bluuebunny/crossref_metadata_2025 huggingface dataset:
Short-Term Foamyviral Transduction with Virions Encoding Recombinant Corrects the Mitomycin C Hypersensitivity of Fancc −/− Progenitor Cells In Vitro and Restores Long Term Repopulating Activity of Fancc −/− Stem Cells In Vivo.
Authors: Si Yue , Leurs Cordula , Srour Edward , Ciccone Samantha , Hanenberg Helmut , Clapp D. Wade
Published: April 2020

Fanconi anemia (FA) is a complex autosomal recessive genetic disorder characterized within the hematological system by progressive bone marrow aplasia, a high propensity to develop acute myeloid leukemia, and hypersensitivity to alkylating agents including mitomycin c. The identification of individual FA genes raises the potential of using gene transfer technology to express/introduce the functional cDNA in/into deficient autologous stem cells. We have previously shown that in the absence of genetic correction with a retroviral mediated Fancc transgene, ex vivo culture of Fancc−/− stem/progenitor cells (HSPC) predisposes uncorrected Fancc−/− HSPC cells to clonal hematopoiesis (Haneline, Blood 2003). Therefore we examined the potential of a helper-free human foamy virus (HFV) derived construct that encodes both the human FANCC and EGFP transgenes to transduce murine Fancc−/− HSC in the absence of prestimulation. In initial experiments, we determined that 40–80% of progenitors were transduced following a single overnight HFV infection using a 20:1 moiety of infection. Subsequent studies demonstrated that HFV efficiently transduced primitive hematopoietic progenitors in G0 and G1 phases of the cell cycle as evidenced both by using multicolor fluorescence activated cell sorting and subsequent culture of sorted cell populations in high proliferating potential (HPP-CFC) and low proliferating potential colony forming assays. Aliquots of HFV transduced cells that were transduced with the construct encoding both Fancc and EGFP, or the reporter transgene only were transplanted into irradiated recipient mice. Four months following transplantation, bone marrow cells were isolated from the reconstituted recipients and clonogenic assays were established in a range of mitomycin c (MMC) concentrations. In these experiments, the MMC hypersensitivity of Fancc−/− progenitors was corrected to wild-type levels. To assess quantitatively the potential of HFV expressed FANCC to correct stem cell repopulating ability, we next utilized the competitive repopulating assay. In two replicate experiments, we determined that the repopulating activity of HFV-transduced Fancc−/− stem cells was comparable to wildtype controls six months following transplantation in primary and secondary recipients. Collectively, these data provide in vivo evidence that the HFV vector is an efficient vehicle for introducing a functional hFANCC transgene into quiescent Fancc−/− HSC in the absence of prestimulation and for complementing the cellular FA defect in vitro and in vivo.
Augmentation of Therapeutic Tumor Vaccine Efficacy by Genetic Engineering of CD4+ T Cell Help for Tumor Vaccine-Reactive CD8+ T Cells.
Authors: Jalla Sanju , McCadden Erin , Wang Jie , Fuchs Ephraim J. , Whartenby Katharine A.
Published: April 2020

Since CD4+ T cell help has been proposed to be required for maintaining the activity of tumor-specific CD8+ T cells, tolerance in tumor-specific CD4+ T cells may seriously impair the efficacy of therapeutic tumor vaccines. To overcome this problem, we devised a strategy to “engineer” CD4+ T cell help by treating tumor-bearing animals with nonmyeloablative conditioning and transplantation of autologous hematopoietic stem cells (HSCs) that have been genetically modified, via lentiviral transduction, to express an antigen containing “foreign” CD4+ T cell epitopes. After hematopoietic reconstitution, animals received the combination of an autologous tumor cell vaccine and an infusion of primed CD4+ T cells specific for the expressed epitopes. Using influenza hemagglutinin (HA) as the model antigen, we first confirmed that transplantation of HA-transduced HSCs led to efficient expression of HA by antigen-presenting cells, as demonstrated by the clonal expansion of adoptively transferred, HA-specific CD4+ transgenic T cells in mice receiving HA-transduced HSCs but not in mice receiving nerve growth factor receptor (NGFR) gene-transduced HSCs. Next, BALB/c mice harboring 13 day old, metastatic 4T1 mammary cancer were treated with removal of the primary, nonmyeloablative conditioning and transplantation of HA-transduced syngeneic HSCs, and following hematopoietic reconstitution, with concomitant autologous tumor cell vaccination and adoptive transfer of in vitro activated, HA-specific transgenic CD4+ T cells. This therapy was successful in curing the majority of tumor bearing mice, and was superior to the same therapy given to mice transplanted with NGFR-transduced stem cells. Finally, we found that the anti-tumor effect of vaccination plus exogenous T cell help was abolished by the adoptive transfer of either CD4+ or CD8+ T cells from tumor-bearing mice, suggesting that tumor-bearing mice contain both potential effectors and suppressors of anti-tumor immunity, the latter of which are abolished by the non-myeloablative conditioning. These results highlight the importance of CD4+ T cell help in the induction of therapeutic anti-tumor immunity.
Possible Involvement of Histone Acetylation in Differentiation of Erythroid Precursors and Proliferation of Granulocytic Precursors.
Authors: Fujieda Atsushi , Katayama Naoyuki , Ohishi Kohshi , Masuya Masahiro , Shiku Hiroshi
Published: April 2020

