bluuebunny/crossref_metadata_2025 huggingface dataset:
Simvastatin Sensitizes Bcl-2-Negative DLBCL Cell Lines to CHOP: A Finding with Possible Clinical Implications for Treatment of Bcl-2-Negative DLBCL?
Authors: Ageberg Malin , Linderoth Johan , Lindén Ola , Jerkeman Mats , Drott Kristina
Published: April 2020

It has previously been shown that addition of rituximab overcomes bcl-2-associated resistance to CHOP in DLBCL patients (Mounier 2003). However, the role of rituximab in the treatment of bcl-2 negative DLBCL is presently unclear. To better understand the molecular mechanisms for CHOP-resistance, the DLBCL cell lines Karpas 422, ULA, WSU-NHL, SU-DHL-8 and SU-DHL-5 were characterised with regard to CHOP- and rituximab-sensitivity. As compared to the other cell lines, Karpas 422 and ULA were CHOP-refractory and WSU-NHL showed an intermediate response to CHOP-treatment. However, SU-DHL-8 and SU-DHL-5 were sensitive to low levels of CHOP. Interestingly, the response to rituximab did not correspond to CHOP-sensitivity, as CHOP-refractory ULA were sensitive to rituximab alone while CHOP-sensitive SU-DHL-8 were refractory. Moreover, when assayed for expression of bcl-2 protein by western blot, SU-DHL-8 and SU-DHL-5 showed a low or undetectable expression while Karpas 422, ULA and WSU-NHL expressed high levels of bcl-2 protein. Hence, low levels of bcl-2 correlated to CHOP-sensitivity and with rituximab-resistance, consistent with results from clinical studies. The HMG-CoA reductase inhibitor simvastatin can induce apoptosis of Hodgkin lymphoma and myeloma cell lines, and statin use was associated with reduction in lymphoma risk in the international case-control study EPILYMPH (Fortuny 2006). Yet, statins have been shown to impair the antitumour effects of rituximab by inducing conformational changes of CD20 (Winiarska 2008), questioning the concomitant use of statins and rituximab. To evaluate the effect of simvastatin on the cell death of bcl-2-expressing and –non-expressing cell lines, ULA, SU-DHL-8 and Karpas 422 were incubated with increasing levels of simvastatin. Interestingly, already at levels as low as 2 μM (corresponding to a daily intake of 10 mg simvastatin), bcl-2-negative SU-DHL-8 cells showed signs of increased cell death. However, bcl-2 expressing ULA and Karpas 422 cells were resistant to levels of simvastatin as high as 50 μM. Moreover, low levels of simvastatin sensitized SU-DHL-8 cells to CHOP treatment. Taken together, our results may suggest that replacement of rituximab with simvastatin during CHOP-treatment of bcl-2 negative DLBCL could improve treatment responses. However, further studies are warranted to bring this notion to clinical reality.
Inhibition Effects of baicalin on Lymphoma Cell Line CA46 in Vitro and in Vivo
Authors: Hu Jian Da , Huang Yi , Chen Yingyu , Wei Tiannan , Liu Tingbo , Lv Lianhuang
Published: April 2020

Baicalin is a traditional Chinese medicine with multiple biological effects. Some researches showed baicalin has anti-tumor effects in solid tumor, such as prostate cancer. In order to investigate its effects on proliferation inhibition and apoptosis induction in human lymphoma cell, we treated Burkitt lymphoma cell line CA46 with baicalin in vitro and in vivo of CA46 xenograft. Baicalin remarkably inhibited the cell proliferation, with an IC50 value of 10μM. Apoptosis was remarkably induced by baicalin in a dose-dependent manner, which was detected by Annexin V FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation respectively. Furthermore, RT-PCR showed that the mRNA expressions of c-myc and bcl-2 in treated CA46 cell decreased in a time-dependent manner. Western-Blot showed that the protein expressions of c-myc, bcl-2, procaspase-3 and PARP(116KD) in baicalin treated CA46 cell were down-regulated, while the expression of PARP(85KD) increased. Based on the results in vitro, we investigated in vivo efficacy of baicalin, alone or in combination with cytotoxic drug VP16, for treatment in CA46 nude mice xenograft. Baicalin with the dosage of 40mg/kg/d and 80kg/mg/d could remarkably inhibit the growth of the tumor compared with control group. Combination of baicalin and VP16 had better anti-tumor effects. Histological examination of tumor samples showed more necrotic cells in treated groups. And obvious apoptosis could be observed by electron microscope. No adverse events were found in treated groups. From above we could conclude that baicalin could efficiently induce proliferation inhibition and apoptosis of CA46 cells in vitro and in vivo, which may be related with the down-regulation of c-myc and bcl-2 expressions, as well as the up-regulation of caspase-3 activity.
Molecular Biological Mechanisms of Cyclosporine a, Tetrandrine and Their Combination on the Reversion of Multidrug Resistance in Leukemia Cell Line
Authors: Chen Bao-An , Guo Jing-jing , Cheng Jian , Gao Feng , Xu Wen-lin , Shen Hui-lin , Ding Jia-hua , Gao Chong , Sun Yun-Yu , Wang Jun , Zhao Gang , Song Hui-hui , Bao Wen , Sun Xin-chen , Cheng Hong-yan , Deng Yu-xia , Li Guo-hong , Liu Li-jie , Chen Wen-ji , Wang Jue-qiong , Yuan Peng , Pei Xiao-ping , Chen Ning-na , Wang Xue-mei
Published: April 2020

Objective Previous researches confirmed that multidrugresistance(MDR) plays an important role in the failure of chemotherapy on malignant tumors. This study was to investigate the molecular biological mechanisms of cyclosporine A(CsA), tetrandrine(Tet) and their combination on multidrug resistance cell line K562/A02.

