bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Human Multiple Myeloma Cells with High-Resolution Magic Angle Spinning Magnetic Resonance Spectroscopy
Authors: Oriol Albert , Barba Ignasi , Barbera Angels , Arús Carles , Garcia-Dorado Jose-Luis , Ribera Josep-Maria
Published: April 2020

Advancements in the pathogenetic pathways in multiple myeloma have led to the identification of several primary and secondary genetic lesions and ultimately to a multiple myeloma genetic classification with prognostic implications. Although disregulation of cyclin activity has been recognized as a key event leading to the multiple myeloma phenotype, little is known about the metabolic consequences of this phenomenon. We have studied intact multiple myeloma cells by high resolution magnetic resonance spectroscopy to establish the metabolomic profiles of different native multiple myeloma cells as compared to other lymphoproliferative disorders.

Multiple myeloma cells obtained from bone marrow aspirates (n =15), blood (n =3) or other biologic tissues (n =2) from 20 multiple myeloma patients and separated by density gradient centrifugation were evaluated and metabolic profiles were correlated with cytogenetic characteristics of the disease and patients clinical characteristics.

Twelve patients were females (60%) with a median age of 65 years (range 50–82). Multiple myeloma monoclonal proteins were IgG (N=9), IgA (N=5) or BJ (N=6). Five of them (25%) had renal insufficiency. Nine patients (45%) had predominantly extramedullar diesase including four cases of plasma cell leukemia. IgH translocations were identified in 5 samples (25%), hyperploidy in 2 (10%), and other or no genetic lesions in 13 (65%), del13 was present in 9 samples (45%) and p53 alterations in 5 (25%).

Bone marrow samples from thirteen patients with conventional multiple myeloma presented a relatively constant metabolic pattern with predominantly lipidic signals and a metilen to metil ratio ranging from 1.9 to 4.9 (median 2.9). No differences in this pattern were observed among subgroups of primary translocations or involvement of Rb and p53 genes. Four patients with plasma cell leukemia and three with predominant extranodal disease presented either non detectable lipid signals (N=3) or a higher metilen to metil ratio ranging from 2.8 to 3.9 (median 3.5). In fact, extranodal or leukemic disease was significantly associated to undetectable lipids (P < 0.031) or the composite variable undetectable lipids or metilen to metil ratio > 3 (P < 0.043). Furthermore, after a median follow-up of 18 months, absence of lipids in the metabolic profile was also associated to a shorter survival (median 0.45 years, 95%CI 0–1.03 versus 3 years, 95%CI 0.95–5.06, P < 0.022).

These results suggest that metabolic profiles of different multiple myeloma genetic subtypes share common and reletively constant characteristics, while cells obtained from patients with plasma cell leukemia or predominantly extramedullar disease present a clearly distinct profile, probably reflecting the metabolic effect of clonal evolution at a genetic level.
The Prognostic Significance of 8p21 Deletion in Multiple Myeloma
Authors: Nahi Hareth , Sutlu Tolga , Alici Evren , Gahrton Goesta
Published: April 2020

In this study, we have investigated the prognostic impact of fluorescence in situ hybridisation (FISH) results for the chromosomal regions 1q21, 6q21, 8p21, 9p21, 13q14, 15q22, 17p13, 19q13 as well as the translocations t(4;14) and t(11;14) in patients with MM receiving ASCT. Randomly selected 65 patients were analyzed by FISH for these regions and hierarchical clustering analysis revealed associations between the occurrences of various chromosomal abnormalities. 58 patients received high-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) and 14 of these patients had monoallelic loss of the 8p21 region. Overall survivals (OS) and progression-free survivals (PFS) were significantly lower in patients with del(8p21). Moreover, multivariate analysis revealed that the occurrence del(8p21) is an independent prognostic factor for both OS and PFS. These findings suggest that the chromosomal region 8p21 could be monitored and used for the prognosis of patients with MM undergoing ASCT.
Alkylator-Induced DNA Damage Response in Multiple Myeloma (MM) Cells Is Amplified by Histone Deacetylase (HDAC) Inhibition, Resulting in Significant Synergism in Cell Death
Authors: Lee Choon Kee , Wang Shuiliang , Huang Xiaoping , Ryder John , Ordentlich Peter , Liu Bolin
Published: April 2020

One of the main mechanisms of action of HDAC inhibitors is the transcriptional reactivation of dormant tumor-suppressor genes through acetylation of histones, thereby inducing apoptosis. Treatment with HDACI has also been shown to induce chromatin destabilization in a transcription independent way. In the current study, we sought to determine whether HDAC inhibition induces DNA damage and amplifies alkylator-induced mitotic cell death in both melphalan sensitive- and resistant-MM cell lines (RPMI8226, 8226/LR5). The IC50 values of SNDX275, a class I HDACI agent, and melphalan on the 72-hour MTT assay were 268.05 nM and 245.94 nM in the RPMI8226, and 309.91 nM and 8657.46 nM in the 8226/LR5, respectively. When combined together at clinically attainable concentrations, the combination index by the Chou-Talalay method ranged from 0.27 to 0.75 for the RPMI8226 and from 0.33 to 0.7 for the 8226/LR5, indicating a powerful synergism. For elucidation of molecular mechanisms, MM1S and RPMI8226 cell lines were investigated for apoptosis, histone acetylation, cell cycle analysis, DNA double strand break and DNA damage response serially in 48-hour culture with SNDX-275 at 500 nM and melphalan at 10 μM, alone and in combination. Cleavage of PARP was seen following treatment with each SNDX275 and melphalan, but was highest at 48 hours with the combination of both. Apoptosis was associated with cleavage of caspases of 8, 3 and 9, which was most intense on combination. Melphalan amplified SNDX275-induced acetylation of H3. In cell cycle analysis by flow cytometry, SNDX275 caused an increase in G0-G1 and a decrease in S and G2-M. Cyclin D1, E2F-1 and p53 on western blot were not affected but expression of p21 increased. Melphalan arrested the cell cycle at G2, increased expression of p53 in the RPMI8226 and of p21 in the MM1S. The combination intensified the increase in p21 in both cell lines and in p53 only in the RPMI8226. Phosphorylation of H2AX, a marker of DNA double strand break, increased in a time dependent manner following each drug, along with an increase in phosphorylation of CHK1 and CHK2, indicative of initiation of DNA damage response. The increase in γH2AX and pCHK1 & 2, however, was considerably higher on combination than each drug alone. Furthermore, morphologic assessment of dead cells by the 48 hours of culture revealed a significant increase in mitotic catastrophe on combination in the MM1S: 0% on SNDX275 alone; 10% on melphalan alone; 43.4% on combination. The current study suggests that HDAC inhibition synergizes with melphalan in MM cells and that intensification of DNA damage is one of the mechanisms. Further studies are necessary to understand the role of HDAC inhibition for induction of mitotic catastrophe.
The Clinical Study of Combination of Bortezomib and Dexamethasone in Treatment of Newly Diagnosed Multiple Myeloma
Authors: Li Juan , Zeng Lijin
Published: April 2020

