bluuebunny/crossref_metadata_2025 huggingface dataset:
CXCL12 and CXCL13 Levels in Cerebrospinal Fluid (CSF) of Patients with CNS Lymphoma.
Authors: Fischer Lars , Kiewe Philipp , Pfeiffer Sebastian , Cakiroglu Hüsniye , Thiel Eckhard
Published: April 2020

Background: The mechanisms leading to development of CNS lymphoma (CNSL) are still poorly understood. Homing of malignant lymphocytes to the CNS may play an important role in the pathogenesis of CNSL. Expression of chemokine receptors CXCR4 and CXCR5 has been shown on tumor cells of primary CNSL. In this study we examined the level of chemokines CXCL12 and CXCL13 which are ligands of CXCR4 and CXCR5 respectively in the CSF of CNSL patients and compared it to non-CNSL controls.

Methods: Lumbar CSF and peripheral blood from 16 patients with primary and one with secondary CNSL, as well as control CSF from 8 patients with hydrocephalus, glioblastoma, peripheral lymphoma without CNS involvement or unspecific neurological symptoms were collected, and after centrifugation supernatant and serum were cryopreserved at −80°C until analysis. Chemokine concentrations were measured using commercially available ELISA kits (CXCL13/BCA-1 and CXCL12/SDF-1α Quantikine Immunoassay, R&D Systems, Minneapolis) according to the manufacturer’s instructions. Results were confirmed by duplicate measurements.

Results: 22 CSF samples and 11 serum samples from CNSL patients and nine CSF samples from controls were evaluated (see table). There was no correlation between serum and CSF chemokine levels in CNSL patients. Median concentrations of both CXCL12 and CXCR13 were higher in patients with CNS lymphoma compared to controls, CXCL13 being significantly raised (p=0.0019). In one CNSL patient three serial CSF samples could be analysed and a ten-fold decrease of CXCL12 during succesfull chemotherapy was found whilst concentration of CXCL13 remained stable.

Conclusion: CXCL12 and CXCL13 can be measured in the CSF of CNS lymphoma patients at higher concentration as compared to non-CNSL patients. Analysis of a larger population is warranted in order to define the role of these chemokines in CNSL and to establish their value for diagnosis and follow up.

Serum CSF CNSL patients CNSL patients controls p range median (range) median (range) CXCL12 1788–2933 pg/ml 481 (55–2079) pg/ml 115 (0–2442) pg/ml 0.067 CXCL13 36–657 pg/ml 457 (18–657) pg/ml 16.9 (0.6–187) pg/ml 0.00019
Clonal Heavy and Light Chain Immunoglobulin DNA in Plasma/Serum of AIDS Lymphoma Patients.
Authors: Wagner-Johnston Nina D. , Nurmemet Dilber , Gocke Christopher , Lin Lan , Lemas Victor M. , Martinez-Maza Otoniel , Ambinder Richard F.
Published: April 2020

Background DNA tumor markers in plasma and serum such as point mutations, hypermethylation of tumor suppressor genes, and viral sequences have attracted much interest. We investigated clonal Ig DNA rearrangements in the plasma of AIDS-related lymphoma (ARL) patients (pts).

Methods We evaluated specimens for Ig rearrangements from pts with ARL using fluorescently-labeled multiplex primers targeting IgH and IgK. PCR products were analyzed by capillary electrophoresis. Re-amplified PCR products from plasma and tumor were cloned and sequenced.

Results Clonal IgH DNA spikes were seen in plasma at baseline in 5 of 10 pts. Clonal DNA was detected in plasma in pts with either early or advanced stage disease, with or without marrow involvement. Presence of a plasma clone was associated with high LDH. Serial specimens were available from 4 pts. In 2 pts who died with primary refractory disease, clonal peaks remained present after the administration of chemotherapy. In a pt with disease that initially responded and then relapsed, clonal DNA disappeared with chemotherapy, then reappeared presaging relapse and death. In 4 pts, matching serum specimens were available. Results were similar in both types of specimens with identical sized rearrangements amplified from matched specimens. Furthermore in 2 pts, the IgH amplification products from plasma and tumor were sequenced and confirmed to be identical. Two pts with positive IgH amplification products were also screened for light chains in the plasma and in both, clonal IgK rearrangements were detected. For purposes of comparison, plasma from healthy donors (n=20) and AIDS pts with Kaposi’s sarcoma (KS) (n=10) were also surveyed. Clonal Ig DNA rearrangements were not detected in any of these.

Conclusions These results are consistent with observations suggesting that free tumor DNA can often be detected in plasma or serum of cancer pts. The absence of clonal Ig DNA in plasma from healthy donors and pts with AIDS KS suggests that detection of such DNA may be highly specific. Identification of the same Ig rearrangement and sequence in plasma and tumor is consistent with this interpretation. Serial monitoring of circulating clonal DNA appears to track—and perhaps anticipate—clinical evidence of disease. These approaches may be especially valuable in ARL where FDG uptake reflective of inflammatory disease or benign lymphoproliferation detected by PET scan often renders interpretation of imaging ambiguous. Similarly, elevated LDH in this population is relatively non-specific.
Host Genetic Variation in the Cell Cycle and NF-κB Pathways and Overall Survival in Mantle Cell Lymphoma.
Authors: Habermann Thomas M. , Maurer Matthew J. , Wang Sophia S. , Morton Lindsay M. , Lynch Charles F. , Hartge Patricia , Cozen Wendy , Severson Richard K. , Scott Davis , Watson David A. , Chanock Stephen J. , Rothman Nathanial , Cerhan James R.
Published: April 2020

Background: Mantle cell lymphoma (MCL) is an incurable non-Hodgkin lymphoma. MCL is characterized by the chromosomal translocation t(11;14) leading to overexpression of cyclin D1 (CCND1), a regulator of the cell cycle. NF-κB plays a central role in key cellular processes, including cell cycle regulation and apoptosis, and it has been shown to be constitutively activated in MCL cell lines and in patient biopsy samples and to play a key role in the growth and survival of MCL cells. In an exploratory study, we evaluated the hypothesis that germline variation in candidate genes from the cell cycle and NF-κB pathways predict overall survival in MCL.

