bluuebunny/crossref_metadata_2025 huggingface dataset:
Perforin Polymorphism A91V and Susceptibility to Childhood Acute Lymphoblastic Leukemia (ALL).
Authors: Mehta Parinda A. , Davies Stella M. , Kumar Ashish , Devidas Meenakshi , Lee Susan , Wilke Tiffany , Elliott James , Villanueva Joyce , Pullen Jeanette , Zewge Yetnayet , Filipovich Alexandra H.
Published: April 2020

Perforin plays a key role in the cytotoxicity of NK cells and cytotoxic T lymphocytes. Genetic mutations in perforin gene (PRF1) give rise to 30–40% of cases of familial hemophagocytic lymphohistiocytosis. We recently studied two boys who had been treated for PreB ALL and subsequently developed HLH. Both showed decreased perforin expression in NK cells and were found to be heterozygous for PRF1 A91V. The functional consequences of the PRF1 A91V (resulting from C272T transition) are controversial; between 3 and 17% of apparently normal people are heterozygous for this polymorphism depending on the population studied. Recent data suggest that A91V results in conformational change that may impair processing of perforin protein to the active form, and that the polymorphism may increase susceptibility to ALL in children (Trambas et al, Blood2005; 106:932; Santoro et al, Haematologica2005;90:697). We have genotyped 655 normal blood donors and 2,272 children with ALL treated on Pediatric Oncology Group 9900 clinical trials. Genotyping was performed using a specific PCR assay (Taqman). Genotypes in the normal US blood donors showed significant variation in allele frequency by race, with a very low frequency of the variant allele in black controls (0.7% compared with 4% in white controls). Allele frequencies did not vary by gender and genotype frequencies were in Hardy Weinberg equilibrium (whites C/C 92%; C/T 8%; T/T 0.2%). In light of the low frequency of the variant allele in blacks, analysis of leukemia cases was restricted to a comparison of white cases and white controls. Analyses were performed for all B-lineage ALL cases combined, and were also further stratified for known genetic subtypes of ALL.

Perforin genotype CC Perforin genotype CT or TT p-value (cases versus controls Controls (n=507) 464 (91.5%) 43 (8.5%) - Cases (all; n= 1321) 1198 (90.7%) 123 (9.3%) 0.58 BCR-ABL 22 (76%) 7 (24%) 0.0048 Trisomy 4,10 238 (93.7%) 16 (6.3%) 0.29 TEL-AML1 250 (90.6%) 26 (9.4%) 0.66 E2A-PBX 24 (92.3%) 2 (7.7%) 0.89 MLL abn. 40 (87%) 6 (13%) 0.29 Hyperdiploid 244 (92.4%) 20 (7.6%) 0.66 Hypodiploid 8 (100%) 0 (0%) 0.40

Taken together, these data indicate that the A91V polymorphism is not associated with an overall increased risk of childhood ALL, in contrast to the previous report. PRF1 A91V was identified with significantly increased frequency in children with BCR-ABL positive ALL. However, this observation includes a relatively small number of cases and the potential association should be explored further, perhaps in adult ALL series in which the frequency of BCR-ABL positivity is likely to be high.
Activating FLT3 Mutations in CD4+/CD8− Pediatric T-Cell Acute Lymphoblastic Leukemias.
Authors: Van Vlierberghe Pieter , Meijerink Jules P.P. , Stam Ronald W. , van der Smissen Wendy , van Wering Elisabeth R. , Beverloo H. Berna , Pieters Rob
Published: April 2020

Activating mutations in the FMS-like tyrosine kinase 3 gene (FLT3) are the most common genetic aberration in acute myeloid leukemia (AML). Internal tandem duplications (ITD) in the juxtamembrane (JM) domain, or point mutations (PM) in the activation loop of the tyrosine kinase domain lead to a constitutive activated state of the FLT3 tyrosine kinase. Recently, FLT3 mutations were identified in a cohort of 69 adult T-ALL patients, showing that this genetic abnormality is not only restricted to myeloid leukemias. To validate the incidence of FLT3 mutations in pediatric T-ALL and investigate its relation to outcome and other clinical and immunophenotypical parameters, we screened 72 diagnostic pediatric T-ALL samples. FLT3/ITD mutations were identified in 2/72 pediatric T-ALLs (2.7%), whereas 0/72 showed point mutations in the kinase domain. Immunophenotypic analysis revealed a similar profile for both FLT3 mutated patient samples, i.e. TdT+, CD2+, CD5+, CD7+, CD4+/CD8−, cytoplasmic CD3+, surface CD3− and CD10−. Although representing early T-cell differentiation stages for both patient samples, these cases seem to have a more advanced immunophenotype compared to the FLT3 mutated adult T-ALL cases, previously described (CD34+, CD4−/CD8−). Both FLT3 mutated patients showed high level LYL1 and LMO2 expression. In addition, both pediatric samples contained a HOX11L2 translocation, which was not present in the FLT3 mutated adult T-ALL cases. The first FLT3 mutated patient suffered a relapse 13 months after initial diagnosis, whereas the other is still in continued complete remission for 61+ months. Interestingly, the relapse material showed no FLT3/ITD mutation, indicating that the FLT3 mutated T-ALL subclone seems to be effectively eradicated by current chemotherapy. These data suggest that the application of FLT3 inhibitors for FLT3-mutated T-ALLs, as recently suggested in literature, may not further improve treatment outcome in pediatric T-ALL.
Successful In Vitro Priming of EBV-Specific CD8+ T Cells Endowed with Strong Cytotoxic Function for Prophylaxis or Treatment of PTLD in EBV Seronegative Hosts.
Authors: Comoli Patrizia , Locatelli Franco , Frasson Chiara , Basso Sabrina , Labirio Massimo , Moretta Antonia , Montagna Daniela , Ginevri Fabrizio , Maccario Rita
Published: April 2020

