bluuebunny/crossref_metadata_2025 huggingface dataset:
G-CSF Increases Hematological Response among Patients with Myelodysplasia Treated with Azacitidine.
Authors: Rossetti James M. , Falke Eric , Shadduck Richard K. , Latsko Joan M. , Kramer Wendy , Lister John
Published: April 2020

Rationale: Myelodysplastic syndrome (MDS) is characterized by peripheral blood cytopenia, a major cause of morbidity and mortality. Two current treatments that may ameliorate cytopenia in MDS are azacitidine (aza) and hematopoietic growth factors. When used alone in MDS, erythropoietin (epo) and G-CSF are reported to increase the hemoglobin level and the neutrophil count, respectively. Additionally, epo and G-CSF may work synergistically to improve anemia and neutropenia. This study examined whether the combination of growth factors and aza would be more effective than aza alone for the treatment of MDS.

Methods: We identified 86 MDS patients treated at our institution who received an average of 10.8 cycles of aza. Forty-nine (49) of these patients also received either epo, G-CSF or both during the course of their aza treatment, while 37 did not receive hematopoietic growth factors. These 2 groups did not differ significantly either in number of cycles of aza received (p=0.34) or FAB MDS subtype (p=0.50). Hematological responses were tabulated by International Working Group criteria and compared using chi-square statistics.

Results: Patients treated with aza alone had a 51% (19/37) overall hematological response rate (OHR). Patients who received aza + epo (A + E) had a 50% (6/12) OHR and when compared to aza alone (A), was not statistically different (p=0.9). Patients who received aza + G-CSF +/− epo (A + G +/− E) had an 84% (31/37) OHR versus 51% (25/49) in patients who did not receive G-CSF (A +/− E) (p=0.003). When the group A + G +/− E was compared to A +/− E there was a statistically significant difference in favor of A + G +/− E in erythroid (77% vs. 37%; p=0.001) and platelet (72% vs. 47%; p=0.04) response, but no difference in neutrophil response (71% vs. 67%; p=0.9).

Conclusion: In patients with MDS treated with aza the addition of G-CSF improved OHR, erythroid and platelet response. Surprisingly, neutrophil response was not improved. The biological basis for these observations suggests an effect upon erythroid and megakaryocytic lineages. We postulate that G-CSF may have an anti-apoptotic effect as has been previously demonstrated when used in combination with epo.
Acceleration of Leukemogenesis in CREB Transgenic Mice by Retroviral Insertional Mutagenesis.
Authors: Sandoval Salemiz , Sakamoto Kathleen , Deepa Shankar
Published: April 2020

Cyclic adenosine monophosphate response element binding protein (CREB) is a 43 KDa transcription factor that promotes cell growth and survival, and is a downstream target of growth factor signaling in myeloid cells. We previously reported that CREB is overexpressed in greater than 60 percent of acute myeloid leukemia (AML) patients and is associated with an increased risk of relapse and decreased survival. Transgenic mice that overexpress CREB in the myeloid lineage develop a myelodysplastic/myeloproliferative (MDS/MPD) syndrome with splenomegaly and aberrant myeloid proliferation after one year, but not leukemia. To accelerate leukemogenesis and identify genes that specifically cooperate with CREB, we infected CREB transgenic and wild type (WT) mice with the retrovirus, MOL4070LTR (kindly provided by Linda Wolff, NCI). MOL4070LTR has been shown to accelerate the onset of leukemia in mice by insertionally activating or mutating cooperating genes. Fifteen newborn CREB transgenic and nineteen control WT mice were injected intraperitoneally with virus (1×105 PFU). The infected mice were monitored at regular intervals for signs and symptoms of disease. We analyzed peripheral blood counts for evidence of blasts and performed physical examination for signs of abdominal distention and lethargy. Three out of the fifteen CREB transgenic mice infected with the virus developed disease at 6 to 7 months of age. WT mice, now 10 to 12 months of age, have yet to develop disease. All three CREB transgenic mice infected with MOL4070LTR had increased white blood cells, hepatosplenomegaly, and abnormal blood smears. One of the three CREB transgenic mice with leukemia developed AML with circulating blasts in the peripheral blood and infiltration of the spleen and liver with myeloblasts by histologic examination. The two other CREB transgenic mice died from acute lymphoblastic leukemia. Our preliminary results demonstrate that CREB transgenic mice but not WT mice infected with MOL4070LTR develop leukemia. We are currently identifying possible oncogenes using DNA from the mouse that developed AML. Our results suggest that CREB has the potential to induce acute leukemia in mice; however, additional mutations are required.
Circulating Endothelial Cells and Circulating Endothelial Progenitor Cells in Polycythemia Vera.
Authors: Hoeg Rasmus T. , Jobe Dean A. , Asp Krista E. , Callister Steven M. , Meyer Lori A. , Mathiason Michelle A. , Cole Craig E. , Bottner Wayne A. , Farnen John P. , Go Ronald S.
Published: April 2020

Background: Angiogenesis and thrombosis, two conditions associated with perturbation of the vascular endothelium and increased CECs, are frequently observed in PV. We performed this study to quantify and characterize CECs in patients with PV and to determine whether CEC profile is associated with thrombotic complications. We also quantified CEPs in a subgroup of patients.

Methods: We used flow cytometry to prospectively analyze CECs and CEPs in the whole blood of healthy patients (n=20) and patients with PV (n=30; 13 treated with hydroxyurea, 12 undergoing phlebotomy alone, and 5 never treated). CECs (CD45−/CD31+/CD146+) were quantified and characterized to determine their apoptotic (annexin stain) or activation (CD106+) states. Cells with CD45−/CD31+/CD133+/VEGF-R2+ immunophenotype were considered CEPs.