Methods K562/A02 cells were treated with cyclosporine A and (or) tetrandrine. The intracellular DNR concentration and the expression of P-glyco-protein (P-gp) were observed by flow cytometry (FCM) assay. The mRNA expression of mdr-1 was measured by fluorescent semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR).

Resulds CsA and Tet (alone or combination) elevated the intracellular DNR concentration in K562/A02 cells(the fluorescence intensity of intracellelar DNR in K562/A02 cells was 60%,65% and 98% respectively of that in K562 cells); The fluorescence intensity of P-gp in K562 and K562/A02 cells was 0.5% and 97.97%. The P-gp expression was down after treated with CsA,Tet and both(75.32%,76.86% and 48.61%); mdr1 mRNA was also down regulated, and the effect of their combination was greater.

Conclusion Multidrug resistance (MDR) can be partially reversed by CsA or Tet, the combination of both drugs shows a great synergistic reversal effect.
Five New Pedigrees with Inherited RUNX1 Mutations Causing Familial Platelet Disorder with Propensity to Myeloid Malignancy (FPD/AML)
Authors: Owen Carolyn J , Toze Cynthia L , Koochin Anna , Forrest Donna L. , Smith Clayton A. , Jackson Shannon C , Poon Man-Chiu , Sinclair Gary D. , Leber Brian , Johnson Peter , Macheta Anthony , Yin John AL , Barnett Michael J. , Lister T. Andrew , Fitzgibbon Jude
Published: April 2020

Familial platelet disorder with propensity to myeloid malignancy (FPD/AML) is an autosomal dominant syndrome characterised by platelet abnormalities and a predisposition to myelodysplasia (MDS) and/or acute myeloid leukemia (AML). The disorder, caused by inherited mutations in RUNX1, is uncommon with only 14 pedigrees reported. We screened 10 families with a history of more than one first- degree relative with MDS/AML and detected inherited mutations in RUNX1 in 5 of these pedigrees. Several affected members had normal platelet counts or platelet function, features not previously reported in FPD/AML. The median incidence of MDS/AML among carriers of RUNX1 mutation was 35%. Individual treatments varied but included hematopoietic stem cell transplantation (HSCT) from siblings before recognition of the inherited leukemogenic mutation. Transplantation was associated with a high incidence of complications including early relapse, failure of engraftment and post-transplantation lymphoproliferative disorder. As acquired trisomy 13 and 21 and FLT3-ITD have all been associated with RUNX1 mutation in sporadic MDS/AML, a combination of single nucleotide polymorphism profiling and mutation analysis was performed to determine whether these secondary genetic events were implicated in the onset of overt malignancy in FPD/AML. Five disease (MDS and/or AML) samples from 4 of our pedigrees with FPD/AML were screened and in all cases, these abnormalities were excluded. Therefore, the secondary mutations that promote MDS/AML in individuals with germline RUNX1 mutations are distinct from those reported in sporadic cases and require further investigation. The small size of modern families and the clinical heterogeneity of the FPD/AML syndrome may have resulted in the diagnosis being previously overlooked. Based on our data, FPD/AML may be more prevalent than previously recognized and therefore, it would appear prudent to screen young patients with MDS/AML for RUNX1 mutation, particularly prior to consideration of sibling HSCT.
Proposal of a New Prognostic Scoring System for Myelodysplastic Syndromes (MDS) Based on the Immunophenotypic Features of CD34+ Myeloid Precursors
Authors: Sandes Alex Freire , Orfao Alberto , López Antonio , Chauffaille Maria de Lourdes L.F. , Oguro Tsutomu , Flores Juan , Matarraz Sergio , Kimura Eliza YS , Silva Marçal CA , Yamamoto Mihoko
Published: April 2020

Introduction. Myelodysplastic Syndromes (MDS) are hemopoietic clonal disorders characterized by a heterogeneous clinical course, which is currently predicted on the basis of clinical, morphologic and cytogenetic data. Only a few reports have studied the impact of flow cytometry immunophenotypic data on the prognosis of the disease.

Objectives: To evaluate the immunophenotypic characteristics of CD34+ myeloid precursor cells in MDS and their association with the prognosis of the disease.

Material & Methods: Overall, 39 patients with MDS - 8 with refractory anemia (RA), 9 RA with ringed sideroblasts (RARS), 12 refractory cytopenias with multilineage dysplasia (RCMD), 2 RA with excess of blasts (RAEB)-1, 5 RAEB-2, 2 with 5q- Syndrome and one unclassifiable MDS - were studied. Bone marrow cells were stained with an extensive panel of MoAbs.

Results: the most frequent phenotypic abnormalities on CD34+ myeloid cells were: CD7+ (36%), CD25+ (33%) and CD56+ (15%) followed by overexpression of CD13 and/or CD33 (33%). Abnormal expression of CD7, CD25, CD13 and CD33 were evenly distributed within the WHO and IPSS subgroups of the disease, although CD56 expression was more frequently detected in low risk IPSS cases. From the prognostic point of view, patients that showed altered expression of CD25, CD56 and CD33 had a shorter median overall survival (OS): 30 vs 109 months (p = 0.0003), 19 vs 102 months (p=0.009), and 21 vs 102 months (p=0.009), for CD25, CD56 and CD33, respectively. Reactivity for CD7 and overexpression of CD13 did not influence OS. A phenotypic score was built where a score of 1 was given to abnormal CD25, CD56 or CD33 expression. Significantly different median OS rates (p<0.04) were found for cases with a score of 0 (101±11 months), 1 (47 ±6 months) and >1 (15 ± 5 months).