OBJECTIVE: To study retrospectively the response and side effects in two groups of patients with newly diagnosed multiple myeloma (MM) receiving bortezomib and dexamethasone regimen (VD) and vincristine combined with pirarubicin and dexamethasone and melphalan regimen (VADM).

METHODS: Nineteen patients were enrolled in a group of VD, receiving bortezomib 1.3mg/m2 on days 1,4,8,11 and dexamethasone 20mg on days 1–4 in a 21-day cycle. Blade Standard was used to evaluate the therapeutic effect and NCI-CTC was used to assess the adverse effect. Thirty-one matched patients with newly diagnosed MM who received VADM were used as a historical control group, receiving vincristine 0.4mg/d and pirarubicin 9mg•m−2•d−1 and dexamethasone 20mg/d and melphalan 12mg/d on days 1–4, with 28 days as a cycle.

RESULTS: During the median 9 months’ follow-up of patients who received VD, there were 73.7% patients(14/19)responding to the treatment, including 9 cases (47.4%) of complete remission or near complete remission. Light-chain type patients who received VD had a higher overall response rate and CR +nCR rate than that of the VADM (P<0.05). The patients receiving VD who had renal inadequacy had an effective rate of 69.6% (5/6), which was similar to that of the others (69.2%, 9/13), and renal function relieve could be shown in the chemotherapy. The main adverse effects were fatigue, diarrhea, peripheral neuropathy, thrombocytopenia, and infection, with incidences of 73.8%, 68.4%, 63.2%, 31.5% and 26.3% respectively. Most of the adverse effects were mild and could be relieved by symptomatic treatments. The most common adverse event in the control group was neutropenia (83.8%), followed by infection(35.5%), vomiting (35.5%) and loss of hair(32.5%).

CONCLUSION: Although there was no significant difference of overall response rate between VD and VADM, VD has higher CR +nCR rate compared with VADM. VD can be tolerant in most patients, and is also safe in patients with renal inadequacy.
Screening JAK2 Exon 12 in JAK2 V617F Negative Patients with Myeloproliferative Disorders Reveals a New Splice Site Mutation
Authors: Bento C. , Estevinho A. , Rapado I. , Grande S. , Matos H. , Coucelo M. , Osorio S. , Albizua E. , Molinero R. , Duarte M. , Menezes C. , Kaeda J. , Martinez-Lopes J. , Ribeiro M. L.
Published: April 2020

Background. The identification of the somatic mutation V617F in exon 14 of the Janus kinase 2 gene (JAK2) has simplified the diagnosis of many patients affected with typical chronic myeloproliferative diseases (MPDs). In patients without the V617F the molecular basis of MPD are still unclear but, recently, mutations in exon 12 of the JAK2 gene have been identified in a minority of patients, associated with a selective increase in erythropoiesis resulting in polycythemia vera (PV) or idiopathic erythrocytosis (IE) (Scott et al, NEJM 2007).

Aim. To determine the JAK2 exon 12 mutational status in a group of JAK2 V617F negative patients with PV or IE.

Methods. Genomic DNA was extracted from peripheral blood leukocytes from 85 MPD patients (PV or IE) included in the present study. All the samples were tested for JAK2 V617F mutation by allele specific polymerase chain reaction (ASO-PCR) and those negative for V617F mutant allele were subjected to real time quantitative PCR (RQ-PCR) using hybridization probes. Subsequently, all JAK2 V617F negative samples by both ASO-PCR and RQ-PCR where subjected to direct sequencing to exclude JAK2 exon 12 mutations.

Results. JAK2 V617F was positive in 78 (91.7%) of the 85 patients, however, in two of these patients the V617F mutation was only detected upon subjecting genomic DNA to RQ-PCR which revealed low levels of the mutant allele, 2.65 % and 3.98%. Analysis of the JAK2 exon 12 in the seven JAK2 V617F negative patients detected three previously described mutations: a duplication (V536-1546) and two deletions (H539-K540del+542K and R541-E543 delinsK)and a previously unreported splice site mutation detected in intron 12 (IVS12nt6 T-C). To our knowledge this was the first description of intronic mutations in the JAK2 gene. No mutations were detected in the remaining 3 (3.6%) patients.

Conclusions. In this cohort study JAK2 mutations were observed in 82 (96,5%), of the 85 patients, of these 78 (95.12%) had the V617F allele. In two cases V617F was detected at low levels (2.65% and 3.98%) only by RQ-PCR, highlighting the need for sensitive techniques to detect somatic mutations. One of the patients, a young male with erythrocytosis and low serum Epo levels, revealed a previously unreported splicing mutation (IVS12 nt6: T-C). This study illustrates the heterogeneity at the DNA level in PV patients which may assist in better understanding the genotype and phenotype relationship in MPD patients and assist in further delineating the role of JAK2 in these disorders.
The Overall Analysis of JAK2 and MPL Mutation Status in Chinese MPD Patients by MassARRAY Assay
Authors: Zhang Sujiang , Li Jianyong , Shi Jingyi , Xia Jun
Published: April 2020