Results: CEC levels in PV patients were higher compared to healthy controls (median 53 cells/mL [range, 11–392] vs 18.5 cells/mL [range, 4–66]; P< .0001). However, the proportions of apoptotic (17.3% [range, 0–87.5] vs 25.9% [range, 0–57.1]; P= .87) and activated (0.7% [range, 0–28.6] vs 0% [range, 0–57.1]; P= .14) CECs were similar between the two groups. In PV patients with high levels (≥2 SD above control mean or ≥53 cells/mL) of CECs (n=14), 5 (36%) developed thromboses. Four (25%) of 16 PV patients with CEC levels similar to healthy controls developed thromboses. The rates of thrombosis between the groups were not statistically different (P= .69). In addition, CEC count, activation, and apoptosis were similar between the hydroxyurea-treated group and the group not treated with hydroxyurea. We detected low numbers of CEPs in PV patients (n=25) that were similar to controls (4 cells/mL [range, 0–100] vs 8 cells/mL [range, 0–60]; P= .34).

Conclusions: CECs, but not CEPs, are significantly increased in PV patients. However, there appears to be no association between CEC number, activation or apoptotic state and the development of thromboses. In addition, hydroxyurea therapy does not appear to effect endothelial cells.
In Vitro Erythroid Cell Expansion Analysis in Polycythemia Vera.
Authors: Yoon Donghoon , Bruchova Hana , Agarwal Archana M. , Prchal Josef T. , Prchal Jaroslav F.
Published: April 2020

Polycythemia vera (PV) is an acquired myeloproliferative clonal stem cell disorder characterized by cytokine hypersensitivity. The erythroid colony forming assay has been useful in PV diagnosis. However, studies aiming to elucidate molecular mechanism in PV pathogenesis often require large numbers of cells from a specific erythroid stage. For this purpose, we had adapted the mouse expansion assay described by Karur et al (Blood, 2006 in E-Pub) and differential display method described by Zhang et al (Blood, 2003, 102; 3938) to develop a human erythroid expansion protocol. In this study, we used peripheral blood mononuclear cells (PB-MNCs) from PV patients and healthy donors, and expanded them along erythroid lineage in 21 day culture. Through the culture, we took samples at 8 timepoints (days 1, 7, 9, 11, 14, 16, 19, 21) to evaluate differentiation patterns. We had generated 5 different regions using CD71 (transferrin receptor) and CD235a (glycophorin A) and characterized these regions by standard morphology analysis. This protocol allowed differentiation of PB-MNCs to all erythroid stages ending with late normoblast, reticulocyte, and mature erythrocytes (which do not survive well in this culture environment).

The erythroid differentiation and proliferation patterns were different between PV and normal. In the first week of culture, there was no significant proliferation difference observed between PV and normal. In contrast, the differentiation progressed more rapidly in PV, likely reflecting the differentiation of Epo independent PV population (since the first week culture contains no Epo). In the second week of culture (Epo present), there was a markedly increased expansion of PV erythroid cells. However, the differentiation pattern of PV resembled that of normal.

In conclusion, we demonstrated that our in vitro expansion method allows expansion of PB-MNCs along erythroid lineage with 60~80% stage homogeneity as to the stage of differentiation in both PV and normal. The differences of differentiation and proliferation pattern between PV and normal reflected the expected biological behavior of erythroid progenitors. Thus, the expansion protocol is useful to study molecular mechanism of PV pathogenesis, which requires large number of erythroid progenitors of various stages.
GATA-1 Protein Levels Are Not Altered and Are Correlated to the Jak-2 Mutation Status in Patients with Idiopathic Myelofibrosis (IMF).
Authors: Wang Jen-Chin , Xiao Ying-Li , Chang Tsong H , Hunis Brian , Astrow Alan
Published: April 2020

GATA-1 is important in megakaryocytopoiesis, and the fact that GATA-1low mice eventually develop myelofibrosis indicates that GATA-1 is important in the pathogenesis of idiopathic myelofibrosis (IMF). Previous study has shown that GATA-1 levels were lower in protein content in CD61+ cells of patients with IMF (Vannucci et al., Am J Pathol 167:849 2005). But the study was done with pooled protein from only three patients. We used ELISA assay (GATA-1 ELISA kit, Panomics, CA) to assay the protein content from nuclear extracts of blood hematopoietic progenitor cells (CD34+cells) and megakaryocytes (CD61+ cells) from patients with IMF and compared the results with those of patients with other myeloproliferative disorders and with controls. CD 61+ cells were obtained from 10-day cultures of blood CD34+ cells in serum-free culture medium with thrombopoietin and stem cell growth factor as described. The results are shown in Table 1. No statistical difference was found in the GATA-1 content in the nuclear extracts in CD34+ or CD61+ cells. We further analyze the GATA-1 levels in relation to the JAK2 status and significant correlation between the levels of GATA-1 levels and positive JAK2 nutation were found in blood CD34+ cells (p=0.01) and subsignificant in CD61+cells (p=0.08) (Table2) .These preliminary results suggest that GATA-1 protein contents are not altered and higher GATA-1 levels are seen in JAK-2 mutation positive patients with IMF.