EBV-seronegative solid organ transplant recipients, or recipients of T-cell depleted hematopoietic stem cell from a EBV-seronegative donor, are cohorts at high risk for the development of EBV-associated posttransplant lymphoproliferative disease (PTLD), and would maximally benefit from an EBV-directed T-cell therapy for prevention or treatment of PTLD. Reported experiences have shown that CTL obtained from EBV-seronegative individuals are almost exclusively HLA-class II-restricted CD4+ cytotoxic T cells, and efficiency of this CTL subset in the control of PTLD has not yet been demonstrated in vivo. Conversely, the central role of CD8+ CTL in the cell-mediated containment of EBV infection is well established. Therefore, we endeavoured to develop a protocol of EBV-CTL priming that would allow optimal expansion and high specific cytotoxic activity of CD8+ CTL. We employed naïve lymphocytes obtained from 5 EBV seronegative patients with end-stage renal failure receiving dialysis while listed for a kidney transplantation (4 children, 1 adult). In particular, we compared the CD8+ CTL priming efficiency of three different modified activation protocols, based on LCL stimulation potentially enhanced by either LCL presentation through DC, or selection of IFNg+ cultured cells, or culture in the presence of rhIL-12 and rhIL-7, to the standard protocol for reactivation of EBV-specific CTL from EBV-seropositive individuals. We found that while all protocols employed, with the exception of IFN-g+ T cell selection, allowed for the amplification of CD8+ and CD4+ mediated EBV-specific cytotoxic T cell responses in the EBV-seronegative adult, only specific EBV-transformed lymphoblastoid cell lines (LCL) stimulation in the presence of rhIL-12 and rhIL-7 was able to reproducibly generate and expand EBV-specific CD8+ CTL endowed with strong cytotoxic activity from truly EBV-seronegative children (mean lysis at a E:T ratio of 10:1: 14±10% with the standard protocol vs. 66±30% with the addition of rhIL-7 and rhIL-12). The lines thus activated showed a higher percentage CD8+ T cells, with less than 10% naïve CD8+/CCR7+/CD45RA+ cells. Overall, the total number of CD8+ central memory cells, and of CCR7- T cell effectors was comparable to that observed in healthy EBV-seropositive controls. In conclusion, stimulation with LCL in the presence of IL-12 and IL-7 activates EBV-specific, highly cytotoxic CD8+ CTL from EBV-seronegative subjects, this opening new perspectives for prevention/treatment of PTLD.
In Vitro Expansion of Autologous Follicular Lymphoma-Specific T Cells for Adoptive Transfer.
Authors: Lee Seung-Tae , Jiang Yun Fang , Cha Soung-Chul , Qin Hong , Kwak Larry W. , Neelapu Sattva S.
Published: April 2020

Advanced stage follicular lymphoma remains an incurable disease with a median survival of 8 to 10 years that has not significantly changed over the last four decades. Therefore, novel treatment options are necessary to improve the clinical outcome in these patients. The observation of spontaneous regressions in a small percentage of patients suggested that augmenting the host immune response could potentially control this malignancy. Strategies using active specific immunotherapy with idiotype vaccines led to induction of clinical and molecular responses in a few patients but have met with only limited success possibly due to the low frequency of antigen-specific T cells induced in the patients. In contrast to active immunization, T cells of a given specificity and function may be selected and expanded in vitro to the desired number for adoptive cell transfer. Towards this goal, we stimulated tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) from five follicular lymphoma patients with CD40 ligand-activated autologous tumor cells at approximately ten-day intervals in the presence of IL-2 and IL-15. After four rounds of stimulations, T cell lines generated from 3/5 patients recognized autologous unmodified tumor cells by producing significant amounts of TNF-α, GM-CSF and/or IFN-γ. By phenotypic analysis, the T cell lines were predominantly CD4+ T cells (> 70%), and intracellular cytokine assay showed that up to 40% of the CD4+ T cells were tumor-reactive. The inhibition of cytokine production by anti-HLA class II but not class I blocking antibodies confirmed that the CD4+ T cells were tumor-reactive. Further characterization revealed that the T cells from one patient recognized autologous tumor but not autologous normal B cells suggesting that they were tumor-specific. While in a second patient CD4+ T cell clones generated from the T cell line by limiting dilution recognized autologous tumor and autologous normal B cells but not autologous monocytes suggesting that they were B cell lineage-specific. We conclude that follicular lymphoma-specific T cells exist and can be efficiently expanded in vitro from both TILs and PBMCs using CD40 ligand-activated autologous tumor cells for adoptive T cell therapy. Additionally, identification of antigens recognized by these T cells could lead to development of novel immunotherapeutic strategies for lymphomas.
Chemo-Immunotherapy with Hyper-CVAD Plus Ritixumab for Adult Burkitt’s and Burkitt’s Type Lymphoma (BL) or Acute Lymphoblastic Leukemia (B-ALL).
Authors: Thomas Deborah A. , Faderl Stefan , O’Brien Susan , Bueso-Ramos Carlos , Cortes Jorge , Garcia-Manero Guillermo , Giles Francis , Verstovsek Srdan , Wierda William , Pierce Sherry , Shan Jiaquin , Brandt Mark , Hagemeister Fredrick , Cabanillas Fernando , Keating Michael J. , Kantarjian Hagop
Published: April 2020