In recent three years the new notable progress associated with myeloproliferative disorder (MPD) was the identification of JAK2 and MPL mutations, which have been confirmed to be the major molecular mechanism and marker of BCR/ABL negative MPD. Multiple techniques including allele-specific PCR, conventional DNA sequencing, pyrosequencing as well as BsaXI restriction analysis have been used to detect these mutations. The previous data of Shanghai Group had demonstrated the sensitivity and preliminary result of JAK2 V617F in 162 Chinese patients with MPD using MassARRAY assay. To generally identify the frequency of these mutations in Chinese MPD patients, we continued to employ this technique-MassARRAY assay to detect JAK2 V617F, JAK2 K539L as well as MPL W515L mutation in a larger scale of Chinese MPD patients. A total of 204 Chinese MPD patients were enrolled in our study. These patients were referred to polycythemia vera (PV) (n=55), essential thrombocythemia (ET) (n=110), primary myelofibrosis (PMF) (n=29) and hypereosinophilic syndrome (HES) (n=10). 2 ml peripheral blood was obtained with informed consent and genomic DNA was isolated. The diagnosis of MPD was established according to the 2001 WHO diagnostic criteria. Finally, 187 patient samples with good signal can be analyzed for JAK2 V617F mutation. Among the available 101 signals of ET patients, 30 were heterozygous mutation and 9 were homozygous mutation. Among 52 PV patients, 25 were heterozygous mutation and 14 were homozygous mutation. Among 25 PMF patients, 9 were heterozygous mutation and 5 were homozygous mutation. None of HES patients was found harboring JAK2 V617F. In addition, we identified a PV patient harboring JAK2 K539L but not V617F mutation (1/52, 1.9%), and an ET patient harboring MPL W515L mutation (1/101, 1%). Both results were further confirmed by sequence analysis. Hence, we concluded that JAK2 V617F underlie the major molecular pathogenesis of Chinese MPD patients especially PV, however, JAK2 K539L and MPL W515L are rare events in these patients.
Poor Survival Predicted by MDM2 Oncoprotein Expression in Diffuse Large B-Cell Lymphoma (DLBCL) with Wild-Type TP53 Gene
Authors: Rocque Gabrielle L. , Møller Michael B. , Colleoni Gisele W. B. , Sánchez-Beato Margarita , Green Tina M. , Thorborg Trine , Piris Miguel A. , Eickhoff Jens C. , Twohig Matthew , Malik Jeffrey T. , Young Allen H. , Oberley Terry D. , Malter James S. , Ferry Judith , Chan Wing C. , Weisenburger Dennis D. , Greiner Timothy C. , Young Ken H.
Published: April 2020

The p53 pathway is known to play a role in pathogenesis and prognosis of human malignancies including diffuse large B-cell lymphoma (DLBCL). The MDM2 protein has been shown to regulate p53 function and stability via a multi-factorial negative feedback loop. MDM2 is involved in transport of p53 out of the nucleus and its subsequent degradation. Additionally, MDM2 binds p53 protein and inhibits its function as a transcription factor. As an integral part of the regulation of the p53 pathway, we hypothesize MDM2 expression contributes to the pathogenesis of DLBCL and affects survival. The purpose of this study is to investigate the potential role of the MDM2 protein in DLBCL by correlating expression of MDM2 with p53 expression, TP53 mutation status and clinical outcome. Immunostains for MDM2 and TP53 gene-product proteins were performed in 133 cases from 6 medical centers. A positive immunostain was defined as nuclear staining in 10% or more of the tumor cells. The TP53 gene was analyzed for mutations with PCR-based and sequencing methods. Patients were treated with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) or CHOP-like regimen. The Kaplan-Meier method was used for survival analysis. MDM2 immunostains were positive in 64 of 133 cases of DLBCL (48%). There was a trend toward poor overall survival (OS) with MDM2 expression in the entire group of patient with DLBCL (p=0.32). Twelve of 24 cases (50%) with TP53 mutations demonstrated MDM2 expression. Within this TP53 mutated group, no significant difference in OS was observed between MDM2-positive and MDM2-negative cases (p=0.32). Of patients with wild-type (WT) TP53, 51 of 107 cases (48%) expressed MDM2. In this subset of patients, the MDM2-positive phenotype predicted for poor 5-year OS (38% vs 67%, p=0.002), and a significantly shorter median disease-free survival (2.3 years vs. >5.0 years; p=0.013). The complete remission rate was 57% in MDM2-positive cases compared to 73% in MDM2-negative cases in the WT-TP53 group (p=0.08). Multivariate analysis confirmed that MDM2 expression was an independent predictor of poor OS in patient with DLBCL with a WT-TP53 gene (HR 2.0, 95% CI 1.15–3.56; p=0.015). This study demonstrates the negative survival impact of MDM2 expression in patients with wild-type TP53, suggesting that MDM2 provides an alternative mechanism of p53 pathway inactivation in DLBCL cases with a WT-TP53 gene.
Diagnostic Role of Flow Cytometry Analysis of Bioptical Samples in the Diagnosis of Lymphoid Tumors
Authors: Cro Lilla , Ferrario Andrea , Zucal Nadia , Gianelli Umberto , Fabris Sonia , Nobili Lucia , Olivero Barbara , Rossi Francesca G , Baldini Luca , Deliliers Giorgio Lambertenghi
Published: April 2020

We reviewed flow cytometric immunophenotyping (FCI) data of 470 tissue suspensions suspected of being involved by lymphoma (371 lymph nodes, 263 surgical biopsies and 108 fine needle aspirations,16 spleens, 6 tonsils, and 77 other tissues) and we have compared corresponding histologic diagnosis. The screening panel of FCI (CD45, CD19, CD3, CD4, CD8, and sIgκ/sIgλ ratio) identified three main groups:

239 cases with demonstrable light chain restriction (sIgκ/sIgλ ratio ≤ 0.5 or ≥4); 37 cases with demonstrable T cell proliferation and polyclonal B cell population; 194 cases with polyclonal B cell and normal T cell populations (146 cases) or with not detectable CD45 reactivity and other leucocyte markers (48 cases).

