bluuebunny/crossref_metadata_2025 huggingface dataset:
“Virtual Flow Cytometry” of Immunostained Lymphocytes on Microscopic Tissue Slides.
Authors: Cualing Hernani D. , Moscinski Lynn C.
Published: April 2020

A method and approach were developed for fully automated measurements of immunostained lymphocytes in tissue sections by means of digital color microscopy and patent pending advanced cell analysis. The validation data for population statistic measurements of immunostained lymphocytes in tissue sections using tissue cytometry is presented. Segmentation of a 512 x 474 RGB image and display of statistical results table took 12 to 15 seconds using in-house developed algorithms. We used a panel of 7 antibodies for validation on 14 cases of mantle cell lymphoma giving percentage positive, total lymphocytes, density staining. An average of 2027 image frames with 810,800 cell objects were evaluated. Antibodies to CD3, CD4, CD8, Bcl-1, Ki-67, CD20, CD5 were subjected to virtual flow cytometry on tissue. The results of tissue cytometry were compared with manual counts of expert observers and with the results of flow cytometry immunophenotyping of the same specimen. The correlation coefficient and 95 % confidence interval by linear regression analysis yielded a high concordance between manual human results (M), flow cytometry results(FC), and tissue cytometry (TC) results per antibody, (r =0.9365 manual vs TC, r =0.9537 FC vs TC). We noted a lower CD8 tumor infiltrating lymphocytes in blastic form than in non-blastic form of mantle cell and a higher Ki67 in the latter subtype than in the former. These results suggest the technology of tissue cytometry could be a clinically valid surrogate for both manual and flow cytometry analysis when only tissue immunohistochemistry is available for diagnosis and prognosis. The application for cancer diagnosis, monitoring, and prognosis is for objective counting of immunostained cells in tissues with percentage results. These results could be also be used in standardization of cut off percentage used in diagnosis; for exemplary tool for leukemic blasts(CD34) >20% for acute leukemia, for determining automated 30 % cut off for positivity for prognostic markers in diffuse large B-cell lymphoma for bcl-2, bcl-6, mum-1, and CD10.
Outcome and Early Prognostic Indicators in Patients with Hematological Disorders Admitted to the Intensive Care Unit for a Life-Threatening Complication.
Authors: Cherif Honar , Hansen Jan , Kalin Mats , Prof Magnus Bjorkholm
Published: April 2020

Background: Appropriately aggressive treatment of haematological malignancies can be complicated by a variety of life threatening events. Despite high hospital mortality rates for such patients admitted to intensive care units (ICU) it is now generally considered to be appropriate to offer intensive care to selected cases, provided there is a reasonable prospect of cure or at least worthwhile palliation.

Aims and Methods: We conducted a retrospective observational study to assess outcome and prognostic indicators in consecutive patients with hematological diseases admitted to the ICU during a 6-year-period.

Results: From 1996 through 2001, a total of 95 patients with hematological diseases and a median age of 57 years (range 16–86) were admitted to the ICU. The median duration of ICU stay was 1 day (mean 4.2 days: range 1–67 days). The Mean Acute Physiology and Chronic Health Evaluation II (APACHE II) score was 20 (± 9). The large majority of patients underwent active treatment of a hematological malignancy (90%) with acute leukaemia (27%), non-Hodgkin lymphoma (24%) and multiple myeloma (10%) dominating. Respiratory failure (46%), sepsis (24%), cardiovascular complications (9%) and bleeding disorders (7%) were the major reasons for ICU admission. A total of 49 patients (51%) had a microbiologically verified infection and 21 (22%) had bacteremia. Crude ICU, 4-week and 6-month mortality rates were 28%, 45%, and 57%, respectively. An APACHE II score > 30 predicted a high short-term mortality rate (p= 0.0001). However, age > 65 years, respiratory failure, bacteremia, and a diagnosis of acute leukemia were not significantly associated with a poor short-term survival (p> 0.05). A total of 30 patients (31%) were alive after a minimum follow up of 3.5 years.

Conclusion: The lower mortality rate as compared with most other series is probably explained by a more liberal attitude towards ICU admission. Not withstanding this, for a substantial proportion of critically ill hematological patients a short time care at an ICU is life saving. Patients with life threatening complications of haematological disease should be offered intensive care unless or until it is clear that there is no prospect of recovery from the acute illness or that the underlying malignancy cannot be controlled.
CLL/SLL Is More Common Than Registry Incidence Suggests: A Population-Based Study in Manitoba, Canada.
Authors: Seftel Matthew D. , Hewitt Donna , Zhang Hui , Turner Donna , Gibson Spencer , Morales Carmen , Johnston James B.
Published: April 2020

Background: The exact incidence of chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) is unknown. In the appropriate clinical setting, peripheral blood immunophenotyping is often sufficient for diagnosis. Cancer registries that rely only on histological or cytological reporting may inaccurately estimate the incidence of CLL/SLL. The province of Manitoba, with a population of 1.2 million people, has a centralized flow cytometry service as well as a provincial cancer registry. We thus had the opportunity to use these large databases to describe the demographic and clinical patterns of CLL/SLL. This has enabled us to test the hypothesis that registry data underestimates the incidence of this disease.

Methods: All patients diagnosed with CLL/SLL between January 1, 1998 and December 31, 2003 were obtained from the Manitoba cancer registry and the central flow cytometry database. Additional clinical characteristics were obtained from a chart review.

Results: 491 patients were diagnosed by flow cytometry. In contrast, cancer registry data reported 345 patients with CLL/SLL, 131 (38%) of which were diagnosed in tertiary care centres. Thus, 146 (30%) patients were not known to the provincial cancer registry. Median age of pts was 71 years (range, 24–97). Based on 2001 Canadian census data, the crude incidence of CLL/SLL in Manitoba is estimated to be 7 per 100 000 persons. Other demographic and clinical data of this population-based study will be presented.