Methods: We genotyped 235 SNPs from 29 cell cycle genes and 447 SNPs from 55 NF-κB genes in 39 MCL patients aged 38–64 years who participated in a population-based study conducted from 1998–2000 through the Surveillance, Epidemiology, and End Results (SEER) cancer registries in Detroit, Seattle, Iowa, and Los Angeles county. Tagging single nucleotide polymorphisms (SNPs) were selected from HapMap. Stage, presence of B-symptoms, first course of therapy, date of last follow-up, and vital status were derived from linkage to registry databases at each study site. To assess the statistical significance of a gene, we first used principal components analysis to capture the genetic variation of the SNPs within a gene. Cox proportional hazards analysis was then used to assess the association between the top principal components from a gene with overall survival.

Results: Through early 2005, there were 22 deaths in 39 patients (56%). The median follow-up of the 17 surviving patients was 56 months (range 36–70). Eleven of the 55 genes (TNFSF14 p=0.0007, NF-κBIA p=0.003, IL1R2 p=0.01, TNFRSF25 p=0.02, TNFSF13B p=0.02, FAS p=0.03, TLR2 p=0.04, TNFSF10D p=0.04, TNFSF10 p=0.06, MAP3K2 p=0.06) in the NF-κB pathway were associated with overall survival. In the cell cycle pathway, only 3 of the 29 genes (CASP5 p=0.06, CASP9 p=0.09, BCL2 p=0.09) were associated with overall survival, and a previously reported SNP in CCND1 (rs603965) showed no association (p=0.5). Permutation p-values for observing at least this number of associated tests in each pathway due to chance (from multiple testing) was p=0.8 for the cell cycle pathway and p=0.03 for NF-κB pathway. The small sample size precluded multi-gene modeling.

Conclusion: In a population series of MCL patients, we found suggestive evidence that host genetic variation in candidate NF-κB pathway genes affects overall survival. These results complement tumor expression data implicating this pathway in the pathogenesis of MCL. In addition, such findings further support the value of developing therapeutic targets from this pathway.
Pifithrin α, a Selective Inhibitor of p53-Mediated Transcription, Augments Apoptotic Killing of Chronic Lymphocytic Leukemia Cells by Nutlin 3a and Cytotoxic Drugs.
Authors: Steele Andrew J. , Prentice Archie , Cwynarski Kate , Hoffbrand A. Victor , Wickremasinghe R. Gitendra
Published: April 2020

The p53 protein plays a key role in triggering DNA damage-induced apoptosis. The classical pathway of p53-mediated apoptosis involves transcriptional upregulation of pro-apoptotic proteins Puma and Noxa. However, recent studies identified a non-transcriptional mechanism, involving direct association of p53 with mitochondrial anti-apoptotic proteins Bcl-2 or Bcl-XL. We studied the relative contributions of transcription-dependent and -independent mechanisms of p53-mediated apoptosis in CLL cells in vitro. The cytotoxic drugs chlorambucil (chl) and fludarabine (flu) were used to induce p53, as was nutlin 3a, which augments p53 levels by inhibiting interaction with its negative regulator MDM2. Using differential detergent fractionation to isolate cytosolic (cyt), mitochondrial (mt) and nuclear (nuc) fractions, we found that a significant proportion (>50%) of the induced p53 was stably associated with mitochondria in cells treated with nutlin 3a (Fig 1), chl or flu. Co-immunoprecipitation studies established that p53 bound to Bcl-2. PFTα, an inhibitor of p53-mediated transcription, blocked upregulation of the known p53 targets p21CIP1, MDM2 and Puma. Surprisingly, apoptosis induction by nutlin 3a (Figure 2), chl or flu, quantified by western blot analysis of cleavage of poly(ADP ribose) polymerase (PARP), was augmented by PFTα. This observation was confirmed by morphological analysis of apoptosis. Cytochrome c release from mitochondria following p53 induction was also enhanced by PFTα. PFTα did not augment nutlin-, chl- or flu-induced killing of CLL cells lacking functional p53. Furthermore, PFTα did not augment killing by the p53-independent drug parthenolide and failed to increase apoptosis of normal T lymphocytes treated with chl or nutlin 3a. Our observations suggest that p53 induces apoptosis of CLL cells principally via its transcription-independent function, involving direct association with mitochondrial Bcl-2. p53’s transcriptional function apparently blocks apoptosis at a point between p53 association with Bcl-2 and subsequent release of cytochrome c. Therefore, therapeutic strategies aimed at blocking p53-mediated transcription may be of value in enhancing the action of agents which induce apoptosis of CLL cells via p53 upregulation.