Discussion: Our results show that flow cytometry immunophenotyping of CD34+ myeloid precursors provides clinically useful information for the prognostic stratification of MDS patients. Further studies including larger number of patients with a longer follow-up are necessary to confirm these results.
Impact of Age on Transfusion Independence Response, Survival, and Transformation to Acute Myeloid Leukemia in Patients with Deletion 5q: A Sub-Analysis of the MDS-003 Study
Authors: Battiwalla Minoo , Fu Tommy , Knight Robert D. , List Alan F.
Published: April 2020

Introduction: Recent data have demonstrated that lenalidomide reduced transfusion requirements and reversed cytologic and cytogenetic abnormalities in patients who have myelodysplastic syndrome (MDS) with the chromosome 5q31 deletion (List et al NEJM 2006). Previous studies have demonstrated that age may impact on survival rates in cancer patients, but the impact of age on patients treated with lenalidomide for deletion 5q [del(5q)] MDS has not been investigated. This sub-analysis investigated the impact of age on transfusion independence (TI) response, survival, transformation to acute myeloid leukemia (AML), and cytopenias in patients with deletion 5q who were treated with lenalidomide.

Methods: Transfusion-dependent, Low/Int-1-risk patients with primary MDS and del(5q) with/without additional cytogenetic abnormalities were treated with lenalidomide 10 mg daily or for 21 days every 28 days. Patients were treated until disease progression, treatment failure, or treatment-limiting adverse events. For this analysis, patients were subdivided into two categories: ≤75 and >75 years of age. The relationship between age and TI response, survival, progression to AML, and cytopenias were analyzed.

Results: A total of 148 patients were included in the study: 103 patients aged ≤75 years and 45 patients aged >75 years (Table). The TI response was 68% for patients aged ≤75 years and 60% for patients aged >75 years (P=0.35). As expected, median survival was shorter for older patients (P=0.01). There was no difference in the progression to AML with 22% progressing for patients aged ≤75 years and 16% for patients aged >75 years (P=0.65). Cytopenias were reported in 84% and 76% of patients aged ≤75 and >75 years, respectively (p=0.42).

Conclusion: Patients aged >75 years had a similar TI response to patients aged ≤75 years of age. Survival was shorter as is to be expected in an older age group with a lower average life expectancy. There was no difference in the rate of progression to AML when compared with patients aged ≤75 years. The rates of cytopenias were not influenced by patient age.

≤75 years (n = 103) >75 years (n = 45) P CI, confidence interval; NA, not yet reached. TI response, n/N (%) 70/103 (68) 27/45 (60) 0.35 Median survival, years (95% CI) NA (3.186–NA) 2.52 (2.13–3.09) 0.01 AML progression, n/N (%) 23/103 (22) 7/45 (16) 0.65 Cytopenias, n/N (%) 87/103 (84) 34/45 (76) 0.42
Activity of Azacitidine (AZA) in Chronic Myelomonocytic Leukemeia (CMML)
Authors: Abdulhaq Haifaa , Haq Bushra , Rossetti James M. , Shadduck Richard K. , Sahovic Entezam , Christianson Dijana , Lister John
Published: April 2020

Background: CMML is a distinct clinicopathologic entity recognized by the World Health Organization (WHO). Treatment of CMML by AZA was approved by the US Food and Drug Administration on the basis of a phase III randomized trial conducted by the Cancer and Leukemia Group B, for the treatment of myelodysplastic syndromes (MDS). Only 14 patients with CMML were included in the pivotal trial.

Methods: We retrospectively reviewed our institutional experience of CMML treatment with AZA. 39 patients were treated between May 1996 and January 2006. AZA was given at 75 mg/m2 subcutaneously daily for seven days every 4 weeks or 100 mg/m2 daily for 5 days every 4 weeks. Patients who received at least 2 cycles of therapy were considered evaluable for response (N=34). All patients were evaluated for toxicity. The modified International Working Group standardized response criteria for MDS were used for evaluation. The response to AZA, duration of response, tolerability and survival are presented.

Results: Patients had a median age of 68.5 years (range: 46–83); male: female ratio was 4.5:1. 28 patients (72%) had a WBC >12,000/μl (myeloproliferative form of CMML). 6 patients had 20–30% blasts in their bone marrow. The International Prognostic Scores were as follows: Low (N=3), Intermediate-1 (N=13), Internediate-2 (N=14), High (N=7), unknown in 2 patients (due to lack of aspirate particles in one and cytogenetics in the other). CMML was secondary to chemotherapy/radiotherapy in 5 patients. Overall response was seen in 14 patients (41%): 5 patients (15%) had a complete response (CR), 9 patients (26%) had hematologic improvement. Mean duration of response was 16.2 months (range: 3–50+); 2 patients are still alive and being treated with CR for >50 months. Average number of cycles to respond was 2.4 cycles (range: 1–6). Splenomegaly was present in 8 patients (20%). Treatment resulted in normalization of spleen size in 1 patient, decrease in 3 patients and no effect in 4 patients. Mean overall survival was 13.2 months (range 1–72). Average survival in responders was 23.5 months (5–72) while average survival in non-responders was 7.48 months (<1–23). Treatment was well tolerated. The most common non-hematological toxicities were nausea and fever.

Conclusions: AZA appears to be effective and well tolerated in CMML.
Disease-Related Mortality in Patients with Lower-Risk Myelodysplastic Syndrome (MDS)
Authors: Dayyani Farshid , Conley Anthony P , Pierce Sherry , Strom Sara , Stevenson William , Borthakur Gautam , Kantarjian Hagop , Garcia-Manero Guillermo
Published: April 2020

