bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Disruption of the SH3 and SAM Domains of HACS1 Enhances Adaptive Immunity.
Authors: Wang Dingyan , Stewart A. Keith , Zhuang Lihua , Xiao Nancy , Hlaing Mawmaw , Shi Changxin , Zhu Yuanxiao , Wang Youdong , Wen Xiao-Yan
Published: April 2020

HACS1 is a SH3 (Src homology 3) and SAM (sterile alpha motif) domain containing adaptor protein that is expressed in activated B and dendritic cells, is up-regulated by Interleukin 4 in the process of B cell activation and likely serves to dampen the immune response. To elucidate the function of HACS1, we generated HACS1 gene knockout mice by deletion of the SH3 and SAM domains. HACS1−/− mice were viable and fertile and had normal bone marrow B cell development and normal splenic T and B cell populations. However, adult HACS1−/− mice had increased numbers of peritoneal B1 cells (IgM+CD5+). Upon LPS plus BCR or TCR stimulations, splenic cells from HACS1−/− mice demonstrated an upregulation of activation markers with increased intensity of CD23 expression or increased population of CD69 positive cells. Purified B220+ splenic B cells from HACS1−/− mice showed increased cell proliferation upon BCR stimulation. Both T helper type 1 (Th1) and T helper type 2 (Th2) humoral responses were enhanced in HACS1−/− mice upon immunization with T cell-dependent antigen NP-KLH, which resulted in increased production of interferon-gamma (IFN-γ), anti-NP IgG2a and IL-4, as well as anti-NP IgG1 and IgE. Upon immunization with T cell-independent antigens such as TNP-LPS and TNP-Ficoll, HACS1−/− mice had increased production of anti-TNP IgM and IgG3 as compared to normal controls. The in vitro maturation of bone marrow-derived dentritic cells from HACS1−/− mice was similar to wild-type mice but HACS1−/− dentritic cells showed increased IL-12 production upon stimulation with anti-CD40 or LPS. There was no significant difference in antigen uptake by cultured dentritic cells from non-immunized wild-type or knockout mice. However, in immunized mice, an increase in antigen uptake in HACS1−/− dentritic cells was observed. We further demonstrate that the HACS1−/− B cells had increased tyrosine phosphorylation in the resting state. As deletion of the SH3 & SAM domains of HACS1 appears to generate a hypersensitive immune response, our results collectively support that HACS1 negatively regulates adaptive immunity.
The CD22 Epitope, Dose, Dosing Interval, and Timing All Effect Efficacy of Anti-CD22 mAb-Based Therapy of NHL.
Authors: O’Donnell Robert , Pearson David , McKnight Hayes , Tedder Thomas , Tuscano Joseph M.
Published: April 2020

Rituximab, a chimeric anti-CD20 monoclonal antibody (mAb) has become integral to non-Hodgkins lymphoma therapy. New, clinically effective, anti-NHL mAb may further improve outcomes. CD22 is a B-lymphocyte-specific adhesion molecule on the surface of most NHL, so it is a promising target for immunotherapy. A panel of anti-CD22 mAbs were developed and tested. Anti-CD22 mAbs that bind the two NH2-terminal immunoglobulin domains of CD22 and specifically block the interaction of CD22 with its ligand were identified. CD22-blocking mAbs induce apoptosis in neoplastic B-cells and are functionally distinguishable from other anti-B-cell, and even other anti-CD22 mAbs. MAbs that do not block ligand-binding have only modest functional effects. HB22.7 is the blocking anti-CD22 mAb chosen for study and characterization in a Raji NHL-bearing nude mouse model. The goal of this study was to characterize the dose, route and schedule of administration that can guide the translation of HB22.7 to a clinical trial. In addition to untreated Raji-bearing nude mice, a control, non-blocking mAb (HB22.27) was compared to HB22.7. The non-blocking mAb had minimal pre-clinical efficacy, whereas HB22.7 improved survival and caused substantial tumor shrinkage. Varying doses of HB22.7 were tested; doses greater than 1.4 mg did not further increase efficacy (or toxicity). An important consideration regarding translation of HB22.7 to clinical trials is the size of tumors to be treated. In this study, tumors less than 200 mm3 had a much higher response rate than did larger tumors. Varying schedules of HB22.7 administration (1.4 mg/dose) were tested; one dose every other week was more effective than more or less frequent dosing. Immuno-PET showed that NHL was effectively and specifically targeted when copper-64-labeled HB22.7 was administered either intravenously or subcutaneously. This study provided preclinical data as to a useful dose, route and schedule of administration of the anti-CD22 mAb, HB22.7
Mathematical Models To Identify Combined Protein and Clinical Biomarkers of Response to Induction Chemotherapy in Acute Myeloid Leukemia (AML).
Authors: Tibes Raoul , Meurice Natalie , Anthony Steven P. , Qiu YiHua , Kornblau Steven M. , Mousses Spyro , Petit Joachim
Published: April 2020

