis. Most patients in CR had persistently detectable WT1 transcripts in BM following induction (92%), consolidation (91%), and ASCT (63%), whereas, clearance of WT-1 in PB was more frequent (49% detectable post induction, 50% post consolidation, and 31% post ASCT). A decrease in WT1 was noted in 67% (12/18 in the BM and 10/15 in PB) following the pretransplant vaccination.

Conclusions: Collection of large quantities of autologous leukemia cells for vaccine production is feasible. Reduction in WT-1 transcript levels following the pre-transplant vaccine suggests anti-leukemic activity of this GVAX® vaccine platform.
The Comparative Analysis of Serum Proteomes for the Discovery of Biomarkers for Acute Myeloid Leukemia.
Authors: Kwak Jae-Yong , Lee Na-Ri , Kim So-Ri , Yim Chang-Yeol , Song Eun-Gi , Kwak Yong-Geun
Published: April 2020

Acute Myeloid Leukemia (AML) develops as the consequence of a series of genetic changes in a hematopoietic precursor cell. However, the definitive diagnostic protein-biomarkers for AML are still unclear, and monitoring these markers has not yet been completely achieved. In our study, to identify the biomarkers for an initial diagnosis, detection of relapse and monitoring the minimal residual disease in acute myeloid leukemia (AML) by a lesser invasive method, serum proteins reflecting alteration in their proteomes were analyzed. We compared two-dimensional electrophoresis patterns of human sera of twelve patients with acute myeloid leukemia with that of normal twelve subjects. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization-time-of flight (MALDI-TOF) and electrospray ionization quadupole time of flight (ESI-Q-TOF) mass spectrometry. Eight proteins that expressed differentially in AML group were found. The expression levels of alpha-2-HS-glycoprotein, complement-associated protein SP-40,40, RBP4 gene product, lipoprotein C-III and an unknown protein were down-regulated in serum of AML patients, whereas the other three proteins including immunoglobulin heavy chain variant, proteosome 26S ATPase subunit 1 and haptoglobin-1 up-regulated. These results suggest that these proteins can be used as less invasive diagnostic and monitoring biomarkers of AML if further studies were done.
Prognostic Significance of Molecular Remission Determined by Highly Specific PCR after Dose-Reduced Allogeneic Stem Cell Transplantation (SCT) in Patients with Multiple Myeloma.
Authors: Zagrivnaja Maria V. , Badbaran Anita , Corradini Paolo , Fehse Boris , Zander Axel R. , Kröger Nicolaus
Published: April 2020

Achieving molecular remission after standard allogeneic stem cell transplantation in multiple myeloma is associated with long term freedom of disease and possible cure. We recently reported a high rate of complete remission after an auto-allo tandem approach using fludarabine (150 mg/m²), melphalan (140 mg/m²) and anti-thymocyte globulin (ATG: 3 x 10–20mg/kg) three months after a cytoreductive autograft (melphalan 200 mg/m²). To determine the incidence of molecular remission we tried to develop allele-specific oligonucleotides (ASO) based upon the clonal rearrangement of immunoglobulin heavy chain genes and ASO - polymerase chain reaction (ASO-PCR) for each patient who achieved complete remission with negative immunofixation after SCT (n=27). The specificity of the ASO-PCR was tested using patient and control DNA. Patient DNA isolated from bone marrow (BM) samples obtained at diagnosis and after allografting at variable time points was used for further molecular analysis. For nine patients we were able to generate specific primers and MRD diagnostics have been performed for a median of two years after SCT. Molecular remission was strictly defined by the absence of any PCR-positivity. In 6 patients nested PCR remained negative after a median follow up of 22 months (range 10–24). Only one of these patients relapsed with extramedullary disease (14 months) still being PCR-negative in his bone marrow. In contrast, all those patients with complete remission but positive PCR (n=3) relapsed (at 11, 14 and 16 months after SCT, respectively) thus underlining the importance of achieving molecular remission after allo-SCT. We suppose that quantitative real-time PCR might be a useful tool to closely monitor MRD kinetics post allo-transplantation in order to verify efficiency of various treatment regimens, e.g. donor lymphocyte infusions.
Results of Conventional Cytogenetics and Interphase FISH (I-FISH) Analyses in Patients with a Clinical and Morphologic Diagnosis of CML: Analysis of 52 Cases.
Authors: Gupta Seema , Berman Ellin , Jhanwar Suresh
Published: April 2020