Conclusion: By incorporating diagnostic immunophenotyping, the incidence of CLL/SLL appears to be higher than that reported by a large Canadian cancer registry. This observation may apply to other local and national jurisdictions, and should be studied further.
Age Significantly Influences Presentation and Patterns of Care in Patients with Newly Diagnosed Diffuse Large Cell Lymphoma (DLCL): Collective Data from 5 Centers Participating in the National Comprehensive Center Network (NCCN) NHL Outcomes Project.
Authors: Rodriguez Maria Alma , Czuczman Myron S. , Friedberg Jonathan W. , Kho Michelle E. , LaCasce Ann S. , Millenson Michael , Auayporn Nademanee , Niland Joyce , Veer Anna Ter , Zelenetz Andrew D. , Weeks Jane C.
Published: April 2020

Background: Older patients with DLCL are underrepresented in clinical trials, and little is known about the impact of age on therapeutic decisions outside the clinical trial setting. Treatment for older individuals can be influenced by comorbidities, possibly limiting the use of proven aggressive therapies. We investigated the effects of age on patterns of presentation and treatment for patients with DLCL.

Methods: Data from the 5 centers participating in the NCCN NHL Outcomes Project were used for analysis. Patients with newly diagnosed DLCL, receiving some or all of their care at the NCCN institutions, with first presention between 7/01/2000 to 6/30/2004 were included. Independent variables evaluated were stage, International Prognostic Index (IPI), Charlson comorbidity score, NCCN center, and age (≤ 60 and >60). Components of IPI were evaluated as separate independent variables. Fisher’s exact tests compared baseline characteristics between age groups. Univariate and multivariate logistic regression models were developed to identify factors associated with the receipt of anthracycline-based first-line therapy.

Results: Of 417 patients presenting with newly diagnosed DLCL, 376 were eligible for analysis. Older patients (>60 years) accounted for 38% (142/376) of our cohort. The median age was 55.6 years; among older patients, over half were over 70. Older patients were significantly more likely to have HI/H IPI scores (52% vs. 27%, p=0.0001) and more than 2 major comorbidities (29% vs. 8%, p=0.0001). Overall, 18% (66/376) participated in first-line clinical trials, with no difference in participation by age (p=0.49). Among the 310 patients treated off protocol, 93% (289) received an anthracycline-containing first-line regimen. Older patients were less likely to receive anthracyclines [87% (105/120) vs. 97% (184/190)]. Among patients receiving anthracyclines, older patients were more likely to receive growth factor support [78% (82/105) vs.54% (100/184)]. In multivariable logistic regression adjusting for stage, comorbidity, and center, age was the only factor statistically significantly associated with treatment choice, with patients over 60 less likely to receive an anthracycline-based first-line regimen (OR=0.29; 95% CI=0.11 to 0.80 p=0.02).

Conclusions: In a large cohort of patients with newly diagnosed DLCL, we found that older patients (>60 years) with DLCL had both higher risk disease, and more comorbidity at diagnosis. In our centers, there was no difference in clinical trail participation between older and younger patients; however, older patients were less likely to receive anthracyclines.
Autoantibodies Against the Fibrinolytic Receptor, Annexin 2, in Antiphospholipid Syndrome.
Authors: Cesarman Gabriela , Ríos-Luna Nina P. , Deora Arunkumar B. , del Carmen Cravioto Maria , Alarcón-Segovia Donato , Sánchez-Guerrero Jorge , Hajjar Katherine A.
Published: April 2020

The association of thrombosis, obstetric morbidity, and hemocytopenias with antiphospholipid antibodies is termed antiphospholipid syndrome. Annexin 2 is a profibrinolytic endothelial cell surface receptor that binds plasminogen, its tissue activator (tPA), and beta-2-glycoprotein-I, the main antigen for antiphospholipid antibodies. Here we evaluate annexin 2 as a target antigen in antiphospholipid syndrome. Serum samples from 434 individuals (206 patients with systemic lupus erythematosus without thrombosis, 62 with antiphospholipid syndrome, 21 with non-autoimmune thrombosis, and 145 healthy individuals) were analyzed by ELISA and immunoblotting for antiphospholipid and annexin 2 antibodies. IgG was purified and the effect of anti-annexin 2 antibodies on human umbilical vein endothelial cell activation and cell surface plasmin generation were evaluated. Anti-annexin 2 antibodies (titer >3SD) were significantly more prevalent in patients with antiphospholipid syndrome (22.6%, venous 17.5%, arterial 34.3% and mixed thrombosis 40.4%), than in healthy individuals (2.1%, p<0.001), patients with non-autoimmune thrombosis (0%, p=0.017) or patients with lupus without thrombosis (6.3%, p<0.001). Anti-annexin 2 antibodies enhanced the expression of tissue factor, a procoagulant protein, on endothelial cells (6.4 fold ± 0.13 SE), and blocked the fibrinolytic cofactor activity of purified placental annexin 2 in a tissue plasminogen activator-dependent plasmin generation assay (19 – 71%), independently of beta-2-glycoprotein-I. Similarly, cell surface plasmin generation on human umbilical vein endothelial cells was inhibited by 34–83%. We conclude that antibodies against the fibrinolytic receptor annexin 2 are significantly associated with thrombosis in antiphospholipid syndrome, and that anti-annexin 2 antibodies activate endothelial cells and inhibit endothelial cell surface-localized plasmin generation. We propose that these mechanisms contribute to the prothrombotic tendency in antiphospholipid syndrome.
Ss Polymorphisms Carried on GPB in Brazilian Malaria Infected and Non-Infected Individuals.
Authors: Castilho Lilian , Lustigman Sara , Reid Marion , Cavasini Carlos E. , Rossit Andrea , Couto Alvaro , Costa Fernando F. , Machado Ricardo D.
Published: April 2020

Background: The present study was carried out to compare the Ss blood group polymorphisms carried on GPB in patients infected by malaria (n=51–100) with blood donors (n=100–170) living under the same conditions in a malaria endemic region of the Brazilian Amazon (Macapá, Belém, Porto Velho and Rio Branco). The blood donors had no clinical signs for malaria, their thick blood film exam was negative and they reported during the interview that they had no prior episodes of malaria.