Prognosis has improved for BL/B-ALL, particularly with short, intensive, multi-agent chemotherapy. Outcome of 26 non-HIV BL/B-ALL patients (pts) treated with hyper-CVAD (fractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone) showed CR rate of 81% with induction mortality 19% owing to systemic fungal infections in pts aged 60 years (yrs) and older [Thomas et al, JCO 17:2461, 1999]. Older age was also an adverse feature predicting relapse with shorter 3 yr survival compared with younger pts (17% vs 77%). To evaluate the addition of rituximab, a CD20 monoclonal antibody, to hyper-CVAD, 31 adults with newly diagnosed HIV negative BL (n=15) or B-ALL (n=16) received hyper-CVAD with rituximab. Their median age was 46 years; 29% were 60 years or older. Course 1 was given in laminar air flow room for pts aged 60 years or older. Rituximab 375 mg/m2 was given intravenously days 1 and 11 of hyper-CVAD courses and days 1 and 8 of methotrexate and cytarabine courses with 16 intrathecal treatments (methotrexate day 2 and cytarabine day 7 of each course). No maintenance chemotherapy was given. No allogeneic or autologous stem cell transplant was planned in first CR. Complete remission (CR) was achieved in 24 of 28 evaluable patients (86%); 3 had a partial response and 1 had resistant disease. There were no induction deaths. The 3-year overall survival (OS), event-free survival, and disease-free survival rates were 89%, 80% and 88% respectively. All nine elderly pts aged ≥ 60 yrs achieved CR with all but one in continuous CR (death in CR from infection); 3-year OS rate was 89%. Nineteen pts (61%) were older than 40 yrs; OS rate was 90% compared with 35% for historical group (48 pts treated with hyper-CVAD alone). Reduction in the relapse rate with rituximab was observed compared with hyper-CVAD alone (7% versus 34% overall, p=0.008; 0% versus 50% for elderly, p=0.02; 11% versus 29% for age < 60 years, p=0.11). Toxicity profile was similar to hyper-CVAD alone. Multivariate analysis of the current cohort and historical group identified age and treatment with rituximab as factors predicting cure. The addition of rituximab to hyper-CVAD appears to improve outcome in adult BL or B-ALL, particularly in elderly patients.
A Phase 2 Study of the Thrombospondin-Mimetic Peptide ABT-510 in Patients with Refractory Lymphoma.
Authors: Levine Alexandra M. , Schwartzberg Lee , Smith Sonali , Belt Robert , Knight Raymond , Carlson Dawn , Hippensteel Richard , Kahl Brad
Published: April 2020

ABT-510 is a potent nonapeptide that mimics the antiangiogenic activity of the endogenous protein, thrombospondin-1. Dogs with spontaneous lymphoma showed a response rate of 17% (4/23). In 2 phase 1 studies in patients with solid tumors, ABT-510 was tolerated at doses up to 260 mg per day. Doses ≥20 mg daily exceeded the pharmacokinetic target of 100 ng/mL for >3 hours. 67 patients with relapsed or refractory lymphoma were randomized to receive ABT-510 10 mg or 100 mg twice daily (BID) by subcutaneous injection. The 10 mg BID arm was discontinued after 15 patients were enrolled to consolidate recruitment to the higher dose. The primary endpoint was tumor response; secondary endpoints included progression-free survival (PFS) and overall survival. Tumor progression was evaluated every 8 weeks by the Cheson criteria; safety was evaluated every 4 weeks. 6 of the 67 patients remain on active therapy. Data are available for 56 patients (32M/24F); the median age is 63 years (range 22–87), and the baseline ECOG Performance Score for all patients ranges from 0–2. All patients were heavily pretreated, with a median of 5 prior treatments (range 1–11). There are 3 partial responses, all in the 100 mg dose group in non-Hodgkins lymphoma (NHL) patients. PFS data are as follows:

Patient Group N 6-month PFS % (90 % CI) 12 month PFS % (90 % CI) All 56 41 (28, 53) 17 (3, 31) 10 mg BID 15 34 (11, 56) 0 100 mg BID 38 48 (33, 64) 24 (8, 41) Hodgkins 11 29 (3, 55) 0 NHL 45 44 (30, 58) 22 (4, 40) Aggressive 17 29 (9, 49) 0 Indolent 27 51 (33, 70) 36 (16, 56)

The most frequent adverse events (AEs) were asthenia (39%), injection site reaction (39%), diarrhea (24%), and anorexia (20%). 19% of patients had Grade 3–4 AEs considered related to ABT-510: anemia (4), thrombocytopenia (2), asthenia, chest pain, hemorrhage, gastrointestinal hemorrhage, leukopenia, and hypercalcemia (1 each). Correlative analyses of circulating endothelial cell data are pending. Based on these preliminary data, ABT-510 appears well-tolerated with biological activity in heavily pretreated patients with lymphoma. Study of ABT-510 in combination with immunotherapy or standard chemotherapy is warranted.
Regulation of Chemoresistance and Immune Resistance of B-NHL Cell Lines by Overexpression of YY1 and Bcl-xL, Respectively: Reversal of Resistance by Rituximab.
Authors: Vega Mario I. , Jazirehi Ali R. , Huerta-Yepez Sara , Bonavida Benjamin
Published: April 2020

We have recently reported that treatment of B-NHL cell lines with rituximab sensitizes the tumor cells to both chemotherapy and Fas-induced apoptosis (Jazirehi and Bonavida, 2005, Oncogene, 24:2121–2145). This study investigated the underlying molecular mechanism of rituximab-mediated reversal of resistance. Treatment of B-NHL cell lines inhibited the constitutively activated NF- κB. Cells expressing dominant active IκB or treated with NF-κB specific inhibitors were sensitized to both drugs and FasL agonist mAb (CH-11)-induced apoptosis. Downregulation of Bcl-xL expression via inhibition of NF-κB activity correlated with chemosensitivity. The direct role of Bcl-xL in chemoresistance was demonstrated by the use of Bcl-xL overexpressing Ramos cells, Ramos HA-BclxL (gift from Genhong Cheng, UCLA), which were not sensitized by rituximab to drug-induced apoptosis. However, inhibition of Bcl-xL in Ramos HA-Bcl-x resulted in sensitization to drug-induced apoptosis. The role of Bcl-xL expression in the regulation of Fas resistance was not apparent as Ramos HA-Bcl cells were as sensitive as the wild type cells to CH-11-induced apoptosis. Several lines of evidence support the direct role of the transcription repressor Yin-Yang 1 (YY1) in the regulation of resistance to CH-11-induced apoptosis. Inhibition of YY1 activity by either rituximab, the NO donor DETANONOate, or following transfection with YY1 siRNA all resulted in upregulation of Fas expression and sensitization to CH-11-induced apoptosis. These findings suggest two complementary mechanisms underlying the chemo-sensitization and immuno-sensitization of B NHL cells by rituximab via inhibition of NF-κB. The regulation of chemoresistance by NF-κB is mediated via Bcl-xL expression whereas the regulation of Fas resistance by NF-κB is mediated via YY1 expression and activity. These findings suggest that drug-resistant NHL tumor cells may be sensitive to immune-mediated therapeutics.
Simultaneous Inhibition of Mcl-1 and XIAP To Induce Apoptosis in AML Cells.
Authors: Carter Bing Z. , Mak Duncan , Schober Wendy , Koller Erich , Dean Nicholas M. , Andreeff Michael
Published: April 2020