Table 1. GATA-1 Levels in Patients with IMF and others IMF MPD Controls GATA-1 Levels: mean± SE of ng/2μg protein; n: numbers of patients CD34+ cells 27.58±3.2 (n=9) 30.57±5.4 (n=4) 28.06±2.9 (n=7) CD61+ cells 58.99±11.53 (n=8) 38.64±18.2(n=4) 41.03±11.3 (n=6) Table 2. Correlation between GATA-1 Levels and JAK2 Status in Patients with IMF JAK2 mutation Positive JAK2 mutation Negative P Value GATA-1 Levels: mean±SE(ng/2μg protein): n: numbers of patients CD34 + cells 35.6±3.4 (n=4) 21.1±2.7(n=5) 0.01 CD61+ cells 73.7±13.0(n=4) 43.8±13.7(n=5) 0.08
JAK2 V617F Mutation in Essential Thrombocythemia.
Authors: Ahn Jeong Yeal , Park Pil Whan , Seo Yiel Hea , Shin Dong-Bok , Lee Jae-Hoon , Yoo Soo-Jin , Bang Soo-Mee
Published: April 2020

Background: Essential thrombocythemia (ET) is thought to reflect transformation of a multipotent hematopoietic stem cell, but its molecular pathogenesis has remained obscure. But tyrosine kinase, especially Janus kinase 2 (JAK2) has been implicated in myeloproliferative disorders other than chronic myeloid leukemia. We investigated the incidence and its correlation with other clinicopathologic variables of JAK2 mutation in patients with ET and reactive thrombocytosis (RT).

Method: JAK2 mutation analysis, using allele-specific polymerase chain reaction, was undertaken on genomic DNA from bone marrow aspirates of 24 patients with ET and peripheral blood in 36 patients with RT.

Results: JAK2 mutation was detected in 11 patients (46%) among the 24 patients with ET and was not found in 36 patients with RT. In patients with ET, older age and leukocytosis were related with JAK2 mutation without statistical significance (p=0.172 and 0.094, respectively). But this mutation was not correlated with sex, hemoglobin, platelet count, splenomegaly, increased cellularity of bone marrow, bone marrow fibrosis and vascular complications.

Conclusions: The current observation strengthens the specific association between JAK2 mutation and ET. At the diagnosis of ET, identification of JAK2 mutation should be incorporated in foundation for new approaches.
Mcl-1 Promoter Insertions as an Independent Prognostic Factor for Treatment Free Survival in Chronic Lymphocytic Leukaemia.
Authors: Hooper Laura , Delgado Julio , Starczynski Jane , Evans Jason , Pepper Chris , Greenwell Pamela , Fegan Chris , Pratt Guy
Published: April 2020

Mcl-1 (Myeloid cell leukaemia 1) is an anti-apoptotic member of the Bcl-2 family of proteins and is known to be important in the biology of Chronic Lymphocytic Leukaemia (CLL). A study by Moshynska et al demonstrated that a 6 or 18 nucleotide promoter insertion within the Mcl-1 promoter region influenced clinical outcome in CLL. In this study the presence of the insertion was also associated with high Mcl-1 mRNA and Mcl-1 protein levels. Subsequently there have been a number of published reports showing that these insertions have no impact at all on survival or any role in the pathogenesis of CLL. Using PCR and WAVE analysis, we analysed both the 6–18bp Mcl-1 promoter insertions in a cohort of a 100 CLL patients and 100 normal controls. Mcl-1 promoter insertions occurred at a similar frequency in the control and CLL groups. Mcl-1 promoter insertions did not correlate with any of the established prognostic factors in CLL (CD38 expression, VH mutation status, LDT, Zap 70 and stage), did not correlate with Mcl-1 protein levels and did not influence overall survival (P values >0.05). However Mcl-1 promoter insertions significantly affected treatment free survival (P= 0.002). This result needs confirming in a larger number of patients as it suggests that promoter insertions identify patients that are more likely to require therapy and again raises the question of its role, if any, in CLL pathology.
Chronic Lymphocytic Leukemia (CLL) in Parents and Children.
Authors: Mauro Francesca R. , Ghia Emanuela M. , De Propris Maria S. , Giammartini Elena , Starza Irene Della , Giannarelli Diana , Giudice Ilaria Del , Santangelo Simona , Brugnoletti Fulvia , Gentile Massimo , Guarini Anna , Foa Robert
Published: April 2020

Chronic lymphocytic leukemia (CLL) is the most frequent form of leukemia in which familial aggregation has been observed. The affected relative is usually a sibling and less frequently a child. In the present study we evaluated whether CLL affecting parents and their children was associated with different clinical features, survival, immunoglobulin (Ig) VH genes repertoire and mutation status. The family history of 2% of the CLL patients diagnosed at our institution was characterized by the presence of a parent or a child affected by CLL. Other lymphoproliferative diseases, such as non-Hodgkin lymphoma, Hodgkin disease and acute lymphoid leukemia, affecting parents or children were also recorded in 3% of cases. Among the 17 identified parent/child CLL pairs there was a higher, though not significant, rate of gender concordant pairs than expected. Children were significantly younger at diagnosis than their parents. No significant differences in stage distribution, clinical course, survival and proportion of unmutated cases emerged between the two generations. In no case the parent and the respective child shared an identical IgVH repertoire. However, a significantly higher than expected usage of VH3-23 (p<0.005), VH3-21 (p<0.005), VH5-51 (p=0.001), VH1-18 (p= .90E-04), VH3-30.3 (p<0.005) genes was recorded. Of interest, a VH5-51 gene usage has been also described by other authors in multiaffected families. By flow cytometry and PCR analysis, no clonal lymphocytes were found in the peripheral blood of 38 young healthy (median age: 37 years) relatives included in the third generation of these multiply affected families. This could be explained not only by the relatively small sample of subjects analyzed but also by their relatively younger age compared to that of the healthy individuals analyzed in other series. In conclusion, the coexistence of CLL in parents and offspring is a relatively uncommon event. The differences in the IgVH gene repertoire could reflect a different ontogeny of these cases. Affected children show a significantly earlier, but not a more aggressive clinical course. These data can be used to offer counselling to CLL patients.
Co-Expression of CD38 with Zap-70 Is Predictive of Treatment Intervention in Patients (pts) with Early Stage Chronic Lymphocytic Leukemia (CLL).
Authors: Coignet Jean-Gabriel , Padmanabhan Swaminathan , Manochakian Rami , Miller Kena C. , Wallace Paul , Block AnneMarie , Chanan-Khan Asher Alban
Published: April 2020