Methods: Blood donors and individuals having only P. falciparum infected were identified and their blood was used for Ss phenotyping in gel cards (DiaMed-AG) and to prepare DNA for Ss genotyping and associations studies. The Ss polymorphisms were genotyped by allele-specific (AS)-PCR.

Results: Comparison between the frequencies of the Ss phenotypes/genotypes in the two studied groups in each endemic area showed a significant correlation between the frequency of individual carrying the S+s+ or S+s- phenotypes/genotypes and incidence of malaria infection in three regions of the study; Rio Branco, Porto Velho and Belem. The presence of the S antigen was significantly more frequent in malaria patients when compared with the blood donors in three regions of the study; Rio Branco (60.8 vs. 38%), Porto Velho (53.1 vs. 38%) and Belem (62 vs. 50%). As a confirmation, we also found that the S-s+ phenotype in the three regions was contributing to resistance to infection with malaria, as it was significantly more frequent in the donor population than in infected individuals. Importantly, there are approximately 1.5 times more copies of GPB in S+s- than in S-s+ RBCs. S+s+ RBCs have an intermediate amount of GPB. When we combined the results from the four regions and analyzed the 64 P. falciparum (Pf) positive individuals vs. the total 63 blood donors, 46 P. falciparum positive individuals (71.8%) carried the S+ phenotype on the GPB whereas in normal donor population only 30 individuals (47.6%) carried the S antigen.. In this analysis, the presence of the S antigen was significantly more frequent in the P. falciparum patients when compared with the blood donors (P = 0.006). Conversely, the S-s+ phenotype in the four regions was contributing to resistance to P. falciparum infection, as it was significantly more frequent in the donor population (52.3%) than in infected individuals (28.1%).

Conclusion: These results support the hypothesis that the expression of the S antigen on GPB is associated not only with incidence of malaria infection, but more importantly, with incidence of P. falciparum infection. These preliminary results open up the opportunity to study the utilization of the GPB invasion pathway, in particular via a domain that contains the S antigen, by the P. falciparum field isolates of Brazil.
Marked Improvement in Detecting the Number of Involved Nodal Areas in Lymphoma, Using 18 F- FDG - PET and CT Scan.
Authors: Chalapud Juan , Sobrevilla-Calvo Pedro , Rivas-Vera Silvia , Altamirano-Levy Javier
Published: April 2020

Positron emission tomography (PET) imaging with 18-fluoro-2-deoxiglucose (FDG) is used increasingly for the initial evaluation and staging of patients with Hodgkin’s lymphoma (HL) and non- Hodgkin’s lymphoma (NHL). However, the degree of concordance of PET and TAC scanning for each nodal and extra nodal site are not well defined. The number of nodal areas involved is a new prognostic factor in follicular lymphomas as was demonstrated in the Follicular Lymphoma (FL) International prognostic index (FLIPI), and their use may be useful for the LNH and HL. In this study, we examined the performance of CT versus FDG-PET scanning, comparing each one of the nodal and extra nodal areas, as it is described in the FLIPI, in a retrospective cohort of lymphoma patients (pts) with HL and NHL. We reviewed the charts of 56 patients with diagnosis of HL and NHL in the initial and relapse staging, in a single tertiary care center. All patients had FDG-PET imaging study, clinical examination and CT scans. The Ann Arbor stage, each nodal site (cervical, mediastinal, axillary, mesenteric, para aortic, inguinal), and extra nodal sites were evaluated on the basis of FDG-PET scanning and were compared with the findings derived from CT. Bone marrow biopsy results were excluded from this initial analysis. The histopathological diagnoses included diffuse large B-cell Lymphomas in 20/56 pts (36%), HL 15/56 pts (27%), anaplastic large cell lymphoma 8/56 pts (14%), FL 5/56 pts (9%), peripheral T-cell Lymphoma 4/56 (7%) and others 7%. Among the 56 pts, 22 (39%) had discordant results between FDG-PET scanning and CT scanning, that lead to a change in stage assignment. Among the discordant cases FDG-PET resulted in upstaging in 18/56 pts (32%), and down staging in 4/56 pts (7%). Forty for pts (79%) had discordant results in the number of nodal areas, among the discordant cases FDG-PET detected more nodal areas in 36/56 pts (64%) and CT in 8/56 pts (14%). The discordant cases were distributed as it is shown in the table.

In conclusion Pet and CT in combination detects more involved nodal areas than each method by itself.