Figure 1 Figure 1. Figure 2 Figure 2.
The Novel HSP90 Inhibitor IPI-504 Synergizes with Bortezomib in Mantle Cell Lymphoma Cells by Targeting Both NF-kappaB Signaling and Unfolded Protein Response and Leading to Increased Mitochondrial Apoptosis.
Authors: Roue Gael , Perez-Galan Patricia , Lopez-Guerra Monica , Villamor Neus , Campo Elias , Colomer Dolors
Published: April 2020

Mantle cell lymphoma (MCL) is an aggressive B lymphoid neoplasm with a mature B-cell phenotype and genetically characterized by the t(11;14)(q13;q32) leading to cyclin D1 overexpression with the consequent deregulation of cell cycle at the G1-S checkpoint. MCL cells present a constitutive activation of the NF-kappaB pathway which leads to the overexpression of several anti-apoptotic regulators. Recently, MCL cells have been shown to express high levels of the chaperone heat shock protein of 90 kDa (HSP90) and to respond well to the ansamycin derivative 17-AAG, an HSP90 inhibitor. We have analyzed the sensitivity to the novel, highly soluble, 17-AAG derivative IPI-504 (Infinity Pharmaceuticals) on a panel of eleven human MCL cell lines and primary cells from MCL patients, which differ in their p53-dependent pathway status, growth characteristics and sensitivity to cytotoxic drugs. We observed that IPI-504 heterogeneously exerted cytostatic effect among MCL samples, with IC50 ranging from 0.06 to 15.4 μM, irrespective of the mutational status of the client protein p53 and/or expression levels of other client proteins such as cyclin D1 and BCL-2. IPI-504 activity involved the downregulation of the client proteins IKKb and phospho-AKT, and consequent inhibition of NF-kappaB signaling. In the most sensitive samples, these events led to the induction of cell death characterized by loss of mitochondrial membrane potential, activation of caspases and phophatidylserine exposure. IPI-504 cytotoxic activity was increased by cotreatment with pharmacological inhibitors of IKK and AKT. Noteworthy, IPI-504 showed remarkable synergistic interaction with the proteasome inhibitor bortezomib, reaching combination indexes (CIs) between 0.53 and 1.094. Efficiency of this latest combination was found to be correlated with the ratio between HSP90 and the co-chaperone HSP70 (P<0.05), whose levels were increased following treatment with both IPI-504 and bortezomib, thus limiting the cytotoxicity. Accordingly, pharmacological inhibition of HSP70 sensitized MCL cells primarily resistant to IPI-504. We compared the modulation of some components of the physiologic unfolded protein response (UPR) among samples harboring different sensitivity to IPI-504 and bortezomib. We observed, in the most sensitive cells, a complete inhibition of the spliced isoform of XBP-1 and phospho-eIF2a after a long time exposure to the combination, a fact that was associated to increased upregulation of the proapoptotic BH3-only protein NOXA and induction of apoptosis. Considering that both bortezomib and IPI-504 have already demonstrated selective cytotoxicity against malignant B cells, combining these two drugs may represent an attractive model for the design of a new and rational combination therapy for MCL.
Targeting Histone Deacetylase 6 and the Aggresome Pathway in Acute Lymphoblastic Leukemia Cells.
Authors: Rodriguez-Gonzalez Augustin , Ch’ng James , Ikeda Alan K. , Lin Tara , Bahrami Bita , Mazitschek Ralph , Bradner James E. , Fu Cecilia , Sakamoto Kathleen M.
Published: April 2020

Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Despite effective chemotherapy, 20% of patients will relapse. Therefore, it is critical that we identify novel therapies to treat ALL. We are studying a new small molecule compound known as tubacin (tubulin acetylation inducer) that selectively inhibits histone deacetylase 6 (HDAC6). HDAC6 binds to polyubiquitinated misfolded proteins and to dynein motor proteins, including alpha-tubulin, thereby recruiting misfolded or unwanted proteins to aggresomes and subsequent degradation by the lysosome. Tubacin was discovered through a chemical genetic screen of a 7,392 small molecule library and was found to induce acetylation of alpha-tubulin by inhibiting one of the two catalytic domains of HDAC6. This inhibition disrupts the interaction of HDAC6 with dynein resulting in marked accumulation of ubiquitinated proteins. Previous work demonstrated that treatment of multiple myeloma cells with tubacin inhibited growth at low micromolar concentrations. Tubacin does not appear to affect global histone acetylation, gene expression, or cell cycle regulation. To determine the effects of tubacin in human ALL cells, we treated both B- and T-cell ALL cell lines with varying concentrations of the drug and performed MTT assays. In T-ALL cells (Jurkat, Loucy), the IC50 of tubacin was found to be 1 to 3 uM, while in B-cell ALL cells (REH, Nalm-6), the IC50 was 2 to 5uM. Tubacin induced apoptosis of Jurkat and Loucy cells stained with Annexin V and propidium iodide. Within 12 hours, we observed increased protein polyubiquitination and PARP cleavage, but no difference in Rb phosphorylation in cells treated with tubacin. Furthermore, tubacin treatment increased acetylation of alpha-tubulin in Loucy and Jurkat cells within 3 hours. To study whether tubacin was toxic to normal hematopoietic cells, we treated human bone marrow cells cultured in methylcellulose containing IL-3, IL-6, and Stem Cell Factor with varying concentrations of tubacin. The IC50 of 20uM was determined from numbers of colonies plated in triplicate. Similarly, treatment of normal human lymphocytes cultured in IL-2, demonstrated an IC50 of 16uM. Finally, we examined the effects of tubacin on growth of primary ALL cells. Bone marrow cells from three patients with B-cell ALL at diagnosis were cultured in tubacin at varying concentrations and MTT assays were performed. In two of the three ALL samples, the IC50 was less than 5uM. Experiments to study the effects of tubacin in mouse models of leukemia are in progress. Our results suggest that inhibition of HDAC6 and the aggresome pathway provides a novel approach to treat ALL.
Leukemogenic FLT3 Mutations Confer Enhanced Proliferation and Survival Properties to Multipotent Progenitor Cells in a Murine Model of Chronic Myelomonocytic Leukemia.
Authors: Lee Benjamin H. , Tothova Zuzana , Levine Ross L. , Anderson Kristina , Buza-Vidas Natalija , Cullen Dana E. , McDowell Elizabeth P. , Adelsperger Jennifer , Frohling Stefan , Huntly Brian J.P. , Beran Miloslav , Jacobsen Sten Eirik , Gilliland D. Gary
Published: April 2020