The apoptotic cascade is regulated by both upstream Bcl-2 and the distal IAP family proteins. Mcl-1, a member of antiapoptotic Bcl-2 family proteins is highly expressed in various cancer cells. It was reported that its expression is elevated at the time of leukemic relapse (Blood1998;19:991–1000). XIAP, the most potent cellular caspase inhibitor, inhibits both the initiator caspase-9 and the effector caspase-3 and suppresses apoptosis. Our previous study showed that decrease of XIAP by its antisense oligonucleotide enhances Ara-C-induced apoptosis (Leukemia2003;17:2081–2089). In this study, we investigated the regulation of Mcl-1 expression, its potential as a therapeutic target, and apoptosis induction by simultaneous inhibition of Mcl-1 and XIAP in AML cells. We found that like survivin, Mcl-1 protein level is decreased by MEK inhibition in HL-60 and OCI-AML2 cells suggesting that Mcl-1 expression is regulated through the MAPK signaling pathway. However, in contrast to survivin levels, no significant differences in Mcl-1 expression in G0/G1, S, and G2M cells were observed in FACS-sorted HL-60 cells. Downregulation of Mcl-1 by its antisense oligonucleotide (Mcl-1-AS, 20408, ISIS Pharmaceuticals) induced cell death accompanied by decrease in mitochondrial membrane potential (MMP), caspase activation and annexin V positivity in HL-60 cells, while control oligonucleotide was not toxic. By itself, MEK inhibitor primarily induced G1/G0 cell cycle block and no apoptosis. When combined with Mcl-1-AS, MEK inhibition further decreased Mcl-1 protein levels and significantly increased Mcl-1-AS induced cell death (from 25.8±1.3% to 40±3.8%, p=0.02). Inhibition of XIAP by its antisense oligonucleotide (XIAP-AS, 102369, ISIS Pharmaceuticals) did not induce significant loss of MMP at 24 hrs and slightly increased annexin V positive cells at 48 hrs (14.5%±2.9% dead cells vs. 6.2%±0.72% in XIAP-NS treated cells). However, when combined with Mcl-1 inhibition, significant increases in loss of MMP at 24 hrs and annexin V positive cells at 48 hrs (p=0.003), more than inhibition of each protein alone, was observed indicating that activating the upstream and downstream apoptotic cascade will amplify the caspase activation loop and more efficiently induce cell death. Our study suggests that Mcl-1 is regulated by the MAPK signaling pathway. Its expression is cell cycle independent. Mcl-1 is essential for HL-60 cell survival and simultaneously downregulation of the upstream anti-apoptotic protein Mcl-1 and the downstream caspase inhibitor XIAP significantly enhances leukemia cell apoptosis.
Effect on NB4 Cells Proliferation, Differentiation and Resistance to Chemotherapy Apoptosis by VEGF-C cDNA Transfection.
Authors: Ding Kaiyang , Bai Xia , Dai Lan , Dong Ningzheng , Ruan Changgeng
Published: April 2020

VEGF-C secreted by kinds of tumor cells play the key role in lymphangiogenesis via activating tyrosine kinase of VEGFR-3, which is one of most important pathway of infiltration and metastasis of tumor cells. In recent years, the effects of VEGF-C on hematological malignant cells are paid attention. In the current study, the recombinant eukaryotic expression plasmid (pcDNA3.1-VEGF-C) and the vacant pcDNA3.1 vector were introduced into the acute promyelocytic leukemia cell line- NB4 cells (VEGFR-3+/VEGFR-2−) by lipofectamine mediation and positive clones were screened by G418. The stable expression of VEGF-C was detected by reverse transcriptase-PCR and Western blotting. The proliferation ability of NB4/VEGF-C cells is analysed by MTT assay. After NB4/VEGF-C cells were induced by ATRA (1μmol/L), the expression levels of C/EBPα gene which can promote myelocytes differentiation and maturation, CD11b on cells surface and morphological variation were analysed by real-time quantitative PCR (RT-QPCR), flow cytometry analysis (FCM), Wright-Giemsa staining respectively. Furthermore, after the NB4/VEGF-C cells were cultured together with Etoposide (200ng/ml) for 48 hours, the numbers of AnnexinV+/PI−cells (apoptotic cells) were determined by FCM to investigate the effect of VEGF-C on the cells apoptosis and expression levels of antiapoptotic bcl-2 gene in these cells were analysed by RT-QPCR. The NB4/pcDNA3.1 cells was used as control during the experiments. From these experiments, NB4/VEGF-C cells can stably secret active protein VEGF-C in the cell supernatants. The cell growth curve shows that the proliferation ability of NB4/VEGF-C cells is stronger compared with NB4/pcDNA3.1cells. The expression levels of C/EBPα gene of NB4/VEGF-C cells after induced by ATRA is only 1/32 that of NB4/pcDNA3.1 cells. Morphological analysis shows that the degree of promyelocytes gradually maturing into myelocytes among NB4/VEGF-C cells is weaker compared with the controls after induced by ATRA. After induced by Etoposide, the percent of apoptotic cells of NB4/VEGF-C cells (7.20±2.52%) is significantly lower than that of NB4/pcDNA3.1 cells (16.07±3.58%)( P=0.005) and the bcl-2 gene expression level of NB4/VEGF-C cells is 2.28 times more than that of NB4/pcDNA3.1 cells. The results implied that the VEGF-C via VEGFR-3 signaling pathway could promote the proliferation of NB4 cells by autocrine pathway and inhibit the NB4cells differentiation and maturation and chemotherapy-induced apoptosis via upregulating bcl-2 gene expression. Thus, VEGF-C/VEGFR-3 signaling loops might play an important role in disease progression and be potential therapeutic target for the treatment of leukemias.
Chromosomal Deletions and Amplifications in Multiple Myeloma Detected by 500K Single Nucleotide Polymorphism Array Analysis.
Authors: Tai Yu-Tzu , Li Cheng , Coffey Rory , Breitkreutz Iris , Li Xian-Feng , Catley Laurence Laurence , Schlossman Robert L. , Richardson Paul G. , Anderson Kenneth C. , Munshi Nikhil C.
Published: April 2020