We have recently developed a risk model specific for patients with lower risk MDS that allows the identification of poor prognosis patients (Leukemia. 2008; 22:538-3). We showed that the majority of patients classified by IPSS as low risk actually have poor outcomes with respect to overall survival. The cause of death (COD) of these patients is not well understood. In order to guide early therapeutic intervention, it is important to identify whether the mortality in this group is attributable to causes related to MDS or whether death occurs due to age-related comorbidities. The aim of the present study was to examine the rate of disease-related deaths in a cohort of 269 deceased patients with low or intermediate-1 disease by IPSS at presentation to a tertiary care center from 1980 to 2006. Autopsy, final progress note, physician letter, or death certificate were used to determine COD. For each patient, we categorized the COD as disease-related vs. non-related. MDS-related death was defined as infection, bleeding, transformation to acute myeloid leukemia (AML), or disease progression. Non-related causes included accidents, coronary events, heart failure, respiratory and renal failure, non-hemorrhagic stroke, and chronic hepatitis. Median age at presentation and at time of death was 66 years (range 19–88) and 67 years (range 21–90), respectively. Overall median survival was 59 weeks (range = 1–831). By FAB classification, all subgroups were identified (RA = 41.3%, RARS = 5.8%, RAEB = 28.6%, RAEB-t = 12.3%, CMML = 12.0%). By IPSS score, most patients were Intermediate-1 (Int-1 = 79% and Low = 21%). Most patients had diploid cytogenetics (58.3%), and the most common cytogenetic abnormality was deletion of chromosome 5 (9.1%). Almost all patients received supportive care only. The COD was identified as MDS-related in 229 of 269 patients (85%). The most common disease-related COD was infection (39% of all deaths) followed by hemorrhage (34%) and transformation to AML (23%). The most frequent non-disease-related COD were cardiovascular events (19 of 40 patients). 8 of 269 patients died of a secondary neoplasm. Our data demonstrates that the majority of patients with low- or intermediate risk MDS will succumb to causes related to their underlying disease. Although these results need to be validated prospectively, early therapeutic intervention could be considered in the management of these patients to improve survival.
Utility of Oligonucleotide Array Comparative Genomic Hybridization to Identify Cryptic Copy Number Alterations in Myelodysplastic Syndromes
Authors: Chung Wendy K , Weisberger James , Sun Yi , Brenholz Pauline , Warren Stephanie , Brown-Kipphut Brigette A , Aradhya Swaroop , LeDuc Charles A , Grodman Marc , Burack Richard , Iqbal M. Anwar , Bennett John M.
Published: April 2020

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematological neoplasms characterized by peripheral cytopenias due to ineffective hematopoiesis and significant cytologic atypia in one or more of the myeloid lineages. There is a variable risk of progression to acute myeloid leukemia, which is dependent on the blast count and certain recurrent cytogenetic abnormalities. Cytogenetic characterization is important in both diagnosis and prognosis, but can be of limited value in lower-risk MDS subtypes because of the low frequency of karyotypic abnormalities detected by chromosome analysis. Recently, array comparative genomic hybridization (aCGH) has been shown to be an important technique in detecting gross and cryptic copy number changes in leukemias. However, there is little data on aCGH application in MDS. We used a custom design 105,000 probe oligonucleotide aCGH platform to analyze 44 bone marrows, in which a diagnosis of MDS was made to determine the clinical utility of oligonucleotide aCGH. All bone marrows were analyzed by morphology, flow cytometry, cytogenetics, and fluorescent in situ hybridization (FISH). Gender matched normal controls were used to assess copy number alterations in MDS samples. We classified significant gains and losses

as a minimum of 10% deviation from the baseline of at least 10 contiguous oligonucleotide probes spanning at least 1 Mb and as not within a previously defined region of copy number polymorphism in the Database of Genomic Variants.

Array CGH was able to detect all cytogenetically identified imbalances present in more than 1 cell, but as expected did not detect any of the balanced translocations or inversions. Of the 24 cases that were cytogenetically normal, 9 (38%) had anomalies detected by aCGH. In 5 (25%) of the 20 cases with at least one cytogenetic abnormality detected previously by chromosome analysis, additional imbalances were identified by aCGH. Recurrent copy number losses detected by aCGH were observed in 13/44 (29.5%) of cases and included 5q21 in 5 cases, chromosome 7 in 2 cases, 12p13 in 3 cases, 13q21-31 in 3 cases, and 20q12-13 in 2 cases. Oligonucleotide aCGH was able to provide extremely high resolution of the boundaries of the gains and losses due to the high oligonucleotide probe density. In 14/44 cases (31.8%), oligonucleotide aCGH provided novel information not provided by conventional cytogenetics and FISH and can be used to precisely define recurrent gains and losses. Our data demonstrates the clinical applicability of oligonucleotide aCGH in the diagnosis of MDS in addition to conventional cytogenetics to detect new cryptic abnormalities which may provide novel targets for therapy.
Serum Proteome Profiling Detects Myelodysplastic Syndromes and Different Expression of Fibrinogen Between RAEB and RCMD
Authors: Zhong Liye , Liu TianHao , Geng SuXia , Lu Zesheng , Weng Jianyu , Du Xin
Published: April 2020

BACKGROUND&OBJECTIVE FMyelodysplastic syndromes (MDS) are among the most frequent hematologic malignancies. The diagnosis of MDS can be difficult, and there is a paucity of molecular markers. The pathophysiology is still largely unknown. Therefore, we investigated whether serum proteome profiling may serve as a noninvasive platform to discover novel molecular markers for MDS and establish the predictive models that may be of help to serologic diagnosis and classification of MDS.

METHODS FSerum samples were collected from 14 MDS patients including to 8 Refractory anemia with excess blasts in transformation (RAEB) and 6 Refractory cytopenia with multilineage dysplasia (RCMD) and 18 non-MDS hematologic malignancies and 8 age- and sex-matched healthy subjects. Serum peptides were separated and purified with a purification kit of magnetic beads, using magnetic beads-based weak cation exchange chromatography (MB-WCX) and MB-IMAC Cu, bases on immobilized metal ion affinity chromatography on the surface of superparamagnetic microparticles. We generated serum proteome profiles by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF- MS) and identified a profile that distinguishes MDS from non-MDS hematologic malignancies and healthy subjects.