Summary of PET/CT correlation with nodal areas Nodal areas Cervical(n) Axillar (n) Mediastinal(n) Paraaortic(n) Inguinal (n) positive total 40 26 26 24 17 Only Positive FDG-PET 16 12 13 7 11 Only Positive CT 7 3 2 2 1 Positive FGD-PET + CT 17 11 11 15 5
Enhancing Neutrophil Differentiation - A Novel Role for the Death-Associated Protein Kinase 2 (DAPK2).
Authors: Rizzi Mattia , Britschgi Christian , Tschan Mario P. , Grob Tobias J. , Huegli Barbara , Leupin Nicolas , Mueller Beatrice U. , Ziemiecki Andrew , Torbett Bruce E. , Fey Martin F. , Tobler Andreas
Published: April 2020

DAPK2 is a 42-kDa Ca2+/Calmodulin-regulated serine/threonine kinase involved in apoptosis. In gene expression profiles derived from in vitro differentiated myeloid leukemic NB4 cells treated with all-trans retinoid acid (ATRA), we found that DAPK2 was decisively induced during differentiation towards neutrophils. DAPK1, a close relative of DAPK2, is inactivated in a number of hematopoietic malignancies (AML, lymphoma, myeloma), and it may play a role during normal and leukemic myeloid cell differentiation. We therefore investigated DAPK2 for its possible role in both normal and leukemic myelopoiesis. Real time quantitative RT-PCR (RQ-PCR) and Western blot analysis of DAPK2 gene expression in primary myeloid cells revealed significantly higher DAPK2 expression in granulocytes (G; n=9) compared with monocytes/macrophages (M; n=8) and CD34+ progenitor cells (CD34+; n=6) (Δ, p< 0.001; figure, left panel). Moreover, significantly increased DAPK2 mRNA levels were also seen when cord blood CD34+ progenitor cells were induced to differentiate towards neutrophils with human recombinant G-CSF (hrG-CSF). In addition, ATRA-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, showed significantly higher DAPK2 mRNA expression paralleled by DAPK2 protein induction. However, during differentiation of CD34+ cells (with hrM-CSF) and U937 cells (with PMA) towards monocytes/macrophages, DAPK2 mRNA levels remained low. DAPK2 expression in primary leukemic cells revealed significantly lower DAPK2 expression levels in AML blasts (AML; n=100) than in samples from chronic myeloid leukemia patients in chronic phase (CML-CP; n=9) (ΔΔ, p< 0.001; figure, right panel).

Figure Figure

Stable lentiviral-mediated expression of wild-type DAPK2 enhanced ATRA-induced granulocytic differentiation of NB4 cells as shown by morphology and by increased CD11b expression. Furthermore, upregulation of mRNA levels of key regulator genes for terminal differentiation, such as C/EBPe, the G-CSF receptor and the secondary granule protein lactoferrin, was also enhanced. Expression of a kinase-inactive DAPK2 mutant did not show these effects, a finding consistent with a role of DAPK2 in granulopoiesis.

Conclusion: we demonstrate for the first time, that DAPK2 expression levels correlate with the degree of granulocytic differentiation, and that DAPK2 upregulation is restricted to granulopoiesis. Furthermore lentiviral-mediated DAPK2 expression enhances granulocytic differentiation. The finding that DAPK2 expression is low in AML and high in CML-CP patients suggests that suppressed DAPK2 expression may contribute to the differentiation block in AML.
CD8+/TCR− Graft Facilitating Cells Enhance HSC Engraftment and Survival Via TNF-α Mediated Prevention of Apoptosis.
Authors: Ildstad Suzanne T. , Rezzoug Francine , Huang Yiming , Wysoczynski Marcin , Schanie Carrie L. , Ratajczak Mariusz Z. , Fugier-Vivier Isabelle J.
Published: April 2020

The use of accessory cells to enhance hematopoietic stem cell (HSC) engraftment could have a significant therapeutic impact, especially when stem cell numbers are limited. The bone marrow (BM) microenvironment is involved in regulation of HSC, allowing production of mature blood cells while maintaining HSC self renewal. To date, the precise identity of specific cells in the microenvironment that exert this regulatory effect on HSC has not been defined. We recently reported that CD8+/TCR− facilitating cells (FC), a subpopulation of BM cells containing predominantly B220+/CD11c+/CD11b− tolerogenic precursor-plasmacytoid dendritic cells, enhance HSC engraftment in allogeneic recipients. Additionally, FC significantly enhance engraftment of limiting numbers of HSC in syngeneic recipients. In the present studies, we investigated the mechanism of FC-mediated enhancement of HSC engraftment. We show for the first time that FC significantly increase HSC survival in vitro and exert an anti-apoptotic effect on HSC via TNF-α. Co-culture of FC with HSC induces production of physiologically relevant low levels of TNF-α by FC. FC from TNF-α−/− mice are impaired in function in vitro and in facilitating HSC engraftment in vivo. Furthermore, neutralization of TNF-α on FC using anti-TNF antibody results in loss of FC function in vitro, confirming a major role for TNF-α in FC function. Notably, co-culture of FC with HSC prevents HSC apoptosis and is associated with significant upregulation of the anti-apoptotic I-κB family member Bcl-3 in HSC. Blocking of TNF-α on FC abrogates the anti-apoptotic effect of FC on HSC and prevents upregulation of Bc1-3 in HSC. Taken together, these findings demonstrate that TNF-α-induced in FC affects highly primitive HSC and identify Bcl-3 as a possible pathway for TNF-α in regulating HSC survival.
Reaction of Fibrinogen with Homocysteine Thiolactone Mimics the Dysfibrinogenemia Produced by Hyperhomocysteinemia.
Authors: Sauls Derrick L. , Lockhart Evelyn , Hoffman Maureane
Published: April 2020