The commitment and differentiation of hematopoietic progenitors are thought to be associated with the restriction of transcriptional accessibility for genes of unselected cell-lineage. Histone acetylation-mediated remodeling of chromatin structures is known to regulate transcriptional activity of genes, and the acetylation level of histones is controlled by the balance between opposing activities of histone acetyltransferases and histone deacetylases (HDACs). In this study, we investigated the role of HDAC activity in the proliferation and differentiation of erythroid and granulocytic precursors, using a HDAC inhibitor FK228 [depsipeptide]. CD36+ erythroid precursors were generated from cord blood CD34+ cells with SCF, flt-3 ligand, TPO, IL-3, IL-6 [5GF], and EPO. These precursors expressed low or undetectable level of glycophorin A (GPA) but mostly expressed CD45. When these precursors were cultured with EPO alone in the presence of FK228 (0.4~0.5 ng/ml), the resulting cells expressed GPA at a low level in comparison with the cells cultured without FK228, and a large population of the GPA+ cells remained to be CD45+. On the other hand, most of cells in cultures without FK228 expressed a high level of GPA and a majority of the GPA+ cells were negative for CD45. These data suggest that FK228 represses the EPO-dependent differentiation of erythroid precursors.

The retardation of differentiation due to FK228 was, however, completely recovered when cells exposed to FK228 were incubated for additional 5 days without FK228. At these concentrations, FK228 did not affect the proliferation of erythroid precursors. Granulocytic precursors were also prepared from CD34+ cells with 5GF and G-CSF. In culture with G-CSF, FK228 did not affect the differentiation of granulocytic precursors but did inhibit their proliferation in a dose-dependent fashion. Histone acetylation induced by FK228 in precursors was confirmed using anti-acetylated histone antibodies. These data demonstrate a distinct and regulatory role for HDAC activity in the proliferation and differentiation of hematopoietic precursors.
High Frequency Gain-of-Function Mutation and/or Over-Expression of Proto-Oncogene C-KIT as Major Contributor to the Leukemogenesis in Human Acute Myeloid Leukemia with t(8; 21).
Authors: Zhou Guang-Biao , Wang Yue-Ying , Yin Tong , Bai Xue-Tao , Liang Wen-Xue , Xu Feng , Liu Yi-Wei , Chen Zhu , Chen Sai-Juan
Published: April 2020

To explore the genetic abnormalities that cooperate with AML1-ETO (AE) fusion gene in the pathogenesis of acute myeloid leukemia (AML) with t(8; 21), we undertook to screen for a number of candidate genes and identified a high frequency mutation of proto-oncogene C-KIT (25/53 cases, 47%). This event seems to be specific to t(8;21) leukemia since no mutation was detected in 57 patients with other types of leukemia. Totally eleven types of mutations were characterized and 6 ones including 3 point mutations in the tyrosine kinase (TK) domain and three types of internal tandem duplication (ITD) in the juxtamembrane (JM) or extracellular (EC) domains were discovered for the first time. Bioinformatics analysis revealed all these mutations might favor the activation of the C-KIT, as proven by TK activity assay. Patients with a mutant C-KIT (mC-KIT) allele had a higher white blood cell count and a poorer prognosis than those bearing wild-type C-KIT. The expression of C-KIT at both mRNA and protein levels was higher in t(8;21) leukemia patients than most other leukemia types. In addition, the TK inhibitor Gleevec suppressed the C-KIT activity, inhibited the proliferation and induced apoptosis of t(8;21) leukemic cells bearing C-KIT mutation or over-expression. Our study thus provides strong evidence to the model of leukemogenesis which requires a cooperation of cytosolic signaling and transcription factor for a full-blown leukemia phenotype to display.
Efficacy and Toxicity of FLAI-G-CSF and Mylotarg for Induction/Consolidation of AML Patients, Not Treatable with Conventional Chemotherapy.
Authors: Malagola Michele , Rondoni Michela , Russo Domenico , Piccaluga Pier Paolo , Paolini Stefania , Bosi Costanza , Gaitani Stavroula , Visani Giuseppe , Martinelli Giovanni , Baccarani Michele
Published: April 2020