RESULTS FA totaI of 146 effective protein peaks were detected at the molecular range of 1.02 tO 10.25 ku, Among which 7 protein peaks were different significantly among MDS patients, non-MDS hematologic malignancies and healthy subjects (P<0.05). There was also different for Peptide mass fingerprinting in MDS patients, and the samples were divided into two groups, which was identical with clinical classification about RAEB and RCMD, using 3-cross validation approach. There was significantly different expression protein between RCMD and RAEB patients, which was identified as a piece of fibrinogen peptide. The expressions of fibrinogen in RAEB subtype patients were higher than RCMD subtype patients.

CONCLUSION F Using the MALDI-TOF-MS technique may help to identify serum proteomic biomarkers related to MDS. The predictive models can discriminate MDS patients from other hematologic malignancies and healthy people effectively and help to identify MDS clinical classification. The different expression of Fibrinogen between RAEB and RCMD may suggested heterogeneity of etiopathogenisis in different subtypeof MDS.
Depleting Autologous Activated CD4+ t Lymphocytes Increased Generation of Colony-Forming-Cells(CFC) in Patients with Myelodysplastic Syndromes
Authors: Zhang Zheng , Li Xiao , Zhang Qianqiao , Tao Ying , He Qi
Published: April 2020

Objective To investigate how autoimmune mechanism playing a role in generation of colony-forming-cells(CFC), bone mononuclear cells(BMNC) from MDS were removed of autologous activated CD4+ T cells in vitro and cultured to find out effect of T cells on MDS hemopietic progenitor.

Methods BMNC from 25 patients with low-grade MDS and 5 normal donors were depleted of CD4+CCR5+ T lymphocytes using magnetic sorting. Depleted and plused CD4+CCR5+ T BMNC were seeded onto methycellulose and the correlation of colony-forming-cell (CFC) number and the polarization of T cells were analyzed, the generation of CFC, the immunophenotype and the clonal cells(which had cytogenetic markers detected by FISH), was compared respectively.

Results ¢Å The capacity of BMNC from 5 healthy donors to generate CFC remained unchanged in the CD4+CCR5+ T lymphocyte-depleted and lymphocyte-plused BMNC. In contrast, cultures initiated with CD4+CCR5+ T lymphocyte-depleted BMNC from patients with low-grade MDS exhibited significantly increased generation of CFC compared with the corresponding lymphocyte-plused cultures, but the lymphocyte-plused cultures had no generation of CFC. ¢Æ The number of CFU-E from the CD4+CCR5+ T lymphocyte-depleted BMNC from patients with low-grade MDS showed significantly correlation with the percentage of Th1 (r=0.52, p≤¼ 0.05), but had no correlation with the percentage of Tc1 and the rate of Th1/Th2 and Tc1/Tc2 (p >0.05); The number of CFC, CFU-G and CFU-GE had no correlation with the polarization of T lymphocyte (P >0.05). ¢Ç The percentage of CD34 in bone nucleated cells of low-grade MDS was higher than that in healthy donors(1.8% vs. 1.0%, P >0.05), that of CD33 in nucleated cells of low-grade MDS was significantly higher than that in healthy donors[(20.3±5.8)% vs.(13.8±1.8)%(P≤¼0.05)], and that of CD13 in nucleated cells of low-grade MDS was significantly higher than that in healthy donors[(21.1±6.4)% vs. (11.6±1.8)%(p<0.05)]. After cultivation, the percentage of CD34 in low-grade MDS nucleated cells decreased to 1.4%(P >0.05), that of CD33 decreased to (12.1±3.7)%(p<0.05), and that of CD13 decreased to (17.1±5.4)%(p<0.05), but the percentage of CD34, CD33 and CD13 had no significantly changed in healthy donors between pre-culture and post-culture. ¢È FISH analysis in 6 patients revealed that +8 clone was increased(from 51% to 61%), but 20q- and -7 clone cell had no significantly changed.

Conclusion In certain subtypes of MDS, selectedly removement of autologous activated CD4T cells can increase the generation of colony-forming-cells(CFC) in vitro, and improve the differentiation of MDS medullary system, but the increased CFC consisting of residual normal hemopoiesis or conal hemopoiesis were still unconcluded.
Site-Specific PEGylation of Factor VIII (PEG-FVIII) Preserves Full Clotting Activity and Extends Therapeutic Efficacy in HemophiliaA Dogs
Authors: Liu Tongyao , Lillicrap David , Zhang Xin , Labelle Andrea , Powell Sandra , Mei Baisong , Murphy John E. , Pierce Glenn F. , Jiang Haiyan
Published: April 2020

To improve the effectiveness of Factor VIII replacement therapy for Hemophilia A, we sought to develop a PEGylated Factor VIII that would effectively treat bleeding episodes, while reducing the frequency of intravenous injections required for prophylaxis. Previously, we found that the site-specific PEGylation of Factor VIII (PEG-FVIII) preserves full clotting activity, prolongs circulating half-life and extends therapeutic efficacy in a number of bleeding models in hemophilic mice. To further characterize its activity, four naïve Hemophilia A dogs were treated with either PEG-FVIII or unmodified BDD-FVIII in a cross-over study design. All treated dogs showed normalized Whole Blood Clotting Time (WBCT), whole blood Thromboelastograph (TEG) profile, and Cuticle Bleeding Time within 30 min from dosing. Pharmacokinetic analysis of the decay of plasma FVIII activity and antigen levels showed that PEG-FVIII achieved 2-fold longer half-life and reduced clearance and volume of distribution relative to BDD-FVIII. Consistently, PEG-FVIII also demonstrated significantly prolonged efficacy relative to BDD-FVIII by measurement of WBCT and TEG. Both BDD-FVIII and PEG-FVIII were well tolerated in naïve HemA dogs, normal hematology and serum chemistry values were observed following administration. However, two naive dogs that received BDD-FVIII and one naive dog that received PEG-FVIII developed detectable neutralizing antibodies to human FVIII as early as on day 9 post-treatment. In summary, consistent with our previously reported findings in hemophilic mice, in comparison to BDD-FVIII, PEG-FVIII demonstrated superior half-life, full activity in stopping acute bleeding and prolonged efficacy in hemophilia A dogs. Taken together, the results support the use of site-specific PEGylation to create a homogeneous therapeutic for both prophylactic and on-demand treatment of patients with Hemophilia A.
Polymorphisms in CD31/PECAM-1 and CD38 Genes Are Associated with Susceptibility to Multiple Myeloma
Authors: Jamroziak Krzysztof , Szemraj Zofia , Szemraj Janusz , Grzybowska-Izydorczyk Olga , Oszajca Katarzyna , Janiszewska Grazyna , Balcerczak Ewa , Mirowski Marek , Urbanska-Rys Halina , Robak Tadeusz
Published: April 2020