Elevated plasma homocysteine (HCys) is associated with atherosclerosis and thrombosis. We have previously found that clots from plasma, whole blood and purified fibrinogen from homocysteinemic rabbits are composed of thin, tightly-packed fibers and are resistant to fibrinolysis. HCys thiolactone is a metabolite of Hcys that can react with lysine residues under physiologic conditions. This reaction introduces a new free sulfhydryl group into the protein. The current study was designed to test the hypothesis that HCys thiolactone modifies key lysines in fibrinogen, thereby causing the alterations in fibrin structure and susceptibility to fibrinolysis that characterize the homocysteine-induced dysfibrinogenemia. We examined the effect of incubation of HCys thiolactone with purified human fibrinogen. The control and homocysteine thiolactone treated fibrinogen (HCys-fibrinogen) samples were added to fibrinogen-deficient plasma, which was then clotted by addition of thrombin and calcium in the presence of tissue plasminogen activator (tPA). Clots from HCys-fibrinogen lysed more slowly than control clots and the magnitude of this effect depended on the concentration of HCys thiolactone used. At 300 μM Hcys thiolactone, the fibrinogen formed clots with characteristics similar to fibrinogen from hyperhomocysteinemic rabbits. These clots 1) had thinner fibers than controls; 2) the reduced fiber thickness could be partially corrected by increasing calcium and 3) were more resistant to fibrinolysis than controls. In addition, the HCys-fibrinogen formed disulfide-stabilized high molecular weight forms, which are also found in the plasma of hyperhomocysteinemic rabbits. Mass spectrometric analysis of Hcys-fibrinogen revealed 12 different HCys-modified lysines - primarily in the D- and alpha-C-domains. Some of these are close to sites of tPA and plasminogen binding and/or plasmin cleavage. Two of them are very close to sites of naturally-occurring prothrombotic mutations that introduce a new cysteine into the alpha-C-domain. A new Cys at these sites mediates mixed disulfide formation with albumin in the plasma - a structural modification that is thought to underlie their prothrombotic effects. Similarly, we find that HCys-fibrinogen forms mixed disulfides with human albumin. Our data suggest that homocysteinylation of fibrinogen could lead to abnormal resistance of fibrin clots to lysis and thus might contribute to the increased risk of cardiovascular disease in hyperhomocysteinemia.
SALL4, a Novel Oncogene Induces Myelodysplastic Syndrome and Acute Myeloid Leukemia Via Wnt/β-Catenin Pathway.
Authors: Chai Li , Cui Wei , Chang Jianchang , Di Chunhui , Amin Hesham , Ma Yupo
Published: April 2020

SALL4, a gene homologous to the Drosophila homeotic spalt, is a zinc finger transcriptional factor essential for human development. In Drosophila, spalt is regulated by the Wnt signaling pathway, a pathway critical for hematopoietic self-renewal of stem cells. We cloned SALL4 and its isoforms (SALL4A and SALL4B). We used immunohistochemistry, to demonstrate that SALL4 was constitutively expressed in primary acute myeloid leukemia cells (French American British, FAB: M1 to M5, N=81). The SALL4 in RNA was quantitative in bone marrow cells derived from acute myeloid leukemia (AML) and compared to non-neoplastic hematopietic cells from a purified CD34 progenitor pool, normal bone marrow and peripheral blood by RT-PCR. SALL4 expression was present in the purified normal CD34+ cells, AML blasts, but absent in mature myeloid cells. We tested the leukemogenic potential of constitutive overexpression of SALL4 in a murine hematopoietic model. All transgenic mice overexpressing SALL4B using the CMV promoter developed hematopoietic disorders, including myelodysplastic (MDS)-like symptoms and an AML transformation that disseminated to peripheral tissues including the spleen, liver, kidney and lymph nodes. The myelodysplastic features in these transgenic mice were pathologically similar to human MDSs and manifested as ineffective myelopoiesis. Dysplatic features such as Pseudo-Pelger-Huet like atypical white cells, and increased immature cells were detectable in the transgenic mouse peripheral blood. A high level of apoptosis and increased immature cells were also evident in transgenic mouse marrows and day-7 colony-forming unit assays in vitro. Both SALL4A and SALL4B were able to bind to β-catenin in vitro and synergistically enhanced the Wnt/β-catenin signaling pathway in a reporter gene assay. Our data suggests that the constitutive expression of SALL4 is causal to the leukemic phenotype. Our model should provide a useful platform to analyze the interaction of SALL4 with Wnt/β-catenin pathway in leukemia stem cells.
Hematopoietic Stem Cells Actively Extrude Imatinib-Mesylate Due to High ABCG2-Transporter Activity.
Authors: Scharenberg Christian , Gessner Michaela , Hoffmann Joerg , Robey Robert , Neubauer Andreas , Brendel Cornelia
Published: April 2020

The tyrosine kinase inhibitor imatinib mesylate (STI571) is a potent therapeutic agent for treatment of chronic myeloid leukemia (CML) due to its specific inhibition of bcr-abl kinase. However, patients with CML always relapse after withdrawal of imatinib therapy. With CML being the paradigm for a stem cell disease, we sought to investigate the influence of imatinib on the most primitive stem cell compartment.

Imatinib has recently been reported to be a high affinity substrate for the ABC-transporters ABCG2/BCRP (breast cancer resistance protein) and MDR1 (P-Glycoprotein). Given the high expression of these ABC-transporters in Hematopoietic Stem Cells (HSC), we examined the influence of imatinib on ABCG2- and MDR1-activity in human and murine HSC by using Hoechst- and Rhodamine-efflux assays. MDR1-mediated Rhodamine-efflux was only mildly influenced by Imatinib. However, addition of imatinib at therapeutic dosages completely abrogated the SP phenotype of total bone marrow after Hoechst 33342 staining. This effect was even more pronounced in cells of the HSC-phenotype, i.e., lineage-negative/AC133+ or c-kit+/Sca-1+/lin− (KSL) of human and murine marrow, respectively. In order to determine the effect of imatinib on stem cells in vivo, we isolated SP cells from 13 CML patients at various stages of disease using FACS. We employed quantitative RT-PCR-analysis for bcr-abl and demonstrated that the majority of CML patients had bcr-abl-negative SP cells, while peripheral blood mononuclear cells were mostly bcr-abl-positive. However, two patients in complete cytogenetic and molecular-genetic remission proved to be bcr-abl-positive within the SP cell population. This supports the notion that continuous imatinib-therapy may not eradicate the malignant stem cell pool and the leukemic clone may be able to expand even during permanent imatinib-therapy.