Treatment of AML is guided by cytogenetics, age, patients’ medical condition and increasingly, new molecular markers. However, no definitive algorithms and biomarkers predicting response to induction chemotherapy exist. We recently showed that distinct protein profiles of AML correlate with cytogenetics, FAB classes and treatment outcome for a class/group of patients as a whole (Tibes, ASH 2005; Kornblau, ASH 2006 and manuscript in submission). To further individualize therapy, we applied a set-based decision modeling methodology based on the Rough Set Theory (RST) to our initial proteomic dataset of 34 (phospho)-proteins for 96 samples from 73 patients (Tibes, ASH 2005). The first model was built based on partitioning discretized protein expression levels of proteins for the 96 samples into 2 outcome categories: complete remission (CR) vs. refractory to treatment; we generated reducts for all proteins and computed pseudo-cores. Several proteins (incl. phospho-NPM1, PTEN, phospho-AKT, p53, cyclin D1 etc.) were able to distinguish outcomes in terms of CR and refractoriness. A second model based on discretized clinical condition attributes (e.g., blast %, FAB, cytogenetics, age, sex, blood counts etc.) yielded the most important clinical reducts. In a third model, protein expression and clinical attributes were combined to generate decision rules to predict outcome to induction therapy for each patient. For proof-of-concept purposes, the data set was divided into a training set and a test set. Prediction accuracy for CR vs. refractory state was in the 75% range for individual patients based on their pre-treatment clinical and proteomic attributes. In conclusion, predictive models obtained with RST yield reducts corresponding to a small but significant number of proteins (6–8) and clinical parameters which can further be used to derive association rules, capable of accurately predicting a patient’s response to induction therapy. The availability of several dozens of distinct decisions rules within each group (CR vs. refractory) based on protein expression and clinical characteristics, allows an individualized approach for each patient. This preliminary study is now being expanded on an independent proteomic dataset of 52 proteins for 258 AML samples (Kornblau, ASH 2006) to be presented at the Annual Meeting. The clinical utility lies in the fact that measuring expression levels of our protein biomarker profiles can be adopted to routine clinical testing (e.g., immunohistochemistry on diagnosis marrows) and potentially help to direct patients towards standard chemotherapy, upfront treatment on a clinical trial or evaluation for early transplant depending on the chance of response and relapse. Lastly, this model can be applied to predict other clinical outcomes (relapse vs. continuous CR), as well as to derive predictive signatures of gene expression datasets.
RB1 Haploinsufficiency Confers a Proliferation Advantage in Myeloma Cell Lines.
Authors: Henry Travis J. , Chng Wee Joo , Carpten John , Bergsagel P. Leif , Fonseca Rafael
Published: April 2020

The retinoblastoma 1 (RB1) gene is a frequent target of mutation or inactivation in neoplastic diseases. However, the current model for RB1-mediated malignancy requires inactivation of both alleles for tumor progression. Single–copy loss of RB1 has been shown to correlate with an approximate 50% reduction in RB1 mRNA expression in patients diagnosed with multiple myeloma and survival in this cohort of patients is significantly reduced. Using siRNA to reduce Rb protein levels to 50% and Rb-expressing adenovirus to restore RB1, variations in proliferation and cell cycle were observed. The KMS–11 adherent cell line with normal Rb protein expression was labeled with carboxyfluorescein succinimidyl ester (CFSE) for tracking of cellular division and RB1 siRNA added to silence 50% of protein expression. Transfection efficiency for siRNA was measured at 95% or greater. Flow cytometric analysis was performed at 72 hours to identify changes in proliferation between controls and Rb knock–down. Using CFSE proliferation analysis software, reduction of Rb protein levels by 50% caused an average increase in the proliferation index from 2.05 to 2.50. Additionally, reduction of Rb protein levels caused an increase in the percentage of cells in S–phase, from 24 to 29%. CFSE and cell cycle experiments were performed in triplicate. Conversely, addition of Rb–expressing adenovirus to MM.1R and U266 cell lines, with mono-allelic and bi-allelic loss of RB1 respectively, resulted in a decrease in proliferation assayed by CFSE and an average decrease in the percentage of cells in S-phase from 29 to 23% compared to controls. No changes in proliferation or cell cycle compared to untreated controls were observed following null adenovirus infection. Western blot and quantitative RT–PCR (qPCR) were used to confirm reduction or addition of RB1 to cell lines. Further, qPCR was used to identify potential activation of the interferon response following addition of siRNA and adenovirus. No changes in annexin V expression were observed following reduction or replacement of Rb as compared to controls. These results suggest the addition of proliferation advantages to tumors with single copy loss of RB1. For malignancies such as multiple myeloma characterized by deletion of 13q14 coupled with an extended period of development, the proliferation advantage associated with RB1 haploinsufficiency may contribute to decreased survival in the deletion 13 cohort.
Ribosomal Proteins Are Overexpressed in Hyperdiploid Multiple Myeloma.
Authors: Weinhold Niels , DeVos John , Hose Dirk , Rossi Jean-Francois , Axel Benner , Mahtouk Karène , Raab Marc S. , Rème Thierry X. , Jauch Anna , Moreaux Jerome , Pantesco Veronique , Goldschmidt Hartmut , Jourdan Eric , Cremer Friedrich W. , Klein Bernard , Moos Marion
Published: April 2020

Multiple myeloma (MM) is proposed to consist of two main pathogenetic groups. While hyperdiploidy (HD) is characterized by multiple trisomies of odd-numbered chromosomes (i.e. 3, 5, 9, 11, 15, and 19), non-hyperdiploid MM (NHD) show frequently one of the several recurrent IgH-translocations. The aim was to compare HD versus NHD by gene expression profiling (GEP). CD138-positive multiple myeloma cells from 74 newly diagnosed MM patients (42 GEP training group (TG), 32 GEP validation group (VG)) were purified by autoMACS-sorting. Sorted cells were analyzed by interphase-FISH with probes specific for 6q21, 8p21, 9q34, 11q23, 13q14, 15q22, 17p13, 19q13 and translocations t(4;14) and t(11;14). HD and NHD were defined by using a copy number score (CS), which was calculated by subtracting the number of probes indicating losses from the number of probes detecting additional copies (CS >0: HD; CS ≤0: NHD). GEP was performed with Affymetrix DNA-microarrays. Nearest shrunken centroid classification (NSCC) was used to discriminate the different groups, using GCRMA-normalized gene expression values. The prediction error was estimated by means of nested cross-validation using 10 repetitions of 10-fold cross-validation within the training set and separately calculated by use of the NSCC classifier of the training set to predict the validation set. Goeman’s global test was used to check the influence of ribosomal protein expression between HD and NHD. In the TG, both HD and NHD were found in 21 patients. The VG comprised 13 patients with NHD and 19 patients with HD. NSCC resulted in a predictor for HD versus NHD of 81 probe sets with a cross-validated misclassification rate of 14.2% for the TG and 26.5% for the VG. Three of the top ten genes were ribosomal proteins, overexpressed in patients with HD. Goeman’s global test further showed that ribosomal proteins are overexpressed in HD (TG: p<0.001; VG: p=0.03). Interestingly, ribosomal protein genes located on even-numbered chromosomes were also overexpressed. In conclusion, overexpression of ribosomal proteins independent of their location on odd- or even-numbered chromosomes indicates more than just a gene dosage effect and therefore a pathogenetic role of the upregulation of the ribosomal machinery in HD.
The JAK2-V617F Mutation Load Remains Stable over Several Years in Most Patients with Myeloproliferative Disorders Analyzed in a Retrospective Single Center Study.
Authors: Theocharides Alexandre , Looser Renate , Hui Hao-Shen , Buser Andreas S. , Gratwohl Alois , Tichelli Andre , Skoda Radek C.
Published: April 2020