Interactions with bone marrow microenvironment play a major role in the control of multiple myeloma (MM) growth and survival. MM clonal plasma cells co-express high levels of a signaling receptor CD38 and its ligand, the platelet endothelial adhesion molecule-1, CD31//PECAM-1. Homotypic and heterotypic interactions through these adhesion molecules (CD31/CD31, CD38/CD31 and CD38/hyaluronate interactions) may be involved in the pathogenesis of MM. The objective of this study was to investigate whether inherited variants in CD31/PECAM-1 and CD38 genes influence on susceptibility to MM. A total of 189 Caucasian patients with MM and 209 healthy controls were genotyped with the use of PCR-based methods. Genotyping was performed to assess allelic frequencies of three non-synonymous single nucleotide polymorphisms (SNPs) in CD31/PECAM1 gene including 373C>G (Leu 125Val), 1688G>A (Ser563Asn) and 2008A>G (Arg670Gly) as well as two SNPs in CD38 gene including 184C>G in intron 1 and non-synonymous 418C>T (Arg140Trp) in exon 3. Analyzing the prevalence of investigated genetic variants, we found that allelic frequencies of two SNPs in CD31/PECAM-1 gene and intron 1 184C>G SNP in CD38 gene differed significantly between MM patients and controls. Concerning CD31/PECAM-1 SNPs, the carriership of 373G allele was associated with the relative risk of MM of 4.5 (p=8×10−10), while in carriers of 1688A allele the relative risk of MM reached 3.0 (p=2×10−6). Regarding CD38 gene, compared to wild-type 184C-allele carriers, the carriers of variant 184G-allele had 3.4-fold increased risk of MM (p=1×1−9). In conclusion, the results of this study suggest that inherited variants in ligand-receptor CD31/PECAM1 - CD38 system contribute to the genetic susceptibility to MM in Caucasians.
Personalized Genetic Monitoring of myeloma using analytic multivariate analysis of total Fully-Sequenced Circulating DNA
Authors: Durie Brian GM , Beck Julia , Jagannath Sundar , Urnovitz Howard B. , Schutz Ekkehard
Published: April 2020

There is no single sequence or breakpoint region linked to all patients with myeloma. It was thus elected to extract, amplify and fully sequence all (total) DNA present in circulating blood (CNA) using pyrosequencing with the Roche/454 sequencer (GSFLX). The origin of circulating DNA was investigated by local alignment analyses using BLAST and compared with the circulating DNA of 47 healthy individuals, aged 18 to 64 years. Thirty-one samples were subjected to total CNA sequencing during the course of the disease for a patient with lambda Bence Jones myeloma sequentially monitored from relapse, through complete response for 38 months and then with development of subsequent relapse. Conventional monitoring included both serum free light chain analyses and whole body CT-PET imaging. For comparative purposes the CNA was categorized by origin, functionality, chromosomal localization and genes, based on public databases (human reference genome build 36.2 and corresponding annotation file seq_gene.md as downloaded from NCBI (ftp.ncbi.nih.gov). The average length of sequenced nucleotides was 185.7 nucleotides and the total number of nucleotides sequenced per sample was 1.2 – 2.6 million. CNA sequences occurring with a frequency significantly greater or less than those observed for healthy controls were evaluated in detail. The next steps were to examine correlations with initial active myeloma, response to treatment, the complete remission state off all therapy, and subsequent relapse. The presence of 10 DNA sequences was highly correlated with the disease course: ZMYM2; TSPAN5; TLL1; PRKD1; ANTXR1; SYT14; PRKCH; MBNL1; EGFR and EDG7. Of these ZYMYM2 was highly correlated with disease reactivation and relapse off therapy. TSPAN5 showed a clear pattern with reduction to background levels during remission, but increase at the very earliest sign of relapse evident on CT-PET. Conversely, other sequences (GRID1; KIF16B) increased substantially during the peak impact of successful therapy. Multivariate analyses were used to identify the best combinations of gene sequences predictive of disease course and outcome. The combination of four genes (GRID1; PRKD1; ANTXR1; GAB1) was sufficient to define the early active disease data points vs the normal controls (odds-ratio OR: 135 [10.6 to 172]; p<.0001). When the resulting model was kept and used as a search engine for the whole 2 years course, the time course followed the treatment success (OR: 54; p=.0017) and failure as well as the final relapse(OR: 13.3; p=.004)

In conclusion: Total sequencing of DNA has proved promising in providing personalized molecular monitoring for myeloma. The details of associated DNA sequences provide insights to the underlying molecular pathophysiology linked to myeloma disease progression, response to successful therapy as well as subsequent relapse. Further testing is ongoing.
Molecular Mechanism of CD56 Expression in Human Myeloma Cells
Authors: Iqbal Mohd S. , Otsuyama Ken-ichiro , Shamsasenjan Karim , Kawano Michio M.
Published: April 2020