The presence of the Philadelphia chromosome (Ph), derived from a balanced translocation between chromosomes 9q and 22q, serves as a hallmark for the diagnosis of CML or Ph-positive ALL. In recent years, I-FISH has become the method of choice to detect Ph-positive clones at diagnosis and during treatment. In addition, I-FISH analysis is also able to identify variant translocations and loss of DNA sequences, some of which are known to be predictors of poor clinical outcome. To determine the contribution of I-FISH in detecting genetic alterations at diagnosis and during therapy, we summarized our experience in a series of patients with CML and Ph+ ALL studied in a single institution between 2000–2004. A total of 52 patients had both cytogenetic and I-FISH analyses available for review. The 52 patients included 30 patients in chronic phase, 10 patients in accelerated phase, 9 patients in blastic phase, and 3 patients with acute lymphoblastic leukemia (ALL). Sixteen of the 52 patients had newly diagnosed CML, and the remaining 36 patients had studies performed while on imatinib. Conventional cytogenetics detected the Ph-chromosome in 44 patients (85%), whereas I-FISH detected Ph chromosome positivity in all 52 patients (100%). In three of the 16 patients with newly diagnosed disease, I-FISH identified variant translocations in the setting of a normal karyotype. Two of these 3 patients were found to be RT-PCR negative for bcr/abl transcripts. Nineteen of 52 patients (37%) had additional abnormalities detected by karyotype. Nine of these 19 patients (47%) had more than one additional cytogenetic abnormality. The most common abnormalities in the Ph+ clone included: 6 patients with 2 copies of the Ph chromosome, 4 patients with trisomy 21, 2 patients with deletion (20q), 2 patients with isochromosome 17, and 2 patients with trisomy 8. Independent clones with trisomy 8 were noted in an additional 2 patients on imatinib therapy. A total of 12 of the 52 patients (23%) had variant signal patterns by I-FISH; loss of DNA sequences on chromosomes 9 and 22 occurred in 4 and 3 cases respectively and a variant signal pattern involving additional chromosomes was seen in 5 patients. While in general we found a good correlation between the proportion of Ph-positive cells detected by conventional cytogenetics and I-FISH, differences between the two types of analyses were observed in 8 patients. Six patients had a normal karyotype (20 cells analyzed) with positive I-FISH analysis (range 3.2–99%). Two of the 8 patients had a small percentage of Ph-positive metaphases with negative I-FISH. These results underscore the significance of utilizing both conventional cytogenetics and I-FISH in CML patients at diagnosis as well as during therapy. The biologic and clinical implications of the disparity between conventional cytogenetics, I-FISH, and in some cases RT-PCR results are not yet fully realized.
FLT3 Internal Tandem Duplications in Adults with De Novo Acute Myeloid Leukemia.
Authors: Xavier Sandra G. , Hassan Rocío , Clementino Nelma C.D. , Tabak Daniel G. , Spector Nelson , Zalcberg Ilana R.
Published: April 2020

FLT3 is a receptor tyrosine kinase involved in the proliferation and differentiation of hematopoietic stem cells. Recently, internal tandem duplication (ITD) mutations of the FLT3 gene have been described in patients with AML and associated with a poor prognosis. The aim of this study was to analyze the prevalence of FLT3-ITD in a series of 90 adults with de novo AML and correlate the presence of this mutation with biological characteristics and clinical response. We analyzed diagnostic peripheral blood or bone marrow specimens from 43 women and 47 men, with a median age of 38 years (16–83). Polymerase chain reaction was performed on genomic DNA using previously published primers for exons 11 and 12. An FLT3-ITD was found in 22/89 patients (25%). It was present in 37% (9/24) of the patients with acute promyelocytic leukemia (APL) and in only 20% (13/65) of the patients with non-M3-AML (p=0.07). The FLT3-ITD was not detected in patients with M6 (n=1) and M7-AML (n=3), nor in patients with the AML1-ETO (n=2) or with the CBFb-MYH11 (n=4) fusion genes. The median WBC counts were higher in FLT3-ITD patients than in those without the mutation (37 X 109/L vs. 27 X 109/L, p=0.43). In APL, FLT3-ITD was found in 5 out of 6 patients with the short PML-RARa isoform, but in only 4 out of 18 patients with the non-short isoform (p=0.01). Univariate analysis showed an association between the presence of FLT3-ITD and both a lower complete remission (CR) rate (41% vs. 64%; p=0.05) and a shorter overall survival (14% vs. 34%; p=0.03). However, FLT3-ITD was not associated with the CR rate (p=0.18) or the OS (p=0.07) in the multivariate analysis. The clinical significance of FLT3-ITD in adult AML remains uncertain, and further investigation is clearly warranted.
Toxicity of ALL Treatment in Adolescents as Compared to That in Younger Children.
Authors: Dijkema Tim , GrootLoonen Jacqueline , Bokkerink Jos , Hoogerbrugge Peter
Published: April 2020