In group 1, 235 cases were then evaluated by means of the following markers: CD5, CD10, CD11a, CD11c, CD20, CD22, CD23, CD30, CD38, CD43, CD79a–b, CD103, CD138, FMC7, and, in group 2, 30 cases were evaluated by means of the following markers: CD1a, CD2, CD5, CD7, CD16, CD26, CD43, CD56, CD57, CD45RA/RO, anti-TCR-ab gd. The complete analysis identified in group 1 four main diagnostic subsets: A (26 cases) characterized by CD5+, CD23+/±, sIg dim (CLL-like); B (36 cases) characterized by CD5+, sIg bright (MCL/CLLv); C (89 cases) characterized by heterogeneous expression of Ig, CD20 and CD43 (lymphoproliferative syndromes CD5−, excluding hairy cell leukemia); D (84 cases) characterized by CD10+, CD20 bright, CD43− (follicular NHL like). In cluster B, FISH analysis for t(11;14) resulted positive in 15/27 cases; in cluster D, FISH analysis for t(14;18) resulted positive in 60/60 cases. Histologic analysis convalidated FCI data in 23 cases of subset A (88%), in 29 cases of subset B (80%) and in 60 cases of subset D (71%). Subset C included 39 cases of LCL and 15 cases of MZL. In group 2, all cases studied expressed an aberrant T phenotype but, with the exception of 1 case of NK proliferation, 1 case of likely Sezary syndrome/Micosis Fungoides (CD4+CD7−CD26−) and 1 case of T lymphoblastic lymphoma (CD3cy+TdT+), in the other 34 cases was not possible identify other peculiar subset. In this group, histologic analysis included 19 cases of NHL T (63%). Considering NHL vs no NHL, sensibility (SE) and specificity (SP) of FCI analysis resulted 88% and 92%, respectively; positive predictive value (PPV) was 96% and negative predictive value (NPV 78%). Considering B-cell NHL only, SE was 91%, SP 95%, PPV 97% and NPV 84%. Even if histology is the basis for the diagnosis of lymphoid tumors, however our study supports that FCI can play an important role mainly in B-NHL diagnosis, combining rapidity of analysis with a multiparametric analysis, also in presence of size limitated samples.
FDG PET in the Staging and Management of Cutaneous T Cell Lymphoma
Authors: Woodward Catherine S , Halkar Raghuveer , Delman Keith , Parker Sareeta , King Nassoma , Lechowicz Mary Jo
Published: April 2020

We aimed to assess the usefulness of 18F-FDG PET in siting lymphoma involvement for CTCL patients. We retrospectively analyzed CTCL patients who had received a lymph node biopsy within approximately one month of a PET scan. Between 2003 and 2007, 19 CTCL patients (7 males, 12 females, age range: 4 – 70, median: 55) underwent a combined 56 PET scans. All studies were performed on GE Discovery LS 8 slice MDCT PET CT scanner or GE Discovery DST 16 slice MDCT PET CT scanner (Milwaukee, WI) and the images were interpreted on a GE AW or Xeleris workstation. The patients underwent whole body imaging from the head to foot at 4 minutes per bed position. Of these 19 patients, eight patients (four men, four women, mean age: 56.6, range: 49 – 70) received a lymph node biopsy following a PET scan that demonstrated elevated focal uptake (SUVs ranging from 2.2 to 9.9) in at least one lymph node. In four of the cases, the biopsied lymph node directly confirmed CTCL involvement sited in that node by PET scan (SUV 2.2 to 6.1). Three cases represented false-positive PET scans which were attributed to category 1 dermatopic lymphadenopathy and post radiation change. Despite these lack in correlations, in one case, PET sited adenopathy (SUV 9.9) in an inguinal node that biopsy pronounced benign. Further studies proved that a sampling error produced a false-negative biopsy. Despite the small sample, the study demonstrates the importance of PET imaging in staging patients with CTCL. Findings suggest that lymph node SUV values ranging from 2.2 – 9.9 often correlate with definite involvement of the nodes, and consequentially progression of the disease. It was found that false-positive PET scans may result from post radiation change or dermatopathic lymphadenopathy, category 1. The sample size was too small to detect differences in SUV and correlation in lymph node involvement. Our study demonstrates the need for improvement in the specificity of FDG-PET scans possibly by two point imaging and accurate localization of lymph nodes by use of an intraoperative probe to further enhance its usefulness in the staging of CTCL. Further prospective study is needed to determine correlation with SUV and lymph node involvement.
A Part of Acute-Type ATLL Cases Has Genomic Imbalance in Common with Lymphoma-Type ATLL
Authors: Nakagawa Masao , Nakagawa-Oshiro Aya , Tsuzuki Shinobu , Utsunomiya Atae , Seto Masao
Published: April 2020

Acute-type Adult T-cell leukemia/lymphoma (ATLL) is classified into four clinical subtypes (Acute, Lymphoma, Chronic and Smoldering). Acute-type ATLL comprises clinically heterogeneous groups because this subtype is defined as a group not classified into three other subtypes. When lymphoma-type progress to show PBL involvement of more than 1%, it becomes acute-type ATLL. We tentatively designate such cases showing leukemic cells in peripheral blood with prominent nodal involvement as “acute-type ATLL-PNI”. Clarification of the molecular basis for clinical heterogeneity of Acute-type ATLL is an important issue for therapeutic strategy. We previously studied acute-type ATLL with fulminating leukemic manifestation (more than 60% leukemic cells in peripheral blood) by array CGH and identified a part of the molecular basis for the ATLL subtype (Oshiro et al., Blood107:4500,2006). Here we analyzed six such cases of “acute-type ATLL-PNI” by means of array CGH consisting of 2304 artificial chromosome clones that cover the whole genome at a 1.3 mega base resolution. Regions of recurrent gain were detected on 2p16.1, 3p26.3-26.1, 3p24.2-24.1, 3p22.2-22.1, 3p14.3, 3p14.2-3q13.3, 3q25.32-26.2, 3q26.31-26.33, 3q27.3-29, 6p25.3-24.1, 7q32.3-33 and 17q23.3-24.2 and regions of recurrent loss on 4q21.22-21.23, 6q13-16.3 and 13q32.1-33.1. We compared the genomic profile of these six cases of “acute-type ATLL-PNI” with that of 49 lymphoma-type ATLL cases previously reported by us (Oshiro et al., Blood, 2006). They were very similar to each other and thirteen of the 15 regions of recurrent genomic imbalance in “acute-type ATLL-PNI” were also found in lymphoma-type ATLL (86.7%). Our findings clearly showed that “acute-type ATLL-PNI” have genomic imbalances in common with lymphoma-type ATLL rather than acute-type. Our array data also indicated that “clinically defined” acute-type ATLL contained leukemic-type and lymphoma-type with leukemic transformation.
MLL/AF10(OM-LZ) Transformed Cells Induce Myeloproliferative Disease-Like Myeloid Leukemia in the Sublethally Irradiated Mice
Authors: Fu Jen-Fen , Shih Lee-Yung
Published: April 2020