Acute myeloid leukaemias (AMLs) usually share a high frequency and a high degree of Pgp-mediated multidrug resistance (MDR), one of the major causes of treatment failure. Patients older than 60 years, as well as patients with secondary AML and patients with relapsed/refractory disease do poorly with conventional chemotherapy. Gemtuzumab ozogamicin (Mylotarg) has shown some in vitro activity against CD33 positive AML blasts. As single agent it induces complete remission (CR) in almost 20 – 30% of cases with mild toxicity. Combinations with chemotherapy look promising. Moreover, several studies proved in vivo efficacy of fludarabine in combinations with cytarabine as anti MDR drug. Fifteen AML patients were treated with an induction regimen including fludarabine (25 mg/sqm/day for 3 days), cytarabine (1 g/sqm/day for 3 days), idarubicine (5 μg/sqm/day for 3 days), mylotarg (3 mg/sqm day +4) and G-CSF (5 mg/day starting on day +9, until hematological recovery). An identical consolidation cycle was administered if at least partial remission (PR) was achieved. Two patients were relapsed after standard induction chemotherapy. Median age at diagnosis was 66 (range 33 – 75). Median white blood cells count was 5.3 x 109/L (range 0.5 – 47.4). Seven out of 15 patients presented with at least 1 adverse karyotype abnormality. Eight patients presented with a secondary AML. Ten out of 15 patients responded to induction cycle (8 CR and 2 PR). The following data regard the induction cycle. The median time to recovery of neutrophils > 1.0 x 109/L was 20 days (range17 - not reached) The median time to recovery of platelets > 20 x 109/L and > 100 x 109/L was 21 days (range 13 - not reached) and 34 (range 19 - not reached) respectively. Two patients developed a grade II WHO FUO and 5 patients developed a grade II-III Gram+ bacteremia. Two patients had radiologically documented fungal infection (lung) and 1 patient had microbiologically documented aspergillosis of maxillary sinus. Non-hematological toxicity was mild. Only one patient developed a grade III WHO hemorragic syndrome (melena). No veno occlusive disease were observed.

Up to now 4 patients completed two cycles of chemotherapy. Three patients out of 15 died (1 progressive disease, 1 multi organ failure after allogeneic bone marrow transplantation, 1 myocardial stroke while in CR) and 7 patients out of 15 are in CR. In conclusion, these preliminary results suggest that FLAI-G-CSF-Mylotarg, as induction course, is an effective regimen with a limited hematological and non-haematologic toxicity and seems to be effective in this high risk subset of patients. A longer follow up and larger series of patients are indeed necessary to draw further conclusions.
Fluorescence In Situ Hybridization (FISH) for the Detection of the (8;14) Translocation in Archival Fine Needle Aspirates of Suspected Burkitt Lymphomas (BL).
Authors: Neto Lara F. , Mendonça Evelina , Cabeçadas José , Mendonça Andreia , Silva Maria G.
Published: April 2020