Genetic aberrations, such as deletions and amplifications are among the major pathogenetic mechanisms underlying malignant transformation and progression. Analysis of chromosomal aberrations is particularly important as amplifications of oncogenes and deletions of tumor suppressor genes are major steps in the “multi-hit” process of tumorigenesis. Genome-wide molecular analyses, such as loss of heterozygosity (LOH) profiling and comparative genomic hybridization (CGH) have significantly enhanced our ability to detect chromosomal aberrations in cancer cells and assess their role in tumorigenesis. The recent introduction of high-density oligonucleotide arrays capable of measuring up to 500K single nucleotide polymorphisms (SNP) loci is able to analyze small areas of gains or losses. Here we describe the use of high-density array representing upto 500K SNPs to not only characterize SNPs but also evaluate genome-wide areas of gains and losses in myeloma. We purified CD138+ myeloma cells from 5 patients and also obtained their peripheral blood mononuclear cells. Purified genomic DNA from these cells was digested with Sty and Nsp restriction enzymes, PCR amplified and hybridized to 500K SNP array. Results from DNA obtained from CD138+ myeloma cells were compared with DNA from normal peripheral blood mononuclear cells from the same patient using the dChip software for LOH and copy number analysis. Areas of deletions and amplifications were identified and correlated with gene expression profile obtained from CD138+ myeloma cells of the same patient. The preliminary analysis identified various areas of amplifications and deletions. The significant areas of structural gains or losses included Chromosome 1, 6, 8, 11, 12, 13, 14, 15, 22, X among others and ranged in size from 1 Mb to the loss of entire chromosome (13q−). In this small sample we have identified recurrent LOH shared by 2 or more samples including 1p21, 6q, 12p13 and 13q. The derived CGH results correlated with known cytogenetic abnormalities in the patients, with identification of additional areas of abnormality not detected by conventional cytogenetics or FISH. These structural changes also corresponded with the results from gene expression profiling of MM cells from same patients, thus validating the technique and its application. A large series of patient samples are now under investigation using 500K SNP chip to define regions of homozygous deletions as well as amplification. In conclusion, these results demonstrate that SNP array analysis provides a powerful new tool for the analysis of allelic imbalance in myeloma. Furthermore, this technique can identify novel candidate loci to study in pathogenesis of myeloma and advance identification of potential therapeutic targets for this fetal disease.
Non-Myeloablative Autologous Hematopoietic Stem Cell Transplantation for Systemic Lupus Erythematosus.
Authors: Statkute Laisvyde , Oyama Yu , Traynor Ann , Verda Larissa , Barr Walter G. , Rosa Robert , Schroeder James , Krosnjar Nela , Quigley Kathleen , Yaung Kimberly , Villa Marcelo , Takahashi Miyuki , Burt Richard K.
Published: April 2020

Manifestations of systemic lupus erythematosus (SLE) may in most patients be ameliorated with medications that suppress the immune system. Nevertheless, there remains a subset of SLE patients for whom current strategies are insufficient to control disease. Here we report results of autologous non-myeloablative hematopoietic stem cell transplantation single arm trial performed at Northwestern University Feinberg School of Medicine between February 1997 and January 2005 involving 50 patients with SLE refractory to standard immune suppressive therapies and either organ- or life-threatening visceral involvement. Peripheral blood stem cells were mobilized with cyclophosphamide (2.0 g/m2) and G-CSF (5 ug/kg/day), enriched ex vivo by CD34+ immunoselection, cryopreserved, and reinfused after treatment with cyclophosphamide (200 mg/kg) and equine anti-thymocyte globulin (90 mg/kg). The primary endpoint was survival, both overall survival and disease free survival. Secondary endpoints include systemic lupus erythematosus disease activity index (SLEDAI), serology (ANA and antids DNA), complement (C3 and C4), and changes in renal and pulmonary organ function assessed pre-treatment and 6 months, 12 months and then yearly for 5 years. Fifty patients were enrolled and underwent stem cell mobilization. Two patients died after mobilization, one from disseminated mucormycosis and another from active lupus after postponing the transplant for 4 months. Forty-eight patients underwent lupus non-myeloablative hematopoietic stem cell transplantation with no treatment related mortality. By intention to treat, treatment related mortality was 2% (1/50). With a mean follow-up of 29 months (range 6 month to 7.5 years ), overall survival was 84%, and probability of disease free survival at 5 years post transplant was 50%. Secondary analysis demonstrated stabilization of renal function and statistically significant improvement (p ≤ .05) in SLEDAI, ANA, anti-ds DNA, complement, and DLCO adjusted for hemoglobin (DLCOadj). In treatment refractory SLE, autologous non-myeloablative hematopoietic stem cell transplantation results in marked amelioration of disease activity, long term disease remission, improvement in serologic markers, and either stabilization or reversal of organ dysfunction.
Enzastaurin (LY317615), an Oral Protein Kinase C β Inhibitor, Induces Apoptosis in Multiple Myeloma Cell Lines.
Authors: Rizvi Mujahid A. , Ghias Kulsoom , Davies Katharine M. , Ma Chunguang , Krett Nancy L. , Rosen Steven T.
Published: April 2020