Chromosomal translocation t(10;11)(p12;q23) resulting in the fusion of MLL gene with AF10 is seen in patients with acute leukemias, especially in acute monoblastic leukemia. The OM-LZ domains of AF10 have been shown to be sufficient for immortalization when fused with 5′MLL, however, the leukemogenesis of MLL/AF10(OM-LZ) is not clear. By retrovirally transducing MLL/AF10(OM-LZ) fusion gene into the murine bone marrow cells, we established six MLL/AF10(OM-LZ)-immortalized cell lines in medium containing IL3, IL6, SCF and GM-CSF. The cytological and immunophenotypic studies showed that all the cell lines were consisted of immature blast-like cells and monocyt/macrophage cells, except that line MA6 also contained about 2% mature granulocytes. The percentage of mature granulocytes in line MA6 raised to 20% upon treatment with G-CSF. Virus integration site (VIS) analysis demonstrated that line MA6 had a VIS at PD-L2 gene. Whether interruption of PD-L2 gene plays a role in releasing of the granulocytic differentiation blockage caused by MLL/AF10(OM-LZ) is under investigation. Nevertheless, line MA6 and the other immortalized cell lines all induced a myeloproliferative disease (MPD)-like myeloid leukemia when they were transplanted into the sublethally-irradiated syngeneic mice. Further studies revealed that the excessive mature granulocytes in the peripheral blood of leukemic mice were generated from the recipient hematopoietic stem/progenitor cells. On the other hand, the hyperleukocytosis found in line MA6 leukemic mice may be partly attributed to the expansion and maturation of the immortalized cells. RT-PCR analysis revealed that the MLL/AF10(OM-LZ)-immortalized cells expressed mRNAs encoding colony-stimulating factors and inflammatory cytokines. The latter were known to be able to stimulate or up-regulate endothelial cells, fibroblasts, T-lymphocytes, granulocytes and monocytes to produce GM-CSF, G-CSF or M-CSF, and, therefore, indirectly regulate myelopoiesis. Our results demonstrated that although the MLL/AF10(OM-LZ)-immortalized cells could not terminally differentiated into granulocytes, they could still induce a MPD by expression and secretion of the colony-stimulating factors and cytokines to over-stimulate the myelopoiesis of the recipient hematopoietic stem/progenitor cells.
Bcl-2 Protects against p53-Induced Apoptosis through Hdm2
Authors: Wergeland Line , Spurgers Kevin B. , Oveland Eystein , Høiby Torill , Cascallo Manel , Honda Tsuyoshi , Lorens James B. , Lozano Guillermina , McDonnell Timothy J. , Gjertsen Bjorn T.
Published: April 2020

Hdm2 is up-regulated in several malignancies including sarcomas and acute myeloid leukemia, where it counteracts the anti-proliferative and pro-apoptotic effect of wild type p53. The anti-apoptotic protein Bcl-2 is often elevated in many tumors with wild type p53 and serves to block p53-induced apoptosis. We demonstrate that the protein level of Hdm2 positively correlates with the level of Bcl-2 and follows the Bcl-2 level in different cell systems. Over-expression of Bcl-2 protects Hdm2 from DNA-damage induced degradation in a dose dependant manner. In addition, modulation of Bcl-2 by shRNA knockdown reduced the Hdm2 protein level in parallel. Consequently, treatment of AML cells with the Bcl-2 small inhibitory molecule HA14-1 attenuated the level of Hdm2. The Bcl-2 level, but not the DNA damage induced Hdm2 degradation, was affected by disruption of the E3 ubiquitin ligase activity of Hdm2. In addition, the DNA-damage induced Hdm2 down-regulation was blocked by disrupted E1 ubiquitin-activation, defect polyubiquitination and by proteasome inhibitors. Finally, we show that Bcl-2 protection from p53-induced cell death requires co-expression of Hdm2 in double null p53/mdm2 mouse embryonic fibroblasts. Our results indicate that Bcl-2 regulates the Hdm2 level and that Hdm2 is a key mediator in Bcl-2 inhibition of p53-induced apoptosis. This is of particular therapeutic interest for cancers displaying elevated Hdm2 and Bcl-2, like sarcoma and acute myeloid leukemia.
Role of PI3-K and Mlck during Activation of Thrombin Receptors
Authors: Gao Haili , Sun Aining , Han Yue , Zhang Wei , Dai Lan , Bai Xia , Wang Zhaoyue , Depei Wu , Ruan Changgeng
Published: April 2020

Object In order to compare the influences of wortmannin on platelet aggregation and platelet membrane surface glycoproteins GPIb expression after thrombin receptors activation, then to investigate the role of phosphatidylinositol 3-kinase (PI3-K) and myosin light chain kinase (MLCK) in the course of thrombin receptors activation.

Methods Peptide SFLLRN (PAR1-AP) and AYPGKF (PAR4-AP)were used for stimulating platelet, then the alterations of platelet aggregation and GPIb were analyzed in the involvement of 100nM wortmannin (inhibitor of PI3-K) and 10μM wortmannin (inhibitor of MLCK).