An acquired somatic mutation in the JAK2 gene (JAK2-V617F) is frequently found in patients with myeloproliferative disorders (MPD). In most studies the JAK2-V617F mutation has been analyzed at single time point. Here we performed a retrospective single center study on 73 MPD patients (36 polycythemia vera (PV), 29 essential thrombocythemia (ET), and 8 primary myelofibrosis (PMF)) from whom at least two blood samples (mean=5, range 2–17) were available with an interval of at least 8 months. The mean follow-up period was 35±17 months (range 8–78 months). The allelic ratio of JAK2-V617F (%T) was determined in DNA from purified peripheral blood granulocytes by allele-specific PCR. In 73 MPD patients studied, 53 (73%) carried the JAK2-V617F mutation (32 PV, 18 ET, 3 PMF). None of the 20 patients negative for JAK2-V617F acquired the JAK2-V617F mutation during the observation period (n=20, mean number of samples=4). In the majority of the JAK2-V617F positive patients (35/53; 66%) the JAK2-V617F allelic ratios remained remarkably stable during the follow-up period (variation ±5%T) (Figure 1 A and B). In 10/53 patients (19%) we observed an increase and in 8/53 (15%) a decrease in JAK2-V617F allelic ratio greater than 5%T. Interestingly, 3/10 patients (1 ET and 2 PV) who showed increase of JAK2-V617F developed secondary myelofibrosis. Twenty six patients (49%) received cytoreductive treatment (hydroxyurea: 24, interferon alpha: 1, anagrelide: 1). Cytoreduction with hydroxyurea did not significantly reduce the JAK2-V617F allelic ratios (Figure 1A) compared to untreated patients (Figure 1B). The only molecular remission was seen in one patient treated with interferon alpha (Figure 1 A). In one patient without cytoreduction JAK2-V617F became undetectable because of transformation to acute myeloid leukemia with blast cells negative for JAK2-V617F. A second patient treated with hydroxyurea showed a pronounced decrease of JAK2-V617F (–47%T in 6 months), but no clinical or laboratory signs of leukemic transformation were present. We conclude that the amount of JAK2-V617F remains very stable in a majority of JAK2-V617F positive patients. Prospective studies will help to elucidate whether increasing JAK2-V617F allelic ratios can predict secondary myelofibrosis or decreasing allelic ratios in absence of cytoreductive therapy (e.g. interferon alpha) are early signs of leukemic transformation.

Figure 1A. Figure 1A. Figure 1B. Figure 1B.
Overexpression of JAK2 mRNA in Chronic Myeloproliferative Disease and Its Implication on Mutation Testing and Biology of Disease.
Authors: Ma Wanlong , Zhang Xi , Jilani Iman , Zhang Zhong , Kantarjian Hagop , Verstovsek Srdan , Giles Francis , O’Brien Susan , Albitar Maher
Published: April 2020

The V617F point mutation in the JAK2 gene is considered the most important molecular abnormality that characterizes patients with non-chronic myeloid leukemia (CML)-myeloproliferative disease (MPD). While it is well-documented that the V617F mutation leads to continuous activation of the JAK2-STAT5 pathway, the levels of JAK2 mRNA expression is not well defined in myeloproliferative diseases. We quantified the levels total JAK2 mRNA as well as V617F mutant mRNA in the plasma of patients with non-CML MPD using RT/PCR, and compared the results with clinical findings and disease behavior. Patients with non-CML MPD (n = 175) had significantly higher levels (P <0.0001) of JAK2 mRNA (median, 1843 pg/ul) than patients with CML (median, 71.53 pg/ul) and normal controls (median, 34.34 pg/ul). In contrast, there was no significant difference (P = 0.15) in DNA levels (copies of JAK2 DNA/ul plasma) between CML patients and non-CML MPD patients. There was no significant difference in JAK2 mRNA levels in the MPD group between patients with or without the V617F mutation. There was no significant difference in JAK2 mRNA levels between patients with polycythemia vera, essential thrombocythemia, or agnogenic myeloid metaplasia, or unclassified MPD (P = 0.09). Survival data was available for only 68 patients; JAK2 mRNA levels in these patients did not correlate with overall survival. In contrast, mutant (V617F) JAK2 mRNA levels in the plasma correlated negatively with overall survival in continuous fashion (P = 0.04) when all patients (with and without the mutation) were considered, as well as when only patients with the mutation were considered (P = 0.03). In contrast, when we quantified levels of mutant JAK2 DNA, 4 of the 68 patients who showed mutation using RNA were negative for the V617F mutation by DNA analysis, and the levels of mutant JAK2 DNA did not correlate with survival. This data suggests that JAK2 mRNA is overexpressed in all patients with non-CML MPD, and levels of mutant JAK2 mRNA may correlate with clinical behavior. Furthermore, when testing for JAK2 mutation, RNA rather than DNA should be used.
Lenalidomide Oral Monotherapy Produces a 53% Response Rate in Patients with Relapsed/Refractory Mantle-Cell Non-Hodgkin’s Lymphoma.
Authors: Tuscano Joseph M. , Lossos Izidore S. , Justice Glen , Vose Julie M. , Takeshita Kenichi , Ervin-Haynes Annette , Pietronigro Dennis , Zeldis Jerome B. , Habermann Thomas M.
Published: April 2020