The detection of the chromosomal translocations involving the myc gene, characteristic of BL, is important in the diagnostic workup of agressive lymphomas given its impact on treatment strategies and prognosis. Recently this has been accomplished using FISH techniques either in paraffin-embedded tissue sections or fresh tumor aspirates. BL has an elevated cellular turn-over and a rapid diagnosis is highly desirable; this, and its high incidence in pediatric populations in whom organ biopsy needs general anesthesia, makes fine needle aspiration (FNA) samples attractive for diagnosis. In some cases, confirmation of the diagnosis may require the analysis of archival pre-stained samples. However, only recent samples have been reported as suitable for FISH analysis. We analysed 18 cytological samples (8 cytospins and 10 smears) obtained by FNA from 16 male and two female patients, median age 7.5 (3 to 48) years old, diagnosed with BL (15), atypical BL (1) or high grade lymphoma with a difficult differential diagnosis with BL (3) between February 2000 and July 2004 on the basis of morphology and phenotype of cytological and/or histological samples. In all cases the presentation and further clinical course suggested the possibility of BL and treatment was administered accordingly. Sixteen smears were May-Grunwald-Giemsa (MGG) stained whereas two were fresh-frozen; the samples had been stored for variable periods (1 day to 53 months). Cytology specimens (6 MGG-stained and 6 frozen) obtained from 12 reactive lymph nodes were used as controls, to determine the cut-off value for positive hybridization signals. After removal of the coverslips with xylene, the slides were destained in a series of water and ethanol washes. Pretreatment with pepsin was followed by the hybridization procedure. The dual fusion t(8;14) fluorescent probe was purchased from Vysis. Eleven out of 18 (61%) samples could be hybridized using this technique. The 7 cases without an interpretable signal were between 9 and 53 months old. The cut-off value for t(8;14) was 0%, meaning that in 12 negative controls the pattern of two fusion signals with or without a remaining signal from der(8) chromosome was never observed. In aggreament to previous publications 8 out of 11 specimens (72%) were positive for the (8;14) translocation, including 3 cases in which a definitive BL diagnosis could not be made by cytology/histology alone. The median percentage of positive cells was 80% (46 and 92%). Although the quality and age of the smears influenced the results, and better signals were observed with frozen specimens, FISH analysis of archival cytopathological material stored for prolonged periods was frequently possible and allowed the diagnosis of 3 difficult cases, whereas it confirmed the morphological results in another 5 patients.
PKC beta II Expression in Lymphoid Malignancies.
Authors: Decouvelaere Anne Valerie , Morschhauser Franck , Copin Marie-Christine , Dumontet Charles M.
Published: April 2020

Protein Kinase C (PKC) is an important enzyme family. Twelve different isozymes have been described, which are implicated in diverse cell responses. Recently, studies have begun to define isoform-specific functions of PKC, and PKC beta II seems to play an antiapoptotic role. PKC beta II expression was analyzed by immunohistochemistry in 119 tumor samples of patients with lymphoid malignancies. The median age of the group was 55 years (range 14–85 y). Immunohistochemical staining was performed on formalin-fixed paraffin-embedded tissue, using the labeled avidin-biotin immuno-peroxidase complex method on the Ventana Benchmark automated immunostainer (Ventana Medical Systems, S.A., Illkirch, France). The sections were incubated with primary antibody (polyclonal antibody cPKC ßII (C-1): sc-210, Santa Cruz Biotechnology, Inc.) for 32 min at 1/100 dilution. The intensity of cytoplasmic and membrane immunostaining was assessed semi-quantitatively by two pathologists independently. Cases were considered “negative” when <10% of cells were stained, and “positive” when ≥10% of tumor cells showed cytoplasmic or membrane staining. Diffuse or heterogeneous staining was also specified.

Intense and diffuse staining was observed in all mantle cell lymphomas (13 cases, 100%), and chronic lymphocytic lymphomas (CCL) (6 cases, 100%). PKC beta II was positive too in most follicular lymphomas (10 cases out of 11, 91%), although the pattern and the intensity of the staining were variable in accordance with the degree of lymphomatous infiltration. Indeed, in the same lymph node, the follicular lymphomatous areas were constantly labelled, whereas residual germinal centers remained negative. A majority of angioimmunoblastic T-cell lymphomas, lymphoblastic T-cell lymphomas and Marginal Zone/MALT lymphomas were labelled with anti-PKC beta II antibody, but the pattern of expression was more heterogeneous in these subtypes. A minority of diffuse large B-cell lymphomas expressed PKC beta II (8 out of 22 cases, including 4 with weak staining). Nearly all plasma cell neoplasms were negative. Nevertheless, 2 cases out of 16 (13%) displayed a diffuse moderate staining. In peripheral T-cell lymphomas, staining for PKC beta II was less frequent, as well as in primary cutaneous B and T lymphomas and pseudo-lymphomas. None of the cases of Hodgkin’s disease (13 cases) and anaplastic large cell lymphoma (5 cases) expressed PKC beta II. However, most of the numerous reactive cells were strongly stained.