FLT3 internal tandem duplication (ITD) activating mutations are among the most common genetic events in acute myelogenous leukemia (AML) and confer a poor clinical prognosis. Despite their known transforming properties, the effects of constitutive FLT3 signaling on multipotent stem and progenitor cells and hematopoietic differentiation are not well understood. Here we report a novel murine model harboring an ITD mutation in the endogenous murine Flt3 locus. These mice exhibit a myeloproliferative disease (MPD) resembling human chronic myelomonocytic leukemia (CMML) and concomitant with the murine phenotype, we identified FLT3-ITD mutations in a proportion of human CMML. Disease was more pronounced in animals that were bi-allelic for the ITD mutation (Flt3ITD/ITD) and these findings correlated with an increase in number, cell cycling and survival of multipotent stem and progenitor cells in Flt3+/ITD and Flt3ITD/ITD mice in a dose-dependent manner. Moreover, Flt3 mutant animals demonstrated expansion of granulocyte-monocyte (GMP) progenitors as well as reduction in erythroid progenitors and a block in normal B cell development. Together, these findings provide novel insights into the consequences of dysregulated FLT3 tyrosine kinase signaling on primitive hematopoietic progenitor quiescence, function and cell fate and how these effects contribute to the pathogenesis of acute myelogenous leukemia.
Identification of Tyrosine Residues of Importance for Survival Signaling through the Scaffolding Protein Gab2 in Both Wild-Type FLT3 and the FLT3-ITD.
Authors: Masson Kristina , Liu Tao , Sun Jianmin , Ronnstrand Lars
Published: April 2020

The receptor tyrosine kinase FLT3 is normally expressed in hematopoietic progenitor cells and has been implicated as a major cause of transformation in acute myeloid leukemia, where it in approximately 30% of cases is mutated and constitutively active. This is in most cases due to duplication of a DNA sequence coding for amino acids in the juxtamembrane region of FLT3, commonly referred to as ITD (Internal Tandem Duplication). In this study we have identified several novel in vivo tyrosine phosphorylation sites that are phosphorylated in wild-type FLT3 upon ligand stimulation and that are constitutively phosphorylated in the FLT3-ITD. We were able to demonstrate that these phosphorylation sites are critical for full phosphorylation of the scaffolding protein Gab2 both in wild-type FLT3 and FLT3-ITD. Y-to-F mutants of either wild-type FLT3 or FLT3-ITD, lacking these tyrosine residues, fail to phosphorylate Gab2 and demonstrate a considerable reduction in phosphorylation of Akt and Erk. Furthermore, FL-dependent survival and proliferation of wild-type FLT3 expressing Ba/F3 cells as well as FL-independent survival and proliferation of Ba/F3 cells transfected with FLT3-ITD was dramatically reduced by mutation of these tyrosine residues. In the case of the FLT3-ITD, this was shown to correlate with strongly reduced STAT5 phosphorylation. To verify the importance of Gab2 in FLT3-ITD signaling, we used siRNA technology to knock down the expression of Gab2 in the human AML cell line MV4-11 that is known to express FLT3-ITD. Knockdown of Gab2 expression led to a dramatic reduction in the phosphorylation of Akt, Erk and Stat5. To summarize, we have identified novel phosphorylation sites in FLT3 and how they link to downstream signaling of survival and proliferation. These findings not only reveal novel phosphorylation sites in FLT3 but also contribute to the understanding of the molecular mechanism by which FLT3-ITD functions in pathological conditions. Future studies are aiming at elucidating the mechanism by which Gab2 mediates phosphorylation and activation of STAT5, which could be a future potential target for therapy in AML with FLT3-ITD.
Sickle Cell Trait, Oral Contraceptives, and the Risk of Venous Thromboembolism.
Authors: Austin Harland , Lally Cathy , Benson Jane M. , Whitsett Carolyn , Hooper W. Craig , Key Nigel S.
Published: April 2020

We recently reported that sickle-cell trait (SCT) is a risk factor for venous thromboembolism (VTE) among African Americans (1). Since current usage of oral contraceptives (OC’s) is an established risk factor for VTE in women of European descent, we investigated the joint effect of OC’s and SCT on VTE risk in the Genetic Attributes and Thrombosis Epidemiology (GATE) study. This case-control study included 57 African American women with an idiopathic, first deep-vein thrombosis and/or pulmonary embolism and 185 controls between the ages of 18 and 49. Subjects were genotyped at codon 6 (E>V) for the hemoglobin S mutation and were interviewed about OC usage. The distribution of cases and controls according to current OC use and SCT is displayed below: The odds ratio of VTE among OC users versus women not currently using OC’s is 2.7 (95% CI: 1.4, 5.1; P = 0.003). However, among subjects with SCT, the odds ratio for OC use is considerably higher (OR = 7.0; 1.1, 45; P = 0.04) than is the odds ratio for OC use among women without SCT (OR = 2.1; 1.0, 4.3; P = 0.05), although the difference in these two odds ratios is not statistically significant (P > 0.20). Nonetheless, the findings indicate that overall, current OC use is a strong risk factor for VTE among African American women. The observation that there is synergy between current OC use and SCT provides some additional support for the finding that SCT is a risk factor for VTE. However, a limitation of this analysis is small numbers and the resultant imprecision. Replication of these findings is thus required before causation can be established.