Background: Mantle cell lymphoma (MCL) is a distinct type of non-Hodgkin’s Lymphoma characterized by being incurable with a low response rate and short progression free survival when treated with conventional chemotherapy agents. Lenalidomide (Revlimid®), an immunomodulatory drug, is approved for the treatment of relapsed/refractory multiple myeloma and myelodysplastic syndromes associated with a del(q5) cytogenetic abnormality. Lenalidomide has also shown activity in chronic lymphocytic leukemia and cutaneous T-cell lymphoma.

Aim: To determine the activity and safety of lenalidomide in relapsed/refractory MCL.

Methods: Patients with relapsed/refractory MCL and measurable disease ≥ 2 cm after at least 1 prior treatment regimen were eligible. Patients received 25 mg lenalidomide orally once daily on Days 1–21 every 28 days and continued therapy for 52 weeks as tolerated or until disease progression. Response and progression were evaluated using the IWLRC methodology.

Results: Fifteen patients with MCL were enrolled. Median age was 67 (45–84) and 7 were female. Median time from diagnosis to lenalidomide was 5.1 (0.7–12.7) years and median number of prior treatment regimens was 4 (2–6). Eight patients (53%) exhibited an objective response (1 complete response (CR), 1 complete response unconfirmed (CRu) and 6 partial responses (PR)), 2 had stable disease (SD) and 5 had progressive disease (PD). All eight responses were in eleven patients having a tumor burden < 50 cm2 and a time since last rituximab therapy of ≥ 230 days. No responses were achieved in four patients having a tumor burden ≥ 50 cm2 or a time since last rituximab therapy of < 230 days. Four of 5 patients (80%) with a prior stem cell transplant responded. Progression free survival [PFS] is 5.7 months and ongoing. Seven patients (47%) required at least one dose reduction with a median time to first dose reduction of 2.3 months (1.2–4.9). Grade 4 adverse events were neutropenia (13%), thrombocytopenia (13%), and thromboembolism (13%). Most common Grade 3 adverse events were neutropenia (33%) and leukopenia (20%).

Conclusion: Lenalidomide oral monotherapy produced a 53% response rate in relapsed/refractory MCL with manageable side effects.
Functional Regulation of TEL through MOZ-Induced Acetylation.
Authors: Maki Kazuhiro , Nakamura Fumihiko , Yamagata Tetsuya , Kitabayashi Issay , Mitani Kinuko
Published: April 2020

TEL, a member of ETS transcription factors, acts as a transcriptional repressor and a tumor suppressor. TEL is inactivated by genomic alterations such as deletion and chromosomal translocation in a variety of human leukemia. TEL also has important roles in the maintenance of hematopoietic stem cells and differentiation of erythroid progenitors. Regulation of its function through phosphorylation and sumoylation has been described in the past literatures. We here demonstrated that TEL is also regulated by acetylation, another mode of post-translational modification. First, immunoprecipitation assay using anti-acetylated lysine (Ack) antibody showed that TEL proteins overexpressed in 293T cells were acetylated in vivo. To determine the acetylated lysine residue(s), we used a set of TEL mutants in which one of 16 lysine residues in the molecule was left with all the others substituted with arginine. We found that TEL was acetylated only at Lys99. To confirm that TEL is acetylated in hematopoietic cells, we established mouse erythroleukemia (MEL) cell clones expressing wild-type-TEL or K99R mutant in which Lys99 was substituted with arginine, and demonstrated acetylation of TEL at the same lysine residue in MEL cells by in vivo acetylation assay using [1–14C] acetic acid. Next, to identify histone acetyltransferase that is responsible for acetylation of TEL, we co-expressed each one of several known histone acetyltransferases such as p300, CBP, P/CAF and MOZ with TEL. The immunoprecipitation assay using anti-Ack antibody showed that only MOZ induced acetylation of TEL proteins among them. Physical interaction between TEL and MOZ was detected in immunoprecipitation assay. In order to get insights into functional modification of TEL through MOZ-induced acetylation, we examined whether the acetylation alters trans-repressional abilities of TEL. We employed pGL2-754TR reporter that has a natural promoter derived from stromelysin-1, one of TEL’s target genes, in luciferase assays. A two-fold decrease in luciferase activities was observed when TEL was expressed. Importantly, co-expression of MOZ attenuated the transcriptional suppression by TEL. Furthermore, to analyze alterations of TEL’s biological functions through acetylation, we employed MEL cells whose erythroid differentiation is accelerated by TEL, and compared the effect of overexpression between wild-type-TEL and its K99R mutant. Interestingly, K99R mutant stimulated erythroid differentiation with a more extent than wild-type-TEL. All these data suggest that molecular and biological functions of TEL might be controlled negatively through MOZ-induced acetylation at Lys99. Being a tumor suppressor, functional inactivation of TEL through protein modification mechanisms could be critical in the development and progression of human leukemia.
DMT1 Expression Is Regulated by DMT1 Associated Protein (DAP) in K562 Cells.
Authors: Okazaki Yasumasa , Yin Hong , Ma Yuxiang , Okazaki Emiko , Yeh Mary , Yeh Kwo-yih , Glass Jonathan
Published: April 2020