Introduction: The results of therapy in young adults and adolescents with ALL has reported to be better if more aggressive pediatric treatment regimens were applied as compared to regimens more commonly used in adults (E.g. deBont, ASH 2003; Boissel, JCO 2003). A point of concern in applying pediatric regimens in young adults is the possible toxicity of these regimens. To address this, we retrospectively evaluated the toxicity of childhood ALL regimens in younger children as compared to older children and adolescents.

Patients: 90 patients treated for ALL in our hospital between 1985 and 2003 were included in the study. Treatment consisted of ‘classical’, multi-agent, pediatric regimens of the Dutch Childhood Oncology Group and the I-BFM, and included dexamethasos (n=47) or prednison (n=4) during induction and maintenance therapy in the majority of the patients. The survival and toxicity data of 66 children aged 3 to 9 years (mean 6 +/− 1 year) were compared with those in 24 children aged 13 to 17 (mean 15 +/− 1 year). Toxicity analysis included infectious complications, renal and liver function, bone density and occurrence of diabetes.

Results: As expected, as older age is a risk factor in childhood ALL, survival was better in the younger group (87% vs. 67%), although the survival in the older group is still better than in most adult-studies (Boissel, JCO 2003, deBont, ASH 2003). Older children showed more corticosteroid-related toxicity: Insulin-dependant hyperglycemia occurred in 25% of older patients, as compared to 0% in the younger group. Also, bone-density was significantly more reduced in patients treated with dexamethason-including maintenance treatment in older patients (Z-score of -2.8) as compared to the younger patients (Z-score of -1.5) at the end of treatment. No differences in renal and liver toxicities were seen between the two groups. Fever with neutropenia occurred more often in the younger patients (NS), and also treatment delay was more often necessary in the younger patients.

Conclusion: Childhood-ALL regimens are regarded as possible beneficial in older patients as compared to ‘adult regimens’. However, major concern is the possible toxicity of these pediatric regimens. Our data show that toxicity of these aggressive regimens is similar in older children/adolescents as compared to the toxicity in younger children. Only reversible corticosteroid-induced toxicity was more prominent in older patients, other toxicities were similar or even less in older patients. These data suggest that ‘pediatric regimens’ are well-tolerable in older patients with ALL.
Kringle IV Encoding Domains in the Apo(a) Gene and Serum Lipoprotein(a) Level Do Not Contribute to Avascular Necrosis of Bone (AVN) in Acute Lymphoblastic Leukemia.
Authors: Bezemer Désirée D.L. , van Beek Robert D. , Meijerink Jules P.P. , de Muinck Keizer-Schrama Sabine M.P.F. , Pieters Rob , van den Heuvel-Eibrink Marry M.
Published: April 2020

Introdution

Symptomatic AVN occurs as a serious complication of treatment in 4%–12.5% of pediatric cases with ALL. The final common pathway for the development of AVN involves a compromise in the blood flow, leading to infarction. The pathogenesis is multifactorial, including elevated intracortical pressure, vascular occlusion, altered lipid metabolism/fat emboli, hemostatic abnormalities and inhibition of angiogenesis. The difference in incidence of AVN in patients treated according to one and the same protocol suggests a genetic variation as a contribution to the biology of AVN. In one report, familial occurence of bone marrow edema syndrome (early phase of AVN) was associated with elevated levels of lipoprotein(a)[Lp(a)]. Lp(a) is a complex of low-density lipoprotein (LDL) and a high molecular weight glycoprotein called apolipoprotein(a)[apo(a)]. The Lp(a) level is inversely correlated with the kringle IV repeat number in apo(a), that is present as a naturally variation in the population.