Current OC use and Sickle Cell Trait SCT OC Case Control Odds Ratio No No 31 138 1.0 No Yes 15 32 2.1 Yes No 4 12 1.5 Yes Yes 7 3 10.4
JAK2 V617F in Patients with Idiopathic Thromboses in Common Locations.
Authors: Abel Gregory A. , DeAngelo Daniel J. , Connors Jean M. , Sholl Lynette M. , McCaffrey Ronald P. , Longtine Janina A.
Published: April 2020

Background: Polycythemia vera and essential thrombocytosis, two of the myeloproliferative disorders (MPD) often characterized by the recurrent somatic missense mutation JAK2 V617F, are associated with an increased risk of arterial and venous thrombosis. Although recent anecdotal reports have suggested the possibility of latent MPDs by finding JAK2 mutations in patients with abdominal venous thromboses (AVT), the usefulness of routine JAK2 V617F testing has not been assessed in a cohort of general hospital patients with idiopathic thromboses in common locations.

Methods: To assess the utility of JAK2 V617F testing in patients undergoing thrombophilia workup, we identified all patients with negative molecular diagnostic tests for the Factor V Leiden (FVL) mutation and the prothrombin gene mutation (G20210A) over the course of one year at the Brigham and Women’s Hospital. We then determined, by medical record review, the nature of the thrombotic events for which they were being evaluated, and tested their nucleated white blood cell DNA for the JAK2 V617F mutation using the amplification refractory mutation system (ARMS)-PCR assay (which has a lower limit of detection of 5% mutant alleles).

Results: From Februrary 2006 to February 2007, 111 patients had negative tests for both FVL and G20210A. Of these, our chart review revealed that 65 had documented thromboses without a history of malignancy or MPD. None of these were found to have the JAK2 V617F mutation. Laboratory hypercoaguability studies for thrombosis by type (from medical record review) were as follows:

Protein C deficiency (%) Protein S deficiency (%) Anti-thrombin III deficiency (%) Antiphospholipid Abs (%) Elevated homocysteine (%) JAK2 V617 mutation (%) None (%) CVA/Stroke (n=10) 0 20 0 10 0 0 70 DVT/PE (n=45) 11 20 2 4 13 0 60 AVT (n=6) 33 33 0 0 17 0 33 Other Arterial Thrombosis (n=4) 0 25 0 0 25 0 50

In terms of clinical risk factors, 27.6% of patients with documented thromboses had had surgery within the prior month, 26.2% were current smokers, and 31.4% of female patients were either pregnant or taking oral contraceptives. 29.2% of all patients had no clinical or laboratory thrombophilic risk factors.

Conclusions: About two-thirds of our general hospital patients with thrombosis and negative tests for FVL and G20210A had another underlying thrombophilic risk factor. Adding JAK2 V617F mutation testing as a routine component of the thrombophilia diagnostic workup is not recommended, as it is unlikely to reveal evidence of a putative latent MPD.
Regulation of Microparticles and Adhesion Molecules in Pregnancy. Diagnostic and Pathophysiologic Implications.
Authors: Hoppensteadt Debra , Cunanan Josephine , Anderson Anita Sylvester , Bergholt Thomas , Berthelsen Jorgen , Fareed Jawed
Published: April 2020

OBJECTIVE In addition to increased levels of coagulation factors and platelet counts,, microparticles are also found in increased numbers in pregnancy and are generated due to cellular activation and consumption. Microparticles may also be capable of amplifying the procoagulant and inflammatory responses by up regulating the adhesion molecules such as P, E and L selectins. This study will provide newer data on the regulation of microparticles in different trimesters of pregnancy and their relevance to the generation of adhesion molecules and tissue factor (TF).

STUDY DESIGN Blood samples from pregnant women at their first prenatal visit and during the second and third trimester (n=50) were collected and citrated plasma samples were profiled for TF (American Diagnostica, Stamford, CT), E, L and P-selection (R &D Systems, Minneapolis, MN) by using commercially available sandwich ELISA methods and microparticles utilizing a functional method from Hyphen Biomed (Neuville-Sur-Oise, France). The results were compiled for each trimester and compared to aged match non-pregnant controls (n=40).

RESULTS During the first trimester all of the mediators were increased in comparison to the controls. The relative increase was mediator dependent. L-selectin remained elevated, at a steady level, throughout the pregnancy. E-selectin showed a decrease in the second trimester with a rebound increase in the third trimester. A gradual increase in the TF and microparticles was evident throughout the pregnancy. The P-selectin levels decreased during the second trimester and stayed at this level for the remainder of the pregnancy.

CONCLUSION Throughout pregnancy the TF levels directly correlated with an increase in the MP levels indicating that TF cause cellular activation leading to the formation of MPs. However, the levels of the adhesion molecules, which were initially elevated, fluctuate throughout the 2nd and 3rd trimester. These results suggest that increased levels of TF and MPs are progressively generated during pregnancy and may be responsible for the observed thrombotic complications and prothrombotic state in pregnancy.
Risk Factors for Venous Thromboembolism (VTE) among Patients with Active Hematological Cancer: A Population-Based Case-Control Study.
Authors: Ashrani Aneel A. , Heit John A. , Schmoll Jeffrey A. , Farmer Sara A. , Petterson Tanya M. , Melton Lee J.
Published: April 2020

Background: Hematological cancer patients are at an increased risk for VTE (RR range = 12–32). However, whether VTE risk among such patients can be further stratified is uncertain.