Introduction: CLL is a heterogeneous disease where advanced stage pts have compromised survival despite treatment, while early stage pts may not require any intervention. Based on current NCI-WG guidelines, most early stage (Rai stage 0 and 1) CLL pts do not require treatment until the development of progressive or symptomatic disease, though eventually over 70% of CLL pts will receive therapy. Currently, there are no markers to predict this subset of pts. Several markers of adverse prognosis, including ZAP-70+, CD38+, un-mutated IgVH gene and cytogenetic aberrations (17p-, 11q-, +12) have been identified. These markers are primarily studied in context of aggressive clinical course and survival outcome. The value of these markers to predict the necessity of treatment intervention in early stage CLL pts has not yet been completely evaluated. We investigated the expression pattern of Zap-70 and CD38 to examine their predictive value in identifying early stage CLL pts who will require therapeutic intervention.

Methods & Results: 93 CLL pts were evaluated since 2002 at our institution. Flow cytometry was used to determine Zap-70 and CD38 expression on CD19+ CLL cells obtained from peripheral blood. Pts were considered to be positive for Zap-70 and CD38 expression if ≥ 20% and ≥ 30% of the cell stained for these proteins, respectively. For the Zap-70 analysis we used similar methodology as described by Crespo et al.1 Thirty-six (19M, 17F) pts had limited stage CLL, based on Rai staging criterion. Median age was 65 years (range 43–86) with stage 0 or 1 observed in 14 and 22 pts, respectively. Median time from diagnosis is 2 years (range <1–17). Increased expression of either Zap-70 or CD38 was seen in 22 and 7 pts, respectively. Five (14%) pts were positive for both. All (100%) pts with concurrent increased expression of Zap-70 and CD38 required treatment (p=0.003), as compared to 30% of patients requiring treatment that were negative for both proteins or positive for only protein. Among the pts that required treatment 4 had 13q- and one had trisomy 12 on cytogenetic analysis.

Conclusion: Our study, demonstrates for the first time, the clinical utility of CD38 and Zap-70 co-expression in determining the probability of treatment intervention in early stage CLL pts. Although the number of pts studied is small, our findings highlight a potentially important use of these markers in the management of early-stage CLL pts. These observations warrant validation in a larger cohort of pts early stage CLL pts as well as correlation with other prognostic markers such as cytogenetic and IgVH gene mutational status.
Variations in Proteasome Enzymatic Activities in Plasma of Patients with Chronic Lymphocytic Leukemia and Their Value in Predicting Clinical Behavior.
Authors: Ma Wanlong , O’Brien Susan , Jilani Iman , Zhang Xi , Kantarjian Hagop , Estov Zeev , Ferrajoli Alessandra , Keating Michael , Giles Francis , Albitar Maher
Published: April 2020

The ubiquitin-proteasome pathway plays a major role in degrading proteins that regulate important cellular processes including cell cycle regulation, apoptosis, DNA repair, and stress response. Proteasomes have 2 active sites each for 3 different peptidase activities: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L) (postglutamyl peptide hydrolytic-like). Different proteasome inhibitors affect each of the activities differently and at different concentrations. For example, NPI-0052 inhibits Ch-L and Tr-L activities at lower concentrations than does bortezomib, while bortezomib inhibits Cas-L at lower concentrations than does NPI-0052. These enzymatic activities are usually measured in normal or tumor cells to monitor therapy with proteasome inhibitors. We developed fluorogenic kinetic assays using peptide-AMC (7-amino 4-methylcoumoran) substrates to measure Ch-L, Tr-L, and Cas-L activities in peripheral blood plasma rather than cells. This approach allowed us to standardize measurements and express enzymatic activity as pmol AMC/sec/mL plasma. We tested Ch-L, Tr-L, and Cas-L activities in the plasma of 226 patients with chronic lymphocytic leukemia (CLL) and assessed their correlations with clinical behavior. Ch-L, Tr-L, and Cas-L activities were significantly (P <0.001) higher in the plasma of patients with CLL (medians: 1.47, 2.44, and 1.38 pmol AMC/sec/mL, respectively) than in healthy volunteers (n = 42) (medians: 0.80, 0.74, and 0.81 pmol AMC/sec/mL, respectively). Although Ch-L and Cas-L activities did not differ significantly between men and women with CLL, Tr-L activity was significantly higher in women (P = 0.01). Rai stage correlated with Ch-L (P <0.001) but not Cas-L or Tr-L activity. Only Ch-L activity correlated with WBC count (P <0.001). β2-microglobulin levels correlated strongly with Ch-L activity (R=0.40, P <0.001) and weakly with Cas-L activity (R=0.25, P = 0.001) but not with Tr-L activity. Ch-L and Cas-L activities were both strong and independent predictor of survival when examined as continuous variables (P=0.02 for both), as well as when the median was used as a cut-off point (P =0.02 and P=0.03, respectively). Both Ch-L and Cas-L activities were independent of β2-microglobulin in predicting survival, but both correlated with each other and were not independent of each other in predicting survival. There was no correlation between Tr-L activity and survival. These data suggest not only that proteasome activity as measured in the plasma of patients with CLL has important prognostic value, but also that CLL patients may benefit from proteasome inhibition therapy that specifically targets Ch-L or Cas-L activities.
Comparison of a Quantitative PCR Method with FISH for the Assessment of the Four Aneuploïdies Commonly Evaluated in CLL Patients.
Authors: Bastard Christian , Fruchart Christophe , Raux Gregory , Parmentier Francoise , Vaur Dominique , Tilly Herve
Published: April 2020