The protein kinase C (PKC) family consists of 11 serine/threonine protein kinase isoforms that are involved with cell proliferation and differentiation, gene transcription, and tumor-induced angiogenesis. The PKC pathway has been shown to play a role in the regulation of cell growth in several hematologic malignancies. However, in multiple myeloma (MM), the role of the PKC pathway has not been extensively studied. Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is an oral inhibitor of the PKC β isozyme. Enzastaurin has been reported to induce apoptosis and suppress proliferation in the HCT116 colon cancer cell line by inhibiting the AKT pathway. The objective of this study was to assess the efficacy of Enzastaurin in inducing apoptosis in MM cell lines and to investigate possible mechanisms of apoptosis. A spectrum of MM cell lines, with unique characteristics (dexamethasone-sensitive, dexamethasone-resistant, chemotherapy-sensitive, chemotherapy-resistant) were treated with Enzastaurin. There is evidence of cell death in all cell lines at clinically significant concentrations (1–3μM) after 72 hours of treatment. The dexamethasone-sensitive MM1.S cell line was used to further assess the effect of Enzastaurin in the presence of dexamethasone, insulin-like growth factor-1 (IGF-1), and IL-6. IGF-1 and IL-6 are potent growth factors for MM and have been observed to blunt the cytotoxic activity of dexamethasone. Dexamethasone and Enzastaurin appear to have an additive effect in the induction of apoptosis. In addition, while IGF-1 slightly decreases the effect of Enzastaurin, IL-6 has no effect. Enzastaurin treatment also induces a decrease in GSK3β and AKTSerine473 phosphorylation. GSK3β phosphorylation is thought to be a reliable pharmacodynamic marker for Enzastaurin activity. In conclusion, these data indicate that Enzastaurin induces apoptosis in MM cells and suppression of the AKT pathway may be one of the mechanisms by which Enzastaurin exerts its anti-myeloma activity.
Novel Hydroxamic Acid-Derived HDAC Inhibitor LBH589 Potently Activates Intrinsic and Extrinsic Apoptotic Pathways, and Induces Tubulin Hyperacetylation in Multiple Myeloma.
Authors: Catley Laurence , Tai Yu-Tzu , Hideshima Teru , Chauhan Dharminder , Weisberg Ellen , Kiziltepe Tanyel , Neri Paola , Tassone Pierfrancesco , Versace Richard , Atadja Peter , Munshi Nikhil C. , Anderson Kenneth C.
Published: April 2020

Histone deacetylase (HDAC) inhibitors are emerging as a promising new treatment strategy in hematological malignancies. LBH589 is a novel hydroxamic acid derivative HDAC inhibitor that inhibits myeloma cell growth at a median inhibitory concentration of less than 10 nM at 48 hours in multiple myeloma (MM) cell lines resistant to conventional therapies, as well as MM patient cells, as determined by MTS assay and tritiated-thymidine uptake. Cell cycle analysis revealed G1 arrest and a significant increase in the sub-G1 populations in these cells. MM.1S myeloma cell proliferation was also inhibited when co-cultured with bone marrow stromal cells, demonstrating ability to overcome the stimulatory effects of the bone marrow microenvironment. Pro-apoptotic effects were confirmed by Western blot. Significant cleavage of caspase 8, caspase 9, caspase 3 and PARP were observed within 24h with 20nM of LBH589 in MM.1S cells, indicating involvement of both the intrinsic and extrinsic apoptotic pathways in LBH589-mediated apoptosis. There was associated up-regulation of p21 and down-regulation of c-myc. Synergistic activity was observed in combination with proteasome inhibitor bortezomib. LBH589-induced HDAC inhibition resulted in histone hyperacetylation at low nanomolar concentrations. Acetylation of alpha-tubulin is a post-translational modification that consists of the addition of an acetyl group to lysine 40, which is reversed by HDAC6, or tubulin deacetylase (TDAC). Tubulin acetylation plays an important role in the differentiation of microtubule structure and function. Furthermore, TDAC constitutively binds both polyubiquitinated misfolded proteins and dynein, recruiting misfolded protein to dynein motors for transport to aggresomes along microtubules, which serves an important defense mechanism against apoptosis in MM cells. Importantly, dose-dependent inhibition of TDAC in MM.1S cells was observed at low nanomolar concentrations of LBH589. These findings provide the rationale for clinical studies with LBH589, either alone or in combination with bortezomib, to overcome drug resistance and improve patient outcome in MM.
Mechanism by Which mTOR Inhibitors Sesitize Multiple Myeloma Cells to Dexamethasone-Induced Apoptosis.
Authors: Yan Huajun , Frost Patrick , Shi Yijiang , Hoang Bao , Sharma Sanjai , Fisher Myrna , Gera Joseph , Lichtenstein Alan
Published: April 2020

Previously we reported mTOR inhibitors such as rapamycin (RAPA) remarkably sensitize multiple myeloma (MM) cells to apoptosis induced by dexamethasone (DEX). In this study we investigated the molecular mechanisms behind this phenomenon. Firstly we excluded the involvement of post-translational modifications of the Related Adhesion Focal Tyrosine Kinase (RAFTK kinase), the stress-activated kinases jun kinase or p38 MAP kinase or the proapoptotic BCL-2 family member protein BAD in the sensitizing effect, since exposure to RAPA did not affect their phosphorylation status. Sensitization was not associated with a RAPA-mediated increase in glucocorticoid receptor reporter expression either. mTOR inhibitors are know to specifically paralyze cap-dependent translation through dephosphorylation of 4E-BP1, therefore we assessed whether the sensitizing effect is due to inhibition of cap-dependent translation. We transfected MM cells with a construct expressing a mutant 4E-BP1, which is resistant to phosphorylation induced by mTOR and acts as a constitutive translational repressor and found MM cells responded to DEX with significantly enhanced apoptosis, mimicking the effect of co-exposure to RAPA. These results indicate sensitization to DEX is mediated by inhibition of cap-dependent translation. A high throughput screening for translational efficiency of apoptosis-regulating transcripts identified several anti-apoptotic proteins whose translation was selectively inhibited by RAPA. Immunoblot assay confirmed a RAPA-induced down-regulated expression of XIAP-1, CIAP, HSP-27 and BAG 3, all which may play a role in the sensitization to apoptosis. Studies in a xenograft model demonstrated synergistic in vivo anti-myeloma effects when dexamethasone was combined with the mTOR inhibitor CCI-779. Synergistic effects were associated with a markedly enhanced degree of MM cell apoptosis in vivo. This study further supports the strategy of combining DEX with mTOR inhibitors in MM and identifies a potential mechanism by which the synergistic effect is achieved.
Role of NPM in the Control of Genetic Stability and Tumorigenesis.
Authors: Grisendi Silvia , Bernardi Rosa , Rossi Marco , Cheng Ke , Khandker Luipa , Manova Katia , Pandolfi Pier Paolo
Published: April 2020