Objective: To test hematological cancer type, stage, stage progression and chemotherapy as potential risk factors for VTE among active hematological cancer patients after controlling for other previously-identified VTE risk factors.

Methods: Using the resources of the Rochester Epidemiology Project, Mayo Clinic Master Diagnostic Index and Mayo Clinic Tumor Registry, we identified all Olmsted County, MN residents with active hematological cancer over the 28-year period, 1973–2000. From this prevalence cohort, we identified 86 patients with no prior VTE (controls) who were matched on age and date of hematological cancer diagnosis to 86 hematological cancer patients with incident VTE over the same time frame (cases). For all cases and controls, we reviewed the complete medical records in the community for baseline and hematological cancer-related characteristics. Hematological cancers were re-staged at the dates of the cancer and VTE diagnosis. We tested these characteristics as potential risk factors for VTE in active hematological cancer using conditional logistic regression.

Results: In an initial multivariate analysis that included body mass index (BMI), hospitalization, and any infection or central venous catheter placement within 90 days prior to the VTE event, VTE was significantly associated with hospitalization (OR=6.70; p<0.001), and marginally associated with any infection (OR=2.18; p=0.09). After adjusting for the above variables, chemotherapy administered within the preceding 90 days was significantly associated with VTE (OR=4.25; p=0.02), while stage progression was marginally associated (OR=4.79; p=0.10). Compared to all other hematological cancer types, acute leukemia (OR=5.95; p=0.01) and non-Hodgkin’s lymphoma (OR=2.61; p=0.01) were associated with VTE. However, after adjusting for BMI, hospitalization, any infection and central venous catheter, only non-Hodgkin’s lymphoma was independently associated with VTE (OR=3.79, p=0.009), while acute leukemia was not (OR=2.66, p=0.35).

Conclusions: Hematological cancer type (in particular, non-Hodgkin’s lymphoma and possibly acute leukemia), recent hospitalization, recent chemotherapy, and possibly stage progression and recent infection, are risk factors for VTE among patients with active hematological cancer.
Treatment of Relapse of AML and MDS after Allogeneic Stem Cell Transplantation Using Low-Dose Chemotherapy, Donor PBSC and GM-CSF: Final Results from a Prospective, Multicenter Phase II Trial by the German Cooperative Transplant Group.
Authors: Schmid Christoph , Tischer Johanna , Stelljes Matthias , Schleuning Michael , Schwerdtfeger Rainer , Hegenbart Ute , Schubert Joerg , Vogel Wichard , Gruhn Berd , Lallinger Christiane , Ledderose Georg , Bornhaeuser Martin , Kienast Joachim , Kolb Hans-Jochem
Published: April 2020

No standard treatment is available for relapse of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) after allogeneic stem cell transplantation (alloSCT). Efficacy of donor lymphocyte transfusion (DLT) was limited, at least in part, by the rapid pace of the disease, overwhelming any allogeneic immune response. However, induction chemotherapy before DLT was complicated by severe toxicity. Based on a pilot trial conducted in Munich, a prospective, multicenter phase II trial for relapsed AML and MDS after alloSCT was initiated in 2000 by the German transplant cooperative group. The study evaluated the sequence of low-dose(ld)AraC for leukemic control (intensive chemotherapy for progressive disease only), transfusion of donor PBSC without immunosuppression as adoptive immunotherapy, and systemic application of GM-CSF. GM-CSF was included, since in combination with other cytokines, it has been able to improve the antigene presentation capacity of myeloid blasts in vitro. Between 2000 and 2006, 41 patients with hematological relapse of AML or MDS >3 months after alloSCT were included. Median age was 47y, 50% had an unrelated donor. Median remission after SCT was 223d (93–1614), median percentage of BM blasts at relapse was 40%. Median follow up was 28 months. Control of leukemic proliferation by ldAraC was achieved in 61%, allowing outpatient care in the majority of these patients. 39% required intensive chemotherapy. Three patients died from infections before donor cell transfusion (DCT), one patient was not transfused due to progressive leukemia. Median time from relapse to DCT was 52 days. 25/34 evaluable patients were found to be free of blasts in BM at d35 after PBSC and were considered initial responders. In an intent-to-treat analysis, overall survival (OS) of the entire cohort at 1 and 2 years from relapse was 41% and 32%. Among initial responders, 1y- and 2y-OS was 68% and 49%. A remission >6months after alloSCT, and control of leukemia by ldAraC prior to DCT were associated with better outcome. At last follow up, 8 patiens were in continous CR, 2 were alive with second relapse, 18 had died from leukemia, and 13 had died in remission or aplasia. In conclusion, ldAraC seems to be effective for initial control of leukemic proliferation in relapsed AML and MDS after alloSCT. A longer duration of post-transplant remission and response to ldAraC may identify patients who will benefit from adoptive immunotherapy. On an intend-to-treat-basis, the overall results of our trial compare favorably to other published strategies; nevertheless, outcome is still unsatisfying and warrants further investigation.
Myeloid Dyspoiesis as Determined by Flow Cytometry Is a More Powerful Predictor of Post-Transplant Relapse with MDS Than the Marrow Myeloblast Count.
Authors: Wells Denise A. , Scott Bart L. , Leisenring Wendy M. , Loken Michael R. , Deeg H. Joachim
Published: April 2020