Four chromosomal defects associated with prognosis have been identified in CLL patients, namely deletions of the 13q13-q14, 11q22 and 17p13 regions and trisomy 12. Due to the low proliferation index of these tumors and to the fact that some defects can be cryptic, the detection of these abnormalities by conventional cytogenetics is difficult. Therefore, the resulting aneuploidies are usually evaluated by fluorescent in situ hybridisation (FISH). As probes are expensive and FISH time consuming, we aimed to compare a quantitative PCR method - quantitative PCR of short fluorescent fragments (QMPSF) - with FISH for the detection of these acquired aneuploidies in a series of 110 patients with Binet stage A CLL. Genes located in the deleted or gained regions were selected as target genes - DLEU2 at 13q14, ATM at 11q22, p53 at 17p13, POU6F1 and MDM2 at 12q13 and 12q15 respectively - and amplified using a method based on the simultaneous amplification of short fluorescent genomic fragments under quantitative conditions. A chromosomal imbalance involving one or several of the four loci was detected in 76 patients (69%) either by FISH or QMPSF or both. A deletion of chromosome 13 was found in 61 patients (55%). A 11q22 deletion was present in 9 patients (8%), a trisomy 12 in 9 (8%) and a 17p deletion in one. The rather low frequency of the three latter defects reflects the fact that only patients with stage A CLL at diagnosis were studied, and neither stage B or C CLL. The 13q deletion was isolated in 53 patients and associated with a second defect in 8: these were six of the 11q22 deletions, one trisomy 12 and the 17p deletion. When the 13q deletion was associated with either a 11q deletion or a trisomy 12 both abnormalities were present in the same proportion of cells. This was not the case for the 17p deletion which appeared to be a secondary event, since, as evaluated by FISH, it was present in 24% of cells whereas the 13q deletion was present in 85% of interphase nuclei. FISH and QMPSF results were identical for 103 of 110 patients. The secondary 17p deletion was not detected whereas all other discrepancies could be explained. This study demonstrates that a single multiplex PCR can replace FISH in CLL patients. However, whereas QMPSF is perfectly adapted to the detection of primary defects, care should be taken when searching for minor clonal evolutions present in a small proportion of tumor cells.
CD200 Is a New Prognostic Factor in Multiple Myeloma.
Authors: Moreaux Jerome , Hose Dirk , Reme Thierry X. , Jourdan Eric , Hundemer Michael , Eric Legouffe , Moine Philippe , Bourin Philippe , Moos Marion , Corre Jill , Mölher Thomas , DeVos John , Rossi Jean-Francois , Goldschmidt Hartmut , Klein Bernard
Published: April 2020

Using Affymetrix microarrays, we identified the expression of the CD200 gene in multiple myeloma cells (MMC) of 112 patients with newly-diagnosed multiple myeloma (MM). The CD200 gene was either absent or present (Affymetrix call) in 22% and 78% of MMC, respectively. CD200 was not expressed by CD14 monocytes, CD15 polynuclear cells and CD3 T cells that were purified from the bone marrow of 5 newly-diagnosed patients. It is also not expressed in 7 osteoclast samples. BM stromal cells from 5 patients with MM expressed CD200, but at a 3.9 fold lower median signal compared to that in CD200present MMC (P = .04). CD200 is a membrane glycoprotein that imparts an immunoregulatory signal through CD200R, leading to the suppression of T-cell-mediated immune responses. Patients with CD200absent MMC have an increased event free survival (24 months) compared to patients with CD200present MMC (14 months), after high-dose therapy and stem cell transplantation. In a Cox-proportional-hazard model, the absence or presence of CD200 expression in MMC is predictive for EFS for patients independently of ISS stage or B2M serum levels. Thus, CD200 is an independent prognosis factor for patients with MM that could represent a new therapeutic target in MM.
The Small-Molecule VEGF-Receptor Inhibitor Pazopanib (GW786034B) Targets Both Tumor and Endothelial Cells in Multiple Myeloma.
Authors: Podar Klaus , Tonon Giovanni , Sattler Martin , Tai Yu-Tzu , LeGouill Steven , Yasui Hiroshi , Ishitsuka Kenji , Kumar Rakesh , Pandite Lini N. , Hideshima Teru , Chauhan Dharminder , Anderson Kenneth C.
Published: April 2020

A critical role for vascular endothelial factor (VEGF) has been demonstrated in multiple myeloma (MM) pathogenesis. Here we characterized the effect of the novel and potent small-molecule VEGF-receptor inhibitor pazopanib on MM cells in the bone marrow milieu. Pazopanib inhibits VEGF-triggered signaling pathways in both tumor and endothelial cells, thereby blocking in vitro MM cell growth, survival, and migration; and inhibits VEGF-induced upregulation of adhesion molecules on both endothelial and tumor cells, thereby abrogating endothelial cell-MM cell binding and associated cell proliferation. We show that pazopanib is the first-in-class VEGF-receptor inhibitor to inhibit in vivo tumor cell growth associated with increased MM cell apoptosis, decreased angiogenesis, and prolonged survival in a mouse xenograft model of human MM. Low-dose pazopanib demonstrates synergistic cytotoxicity with conventional (melphalan) and novel (bortezomib, immunomodulatory drugs) therapies. Finally, gene expression and signaling network analysis show transcriptional changes of several cancer-related genes, in particular cMyc. Using siRNA, we confirm the role of cMyc in VEGF-production and -secretion, as well as angiogenesis. These preclinical studies provide the rationale for clinical evaluation of pazopanib, alone and in combination with conventional and novel therapies, to increase efficacy, overcome drug resistance, reduce toxicity, and improve patient outcome in MM.
Predictive Factors of Early Mortality in Patients with Multiple Myeloma (MM). Multicentric Study in Chile (1998–2002).
Authors: Conte Guillermo F. , Figueroa Gaston L. , Lois Vivianne I. , Cabrera Maria E. , Leon Alvaro R. , Garcia Hernan L. , Rojas Hernan H.
Published: April 2020

There is no data about the prognosis of patients with MM in Chile, except mortality rate records. The objective of this study is to know the clinical features, survival rates and factors related to early mortality of cases with MM treated in six large medical centers in Chile.