CD56 (NCAM1), one of the non-B cell lineage markers, is frequently detected on primary myeloma cells from more than 80% patients with overt myeloma. Microarray analysis of the CD56(+) myeloma cell lines showed markedly increased expressions of transcription factors involved in the neuronal cell lineage compared to the CD56(−) myeloma cell lines. There are three important binding sites of transcription factors such as HOX, SOX and PAX, present in the CD56 promoter region. Furthermore, ChIP analysis showed the specific bindings of HOXB9 and SOX1 in the CD56 promoter region of both the CD56(+) and CD56(−) myeloma cell line, while there was no significant difference observed in the PAX8 expression between the CD56(+) and CD56(−) myeloma cell lines. RT-PCR and western blot analysis confirmed that HOXB3 and HOXB9 as well as some of their potential cofactors were up-regulated in the CD56(+) myeloma cell lines with the concomitant down-regulation of the SOX1 and OCT3/4. First, we focused on the role of down-regulation of SOX1 in the CD56(−) myeloma cell line by the shRNA transfection. However, shRNA against SOX1 failed to induce CD56 expression in CD56(−) myeloma cell line. Therefore, these results indicate that the down-regulation of SOX1 alone may not be enough for the CD56 induction which might also require the overexpression of either HOXB9 or HOXB3, and the further study in the molecular mechanism of CD56 expression might contribute to the understanding of complex network of the interaction of transcription factors in human myeloma cells.
Biological Significance of Constitutive NF-κB Activity Levels in Human Myeloma Cell Lines
Authors: Otsuyama Ken-ichiro , Amin Jakia , Shamsasenjan Karim , Iqbal Mohd S.
Published: April 2020

To elucidate the mechanism of constitutive NF-κB activity in human myeloma cell lines, constitutive expression of all the NF-κB family, RelA (p65), c-Rel, RelB, NF-κB1 (p105/p50) and NF-κB2 (p100/p52), genes in different human myeloma cell lines, such as IL-6-independent (AMO1, NOP2), IL-6-dependent (ILKM2, ILKM8) and IL-6-responsive cell line (U266), were first checked by RT-PCR and western blot analysis. In the electrophoretic mobility shift assay (EMSA) for κB main consensus, total DNA binding activity levels for NF-κB were higher in IL-6-dependent, IL-6-responsive and IL-6-independent human myeloma cell lines respectively. Supershift analysis with anti-NF-κB family antibodies and immunoprecipitation followed by western blotting, confirmed that p50/p50, p50/RelB and p50/p52 as the principal player responsible for constitutive activation of NF-κB in IL-6-independent cell lines. However, beside these major dimers, p50/p65 dimer was also involved in the constitutive NF-κB activation in IL-6-dependent and IL-6-responsive cell lines. Moreover, on the basis of microarray data in human myeloma cell lines, we confirmed that the expression levels of NF-κB target genes, such as NFKB2, RELB, NFKBIA, MALT1 and CD54, showed correlation with the level of DNA binding activity for NF-κB. Therefore, these data suggest that the difference of total DNA-binding activities for NF-κB partly depended on the different p50/p65 activity among the IL-6-independent, IL-6-dependent and IL-6-responsive human myeloma cell lines, and further studies in the different NF-κB-dimers activity levels might contribute to the clarification of the regulation of NF-κB target genes expression in human myeloma cell lines.
Identification of An Atypical Paraprotein with Inconsistent M-Spike Quantitation in Four Patients with Waldenstrom’s Macroglobulinemia
Authors: Brunker Patricia A. R. , Glidden Elizabeth , Ghobrial Irene M. , Lindeman Neal I.
Published: April 2020

The densitometric quantitation of the M-spike in patients with a monoclonal gammopathy is a standard method used to monitor diseases such as multiple myeloma and Waldenstrom’s macroglobulinemia. Typically, M-spike quantitation shows synchronous parallel trends with other measures of disease status, such as nephelometric immunoglobulin heavy chain measurements, and with clinical condition. However, we report a series of four patients with Waldenstrom’s macroglobulinemia and IgM paraproteins with atypical appearing M-spikes that were significantly discordant with the total IgM measurements and clinical condition (Figure 1). Each of these patients had paraprotein bands that were extremely narrow and intense with slight migration from the sample application site (Figure 2). Although similar band morphology may be seen with cryoglobulins, all of these patients had repeatedly negative cryoglobulin tests. Pre-treatment of these samples with beta-mercaptoethanol (BME) to reduce disulfide bonds led to normal-appearing and migrating M-spikes that, when quantitated, gave precise results that were consistent with the total IgM measurements. This technique has permitted continued use of the M-spike to monitor disease progression in these patients, and demonstrates the importance of careful visual inspection of the SPEP gels prior to performing M-spike quantitation in the clinical laboratory.

Figure 1: Figure 1:. Figure 2: Figure 2:.
Impact of Chromosome 13 Deletion and Plasma Cell Load on Long-Term Survival of Patients with Multiple Myeloma Undergoing Autologous Transplantation
Authors: Paul Esbjörn , Sutlu Tolga , Alici Evren , Gahrton Goesta , Nahi Hareth
Published: April 2020

High-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) is the most common treatment for patients under 60–65 years of age with multiple myeloma (MM). In this study, we present a retrospective analysis of the prognostic impact of different factors in patients who have received this treatment as first line therapy in our centre. Abnormalities in chromosome 13 were identified by fluorescence in situ hybridization at the time of diagnosis. The median OS and PFS from transplantation time in the whole group of 193 patients were 90 and 48 months respectively. The median follow-up was 65 months (range: 6–186 months). The complete remission (CR) rate in patients with and without del(13) was 31% and 40% respectively whereas the median OS in patients with del(13) was 58 months but not reached in patients without del(13) (p=0.006). The PFS was 26 months in patients with del(13) and 84 months in those without del(13) (p=0.001). The transplantation related mortality was 2.5% both in the absence and presence of del(13). Patients who achieved CR following ASCT had longer OS and PFS when compared to those who only achieved partial remission. Thus, this study confirms the role of del(13) as a marker of poor prognosis. Multivariate analysis showed that the existence of del(13) was the only single independent factor effecting survival (p=0.001). In patients without del(13), the prognostic impact was even stronger when combined with the plasma cell load in the bone marrow (p=0.020), whereas the plasma cell load had no effect on survival of patients with del(13). Overall, the absence of del(13) in combination with low plasma cell infiltration at diagnosis predicts the best survival.
NF-Kb Localization in Multiple Myeloma Plasmacells and Mesenchimal Cells
Authors: Conticello Concetta , Giuffrida Raffaella , Adamo Luana , Anastasi Gabriele , Iannolo Gioacchin , Salomone Edvige , Memeo Lorenzo , Amato Gabriella , Gorgone Ausilia , Romano Alessandra , de Maria Ruggero , Gulisano Massimo , Giustolisi Rosario , Di Raimondo Francesco
Published: April 2020