While iron is essential for cell growth and survival excess iron through oxidative stress may produce hepatitic cirrhosis and hepatocellular carcinoma, diabetes mellitus, and cardiomyopathy. Iron is absorbed across the duodenum with transport across the brush border mediated by DMT1 and across the basolateral surface by ferroportin with mechanisms that are inversely regulated by body iron concentrations. We have identified in rat intestine DAP, a novel protein that binds to the C-terminus of DMT1 (IRE) but not to the C-terminus of the non-IRE isoform (Blood, Nov 2004; 104: 53). DAP is a 526 amino acid protein that has been previously described as binding to the peripheral benzodiazepine receptor, an intrinsic mitochondrial protein involved in steriodogenesis and possibly in protoporphyrin IX transport into the mitochrondria. To investigate if DAP may have a role in regulation of intracellular iron transport DAP expression was down regulated using a vector containing a siRNA for DAP transfected into K562 cells by electroporation. Expression levels of DAP, transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1) and ferritin were examined by western blot and quantitative quantitivative PCR assays from days 1 to 6 after transfection. Following transfection with the DAP siRNA DAP mRNA levels were decreased 50% by day 1 with DAP protein levels decreasing by 50% at day 3. The DAP siRNA also decreased DMT1 protein expression by about 50% for the DMT1 (IRE) protein but had no effect on the protein derived from the non-IRE isoform. The leels of DMT1 mRNA were not affected by DAP siRNA. The decrease of DAP expression was not associated with any change in TfR1 or ferritin expression, suggesting that altered levels of DAP did not affect intracellular iron pools. Transfection with the DAP siRNA resulted also in more protean effects decreasing cell proliferation, the transition from S-phase to G2 in cell cycle, and protein synthesis. These data are consistent with DAP regulating DMT1 expression in K562 cells by modulating turnover of DMT1 (IRE) protein and also having more global effects on cellular metabolism.
Comparison of Homeostatic Reticulocyte Aging between Normal and Belgrade Rats Using Flow Cytometry.
Authors: Wiczling Pawel , Krzyzanski Wojciech
Published: April 2020

The purpose of this work was to obtain and compare reticulocyte age distribution of normal and Belgrade (b) rats. The Belgrade rat has a microcytic, hypochromic anemia associated with abnormal reticulocyte iron uptake. Samples of blood were drawn from two homozygous rats (b/b) and three heterozygous rats (t/b). Cells were cultured over 12 hours and sampled at 3 hour intervals. The reticulocyte count was performed using a staining solution composed of thiazole orange and LDS 751, a DNA binding agent. LDS 751 was used to separate RBC form nucleated cells. The differentiation between mature RBC and reticulocytes was done by means of presence of residual RNA, which also served as a marker of reticulocyte age. The in vitro changes of the fluorescence signal of the reticulocyte population allowed us to obtain the unique relationship between the signal and the age for an individual cell based on the previously developed structured population model of cultured reticulocytes. This relation was further used to transform the in vivo signal distribution into the age distribution. The obtained reticulocyte age density was used to characterize age dependence of reticulocyte appearance in blood and death. The Belgrade rats have reticulocytosis of 13.6 ± 4% whereas control rats have reticulocytes in normal range of 2.15 ± 0.4%. The aging of reticulocyte was characterized by a relative time, RT, that a reticulocyte needs to become a mature RBC. For the normal reticulocytes the RT that an average reticulocyte spent in blood is 0.84 ± 0.02 days and ranges from 0 to 2.0 days. For Belgrade rats the time an average reticulocytes in blood needs to become a mature RBC is 1.18 ± 0.08 days and ranges from zero 0 to 2.2 days. In conclusion, the Belgrade reticulocytes are younger by about 0.4 days. The RT distributions for Belgrade rats are wider than for normal rats. Additionally, the increased death of Belgrade reticulocytes in circulation is observed. In this work the new method of studying aging of cells was successfully applied for anemic rats. It allowed us to characterize in vivo aging of normal and Belgrade reticulocytes.
Role of Gamma-Carboxyglutamic Acid (GLA) Domain in Phosphatidylserine (PS)-Regulated Activity of Factor IXa.
Authors: Majumder Rinku , Craft Judson , Rezaie Alireza , Barry R. , Monroe Dougald
Published: April 2020

Both factors Xa and IXa are vitamin-K-dependent serine proteases that consist of disulfide-linked heavy and light chains. The heavy chain contains the serine protease active site, while the light chain contains the GLA (gamma-carboxyglutamic acid) domain, EGF-I (epidermal growth factor-like region) and EGF-II domains. Effect of PS on FXa: We have extensively studied the interaction of factor Xa with 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS). C6PS is a soluble analogue of phosphatidylserine, which is present in platelet membranes; it serves as a model for the effect of membrane-bound PS on factor Xa activity and structure. We located three C6PS binding sites to different domains of factor Xa using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements, showing that :

the Gla domain binds C6PS only in the absence of calcium (kd ∼ 1 mM); a calcium-dependent, regulatory, PS-binding site exists in the EGFN domain when linked by calcium to the Gla domain; and a second calcium-requiring site exists in the EGFC-catalytic domains and shares residues with the substrate recognition site.

We have shown that full functional response to C6PS requires linkage of the Gla, EGFNC, and catalytic domains in the presence of calcium. Recently we have observed that the proteolytic activity of des EGFN factor Xa is not affected in the presence of C6PS, locating more precisely a significant component of the PS-triggered regulatory site to the EGFN domain. Efect of PS on FIXa: We have previously reported that C6PS induces a calcium-dependent conformational change in factor IXa that regulates the amidolytic and proteolytic activities. We have also shown that factor IXa binds 2 molecules of C6PS. Here we have examined the role of the GLA domain in this C6PS regulation of factor IXa using a GLA-domainless variant of factor IXa (GD-IXa). In the absence of the GLA domain, binding to C6PS has no effect on the rate of proteolytic or amidolytic activity of GD-IXa toward both factor X and synthetic substrates. The binding of C6PS to GD-IXa studied using intrinsic tryptophan fluorescence (Kd = ∼50μM) was different from that for native factor IXa (Kd = ∼2μM). The critical micelle concentration (CMC) of C6PS under the conditions of these experiments (∼300μM) was much greater than the C6PS concentrations used in our experiments. We conclude that:

C6PS does bind to GD-IXa, but at a reduced affinity compared to factor IXa; C6PS does not regulate the amidolytic or proteolytic activities of factor IXa in the absence of GLA domain.