Method: Retrospective demographic data, clinical features and survival rate records of patients with MM were collected between 1998 and 2002. Survival curves were generated and a multivariate analysis of factors associated to early mortality was carried out.

Results: Data of 245 patients was collected. A 51.8% of them corresponded to IgG myeloma, 25.3% to IgA and a 6.1% to light chains. Distribution according Durie and Salmon was: Stage I: 8.1%; Stage II: 12.7%; Stage III: 60.7% and 18.4% without information. It is pointed out that, among clinical features, a 50% of the cases presented anemia (Hb <10gr/dL), a 30% renal failure (creatininemia >2 mg/dL) and a 28% hypercalcemia (>10.5 mg/dL). The median survival was 33 months. A 20% of patients died within the six first months after diagnosis. The multivariate analysis identified three factors associated to early mortality (survival <6 months): males (p=0.016), beta2-microglobulin >3.5 mg/L (p=0.021) and albuminemia < 3.5 gr/dL (p=0.016).

Conclusions: It was observed that, in Chilean population, patients with MM presented a short survival time, and a significative proportion of patients (20%) died within the first six months after the first diagnosis. More than a half of cases were diagnosed at an advanced stage. (Durie and Salmon: Stage III). Three factors associated to early mortality, two of which (beta2-microglobulin and albuminemia) are the foundations of the new international staging system, might be identified.
Flow Cytometric Disease Monitoring in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation: A Retrospective Study.
Authors: Liu Hong , Yuan Constance M. , Braylan Raul C. , Chang Myron N. , Wingard John R. , Moreb Jan S.
Published: April 2020

The persistence of abnormal neoplastic plasma cells (APC) detectable in the bone marrow by flow cytometry at more than 3 months after autografting for multiple myeloma (MM) has been reported to predict early disease progression. In this study, we retrospectively reviewed the flow cytometric data from bone marrow aspirates of MM patients before and after autologous stem cell transplantation (ASCT). Light scatter properties and CD38 expression were used to identify plasma cells, and CD19/CD45/CD56 further distinguished normal plasma cells (NPC) from APC. Conventional response criteria (Blade criteria) and survival data were also collected. Forty-seven (47) patients treated with the same conditioning regimen were screened. Median follow up from ASCT was 19 months. After ASCT, 66% (31/47) patients achieved complete remission (CR)/very good partial remission (VGPR), as compared to only 36% (17/47) prior to ASCT. In 39 patients with data before and after ASCT, all 39 (100%) had a detectable abnormal plasma cell population identified phenotypically by flow cytometry prior to ASCT. Of these patient, 18/39 (46%) had greater than 30 APC and these patients had significantly shorter PFS independent of other covariates (1-sided P=0.036, 2 sided P=0.072, logrank). Twenty-six out of 39 patients (67%) also had detectable NPC. Following ASCT, the number of patients with detectable NPC increased to 35/39 (89%), while 3/39 (8%) had no detectable NPC and 1/39 (3%) had neither NPC or APC. The proportion of APC decreased significantly after transplant (81% prior to transplant vs. 59% post-transplant, P=0.008, 2 tailed t-test). Patients with a APC to NPC ratio < 1 post transplant has higher PFS rate at 2 year (54%) when compared to patients with higher APC/NPC ratio (29% PFS at 2 year), however, the difference is not statistically significant. In addition to the presence of APC, the ratio of APC to NPC, age, beta-2 microglobulin levels, and the presence of normal immunoglobulin levels were analyzed. Patients who achieved CR/VGPR after transplant had significantly longer PFS (23 months vs. 11 months, P=0.03). All other covariates were not found to be significant. Because only 10 deaths were observed, covariate analysis for OS was not feasible. In conclusion, the recovery of NPC after ASCT is seen in a substantial propotion of patients with a trend towards better PFS in patients with low APC/NPC ratio. On the other hand, the presence of a significant population of APC (> 30) prior to transplant appears to correlate with poorer PFS in MM patients.
Genotype-Phenotype Associations in Patients with Severe Congenital Neutropenia.
Authors: Rosenberg Philip S. , Stein Steven , Rodger Elin , Bolyard Audrey Anna , Bonilla Mary Ann , Dror Yigal , Kannourakis George , Newburger Peter E. , Boxer Laurence A. , Alter Blanche P. , Dale David C.
Published: April 2020

BACKGROUND: G-CSF therapy has reduced sepsis mortality in patients with severe congenital neutropenia (SCN), revealing a syndromic predisposition to myelodysplastic syndrome and acute myeloid leukemia (MDS/AML). The MDS/AML risk appears to be higher in SCN patients who are less-responsive to therapy; however, the genetic determinants of susceptibility remain to be elucidated.

METHODS: We studied 82 North American and Australian patients with SCN on long-term G-CSF enrolled in the Severe Chronic Neutropenia International Registry. These patients had prospective follow-up, sufficient banked DNA for genotyping, and validated clinical data.