Furthermore, these data imply that the detoxifying function of ABC transporters on stem cells may be altered under imatinib treatment. Using HEK-293 cells transfected with ABCG2, we demonstrate that imatinib reverses mitoxantrone-resistance - potentially due to high affinity substrate inhibition.

In summary, our data suggest the novel resistance mechanism of imatinib being actively extruded from primitive leukemic stem cells, and that addition of chemotherapeutic agents to imatinib therapy may facilitate the eradication of bcr-abl-positive stem cells in chronic myeloid leukemia.
The Role of RUNX1 DNA-Binding Mutations in Acute Myeloid Leukemia.
Authors: Cammenga Jorg , Putz Gabriela , Niebuhr Birte , Horn Stefan , Bergholz Ulla , Buchholz Frank , Stocking Carol
Published: April 2020

The RUNX1 gene encodes an alpha subunit of the core-binding factor (CBF), an important heterodimeric transcription factor in hematopoietic ontogeny and development, and is one of the most frequently disrupted genes in acute leukemia. In addition to its involvement in several translocations, the RUNX1 gene is often subject to deletions or point mutations in acute myelogenous leukemia (AML). Interestingly, in addition to complete loss-of-function mutations, many of the alterations involve missense point mutations within the Runt domain that disrupt DNA binding activity (DB-mutants). In vitro assays have suggested that these DB mutants have a dominant-negative (DN) activity, presumably due to their ability to bind and sequester CBF beta but inability to bind DNA. A strict correlation between the type of mutation and its monoallelic or biallelic incidence is not apparent even though a DN mutant should only affect one allele while a loss of function mutation should affect both alleles. It has been hypothesized that loss of one allele (haploinsufficiency) is sufficient for loss of tumor suppressor activity but the relative high incidence of specific DB mutations suggests a more complex scenario. We thus sought to determine if expression of DB mutants in murine bone marrow (BM) resulted in a similar phenotype as the loss of Runx1, or if these mutations are associated with a gain-of-function. Two RUNX1-DB mutants were thus evaluated using the established retroviral transduction/transplantation mouse model. Between 3 and 6 months after transplantation, peripheral blood, spleen and BM cells were analyzed. Long-term repopulating cells expressing RUNX1 DB-mutants were able to contribute normally to both myeloid and lymphoid compartments, although a disproportionate increase in the B-cell compartment was observed in 3 out of 10 mice. Surprisingly, and inconsistent with a DN activity, disruption of normal T-cell or megakaryocytic development was not observed in the mice, in contrast to Runx1−/+ mice. Significantly, however, replating assays in vitro demonstrated that RUNX1-DB mutants lead to a significant increase in self-renewal activity, in contrast to BM cells of floxed Runx1 mice expressing the Cre recombinase, which showed a less dramatic effect on self-renewal. Colonies derived from CFU-Cs expressing RUNX1-DB mutants were composed of dysplastic granulocytic and monocytic cells, with an increasing number of immature blasts after multiple replatings (>7), whereas residual colonies from Runx1fl/− BM receiving CRE showed a different morphology with more mature cells. Thus our data suggest that RUNX1-DB mutants do not act in a dominant negative fashion to inhibit normal RUNX1 function, but impart a gain-of-function that results in impaired myeloid differentiation and increased self-renewal potential, consistent with its association with AML.
Dominant and Pharmacologically Sensitized ENU Mutagenesis Screens Uncover Novel Regulators of Hematopoiesis and Model Hematopoietic Disease.
Authors: Stanford William L. , Anderson Nicole M. , Milenkovic Zorana , Zitano Lia , Adamson Lee , Wang Chen , Cybulsky Myron , McNagny Kelly , Paulson Robert F. , Barber Dwayne L. , Minden Mark D.
Published: April 2020

To generate new mouse models of human hematopoietic disease and increase our knowledge of the genetic networks that control hematopoiesis, we are performing dominant (generation 1 or G1) and pharmacologically-sensitized forward ethylnitrososurea (ENU) mutagenesis screens. Mice are phenotyped by saphenous vein peripheral blood analysis using an automated hematological analyzer. ENU is ideally suited to generating models of human disease and annotating gene function because the spectrum of mutations (point mutations generating leading to subtle amino acid substitutions, splicing errors, or premature termination) are similar to those often found in human disease. Furthermore, null mutations often do not represent the full extent of a gene’s function, requiring multiple alleles to fully define gene function. While dominant mutations can unequivocally cause some human diseases, often mutations in multiple genes interact and contribute to disease progression. Thus, we have developed sensitized screens that induce transient cytopenias using various pharmacological agents (5-fluorouracil, phenylhydrazine, and hydroxyurea) and analyzing the recovery in peripheral blood levels of red blood cells, white blood cells and platelets. This strategy enables identification of hematopoietic mutants that do not present abnormal blood cell counts in a homeostatic state. The induced cytopenia recovery assay is also being used as a secondary phenotyping assay for some of our G1 dominant mutants. The combined dominant and sensitized screens have yielded 14 heritable dominant mutants to date plus four additional mutants in hereditary testing. The array of mutations that we are analyzing are models for the following diseases: polycythemia, thrombocythemia, leukocytosis, anemia, and thrombocytopenia. I will discuss the progress of the mutagenesis screen and several ENU mutants, including a novel mutation in the protein tyrosine kinase Jak2, leading to thrombocythemia. This point mutation in the protein kinase domain will help us to dissect the recently discovered role of Jak2 in Myeloproliferative Diseases including Essential Thrombocythemia.
Differentiation of Human Embryonic Stem Cells into Mesenchymal Stem Cells.
Authors: Olivier Emmanuel N. , Rybicki Anne C. , Bouhassira Eric E.
Published: April 2020