Several reports have clearly demonstrated that the activation of Nuclear factor-kappa B (NF-kB) is essential for the pathogenesis of MM because it regulates the expression of many proteins involved in cell survival, proliferation, and resistance to chemotherapeutic drugs. We therefore analyzed by immunohistochemistry, immunofluorescence and western blot analysis the nuclear localization of NF-κB in MM cells derived from 60 different patients with MM at presentation and in relapse, as well as in three myeloma cell lines (XG1, RPMI 8226, KMS-18). Surprisingly, nuclear localization (the active form) of NF-κB was detected in only one MM sample from a refractory MM patient and in two samples from relapsed patients, while all the other samples, including the MM cell lines, exclusively express the cytoplasmic (inactive) form of NF-κB. We next analyzed the NF-κB status in mesenchymal cells from MM patients and we found that NF-κB was clearly present in the nucleus. We have also analyzed the sensitivity of MM primary cells to different doses of the proteasome inhibitor Bortezomib, which is described to antagonize NF-κB activity. We found a consistent dose- and time-dependent antitumor activity against both chemoresistant and chemosensitive myeloma cells in all the samples analyzed, independently of NF-κB localization. In conclusion, contrary to the current dogma that indicates a constitutive activation of NF-kB in MM cells, we demonstrated that in a steady state, especially in patients at diagnosis, NF-kB resides in the cytoplasm (the inactive form) but the cells maintain the sensitivity to Bortezomib, thus indicating that block of IkB degradation is not the only mechanism responsible for proteasome inhibitor induced-apoptosis.
Decreased Expression Levels of CD54 Associated with Decreased Activity of CXCL12 in Primary Myeloma Cells
Authors: Kawano Michio M. , Iqbal Mohd S. , Otsuyama Ken-ichiro , Shamsasenjan Karim , Amin Jakia , Islam Abul , Abroun Saeid
Published: April 2020

In order to evaluate NF-kB activity in the primary myeloma cells, we examined the expressionlevels of CD54 by a flow cytometer. First, we confirmed that the expression levels of CD54 showed good correlation with the levels of DNA binding activity for NF-kB in human myeloma cell lines. Primary myeloma cells unexpectedly revealed lower expression levels of CD54 compared to normal BM plasma cells, while plasma cells from MGUS had high expression levels of CD54 as same as normal BM plasma cells. Furthermore, the expression levels of CD54 on these plasma cells showed the significantly positive correlation with the plasma levels of CXCL12 (SDF-1α) in their BM aspirates, and the expression levels of CXCR4, the receptor for CXCL12, decreased on primary myeloma cells compared to normal BM plasma cells. It was also confirmed that addition of CXCL12 in the in vitro culture significantly induced up-regulation of CD54 expressions in primary myeloma cells. Furthermore, myeloma cells with lower expressions of CD54 were unstable in the in vitro culture, resulting in the marked reduction of viable cell number; this was also the case where cell lines with lower or no expression of CD54 were sensitive to H2O2 treatment. Therefore, these data suggest that primary myeloma cells have lower expression levels of CD54, possibly lower NF-kB activities and different stability of in vitro survival from normal BM plasma cells, depending on expression levels of CD54.
Long-Term Arsenic Trioxide (ATO) Treatment Changes Multiple Myeloma (MM) Cells Micro-RNA and RNA Expression Profiles
Authors: Wen Jianguo , Feng Yongdong , Ballon Douglas J , Chang Chung-Che
Published: April 2020

Although the preliminary results of treating refractory and relapsed multiple myeloma patents with ATO are promising, the data suggest that the resistance to ATO treatment frequently occurs (57 to 79%). We treated MM cells RPMI8226 with long-term low dosage ATO (0.5 μM) and harvested the ATO resistant cells. ATO resistant cells IC50 is 4 times higher and ATO resistant cells formed 2 times more colonies with 0.75 μM ATO treatment than parental RPMI8226. P-p38 MAPK, Bcl-2, XIAP and survivin were elevated to a relatively high level in ATO resistant cells under continuous ATO stress. We have established a custom designed miRNA microarray printed with a comprehensive panel of miRNAs in miRBase version 9.0 (238 mmu-mirs) and miRNA predictions including 2617 MED-MIR predictions, 321 ‘Cand” predictions discovered through a strategy based on pylogenetice-shadowing, 129 ‘MIR” predictions discovered through a search for miRNA targets in 3′ UTR sequences. We used this miRNA array to probe the miRNA expression profiles of RPMI8226 and resistant cells and observed differences in miRNA expression between the two lines. We also performed oligonucleotide microarray analysis using HG-U133 plus 2.0 array to screen the differential gene expression. Results showed that ATO resistant cells repressed the expression of has-miR-181a, has-miR-181b, has-let-7i, has-miR-19b, has-miR-18a, has-miR-20a and has-miR222. On the other hand, LMAN1, NPGPx, HSP73 and EIF1 genes expression were up-regulated. These data revealed the preliminary mechanism how MM cells adapt to ATO and provide potential targets to overcome the ATO in MM therapy.