Results Platelet activation was influenced by either concentration of wortmannin in response to PARs stimulation. Platelet aggregation was apparently inhibited by 10μM wortmannin following both peptides, and slightly inhibited by 100nM wortmannin only upon PAR1-AP activation. In addition, GPIbα interalisation is partly inhibited by 100nM wortmannin in response to PAR1 (P <0.05 at 1, 2, 5min) and PAR4 (P <0.05 at 2, 5, 10min) activation. Meanwhile, 10μM wortmannin induces little alteration for GPIbα centralisation in the course of PAR activation, with a delayed restoration of surface GPIbα observed upon PAR1-AP activation, and no change of GPIbα redistribution exists upon PAR4-AP activation.

Conclusion All the results confirm different roles of PI3-K and MLCK in the course of thrombin receptors activation. PI3-K accelerates the short course of GPIb centralisation for two PARs signal pathways, while MLCK inhibites the restoration of GPIbα in PAR1 pathway
Evaluation of the Role of Flow Cytometry as a Diagnostic Tool for Hereditary Spherocytosis
Authors: Pati Nalini K , Sartor Mary M , Wong Sue , Nemet Adrienne , Bradstock Kenneth Francis
Published: April 2020

Flow cytometric analysis of eosin-5′-maleimide-labeled red blood cells has been proposed as a method of identifying hereditary spherocytosis (HS). The flow cytometric test measures the fluorescence intensity of intact red cells labelled with the dye eosin-5-maleimide (EMA), which reacts covalently with Lys-430 on the first extra cellular loop of band 3 protein. Patients with HS have reduced fluorescence compared to other patient groups and normal controls. The aim of the present study was to assess the utility of flow cytometry in the diagnosis of hereditary spherocytosis. Fresh peripheral blood from 40 normal controls was collected in Lithium Heparin, stained with the dye EMA and analysed by flow cytometry. The normal range was established as 55–74 channel numbers. On 7 known cases of Hereditary Spherocytosis the MFI range was 35.6–44.6 channel numbers, and therefore samples falling into this range were considered to be positive for a diagnosis of HS by EMA. Equivocal results were defined when the MFI was in the range of 45–54 channel numbers. A total of 98 samples were sent for investigation or exclusion of HS. Clinical problems were grouped into the following categories: peripheral blood spherocytosis with negative Coomb’s test and no family history of HS, positive family history of HS, Neonatal hyperbilirubinaemia, haemolytic anaemia of unknown origin.

Indications Coomb’s −ve, Spherocytosis, no FH (n=50) (51%) Positive family history (FH) (n=18) (17%) Neonatal hyperbilirubinemia (NNH) (n=8) (8%) Haemolytic anaemia (HA) (n=16) (17%) Results of EMA Pos Neg Equi Pos Neg Equi Pos Neg Equi Pos Neg Equi Number 19 17 13 9 7 2 2 4 2 0 14 2 Percentage 38 34 26 50 39 11 25 50 25 0 87.5 12.5

Within this cohort the group with FH of HS had the highest positive and least equivocal results. The group with suspected HS (peripheral blood spherocytosis with negative coomb’s test) resulted in positive in 38% and equivocal in 26% cases. The group with NNH had positive results in 25% of cases and the group with HA had no positive results. Equivocal results occurred in 20% of cases suggesting further investigations are required to confirm or exclude HS. The EMA dye method by flow cytometry is a useful test for diagnosing red cell membrane abnormalities due to band 3 defects, but the assay produces a substantial number of equivocal results, and has poor utility in the investigation of haemolytic anaemia of unknown origin.
The Antioxidant Effects of Capparis Ovata and Deferasirox in Patients with Thalassemia Major
Authors: Canatan Duran , Duman Handan
Published: April 2020

Introduction: Iron overload and auto-oxidation of unpaired globin chains is the main cause of oxidative stress in thalassemia. The high levels of MDA in iron overloaded thalassemic patients is the best sample of oxidative stres. Generally decreased iron burden was associated with decreased oxidant damage. In vitro studies were shown that iron chelators such as deferoxamine and deferipron neutrolyse intraselluler free iron and inhibits oxidation. We aimed to show the antioxidant effects of capparis ovatta and deferasirox in thalassemic patients.

Material-Method: A total number of 40 thalassemia major patient aged between 7–30 years, who has been taken regular transfusion (15 cc/kg/month to maintain Hb >10 gr/dl) and chelation therapy (30 mg/kg/day ICL-670) are involved. They were separated into two groups as control and study group randomly. Both study and control group were followed by transfusion and chelation therapy. In study In addition study group have been taken capparis marmelade at the breakfast with a dose of a dessert-spoon (12.5 gr) younger than 10 years and a soup-spoon (25 gr) older than ten years for 6 months. Hematological and biochemical parameters, ferritin at every month and oxidative-antioxidant status (MDA, CAT, Gpx, SOD) were measured at the beginning and at the end of the study.

Results: Serum ferritin and MDA levels declined significiantly in both groups (for ferritin; control group p= 0.00; study group p=0.00) during the study but a much more decrease occured at MDA levels in the cappari given group (p=0.02). There was no statistically significant difference between the groups at the initial and last SOD CAT, GPX, SOD levels. Further more in the study group a significant decrease in liver function tests was occured (AST p= 0.05, ALT p= 0.01).