Mesenchymal stem cells (MSC) are multipotent progenitors that contribute to the formation of many connective tissues including fat, bone, cartilage and muscle. Because of this great versatility MSCs have a large therapeutic potential particularly in the areas of cell therapy and regenerative and reconstructive medicine. MSCs are rare cells that can be isolated from tissues such as bone marrow, cartilage and muscles. Since no unique marker characteristic of MSCs has been identified, investigators have relied on a series of functional and morphological criteria to identify them. These criteria include growth on plastic, resistance to trypsin, presence of specific cell surface antigens and potential to differentiate into adipocytes, chondrocytes and osteoblasts.We report here a method to reproducibly differentiate human embryonic stem cells (hESCs) into MSCs by pre-differentiating for 8 days hESCs growing on MEF, mechanically dissociating the differentiated colonies, replating the differentiated colonies in DMEM, 10% FBS and 7.5% CO2 for 4 to 8 weeks until a thick multi-layer epithelium-like sheet of cells develop; and dissociating these multi-layer structures with a combination of proteases. The cells obtained with this procedure are morphologically similar to MSCs, are contact-inhibited, can be grown in culture for about 20–25 passages and have a gene expression profile, as determined by cDNA micro-array, similar to published profiles of mesenchymal stem cells. Immuno-phenotyping of these hESC-derived MSCs revealed a immuno-profile similar to bone marrow MSCs (negative for CD34, CD45 and positive for CD13, CD44, CD71, CD73, CD105, CD166, HLA ABC, SSEA4 and TRA1-85). Functional differentiation experiments revealed that hESC-derived MSCs can be differentiated into osteocytes and adipocytes as demonstrated by staining with alizarin red and oil red O. Finally, we have also shown that hESC-derived MSCs can support the growth of undifferentiated hESCs cells and of CD34+ hematopoietic cells.The ability to produce MSCs from hESCs should prove useful to produce large amount of genetically identical and genetically modifiable MSCS that can be used to study the biology of MSCs and for therapeutic applications.
Up-Regulation of NK Cell Activating Receptors Associated with Elevated IL-15 Levels Post-Transplant.
Authors: Boyiadzis Michael , Carson Jesse , Allen Kenton , Memon Sarfraz A. , Dean Robert A. , Odom Jeanne , Bishop Michael R. , Gress Ronald E. , Hakim Frances T.
Published: April 2020

NK cells express diverse activating and inhibitory receptors, which collectively determine the NK cytotoxic response. Because NK cells can be potent anti-tumor effectors in the post transplant period of minimal residual disease, we investigated the receptor expression on NK cells following non-myeloablative, HLA-matched, allogeneic hematopoietic stem cell transplant (HSCT). We focused on activating receptors, comparing the expression of natural cytotoxicity receptors (NCR) and C-type lectin receptors on circulating NK cells at one, three, six and twelve months following HSCT with that of the donor-derived mobilized stem cell apheresis. During the first post-transplant month, the absolute numbers of NK cells recovered to normal levels in the 14 patients studied, but the subpopulation of CD56++(bright) CD16− NK cells increased disproportionately compared to the more common CD56+(dull) CD16+ NK cells. By six months the proportions of the NK cell subsets normalized to pre-transplant levels. At one month post transplant, the median percentage of NK cells expressing the anti-tumor NCR NKp46 increased from 15 to 64% and that expressing NKp30 increased from 23 to 40% as compared to the donor’s NK cells; expression remained elevated during the first year. Expression of NKG2D, the homodimeric activating C-type lectin receptor, similarly increased post-transplant. CD94 was also upregulated on NK cells, but the activating heterodimeric partner of CD94, NKG2C, did not change significantly. To investigate these shifts in NK populations and receptor expression, we investigated the effect of cytokine milieu post transplant on these shifts in NK populations and receptor expression. We have determined that plasma levels of IL-15, a cytokine critical in NK differentiation and expansion, increase more than 50-fold in these HSCT patients from pretreatment to the day of transplant and decline in the first weeks post transplant, inversely proportional to NK recovery. When NK cells were cultured with rIL-15, we observed a disproportionate expansion of CD56++ NK cells and a rapid up-regulation of the NCR and NKG2D receptors, similar to the changes observed post-transplant. These data suggest that the cytokine milieu of transplant, in particular elevated levels of IL-15, may contribute to anti-tumor efficacy post transplant by affecting the recovery of NK subsets and modulating expression of activating receptors.
High Avidity Cyclin E-Derived Peptide-Specific CTL Contribute to Induction of Remission after Stem Cell Transplantation without Associated Graft-Versus-Host Disease.
Authors: Ishiyama Ken , Kondo Yukio , Wieder Eric , Lu Sijie , Molldrem Jeffrey
Published: April 2020