In conclusion, the intensity and the staining pattern of anti-PKC beta II antibody varied greatly according to the type lymphoid malignancy. These results highlight a significant correlation between PKC beta II expression and some particular type of lymphomas. In our series, the highest level of expression was found in mantle cell lymphomas and CLL.
Subset of T-Cell Prolymphocytic Leukemia Expresses CD117. Potential Target for Therapy.
Authors: Gorczyca Wojciech , Dong Henry Y.
Published: April 2020

Introduction

Excluding non-hematopoietic lesions, CD117 (c-kit) is expressed by erythroid, megakaryocytic and myeloid precursors, mature mast cells, subset of plasma cell neoplasm and occasional precursor lymphoblastic tumors. C-kit plays important role in T-cell development around birth, but the expression of CD117 in mature T-cell lymphoproliferations is rare. We present flow cytometry phenotypic data of 28 cases of T-cell prolymphocytic leukemia (T-PLL).

Material and methods

Multiparameter 4-color FC analysis was performed on peripheral blood and/or bone marrow aspirate samples using the following antibodies: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD13, CD14, CD16, CD19, CD20, CD25, CD33, CD34, CD38, CD45, CD56, CD64, CD79a, CD117, TCRab/γδ, TdT, and HLA-DR. Fresh bone marrow aspirates and/or peripheral blood films and cytospin preparations from FC samples were stained with Wright-Giemsa.

Results

T-PLL was diagnosed 28 cases. The ages ranged from 43 to 92 years. Normal expression of pan-T cell antigens (CD2, CD3, CD5, CD7) was present in 57%. Abnormal expression of one, two and three antigens was noted in 21%, 11% and 11%, respectively. 4 cases showed aberrant expression of CD117. Those cases were always CD8-positive (50% of T-PLL were CD4+). There were no pan-myeloid (CD13, CD33) and blastic (TdT, CD34) markers present. Molecular studies confirmed clonal rearrangement of TCR.

Discussion

T-PLL is rare mature T-cell neoplasm characterized by marked lymphocytosis of small to medium-sized cells with conspicuous nucleoli. The response to conventional chemotherapy in T-PLL, similarly to other mature T-cell lymphoproliferations (except ALK+ anaplastic large cell lymphoma) generally is poor. While the use of alemtuzumab (Campath-1H) has improved remissions, the disease remains incurable. Novel treatment approaches, tailored to individual patients, and based on pathologic, phenotypic and molecular features will be crucial to improve survival for the patients with this disease.

Based on the flow cytometry analysis of 28 cases of T-PLL we identified 4 cases with co-expression of CD117 (14%). When present, it is restricted to CD8+ tumors. We speculate that the expression of CD117 (c-kit) in these patients apart from its diagnostic utility, may serve as a criterion for a combination therapy with Campath-1H and imatinib mesylate.
Serum Interleukin-18 Levels Are Associated with Response to Treatment and Survival in Patients with Aggressive Non-Hodgkin’s Lymphoma.
Authors: Goto Naoe , Tsurumi Hisashi , Takemura Masao , Hara Takeshi , Sawada Michio , Yamada Toshiki , Kasahara Senji , Kanemura Nobuhiro , Takahashi Takeshi , Seishima Mitsuru , Takami Tsuyoshi , Saito Koshiro , Moriwaki Hisataka
Published: April 2020