Conclusion: Our findings suggest that combination of capparis with deferasirox may be usefull for decreasing the effect of oxidative damage.
Vaccination with Leukemia Cell Expression Membrane Bound GM-CSF Overcomes the Host Immunosuppression Induced by Leukemia
Authors: Ling Di , Arlinghaus Ralph B. , Ling Xiaoyang
Published: April 2020

Immunotherapy of leukemia involves stimulating host-cell mediated immunity by facilitating immune recognition of leukemia cells, which are normally weakly immunogenic. We previously showed that vaccination with membrane bound GM-CSF leukemic cells protects mice from leukemia challenge (Ling et al., Oncogene, 2007). In these studies, after addition of a transmembrane domain to the original GM-CSF coding sequence (tmGM-CSF), the construct was transduced into murine leukemia cells (WEHI-3B), which was shown to be more than 98% on the cell surface. Vaccination with lethally irradiated tmGM-CSF expressing murine leukemia cells prevents leukemia in immunocompetent mice (BALB/c), as 100% of vaccinated BALB/c mice were protected from leukemia (Ling et al, Oncogene 2007). No protection was observed by vaccination of nude mice, indicating that adaptive immunity is involved in the protective response. In the present studies, we extended our original observation and provided evidence to show that leukemic mice undergo immunosuppression and that vaccination with leukemia cells expressing cell surface tmGM-CSF overcomes immunosuppression. Vaccination with lethally irradiated leukemia cells expressing cell surface tmGM-CSF overcame the immunosuppression induced by leukemia development, as normal levels of CD4+/CD25+/Foxp3+ T-regulatory (Treg) cells were maintained in spleens and thymus after challenge with leukemia cells. In contrast, the Treg population was significantly increased in leukemic mice vaccinated with leukemia cells lacking cell surface tmGM-CSF (p<0.001) after leukemia challenge, and these mice had a lower CD8+/Treg cell ratio (p<0.01). The ratio of CD8+/Treg cells was higher in tmGM-CSF/GFP vaccinated mice than in GFP vaccinated mice (p<0.001), which in-turn leads to a more effective CD8+ T-cell response. DC levels were also increased from normal levels in mice vaccinated with tmGM-CSF+ leukemia cells compared to control vaccinations. These results suggest that vaccination with leukemia cells expressing GM-CSF on their cell surface leads to an effective cell-mediated immune response in the vaccinated host by overcoming an impaired host cellular immunity induced up-regulation of Treg cells caused by the leukemia process. This strategy has potential for use in the treatment of various human leukemias.
High Levels of Acetylated Low Density Lipoprotein Uptake and Low Tie2 Promoter Activity Distinguishes Sinusoids from Other Vessel Types in the Murine Bone Marrow
Authors: Li Xiaomiao , Hu Zhongbo , Jorgenson Marda , Slayton William
Published: April 2020

Background: The bone marrow contains a variety of blood vessels that have different functions in maintaining the bone marrow as the major blood producing organ in adulthood. For instance, arterioles function to control the flow of blood into bone marrow compartments, and the sinusoids serve as a conduit to the blood stream and niches for megakaryocyte development. Most current studies of the bone marrow vasculature, including studies quantifying changes in the marrow vascular by microvascular density, do not differentiate between different types of marrow vessels. Recognizing the changes in different types of blood vessels has important physiologic implications. Here we report a new method to distinguish sinusoids from arterioles in the murine bone marrow.

Methods and Results: We used transgenic mice with GFP expressed downstream of the Tie-2 promoter, combined with in vivo acetylated low-density lipoprotein (Ac-LDL) uptake method to differentiate sinusoids from arterioles. We found that Ac-LDL was specifically endocytosed by sinusoids, and Tie-2 expression was more pronounced in the arteries, arterioles, and transitional capillaries. Combining these two functional endothelial markers and using confocal microscopy to obtain three dimensional images, we identified transitional zones where arterioles emptied into the sinusoids.

Conclusions: These results demonstrate that the marrow vasculature and specific endothelial cell types are functionally heterogeneous. Methods to study changes in the marrow vasculature and particularly the vascular niche, a function of sinusoids, need to take into account this heterogeneity.
Endogenous Nitric Oxide Synthesis Inhibitor Asymmetrical Dimethyl-L-Arginine (ADMA) and Neoplasms in Children
Authors: Doroszko Adrian , Niedzielska Ewa , Wojcik Dorota , Wojcik Elzbieta , Szuba Andrzej , Andrzejak Ryszard
Published: April 2020

Background: Neoplasms in children are uncommon diseases however they have broad impact on the society. Despite novel therapeutic strategies for children suffering from leukemia or solid tumor, the mortality rate of the disease is still high. The endogenous nitric oxide synthesis inhibitor asymmetrical dimethyl-L-arginine (ADMA) is considered to be a strong cardirovascular risk factor and may lead to new therapeutic strategies. Our previous findings indicate that elevated plasma level of ADMA is correlated with worse prognosis in adult patients with NHL. There are no data available pointing at metabolites of NO-pathway nor clinical evidence as prognostic factor of oncological treatment in children.

Aim: To investigate the relation between elevated plasma level of NO-pathway metabolites and well established markers of clinical stage of the disease.

Material and method: 12 children with acute leukemias (ALL, AML), 13 with solid tumor (PNET, osteosarcoma, Wilms’ tumor, RMS) during hemato-oncological treatment were recruited to a pilot study and N=15 healthy children to a control group. Plasma concentrations of ADMA, SDMA, L-Arg was determined. Relationship to clinical stage of the disease, biochemical and blood markers was tested.

Results Table 1: Differences in mean plasma concentrations of arginine, ADMA and SDMA in pts with leukemia vs pts with solid tumor vs control group

Control:N=15 Mean ±SD Median ±SEM Leukemia;N=12 Mean±SD Median ±SEM Solid tumour; N=13 Mean±SD Median ±SEM P L-Arg 68,58±24,29 60,19±6,27 31,28±8,63 30,82±2,49 49,59±10,81 49,18±3,00 0,0000 ADMA 0,62±0,10 0,61±0,02 1,17±1,10 0,53±0,32 0,98±1,10 0,46±0,31 NS SDMA 0,44±0,18 0,39±0,04 1,18±1,10 0,80±0,32 1,56±1,02 1,09±0,28 0,0024 ARG/ADMA 112,8±46,28 107,4±11,94 67,40±54,31 69,48±15,68 228,82±451,44 117,35±125,21 NS

Table 2:Significant correlations in group of pts with solid tumor and in patients with leukemia