We showed previously that the presence of aberrant marrow myeloblasts, as determined by flow cytometry immediately pre-transplant had predictive value for post-transplant outcomes in patients undergoing hematopoietic cell transplantation (HCT) for Myelodysplastic Syndrome (MDS). The characteristics of phenotypically abnormal myeloblasts in MDS include decreased expression of CD45, presence of non-lineage lymphoid antigens including CD56, and differences in the intensity of various myeloid antigens in comparison to healthy controls. In the present study, the bone marrow aspirates of 156 patients with MDS were analyzed before HCT for the presence of abnormal myeloblasts with the aim of determining whether myeloid dyspoiesis by flow cytometry was predictive of post-transplant outcomes, specifically in patients who were considered good risk by currently accepted criteria. All patients received ”myeloablative” conditioning, which in most patients (78%) consisted of busulfan (targeted) and cyclophosphamide followed by HLA-identical related (52%), HLA-matched unrelated (39%), or alternative donor (9%) stem cell infusions. In agreement with our initial report, patients with severe flow scores (≥4) had an increased hazard of relapse (HR=2.7, 95% CI_1.1–6.3, p=0.017) in comparison to patients with normal flow scores. In addition, even among patients with less than 5% marrow myeloblasts, dyspoietic characteristics of the blasts, as assessed by flow cytometry criteria were associated with an increased hazard of relapse (HR=4.0, 95% CI 1.4–12.1, p=0.013) as compared to patients without dyspoiesis by flow. The cumulative incidence of relapse in MDS patients with flow cytometrically normal marrow myeloblasts was 11.7%, compared to 28.1% in patients with less than 5% but phenotypically aberrant myeloblasts; the latter was not different from a relapse incidence of 31.4% in patients with 5% or more abnormal marrow myeloblasts. Furthermore, patients with intermediate-1 risk disease by the International Prognostic Scoring System, who showed flow cytometric aberrancies were at significantly increased risk of relapse (HR=4.2, p=0.01) in comparison to patients with intermediate-1 risk disease and no or mild dyspoiesis by flow cytometry. Thus, the presence of dyspoietic changes was more relevant than the proportion of marrow myeloblasts in predicting the risk of relapse, suggesting that flow cytometry is a powerful tool to select high risk patients from cohorts of patients who by established criteria would be considered to have good risk MDS.
High Incidence of Fatty Liver and Insulin Resistance in Long-Term Adult Survivors of Childhood Stem Cell Transplantation Recipients.
Authors: Tomita Yuichiro , Ishiguro Hiroyuki , Hyoudou Hiromi , Sinagawa Tsuyoshi , Yasuda Yukiharu , Shimizu Takashi , Morimoto Tsuyoshi , Shinohara Osamu , Yabe Hiromasa , Ando Kiyoshi , Kato Shunichi
Published: April 2020

[Introduction] An increasing number of long-term surviving stem cell transplantation (SCT) recipients have recovered from their primary disease but are at risk of developing failure of endocrine organs. Among the complications that have been described following SCT, metabolic syndrome (MS) has occurred in long-term survivors. Mechanism of MS as the late complication, however, is not completely elucidated.

[Patients & Methods] We investigated 52 recipients (30 males and 22 females) who underwent SCT during childhood or adolescence. The median age at SCT was 10.5 years (range 0.9–15.9 years), and the median follow-up duration after SCT was 15.0 years (range 6.7–24.7 years). Obesity was evaluated by analyzing changes in body mass index (BMI) and abdominal circumference. Lipid and glucose profiles were evaluated by the presence of fatty liver (hepatic steatosis), TG, T-Chol, HDL-C, LDL-C, adiponectine, leptin and HOMA-R, 75g OGTT, respectively.

[Results] 11 males (37%) and 11 females (50%) were diagnosed as fatty liver by abdominal ultrasound and computed tomography and biopsy was performed in selected patients. However only one male recipient satisfied MS criteria in all recipients. Among the 52 recipients, 9 males (30%) and 15 females (68%) indicated under 18.5 kg/m2 of BMI, whereas recipients with over 25 kg/m2 of BMI were two males and one female. The levels of TG and T-Chol were significantly higher in recipients with fatty liver compared to recipients without fatty liver (TG: 111mg/dL, range 49–751 vs 78, 41–219, p <0.05, TC; 213mg/dL, 166–294 vs 175, 82–250, p <0.05). In addition, the level of leptin was significantly increased in recipients with fatty liver compared to recipients without fatty liver (male: 9.9ng/mL, 2.4–20.8 vs 4.2, 1.9–10.4, p <0.05, female: 18.0, 5.6–25.6 vs 7.9, 3.3–13.2, p <0.05, respectively). On the other hand, the level of adiponectin was significantly decreased in recipients with fatty liver compared to recipients without fatty liver (male: 4.8 μg/mL, 2.6–8.9 vs 8.5, 5.0–29.5, p <0.001, female: 4.1, 2.1–18.5 vs 11.4, 7.1–19.7, p <0.05, respectively). The level of HOMA-R was significantly increased in recipients with fatty liver compared to recipients without fatty liver (2.3, 0.5–12.3 vs 1.2, 0.6–3.7, p <0.05, respectively). Moreover, 75g OGTT indicated that insulin resistance significantly appeared in recipients with fatty liver.