Methods

We investigated the Lp(a) levels and the genetic variation in the Apo(a) gene in pediatric patients with and without AVN. DNA from mononuclear cells was isolated from 10 ALL patients with AVN (group I) and 12 patients without AVN (group II), all matched for age, gender and protocol. AVN is defined as pain of the extremities, not related to treatment with vincristine, confirmed by finding typical abnormalities on MRI. The control group consisted of 13 healthy Caucasian subjects (group III). To detect the number of kringle IV repeats pulsed field gel elctrophoresis (PFGE) and Southern blot of genomic DNA was performed. Serum Lp(a) levels were determined by immuno-nefelometric detection method.

Results

The median number of kringle IV repeats (mean of both alleles) did not significantly differ between the three groups: 25 (range 16–37, group I), 24 (range 13–32, group II), 21 (range 10–36, group III). The median Lp(a) levels in ALL patients with AVN versus ALL patients without AVN were respectively 0.085 g/L (range <0.02–0.848) and 0.067 g/L (range <0.02–0.362). Again the difference was not significant.

Conclusion Our results suggest that Lp(a) and genetic polymorphism of apo(a) do not play a mayor role in the pathogenesis of AVN in pediatric ALL.
Differential Expression of VEGF and Its Receptors in the Primary Cells of Various Risk Classified Acute Lymphoblastic Leukemia Patients.
Authors: Liang Aibin , Li Li , Gu Long-Jun , Jiang Hua , Ye Hui
Published: April 2020

Expression of VEGF and its receptors (Flt-1, KDR) were assayed by means of ELISA and RT-PCR on healthy donors (20 cases), patients under remission (20 cases), patients of standard risk (29 cases) and high-risk group cases (12 cases). Meanwhile, the clinical data of all the patients enrolled in this study were collected and then analysed with the detection of VEGF and its receptors. Results showed that expressions of VEGF and its receptors are diversely detected in 4 groups studied. VEGF and its receptors are expressed higher in high risk group than standard risk group, however, compared to remission group and healthy donors standard risk patients expressed higher VEGF and its receptors. Concordantly, the clinical data are in line with the expressions of VEGF and its receptors on all leukemia samples studied. Patients with higher expression of VEGF and its receptors often have higher peripheral white blood cell counts, severe liver infiltration, higher LDH value and poor treatment response. We also found that expression of VEGF and its receptors are not associated with age, sex, sample status and cytogenetic characteristics. This is demonstrated that ALL patients with highly expressed VEGF and its receptors are usually with higher tumor burden, especially in high-risk cases. Detection of VEGF and its receptors might be one of prognostic marker for ALL treatment.
Hyaluronan in the Bone Marrow of Healthy Individuals and Patients with Acute Myeloid Leukaemia.
Authors: Sundstrom Gunnel M. , Dahl IngerMarie S. , Hultdin Magnus , Wahlin Anders , Engstrom-Laurent Anna
Published: April 2020

In the connective tissues the two major structural components are collagens and glycosaminoglycans (GAGs). When assessing pathological processes in the extracellular matrix, e.g. fibrosis, attention is primarily directed to the collagens. Hyaluronan (HYA) is a GAG of great interest not only as a tentative marker of early fibrosis but also of importance for the disease process in various malignancies.

Acute myeloid leukaemia (AML) is a clonal disorder characterised by abnormal proliferation of myeloid cell precursors. Research has mainly focused on the cellular events but the bone marrow matrix has attracted minor interest.

In this study bone marrow biopsies were obtained from 35 newly diagnosed AML patients and 30 healthy individuals. The bone marrow sections were analysed histochemically for HYA and reticulin (type III collagen).

The HYA staining was significantly stronger, p=0.003, in the AML patients compared to the healthy controls. Only one patient demonstrated abnormal reticulin staining score, but in the group of 8 patients with antecedent myelodysplastic syndrome (MDS), the reticulin staining score was significantly higher, p=0.003, compared to the patients with de novo AML. There was a significant correlation between the HYA and reticulin staining scores, p=0.005, in the AML patients as was seen in the control group, p=0.0005, indicating that the variables HYA and reticulin are not independent. No correlation was found between HYA grading and lactate dehydrogenase (LDH), number of blast cells, sex, age or antecedent blood disease before AML diagnosis. In the control group there was a significant correlation, p=0.042, between HYA and age.

Our findings establish that there is a deposition of HYA in bone marrow in healthy individuals and that HYA is increased in AML patients. It is discussed whether HYA could be an early marker of impending fibrosis and also exert important effects on cellular events in the disease process.