LEUKEMIA; N=12 R p ADMA & Ca 0,78 0,008 SDMA & Ca 0,75 0,011 SDMA & CRP −0,78 0,021 Arg/ADMA & K −0,72 0,019 Arg/ADMA & Ca −0,64 0,043 Arg& MONOCYTE 0,65 0,043 ADMA & TOT.BILIRUB. −0,92 0,000 ADMA & TOT.PROTEIN 0,75 0,032 SDMA & EOSINOPHILE 0,73 0,025 SDMA & TOT.BILIRUB −0,82 0,007 Arg/ADMA & TOT.BILIRUB. 0,81 0,009 SOLID TUMOR; N=15 R p Arg &RBC 0,70 0,016 Arg & Hb 0,64 0,035 Arg & LYMPHOCYTE 0,81 0,003 Arg & EOSINOPHILE 0,76 0,011 ADMA &MONOCYTE −0,78 0,008 ADMA & TOT.PROTEIN 0,62 0,040 ADMA & Hb 0,64 0,035 Arg/ADMA &MONOCYTE 0,82 0,004 Arg/ADMA & BASOPHILE 0,65 0,043

Conclusions:A lower plasma level of L-Arg was found both in pts with leukemia and solid tumors. Higher plasma concentration of SDMA was found in patients with leukemia and solid tumor compared to the control group. Further studies are necessary to clarify the significance of these findings.
Interleukin-21 Induces Cell Cycle Arrest and Apoptosis of Diffuse Large B-Cell Lymphomas (DLBCL) Via Activation of STAT3 and Upregulation of C-Myc
Authors: Sarosiek Kristopher A , Nechushtan Hovav , Avisar Eli , Lossos Izidore S
Published: April 2020

IL-21, a recently discovered member of the IL-2 cytokine family, has been shown to have diverse regulatory effects on B-cells including the induction of antibody secretion, differentiation, or apoptosis depending on the cellular milieu and activation status. However, the effects of IL-21 on B-cell neoplasms such as DLBCL are largely unknown. Our research has uncovered the widespread expression of the IL-21 receptor (IL-21R) in B-cell lymphomas including DLBCL. Our results confirmed reports that IL-21R stimulation results in potent phosphorylation of STAT-1 and -3 and weak activation of STAT-5. However, our findings also show that treatment of DLBCL cell lines with IL-21 induces cell cycle arrest and apoptosis. The cell death is caspase-dependent and evident in a majority of DLBCL cell lines. To further examine the potential therapeutic applicability of IL-21, we assessed the effects of IL-21 on primary DLBCL tumor cells and in vivo DLBCL mice models. In primary tumors, IL-21 induced apoptosis in five of five DLBCLs compared to two of three follicular lymphomas, and two of seven chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B-lymphocytes. In xenograft DLBCL tumors, in situ IL-21 injections induced tumor regression and dramatically extended the overall survival of mice (P<0.001). To elucidate the mechanism of IL-21-induced cell death, microarray analysis was performed on endogenously sensitive or resistant DLBCL cell lines as well as an RCK8 cell line that acquired IL-21 resistance following chronic exposure to the cytokine. The most striking difference between IL-21 sensitive and resistant cell lines was observed in the expression of c-Myc, a known target of STAT-3, which was only induced in cell lines undergoing apoptosis upon IL-21 treatment. We have shown that the cell death associated with IL-21 treatment was prevented by utilizing c-Myc specific siRNAs and shRNA. Interestingly, c-Myc upregulation and cell death was also prevented upon knockdown of STAT-3 with siRNAs. Stimulation of the STAT-3-cMYC signaling pathway led to decreases in levels of anti-apoptotic proteins Bcl-XL and Bcl-2, which are bone-fide targets of c-Myc. Our results delineate a new IL-21-induced pro-apoptotic signaling pathway involving STAT-3 and c-Myc, which are usually considered as anti-apoptotic effectors in cancer cells. Furthermore, our findings demonstrate that IL-21 is a highly potent anti-DLBCL agent in vitro and in xenograft DLBCL models and warrant initiation of clinical studies in patients.
Hematopoietic Stem Cell Gene Therapy For Wiskott- Aldrich Syndrome
Authors: Witzel Maximilian GW , Braun Christian J. , Boztug Kaan , Paruzynski Anna , Albert Michael H. , Schwarzer Adrian , Modlich Ute , Beier Rita , Göhring Gudrun , Naundorf Sonja , Kühlcke Klaus , Rose Martina , Fraser Christopher , Mathias Liesl , Ferrari Rudolf , Abboud Miguel R. , Al-Herz Waleed , Kondratenko Irina , Maródi László , Schlegelberger Brigitte , Baum Christopher , Schmidt Manfred , von Kalle Christof , Klein Christoph
Published: April 2020

Wiskott Aldrich Syndrome (WAS) is a life-threatening primary immunodeficiency disorder characterized by susceptibility to infections, hemorrhage and malignancies. We here report the clinical, immunological, and molecular results of ten patients treated with transfusion of autologous genetically corrected hematopoietic stem cells between 2006 and 2009. After hematopoietic stem cell gene therapy with a gamma-retroviral vector, we observed phenotypical and functional reconstitution of lymphoid, myeloid cell lines and platelet numbers in 9/10 patients. Of note, patients had sustained correction of T-cell proliferation, immunological synapse formation and NK-cell mediated lysis, capacity to generate specific antibodies, and platelet production and size. All nine patients with sustained stem cell engraftment had marked clinical improvement with respect to infections, bleeding, and autoimmunity. Comprehensive analysis of retroviral insertions sites revealed a polyclonal hematopoiesis with more than 120.000 recurring loci. However, 5/10 of patients developed T-ALL (mean 1054.2d post GT; range 488-1813d post GT), each associated with a retroviral insertion in close proximity to the LMO2 locus. In 3/5 of patients with T-ALL (treatment according to AEIOP 2009 protocol) allogeneic HSCT due to poor initial response, minimal residual disease relapse, or cytological relapse was indicated. Further, one patient developed MDS like bone marrow changes and consecutively AML (1165d post GT), HSCT was performed in remission. Retrospectively, MDS1 clonal contribution to insertion sites showed a slow increase over the period of 22 month in this patient. GT has proven to revert the cellular and clinical phenotype of WAS but remains associated with considerable risk for insertional mutagenesis. Refining gene therapy strategies is an important goal for patients with WAS.

Disclosures:

Naundorf: EUFETS GmbH: Employment. Kühlcke:EUFETS GmbH: Employment.