RESULTS: Fifty-two patients (63%) were positive for germline mutations in neutrophil elastase (ELA2); the remaining 30 patients (37%) had no detectable mutations. Prior to G-CSF therapy, blood cell counts were significantly different between ELA2 positive and negative patients. Compared with ELA2 positive patients, ELA2 negative patients had significantly higher median absolute neutrophil count (ANC) values (158 versus 53 cells/uL, P=0.02), significantly lower monocyte and eosinophil counts (P<0.001), and significantly lower platelet counts (P=0.002). ELA2 negative patients were maintained on significantly lower doses of G-CSF (5.2 versus 9.9 ug/kg/day, P=0.02), consistent with higher baseline ANC values. However, ELA2 negative patients were significantly less responsive to G-CSF therapy. Compared with ELA2 positive patients, on therapy, ELA2 negative patients had significantly smaller median increases in ANC values (1200 versus 2700 cells/uL, P=0.02); the difference remained significant after controlling for individual G-CSF dose (P=0.04). ELA2 negative patients had 5 MDS/AML events in 132 person-years, versus 8 MDS/AML events in 373 person-years among ELA2 positive patients. Using the Cox proportional hazards model, the hazard of MDS/AML was 3.1-fold higher in ELA2 negative patients compared with ELA2 positive patients. This association was of borderline statistical significance (P=0.08). No patient in either group died of sepsis. We found 34 distinct mutations in 52 ELA2 positive patients. The mutation sites were not clustered in the 8 ELA2 positive patients who developed MDS/AML. We found no associations of phenotype or outcome with specific ELA2 mutations; however, the numbers were limited.

CONCLUSIONS: SCN patients without mutations in ELA2 constitute a clinically distinct subgroup. ELA2 negative patients are significantly less responsive to G-CSF therapy than ELA2 positive patients, and they may be at substantially higher risk of leukemia. Additional studies are needed to confirm the latter association and identify the causative gene or genes.
Comparison of Myeloma Cells Killing Efficiency by Geldanamycin and Its Analogs.
Authors: Zebzda Anna , Jurczyszyn Artur , Czogala Wojciech , Lesko Ewa , Gozdzik Jolanta , Skotnicki Aleksander B. , Majka Marcin
Published: April 2020

Multiple Myeloma (MM) is the second most commonly diagnosed hematologic malignancy and still an incurable disease with dismal prognosis and average survival of 3 – 4 years. None of conventional therapies gives total cure or long survival, so it is urgently necessary to find new treatment strategies. Heat shock protein 90 (HSP90) is a potentially good therapeutic target, because it interacts with diverse and wide panel of client proteins that are important for proliferation and survival of tumor cells. Geldanamycin (GA) and its analog 17-allylaminogeldamycin (17AAG) are HSP90 inhibitors and were shown to block various tumor cells growth including MM cells. 17AAG has been already tested in clinical trials and showed some antitumor properties. There are, however, other inhibitors belonging to geldanamycin family such as: 17DMAG, 17DMAP and 17AEP which have not been tested against multiple myeloma cell lines and primary samples.

In this study we looked at the influence of GA and its analogs on proliferation and apoptosis of MM cells. We found that GA at 100 nM concentration inhibited growth of MM cells by 80%, 17AEP by 75%, 17DMAG and 17DMAP by 70% and 17AAG by 30%. Primary MM cells were more resistant and at 100 nM concentration we observed 30% inhibition with GA and 17DMAG. At the same time 17AAG did not effectively inhibit proliferation of primary MM cells.

We also analyzed influence of 100 nM concentration of GA and analogs on the apoptosis of MM primary cells and cell lines after 6 h and 24 h of incubation. Percentage of Anexin V and propidium iodine double positive geldanamycines treated primary MM cells was similar to the control after 6 h but change after 24 h. In comparison to control, number of apoptotic cells increased by: 175% for 17DMAG, 140% for GA, 80% for 17DMAP and 17AEP, no changes in number of apoptotic cells were observed for 17AAG. Similar observation was made for MM cell lines and increase in number of Anexin V positive cells was noticed. Again, percentage of Anexin V positive cells did not increase after treatment with 17AAG and remained at the control level. We also observed, similarly to others, substantial block in G1 and G2/M phase of cell cycle in cells treated with GA and its analogs. In conclusion, besides to 17AAG other HSP90 inhibitors could be considered as potential therapeutics for treatment of MM. Particularly, based on our data, we suggest that 17DMAG and 17AEP are new HSP90 inhibitors that should be tested in a clinic because they exert strong MM cells growth inhibition.
Bone Marrow Adipocytes Influence Multiple Myeloma Development by Secretion of Different Growth Factors and Chemokines.
Authors: Caers Jo , Belaid Zakia , Deleu Sarah , Defresne Marie Paule , Vanderkerken Karin
Published: April 2020

In multiple myeloma (MM) monoclonal plasma cells accumulate in the bone marrow (BM) where they interact with neighboring cells to receive supportive signals. Since BM adipocytes are known to participate in normal and malignant hematopoiesis, we presumed interactions between BM adipocytes and myeloma cells. Using an adipocytic cell line and primary isolated BM adipocytes, we performed functional assays indicating that adipocytes support MM cell development by affecting proliferation, apoptosis, cell migration and adhesion. We subsequently analyzed the secretion of different cytokines by BM adipocytes and analyzed the contribution of a specific adipokine, leptin, to these processes. We confirmed the expression of leptin by adipocytes and leptin receptor by myeloma cells. Immunostaining showed strong presence of leptin receptor on MM cells in 13 of 28 patients tested. This expression was not correlated to disease stage. On cell proliferation, leptin acted as a moderate growth factor. We conclude that, next to the earlier described interactions within the BM, adipocytes influence MM cell behavior by affecting proliferation, apoptosis and migration and that part of these effects may be contributed to leptin.
Over Expression of YY1 and RKIP in Multiple Myeloma (Cell Lines and MM Tissues).
Authors: Baritaki Stavroula , Huerta-Yepez Sara , Katsman Alina , Yeung Kam C. , Chatterjee Devasis , Chen Haiming , Berenson James , Bonavida Benjamin
Published: April 2020