This could be due to two C6PS sites existing in FIXa, with the regulatory site requiring the Gla domain (as observed for FXa), or to a requirement for the Gla domain for the regulatory activity of a single site. Future studies looking at the stoichiometry of binding of C6PS with GD-IXa will help distinguish between these possibilities.

Supported by Supported by NHLBI (HL 072827).
Overall Survival in Erythrodermic Cutaneous T-Cell Lymphoma: An Analysis of Prognostic Factors in a Cohort of Erythrodermic Cutaneous T-Cell Lymphoma Patients.
Authors: Vidulich Kelley A. , Talpur Rakhshandra , Bassett Roland L. , Duvic Madeleine
Published: April 2020

Most common cutaneous T-cell lymphomas (CTCL) are Mycosis fungoides (MF) and a leukemic, erythrodermic (E) variant known as Sézary Syndrome (SS). Blood (B) criteria were not previously considered for staging purposes and are evolving. E-CTCL can be divided into E-MF versus SS based on new criteria, B0-B2, published by the ISCL guidelines. Historically, overall survival (OS) of “SS” patients is 2–4 years. To determine if hematologic staging and prognostic variables affects overall survival, we retrospectively studied 1197 MF/CTCL patients seen at MDACC since 1987 from which were identified 124 (10%) patients with E-CTCL. Median age at diagnosis was 63 (range 0–89), 71 males and 53 females. Hematologic (H) stage was based on quantitative Sézary cell counts (manual or by flow as absolute CD4+26−). Overall survival curves were estimated by Kaplan and Meier and compared using log-rank tests. Median OS all 124 E-CTCL patients was 5.1 years [range=0.4–18.6]. We also considered patients divided by the H0–H4 staging system (Russell-Jones 2000). For H0–H2, OS was 7.6 years, H3 was 5.4 years, and H4 was 2.4 years (p=0.011). Treatment with systemic steroids, advanced age, increased serum LDH at presentation, WBC > 20,000 were significant prognostic factors. Large cell transformation (p=0.758), positive T-cell receptor gene rearrangement in the skin (p=0.54), stage of disease (p=0.955), prior exposure to multiple treatments (p=0.953), and CD4:CD8 ratio (p=0.068) were not significant. In conclusion, we provide evidence that absolute Sézary cell counts can be used to define three groups of patients with E-CTCL with different overall survival patterns and propose a modification to the ISCL B ratings as follows: B0, absolute Sézary cell count < 1.0 KuL−1; B1: absolute Sézary cell count ≥ 1.0–10.0 KuL−1; and B2: absolute Sézary cell count >10.0 KuL−1. Serum LDH and CD4/8 ratio may be used to estimate tumor burden, as they correlate with Sézary counts.
Race and Non-Hodgkin’s Lymphoma: Adverse Impact of Race and Treatment Delays on Survival. A SEER-Medicare Population Study (1995–2003).
Authors: Vance Katisha T. , Kilgore Meredith L. , Yun Huifeng , Gary Lisa C. , Foran James M.
Published: April 2020

Background: Despite a lower incidence of non-Hodgkin’s lymphoma (NHL) and specifically diffuse large B-cell lymphoma (DLBCL), the most common histologic subtype, previous studies suggest that blacks have a higher disease-specific mortality from NHL than whites. To study this disparity, we looked at the impact of race, treatment delays and the use of rituximab on survival in NHL and DLBCL. To control for access to care, we focused on a population of patients with Medicare benefits.

Methods: After establishing a SEER-Medicare Data-Use Agreement and obtaining IRB approval, we used the linked SEER-Medicare database for NHL patients diagnosed from 1995 through 2002, with claims data through 2003. Multivariable logistic regressions were used to assess the difference between race and rates of survival, NHL histology, and the likelihood of receiving treatment within 90 days and of receiving rituximab at any time during the course of NHL treatment.

Results: Among 41,704 NHL cases, 35,014 (85%) cases were in whites and 2,287 (5.5%) were in blacks. Of the 14,831 total cases of DLBCL, 12,449 (84%) were in whites and 715 (4.8%) were in blacks. Despite a lower incidence of NHL, blacks had a lower 1-year survival for NHL compared to whites (OR 0.68 [95% CI 0.61–0.76]). Blacks also had a lower 1-year survival for DLBCL compared to whites (OR 0.63[95% CI 0.53–0.75]). Patients treated within 90 days of diagnosis had improved 1-year survival for NHL (OR 1.10 [95% CI 1.03–1.18]) and for DLBCL (OR 1.60 [95% CI 1.47–1.75]). When controlling for age, gender and income, blacks were less likely than whites to be treated within 90 days of a NHL diagnosis (OR 0.599 [95% CI 0.526–0.681]). The use of rituximab was associated with improved 1-year survival for NHL (OR 3.67 [95% CI 3.37–4.00]) and specifically for DLBCL (OR 3.01 [95% CI 2.66–3.40]). However, blacks were significantly less likely to receive rituximab for NHL (OR 0.55 [95% CI [0.47–0.63]) and specifically for DLBCL (OR 0.71[95% CI 0.57–0.90]). An analysis of two and three year survival for NHL and DLBCL yielded similar results for the impact of race, treatment delays and use of rituximab.