Cyclin E1 (CCNE1) and cyclin E2 (CCNE2) are cell cycle genes that are overexpressed in AML, ALL, and CML, and in solid tumors such as breast cancer, lung cancer, and gastric cancer. We reported that two homologous nonameric peptides from CCNE1 (CCNE1144-152, ILLDWLMEV) and CCNE2 (CCNE2144-152, ILLDWLLEV), which differ by a single amino acid at position 7, have equal binding affinity for HLA-A2 and that CCNE1- and CCNE2- specific CTL elicited from healthy donors kill ALL and CML cells that overexpress CCNE1 and CCNE2. Interestingly, CCNE1/A2 and CCNE2/A2 tetramers bind to both T-cell receptors (TCR), but the longer tetramer dissociation t1/2 of CCNE1/A2 compared with CCNE2/A2 tetramer suggests CCNE1 is the more dominant epitope. To determine the clinical significance of CTL immunity to these self-epitopes, we studied peripheral blood mononuclear cells from 18 patients before and after allogeneic stem cell transplantation (SCT) using CCNE1/A2 and CCNE2/A2 tetramers. An increase in the absolute number of circulating CCNE1- or CCNE2-CTL after SCT was considered evidence of an immune response (IR). Of the18 patients, there were 10 with CML and 8 with ALL, and all were HLA-A2+ and had sufficient blood samples cryopreserved from various time points before SCT and 3–12 months after SCT for analysis. Ten patients, including all 8 ALL patients, were in complete remission (CR) prior to SCT and 5 patients achieved CR after SCT. An IR to CCNE1 was found in 12 of 18 (67%) patients and an IR to CCNE2 was found in 14 of 18 (78%) patients. All 12 patients who had an IR to CCNE1 also had an IR to CCNE2, reflecting possible cross-recognition of both peptides by the same CTL clones. By Chi-square analysis, IR to either CCNE1 or CCNE2 did not correlate with diagnosis (CML vs. ALL), source of the graft (matched related donors vs. mismatched donors), or disease status prior to SCT (remission vs. no remission). Six patients developed acute graft-versus-host disease (aGVHD) and 12 developed chronic graft-versus-host disease (cGVHD). IR to either CCNE1 or CCNE2 occurred more frequently in patients without aGVHD than in the patients with aGVHD (92% vs. 50%, p<0.05), although no significant difference in the IR frequency was observed amongst those who developed cGVHD versus those that did not. Of 8 CML patients who were not in CR prior to SCT, IR to either CCNE1 or CCNE2 occurred more frequently in patients that achieved CR compared to those that did not achieve CR after SCT (100% vs. 33%, respectively; p<0.04). These results provide clinical evidence that both CCNE1 and CCNE2 peptides are novel leukemia-associated antigens, and they justify future clinical trials to determine whether CTL immunity can be enhanced against these antigens in patients with ALL and CML.
Impact of Recovery of CD4 and CD8 Lymphocyte Subsets on Acute GVHD after Reduced Intensity Conditioning (RIC) Allogeneic Stem Cell Transplantation (allo-SCT).
Authors: Mohty Mohamad , Gaugler Beatrice , Faucher Catherine , Sainty Danielle , Arnoulet Christine , Gastaut Jean-Albert , Viens Patrice , Olive Daniel , Blaise Didier
Published: April 2020

RIC regimens for allo-CST are being explored with good results concerning feasibility and engraftment. However, little is known about the immune recovery pattern in these patients, especially the different CD4 and CD8 lymphoid T cell subsets. Here, we assessed at different time points after allo-SCT, the kinetic of recovery of naïve (CD45RA+/CD27+), central memory (CD45RA−/CD27+), and terminally differentiated (CD45RA+/CD27−) CD4+ and CD8+ T lymphocytes in 64 patients from a single center, receiving HLA-identical RIC allo-SCT. Patients and graft characteristics are: age 48 y (27–63), diagnoses: 22 myeloid malignancies (34%), 22 lymphoid malignancies (34%) and 20 metastatic solid tumors (31%). 51 pts (80%) were considered as high risk. 49 pts (77%) received a fludarabine, busulfan and ATG-based RIC, while 15 pts (23%) received a low dose irradiation-based RIC. 91% of patients received a PBSC graft, with 42 (66%) receiving CSA alone for GVHD prophylaxis and 22 (34%) receiving CSA and MMF. 24 pts (38%) developed grade 2–4 acute GVHD at a median of 49 d (26–85). In this series, in contrast to CD4+ T cell subsets, CD8+ T cell subsets had a progressive and sustained recovery in the first 3 months after allo-SCT, with acquisition of functional markers such as 2B4 and perforin. Among the different subsets analyzed, the recovery of naïve CD4+ T cells, and central memory CD4+ and CD8+ T cells, measured at day 28 after allo-SCT and before onset of grade 2–4 acute GVHD, showed a significant correlation with the risk of grade 2–4 acute GVHD (P=0.001; P=0.002 and P=0.05 respectively). Patients developing grade 2–4 acute GVHD recovered a median of 47 naïve CD4+ T cells/μL prior to onset of GVHD as compared to 11 cells/μL in patients with grade 0–1 acute GVHD (Figure). Naïve CD4+ T cell levels significantly decreased after appropriate acute GVHD treatment. In a Cox multivariate analysis taking into account all relevant risk factors for acute GVHD, early recovery of naïve CD4+/CD45RA+/CD27+ T cells in the first month after allo-SCT was the strongest parameter significantly predictive of grade 2–4 acute GVHD development (P=0.006; RR=4.0; 95%CI, 1.5–11.0). Interestingly, there was a significant correlation between the total number of CD4+ T cells infused with the allogeneic graft and the early recovery of naïve CD4+ T cells (P=0.001), suggesting that graft manipulation might represent an attractive tool towards harnessing alloreactivity after RIC-allo-SCT.

Figure Figure