There is a need to identify underlying molecular mechanisms and gene products (targets for intervention and biomarkers) involved in MM chemo- or immuno-resistance. Recent findings from our laboratory have reported high expression of the transcription factor Yin Yang 1 (YY1) in several tumor types and have demonstrated its fundamental role in tumor cell chemo/immuno-resistance (Gordon, S., et al., Oncogene25:1125, 2006). In contrast, low levels of the Raf kinase inhibitor protein (RKIP) has been shown to play a pivotal role in the negative regulation of cell survival signaling pathways, and has been considered a metastasis tumor suppressor. The overall objective of our studies is to examine whether MM cell lines and tissues derived from MM patients present deregulated expression patterns of RKIP and YY1, and to demonstrate the roles of YY1 and RKIP in MM pathogenesis and response to various therapies. In the present study, the myeloma cell lines RPMI-8226 and MM-1S were screened for RKIP and YY1 expression at the protein and m-RNA levels. In addition, fresh tissues from MM patients were also examined for RKIP and YY1 expression by IHC and their patterns compared with those observed in normal bone marrow cells. The findings demonstrate that both MM cell lines express remarkably higher levels of RKIP protein and transcripts compared to other tumor cell lines examined (lymphomas and prostate cancer), as assessed by western, RT-PCR and IHC. There was significantly higher expression of RKIP in MM patient’s samples compared to normal bone marrow cells. YY1 protein and m-RNA levels were detected in the MM cell lines; however, they were lower compared to Ramos and PC-3 cells. YY1 expression in MM tissues was significantly elevated compared to normal bone marrow cells. RKIP was basically present in the cytoplasm while YY1 was mainly accumulated in the nucleus. In contrast, normal bone marrow cells presented primarily cytoplasmic YY1 and RKIP expression. The YY1 expressing population was the CD38+/CD138− cell subset, which has been reported to be more susceptible to apoptosis (Mitsiadis, S.C., et al., Blood 98:795, 2001). Overall, the present findings support the potential involvement of two new gene products, such as YY1 and RKIP, in MM pathogenesis and their implication in regulating MM resistance to conventional therapeutics. Moreover, these gene products suggest their potential use as new therapeutic targets and/or biomarkers in multiple myeloma. Future studies with large cohorts of tissues will elucidate the importance of the above findings and will give new insights in the unexpected RKIP over expression and activity in multiple myeloma.
Evaluation of a 5-Color Flow Cytometric Technique for Immunophenotyping and Quantitation of Plasma Cell Myeloma in Post-Therapy Bone Marrows.
Authors: Isaacson Tove , Jakubowiak Andrzej , Stoolman Lloyd , Kota Usha , Finn William , Ross Charles
Published: April 2020

Multiparameter flow cytometry is a useful tool for comprehensive immunophenotyping of plasma cell myeloma, and has been proposed as a sensitive method for the evaluation of minimal residual disease in patients following treatment. This study aimed to assess the value of flow cytometry in quantitation of residual disease, in comparison to routine morphologic examination of first-pull bone marrow aspirate smears, in myeloma patients post-therapy. Heparinized bone marrow aspirates were obtained from 27 treated patients with plasma cell myeloma. Cells were prepared for 5-color flow cytometric analysis within 24-hours of specimen draw. Surface membrane staining with anti-CD19, CD20, CD38, CD45, CD56, and CD138 was followed by ammonium chloride lysis of red cells. Fixed and permeabilized cells were analyzed for cytoplasmic light chains to confirm clonality. Data were acquired using an FC500 flow cytometer (Beckman-Coulter), analyzed with CXP software with plasma cells isolated based on bright CD38+ or CD138+ expression. A median of 97,639 cellular events (range 14,279 to 262,508) were collected per analysis. Flow cytometric enumeration of plasma cells was compared to 500-cell differential counts of Wright-Giemsa-stained first-pull aspirate smears from the same cases. The median plasma cell count as determined by flow cytometry was 0.5% (range 0–7.9%). The median plasma cell count estimated by morphologic review was 8.0% (range 0–84.4%). Flow cytometry underestimated the plasma cell content in all but one case. Clonal plasma cells expressed CD38 and CD138 in all cases; 87.5% (21/24) coexpressed CD56, 25% (6/24) coexpressed CD45, and 4.2% (1/24) coexpressed CD19. None was positive for CD20. Although detection of minimal residual disease after therapy for acute leukemia is routinely achieved by flow cytometric analysis, successful quantitation of minimal residual disease in treated myeloma patients using flow cytometry remains limited as it usually underestimates the plasma cell content of bone marrow samples compared to routine morphology of first-pull aspirates. We have observed that this holds true for both pre-treatment and post-treatment specimens. Causes for the discrepancy may include hemodilution of second-pull aspirates used for flow cytometry, fragility and loss of plasma cells during preparation for flow cytometry, and incomplete disaggregation of plasma cells from bone marrow spicules. With improved outcome of treatments, better and more reliable methods of detection of minimal residual disease are needed for optimal prognostic stratification. We are currently validating alternative methods, which may offer more sensitivity while at the same time allow more objectivity, for assessing the amount of minimal residual disease in myeloma patients.