Figure Figure

[Conclusion] These investigations emphasize the need of for long-term follow-up of endocrine and metabolic profiles in adult survivors of childhood SCT recipients.
CD55−CD59− Granulocytes in Patients with Aplastic Anemia Are Clonal Populations Derived from Single PIG-A Mutant Stem Cells without Any Proliferative Advantage.
Authors: Mochizuki Kanako , Sugimori Chiharu , Qi Zhirong , Lu Xuzhang , Nakao Shinji
Published: April 2020

Small populations of CD55−CD59− blood cells are detectable in approximately 50% of all acquired aplastic anemia (AA) patients and the presence of such PNH-type cells are also associated with a good response to immunosuppressive therapy (Sugimori C, et al. Blood 2006). In most patients showing 0.1% to 1.0% PNH-type cells at the diagnosis of AA, the PNH-type cell proportion remains unchanged over 3 years even after successfully responding to immunosuppressive therapy (Mochizuki K, et al. ASH 2006). Although these findings suggest that small populations of PNH-type cells are derived from a limited number of PIG-A mutants without any proliferative advantage, this hypothesis has not yet been verified at the molecular level. To appropriately address this issue, we studied 3 patients with AA who showed 0.14 to 1.6% PNH-type granulocytes. The CD55−CD59− granulocytes were sorted from these patients 2 different times at a minimum of 6 month intervals and then they were subjected to a PIG-A gene analysis. Five exons were amplified using 6 different primer sets and each amplified product was then subcloned into E. coli. At least 5 transformed clones for each amplified product were randomly plucked and subjected to sequencing. Single mutations were thereafter detected in all 3 patients as shown in Table 1. The same single mutations were then detected in the CD55−CD59− granulocytes from the patient obtained 6 months after the first examination. Two patients were in a state of hematologic remission at from 1 to 7 years after the first examination of their peripheral blood and the proportion of PNH-type cells remained stable during this period in all patients. Response to immunosuppressive therapy was not evaluable in one patient because he rejected ATG therapy. These findings indicate that PNH-type granulocytes from patients with AA are therefore clonal populations derived from single hematopoietic stem cells (HSCs) with a PIG-A mutation. If an AA patient has many HSCs with PIG-A mutations before the development of AA, then the immune system attack against HSCs should allow for the survival of the PIG-A mutants leading to the generation of a polyclonal PNH-type cell population. The presence of clonal PNH-type cells at the time of AA diagnosis suggests that the number of HSCs with a PIG-A mutation in healthy individuals may therefore be much lower than we expected and the paucity of PIG-A mutant HSCs may therefore account for the absence of increased number of PNH-type cells in approximately 20% of all AA patients who apparently respond to immunosuppressive therapy.

Table 1 Patient Age Gender Proportion of PNH-type granulocytes PIG-A mutation Response to IST 1 64 M 0.147% 593 bp (exon 2) T insertion PR 2 82 M 1.629% 3′splice site (intron 1) G to A change PR 3 76 M 0.161% 276 bp (exon 2) G deletion not evaluable
Decreased Numbers of Tregs in Aplastic Anemia Is Detected by Immunohistochemistry and Flow Cytometry.
Authors: Serio Bianca , Peerwani Ziad , Tiu Ramon , Powers Jennifer , Hsi Erik , Maciejewski Jaroslaw M.D.
Published: April 2020

Idiopathic aplastic anemia (AA) is characterized by immune-mediated destruction of hematopoietic stem cells, leading to peripheral pancytopenia. Immune pathogenesis in AA is supported by experimental data, as well as clinical observations and may be related to the breach of peripheral or central tolerance. Regulatory T cells (Treg) constitute one of the most important mechanisms of central tolerance engaged in the down-modulation of autoreactive T cells. Tregs have been found to be reduced in several autoimmune diseases and decreased frequencies of Tregs were also reported in AA and MDS. Overexpression of the high affinity IL-2 receptor alpha chain (CD25) and the forkhead family transcription factor P3 (FoxP3), required for the development and function of Tregs, serve as phenotypic markers for Tregs. We investigated Treg levels in a cohort of AA patients (N=21) and healthy individuals (N=15); flow cytometric quantification of Treg was carried out after surface/intracellular staining of whole blood for Treg markers (CD3, CD4, CD25, FoxP3). After proper gating (light scatter properties, CD3, CD4, CD25), CD4+ T cells were subdivided into CD25−, CD25int and CD25hi populations, and the co-expression of CD25hi and Foxp3 was analyzed. In comparison to controls, AA patients (N=12) show not only lower frequencies of CD4+CD25hi+ T cells within the total lymphocyte population (median 0.07% vs. 0.21%; p=.03), but also absolute lower absolute numbers (1.31/uL vs. 5.78/uL, p=.0002). Similarly, CD4+CD25hi+FoxP3+ T cells were found to be depressed in untreated AA patients in comparison to controls (median 0.07% vs. 0.21% and 1.06/uL vs. 4.76/uL; p=.03 and p=.003). While Tregs were lower in patients with active disease unresponsive to immunosuppressive treatment (responder 0.1% vs non responder 0.07%, CD4+CD25hi Tcells, p=.02), serial testing performed in 6 patients treated with ATG/CsA did not reveal correlation between hematologic improvement and recovery of Treg numbers. When double immunohistochemical staining for CD3 and Foxp3 was performed in pre-treatment bone marrow core biopsies of AA patients (N=3) and controls (N=2) a mean of 3 CD3+Foxp3+ cells/10 high power fields (hpf) were counted (vs. mean 28/10 hpf, p<.05 in controls), suggesting that lower numbers of Tregs were also present in the bone marrow of AA patients. In conclusion, our results suggest that Tregs are decreased in blood and marrow of patients with idiopathic AA, consistent with the breach of peripheral tolerance in AA. In addition to flow cytometry, immunohistochemical staining of histologic specimens can be used for the quantitative analysis of Tregs in bone marrow failure syndromes and other immune-mediated conditions such as GvHD.