Conclusions: Blacks are diagnosed with NHL and specifically DLBCL less frequently than whites but have significantly lower 1-year survival rates. Blacks were significantly less likely to begin treatment within 90 days of a NHL diagnosis. In addition, blacks were significantly less likely to receive rituximab despite presumed equal access to healthcare with Medicare benefits. The reason for this racial disparity is unclear. Studies to further characterize the disparity are needed.
Genomic Profiling of Different Subtypes of B-Cell Non-Hodgkin’s Lymphoma Using High-Density Single Nucleotide Polymorphism (SNP) Microarrays.
Authors: Kato Motohiro , Nakazaki Kumi , Takeuchi Kengo , Yamamoto Go , Nannya Yasuhito , Sanada Masashi , Muto Satsuki , Chiba Shigeru , Mori Shigeo , Kobayashi Yukio , Kurokawa Mineo , Ogawa Seishi
Published: April 2020

B-cell non-Hodgkin’s lymphoma (B-NHL) is a constellation of mature B-cell neoplasms characterized by distinct pathological and molecular genetic features, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), MATL-type lymphoma (MALT-L). These B-NHLs undergo a wide variety of genetic alterations, including gains and losses of genetic materials, as well as abnormalities in allelic composition, and these alterations may collectively comprise unique genomic profiles specific to different subtypes of B-NHL, which are tightly linked to the pathogenesis of each B-NHL subtype. Recently, we have developed a robust method (CNAG/AsCNAR) to detect allelic imbalances and other copy number changes in cancer genomes using SNP-genotyping microarrays (Nannya et al. Cancer Res, 2005 and Yamamoto et al. Am J Hum Genet, 2007). Without depending on the availability of constitutive genomic DNA, it enables sensitive detection of allelic imbalances including loss of heterozygosity (LOH) as well as copy number (CN) alterations in the face of 70–80% of normal cell components. In the current study, we performed SNP-chip/CNAG/AsCNAR analysis of 171 B-NHL specimens, including 65 cases of DLBCL, 61 cases of FL, and 45 cases of MALT-L in order to comparatively investigate the unique genomic profiles of different B-NHL subtypes. A large number of gains and losses of chromosomal/genomic segments as well as allelic imbalances were identified. While individual genomic profiles are substantially variable among different cases, they collectively showed a characteristic genomic profile in each B-NHL subtypes; +1q, +2p and +18 are common to DLBCL and FL, while +3 and +11q were more frequently found in DLBCL and +11p was more characteristic to FL. MALT-L also showed +3 and +18, but rarely had +1q and +2p. CN neutral LOH due to mitotic recombination (uniparental disomy; UPD) events are frequently found in DLBCL (70%) and FL (54%), but less common in MALT-L. UPD most commonly involves 1p, 1q, 6p, 9p and 12q. In total, 29 loci of high-grade amplification were identified. Among these recurring amplifications were observed at 1q and 2p, which involves FCGR2B, and cRel genes, respectively. These amplifications were detected in both DLBCL and FL, but rare in MALT-L. Total 14 loci of homozygous deletion were also detected and differentially distributed among different B-NHL subtypes. In conclusion, SNP-chip with CNAG/AsCNAR analysis revealed characteristic genomic signatures of distinctive B-NHL subtypes, implicating their unique pathogenesis.
Red Blood Cell Derived Microparticles Are Thrombogenic in Mouse Models of Atherosclerosis.
Authors: Isobe Hirotaka , Perkmann Thomas , Breuss Johannes M. , Binder Christoph J. , Boulanger Chantal M. , Binder Bernd R.
Published: April 2020

In normal individuals microparticles (MPs) derived from leukocytes, erythrocytes, platelets and endothelial cells are found in the circulation. When the thrombogenicity of microparticles was measured in vitro by their capacity to generate thrombin (thrombin generating activity, TGA) in normal control plasma we found the highest in vitro activity to be generated by red blood cell derived MPs and the lowest by monocyte derived MPs. While all MPs exhibited Annexin V binding, indicating the presence of negatively charged phospholipids on the surface, only monocyte and smooth muscle cell (SMC) derived MPs expressed tissue factor and their TGA was dependent on the “extrinsic” factor VII dependent pathway. All other MPs utilized the “intrinsic” factor XI, factor IX and VIII dependent pathway. In vivo, however, only MPs activating the “extrinsic” pathway were active as shown by TAT formation upon injection of MPs in wt C57/bl6 mice (3 minutes after injection). When, however, not directly thrombogenic MPs derived from red blood cells were co-injected with tissue factor bearing MPs, TAT formation was significantly enhanced, indicating that non-directly thrombogenic MPs need tissue factor “priming” to become thrombogenic. To prove this hypothesis, we injected red blood cell (RBC) derived MPs (106–108) into wild type C57BL/6 mice and into ApoE−/− or LDLR−/ − deficient mice, fed a high lipid diet. In wild type C57BL6 mice red blood cell derived MPs did not significantly increase TAT complexes as compared to PBS (RBC derived MPs 108 6.2±0.8 vs. PBS 4.1±1.3 mg/L). When, however, such RBC derived MPs were injected into LDLR−/− mice fed a high lipid diet, TAT complexes significantly increased (PBS 4.4±0.5 vs RBC derived MPs 108 15.8±1.9mg/L). Similar results were obtained also in ApoE−/− mice. This indicates that tissue factor “priming” also occurs in atherosclerotic mice, likely by tissue factor expressed on the plaque or by plaque derived tissue factor bearing MPs. To determine whether local tissue factor expression causes also local fibrin formation upon injection of red blood cell derived MPs, the aortic arch of such mice were analyzed for local fibrin deposition after injection of red blood cell derived MPs. In fact red blood cell derived MPs did not only induce higher TAT complexes in LDLR−/− mice but also increased the number of fibrin positive plaques as compared to PBS. This indicates that red blood cell derived MPs are thrombogenic in animal models of atherosclerosis. When MPs isolated form the arterial blood of atherosclerotic patients undergoing carotid endarterectomy were analyzed, it was found that MPs from symptomatic patients were more thrombogenic and that this increased thrombogenicity was due to increased number of red blood cell derived MPs. This indicates that red blood cell derived MPs might also cause thrombus formation in atherosclerotic patients.
Predictors of Diagnosis Delay and Complications for Newly-Diagnosed Myeloma Patients.
Authors: Friese Christopher R. , Abel Gregory A. , Magazu Lysa A. , Neville Bridget A. , Richardson Lisa C. , Earle Craig C.
Published: April 2020