Nucleophosmin (NPM, B23, NO38) is a nucleolar protein directly implicated in cancer pathogenesis as the NPM gene is found mutated and rearranged in a number of haematological disorders. In addition, the region of chromosome 5 where NPM maps to (5q35) is deleted in a proportion of de novo human myelodysplastic syndromes (MDS), and loss of chromosome 5 is extremely frequent in therapy-related MDS (t-MDS). NPM is a multifunctional protein, and its role in oncogenesis is controversial as NPM has been attributed both oncogenic and tumour suppressive functions. To study the function of Npm in vivo, we generated a hypomorphic Npm mutant series [Npm+/− < Npmhy/hy < Npm−/−] in the mouse, and we found that Npm is essential for embryonic development and the maintenance of genomic stability. Npm−/− and Npmhy/hy mutants display aberrant organogenesis and die between E11.5 and E16.5 due to severe anemia resulting from defects in primitive haematopoiesis. Furthermore Npm inactivation leads to unrestricted centrosome duplication and genomic instability. We demonstrate that Npm is haploinsufficient in the control of genetic stability and that Npm heterozygosity accelerates oncogenesis both in vitro and in vivo. Importantly, Npm+/− mice develop a haematological syndrome with features of human MDS. Our findings unravel an essential developmental role for Npm and implicate its functional loss in tumourigenesis and MDS pathogenesis.
The Cyclin-Dependent Kinase Inhibitor p57KIP2 Functions as a Tumor Suppressor Gene in Human Leukemia.
Authors: Kuang Shao-Qing , Ling Xiaoyang , Sanchez-Gonzalez Blanca , Yang Hui , Garcia-Manero Guillermo
Published: April 2020

p57KIP2 is a cyclin-dependent kinase inhibitor with a role in the regulation of cell cycle progression, proliferation, differentiation, and development. Disruption any of these biological processes could result in malignant transformation. We have previously reported that epigenetic inactivation of p57KIP2 by aberrant DNA methylation is associated with a worse prognosis in patients with adult acute lymphocytic leukemia (Blood2003;101:4131–6 and JCO2005;23:3932–9). To further study the role of p57KIP2 in human leukemia we have developed several knock-in and knock-down cellular models of p57KIP2 in human leukemia cell lines. First, we established the methylation/expression patterns of p57KIP2 in 21 leukemia cell lines of both myeloid and lymphoid origin and of different phenotypes. We found that p57KIP2 was methylated and silenced in all except 6 (28%) cell lines. Methylation of the p57KIP2 promoter region correlated with gene silencing, and lack of protein expression, and treatment of methylated/silenced cell lines with a 5-aza-2′-deoxycytidine (DAC), a demethylating agent, resulted in p57KIP2 gene and protein re-expression. In unmethylated p57KIP2 cell lines, p57KIP2 mRNA was rapidly up-regulated by stimulation by growth-inhibitory signals such as TNF alpha, LPS and TGF beta, and when cell-cycle progression was arrested. In contrast, treatment with dexamethasone, DAC or the histone deacetylase inhibitor SAHA did not have such an effect. The reverse was observed in methylated cell lines. Transient transfection of p57KIP2 in silenced cell lines resulted in cell-cycle arrest at the G0/G1 phase. Stable transfection of p57KIP2 using a lentivirus system in Raji cells, in which p57kip2 is methylated and silenced, resulted in the induction of apoptosis and the suppression of cell growth in soft agar. Stable transfection of p57KIP2 in Jurkat cells, in which p57KIP2 is unmethylated and expressed, resulted in partial inhibition of cell growth, and increased susceptibility to the induction of apoptosis by staurosporine. Knock-down of p57KIP2 using an shRNA lentivirus in K562 cells, in which p57kip2 is not methylated and is expressed, promoted cell growth and proliferation in soft agar. These studies provide evidence that p57KIP2 has the properties of a bona fide tumor suppressor gene in human leukemia and provide further weight to the notion that p57KIP2 has an important role in human leukomogenesis.
MLL-AF9 and FLT3 Cooperation during Myeloid Leukemogenesis: Development of a Model for Rapid Testing of New Therapeutics.
Authors: Stubbs Matthew C. , Krivtsov Andrei , Kim Yong-mi , Wright Renee D. , Feng Zhaohui , Agarwal Jyoti , Kung Andrew L. , Armstrong Scott A.
Published: April 2020

Human leukemias harboring chromosomal translocations involving the Mixed lineage leukemia (MLL, HRX, ALL-1) gene possess high-level expression, and frequent activating mutations of the receptor tyrosine kinase FLT3. We used a murine bone marrow transplant model to assess cooperation between MLL translocation and FLT3 activation, and demonstrate that MLL-AF9 expression induces leukemia in approximately 70 days whereas the combination of MLL-AF9 and FLT3-ITD does so in less than 30 days. In both cases, the resulting disease is a highly aggressive, clonal, acute myelogenous leukemia. Detailed immunophenotypic analysis demonstrates expansion of an IL-7Rα− Lin− Sca-1− c-Kit+ CD34int. granulocyte macrophage progenitor (GMP)-like population in both the MLL-AF9 and MLL-AF9/FLT3-ITD induced leukemias. Other progenitor populations such as IL-7Rα− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIlo common myeloid progenitors (CMPs) and IL-7Rα− Lin− Sca-1− c-Kit+ CD34− FcγRII/III− megakaryocyte erythroid progenitors (MEPs) are absent in the bone marrow from leukemic mice. Secondary transplantation of splenic cells from diseased mice established that leukemic stem cells are present at a very high frequency of approximately 1:100 in both diseases. Cooperation between MLL-AF9 and FLT3-ITD was further verified by real-time assessment of leukemogenesis using non-invasive bioluminescence imaging. We developed a transgenic mouse model that expresses luciferase under control of the ubiquitin promoter to provide luminescent bone marrow cells that can be used as donor cells in bone marrow transplant assays. In these experiments, the development of leukemia was followed in secondary recipients via bioluminescent imaging. We used this model to confirm cooperation of MLL-AF9 and FLT3-ITD, and to demonstrate that MLL-AF9/FLT3-ITD induced leukemias are sensitive to FLT3 inhibition in a 2–3 week in vivo assay. These data (1) show that MLL-AF9 induces acute myelogenous leukemia with a high frequency of leukemic stem cells and expansion of a GMP-like progenitor population, (2) demonstrate that activated FLT3 cooperates with MLL-AF9 to accelerate leukemogenesis, and (3) provide a new genetically-defined model system that should prove useful for rapid assessment of potential therapeutics in vivo.
Endogenous Thrombin Potential (ETP) for Assessing the Risk of Recurrent Venous Thromboembolism.
Authors: Eichinger Sabine , Hron Gregor , Weltermann Ansgar , Kollars Marietta , Kyrle Paul A.
Published: April 2020

Venous thromboembolism (VTE) is a multifactorial chronic disease. The number and severity of risk factors determine the risk of recurrence. A laboratory method that measures the overall thrombophilia is required.

In a prospective cohort study, we measured ETP in 778 patients with a first unprovoked VTE. Patients were followed after discontinuation of anticoagulants for an average of 49 months. The study endpoint was recurrent symptomatic VTE. ETP was determined by a commercially available (research use only) assay (Dade Behring, Marburg, Germany) in platelet poor plasma by use of a chromogenic substrate and automatic registration as well as computer assisted calculation of thrombin generation over time. Patients with recurrence had higher ETP than those without recurrence (104.2% ± 15.4% vs. 101.4% ±14.2%. Patients with ETP ≥ 100% had an almost two-fold higher relative risk (RR) of recurrence than patients with lower levels (RR 1.6, 95% CI 1.0 – 2.5). At 4 years, the cumulative probability of recurrence was 14.4% in patients with ETP ≥ 100% and 6.1% in those with lower levels (p = 0.05). Patients with ETP ≥ 100% had higher clotting factor levels (Table). ETP was significantly increased in heterozygous carriers of factor II G20210A as compared with patients with wild type factor II (128% ± 18% vs. 100 ± 12%, p < 0.001).

Patients with a first unprovoked VTE and an ETP ≥ 100% have an increased risk of recurrence.

Table Characteristics of 778 Patients with VTE ETP < 100% ETP ≥ 100% p-value Men, no. (%) 160 (42%) 174 (44%) n.s. Age at first VTE (yrs) 46 ± 17 47 ± 14 n.s. Factor V Leiden, no. (%) 124 (32%) 108 (28%) n.s. Factor II G20210A, no. (%) 4 (1%) 49 (13%) < 0.001 Factor VIII (IU/dL) 162 ± 46 168 ± 45 0.09 Factor IX (IU/dL) 113 ± 24 123 ± 27 < 0.001 Factor XI (IU/dL) 102 ± 21 110 ± 24 < 0.001
Deep Vein Thrombosis during Pregnancy in Double Homozygous Carrier of Factor V Leiden and Prothrombin G20210A.
Authors: Martinelli Ida , Battaglioli Tullia , Beretta Angelo L. , Bianchi Marina , Mannucci Pier Mannuccio
Published: April 2020

Background. Factor V Leiden and prothrombin G20210A are gain-of-function mutations leading to an increased risk of venous thrombosis. They are the most common causes of thrombophilia, being present in their heterozygous form in 15–20% for factor V Leiden and 6–15% for prothrombin G20210A of unselected patients with venous thrombosis. The prevalence of each mutation in the Italian general population is 3%. Homozygous carriers for each mutation are rare, and double homozygous carriers, expected in 1 in 1.200.000 individuals, has never been described in the literature so far.

Patient. On March 3, 2005 a 30-year-old woman was admitted to the Unit of Internal Medicine, Valduce Hospital, Como, for swell and pain in the left leg. She was pregnant at the 25th gestational week. Ultrasound examination diagnosed a deep vein thrombosis of the left external and common iliac veins. She had no symptoms of pulmonary embolism. She was given nadroparin 6000 UI b.i.d. (her weight was 63 kg), elastic stockings, and was discharged on March 9, 2005. Because of worsening of the swell in the left leg, she was re-admitted on March 12, 2005 and the ultrasound examination showed an extension of the deep vein thrombosis to the left femoral-popliteal venous system. The dose of nadroparin was increased to 7000 UI b.i.d. Thrombophilia screening revealed a double homozygosity for factor V Leiden and prothrombin G20210A. Her past personal history was negative for thrombosis, but she was never exposed to high risk situations (surgery, bone fractures, prolonged immobilization, oral contraceptive use, long-haul flights) apart from a previous pregnancy which ended in miscarriage at the 6th gestational week. On June 14, 2004 she had an uneventful vaginal delivery after two hours of labour, with the last nadroparin injection in the evening before, The newborn was a female who weighted 3500 g. Her family history was also negative for thrombosis in first- and second-degree relatives despite both parents were double heterozygotes for factor V Leiden and prothrombin G20210A and brother and sister were homozygotes for factor V Leiden and heterozygotes for prothrombin G20210A.

Conclusions. The relative risk of venous thrombosis in double homozygous for factor V Leiden and prothrombin G20210A is unknown but is predicted to be extremely high. However, the patient described here had her first episode during a trigger situation, i.e., pregnancy, and severe thrombophilia seems to run an uneventful cause in her family in spite of repeated exposures to high risk situations. Hence, we shall advise the patient to stop oral anticoagulant therapy, started soon after delivery, one year after the thrombotic event.