Background: The increasing incidence in multiple myeloma (MM), coupled with improved efficacy of therapies, warrant population-based studies of diagnosis delay which could lead to disease-related complications.

Methods: We analyzed tumor registry data and provider claims in the SEER-Medicare database, a geographically and racially diverse sample of Medicare enrollees with confirmed MM. Using extant selection criteria to study eligible MM patients, we identified claims with anemia (An) or back pain (BP) codes, conditions related to MM. We estimated the period of time between first claim for An or BP and confirmed MM diagnosis. We defined delay as ≥98 days (the median) between initial An or BP claim and MM diagnosis, and estimated logit models to predict sociodemographic and clinical factors associated with delay. We also estimated logit models to predict the likelihood of renal or skeletal complications following MM diagnosis. The study period was one year before, through six months following, diagnosis. Analyses were adjusted for a known 30-day lag time in MM diagnosis dates in SEER.

Results: From the analytic sample of 5,185 patients, 3,600 had a diagnosis for An (2,576) and/or BP (2,012) prior to MM diagnosis. Male gender (OR 0.73, 95% CI 0.64–0.84) and northeast region (0.79, 0.65–0.96) decreased the likelihood of delay. Over 60% of patients had ≥ 1 Charlson comorbidity; higher Charlson scores and the presence of both An and BP increased likelihood of delay, but were strongly correlated, and an interaction term with these variables in the model was significant. Of the 5,112 patients with no history of renal or skeletal complications, 1,742 (34%) experienced a complication within six months after MM diagnosis. After adjustment for gender, age, race, and subsequent chemotherapy use, patients with: higher Charlson scores (1.17, 1.03–1.33); initial diagnosis during an inpatient stay (2.40, 1.92–3.00), and; no UPEP, SPEP, or bone marrow biopsy as part of the MM workup (1.68, 1.48–1.92) were more likely to experience a complication (29% of patients had neither PEP nor biopsies recorded). Delay between An or BP diagnosis and MM was not associated with likelihood of complication (0.96, 0.91–1.01).

Conclusions: Despite the presence of two classic signs and symptoms of MM, gender and geography significantly influence diagnosis delays. The surprising finding that both anemia and back pain delay diagnosis is partly explained by increased comorbidity in these patients. These findings highlight the importance of considering MM as part of the routine workup for anemia in elderly patients. The existence of comorbidity in these patients may obscure the clinical presentation, resulting in delayed diagnosis. Underuse of common tests to diagnose myeloma may result in poor outcomes, and could be a focus of quality improvement efforts.
Annexin 2 Mediates Plasminogen-Related Recruitment of Neovascular Mural Cells in Lymphoma Angiogenesis.
Authors: Hajjar Katherine A. , Ruan Jia , Sui Guangzhi , Deora Arunkumar B. , Church Sarah , Cohen-Gould Leona , Rafii Shahin , Lyden David C. , Ling Qi
Published: April 2020

Malignant tumor progression requires the development of tumor-associated blood vessels through co-option of nearby host vascular cells, recruitment of marrow-derived progenitor cells, or both. The annexin 2 (A2)/p11 heterotetramer is a cell surface co-receptor for the fibrinolytic proteins plasminogen (Plg) and tPA, and strongly augments the catalytic efficiency of vascular cell plasmin generation. We showed previously that A2−/ − mice display impaired postnatal angiogenesis in the corneal pocket, oxygen-induced retinopathy, and Matrigel plug assays. Here, we examined neovascularization of experimental lymphoma in mice with fibrinolytic deficiency states. For both EL4, a T cell lymphoma, and B6RV2, a B cell lymphoma, growth of implanted tumor cells was rapid in wildtype and tPA−/ − mice, but nearly flat in both A2−/ − and Plg−/ − mice. Immunohistochemical staining of EL4 tissue on day 8 revealed reduced vascular density, frequent intravascular fibrin thrombi, and dilated microvessels in A2−/ −, compared with A2+/+, mice. Electron microscopy and immunofluorescence revealed a striking paucity of pericytes and secondary dropout of endothelial cells within A2−/ − tumor microvessels. Flow cytometric analysis of circulating progenitor cells showed reduced recruitment of VEGFR1+/CD11b+ hematopoietic precursors from bone marrow to blood in A2−/ − versus A2+/+ tumor-bearing mice. In lethally irradiated A2−/ − mice, moreover, tumor growth was rescued completely upon engraftment with A2+/+ marrow. Transplantation of green fluorescent protein (GFP)-labeled A2+/+ bone marrow revealed the appearance of abundant marrow-derived CD11b+/CD68+ cells closely apposed to developing tumor neovessels. Many of these cells also express the pericyte marker, NG2. Transplantation of A2+/+ marrow, finally, restored the investment of tumor microvessels with α-smooth muscle actin-positive pericytes. These data suggest that A2 contributes critically to tumor angiogenesis in experimental lymphoma in mice, by enabling recruitment of A2+ myelomonocytic cells that promote neovascular stabilization by pericyte-like cells. The data suggest further that targeting the A2/p11 system could offer a novel strategy for the treatment of malignant lymphoma.