Interleukin-18 (IL-18), originally designated as an interferon (IFN)-gamma inducing factor, is a cytokine which induces cytotoxic NK cell activity and stimulates T cells to produce IFN-gamma, IL-2, and GM-CSF. Increased serum IL-18 levels have been found in patients with acute lymphoblastic leukemia, chronic myelocytic leukemia, multiple myeloma and non-Hodgkin’s lymphoma (NHL). The aim of the present study was to assess the prognostic significance of serum IL-18 in aggressive NHL. Consecutive 99 previously untreated patients with aggressive NHL (diffuse large B-cell lymphoma; 84 patients, peripheral T-cell lymphoma; 15 patients) prospectively participated in this study between 1997 and 2003. The patients were treated with 6–8 cycles of CHOP or THP (pirarubicin) -COP regimens. To evaluate serum levels of IL-18, venous blood samples were drawn from patients immediately before the initiation of treatment. In all patients with aggressive NHL, the mean ± SD of serum IL-18 level was 1200.8±151.5 pg/ml (range 96–6603.5) with a median of 556.75 pg/ml. Several poor prognostic features such as poor PS, multiple extranodal sites, advanced disease (clinical stage III/IV), existence of B-symptom, and high soluble interleukin-2 receptor (sIL-2R) were strongly associated with a high serum IL-18 levels. An increased LDH and gender were weakly associated with a high serum IL-18. Histology and age were not associated with serum IL-18 levels. The median serum IL-18 levels of the different IPI risk groups were; 321 pg/ml for the L risk; 442 pg/ml for the LI risk; 557 pg/ml for the HI risk; 1532 pg/ml for the H risk, respectively (P<0.005). The CR rate of patients with an IL-18 level of less than 700 pg/ml was better than that of 700 pg/ml or higher (77% and 50%, P<0.01). Patients with high IL-18 (700 pg/ml and over) at onset had a significantly lower survival rate (5-year: 25%) than those with low IL-18 (below 700 pg/ml) (68 %) ( p<0.0001). Multivariate analysis employing IL-18 and some conventional prognostic factors demonstrated that IL-18, performance status and the number of extranodal sites were significantly poor prognostic factors for both overall survival (OS) and event free survival (EFS). The results suggest that a high serum IL-18 level predicts a poor prognosis in aggressive NHL and may be a useful biomarker for selecting appropriate treatment.
Cellular Immunodeficiency in Untreated Lymphoma Patients.
Authors: Jentsch-Ullrich Kathleen , Gerber Annegret , Reppin Regina , Koenigsmann Michael , Mohren Martin , Franke Astrid
Published: April 2020

The association of lymphoma with a plethora of secondary immune defects is well established. However, most investigations were performed many years ago and little is known about disease specific alterations of the immune system. The availability of new diagnostic methods and a new classification of hematologic diseases (WHO 2000) makes a newly designed study of the immuno-dysfunction in this patient group worthwhile. From January 2003 to August 2004 50 patients with newly diagnosed malignant hematologic diseases - diffuse large cell lymphoma (DLCL) (n = 15), acute myeloid leukemia (AML) (n = 11), mantle cell lymphoma (n = 6), follicular lymphoma (FL) (n = 5), Hodgkin’s disease (HD) (n = 8) and chronic lymphocytic leukemia (CLL) (n = 5) - were analyzed. Parameters of cellular and humoral immunity, including CBC, FACS analysis of lymphocyte subpopulations, cytokines sIL2-R, IL-6, IL-10, TNF-a, serum complement components C3, C4, CH50 and total concentrations of serum immunoglobulin IgG, IgM, IgA were measured. At diagnosis patients with AML had severe cellular immunodeficiency (neutro-, monocyte- and lymphopenia). Infectious complications occurred in 8 of 11 AML patients (73%). Patients with DLCL and MCL had predominantly increased CD3+/CD8+ lymphocyte subsets (median 0,26/m l) with an infection rate from 30%. However, the rate was significantly higher if CD4+ depletion (43%) or CD19+ depletion (40%) were additionally present. High IL-2, IL-6, IL-10 and TNF-a levels were mostly observed in FL and MCL. Elevated TNF-a levels were also associated with infections (43%) and advanced stage or B-symptoms. IL-10 was found to correlate with mortality and relapse risk in all patients (overall: 18% and 20% respectively, increased IL 10 level: 39% and 33% respectively). Serum complement levels were normal to slightly elevated in all patients. Very high levels (CH50 > 160) were found in patients with Hodgkin’s disease (5/8 patients, 63%) and decreased immunoglobulin levels occurred in CLL patients only, none of whom had infections during follow up. These results indicate that untreated patients with DLCL and MCL have a significant lower CD8+T-lymphocyte cell count. In contrast to previous studies we did not found a predominant depletion of CD4+T-lymphocyte. A combination of CD4+/CD8+ T-cell deficiency or T-/B-cell deficiency was strongly associated with infections, thus antibiotic prophylaxis seems warranted in this patient group. Additionally IL-10 may serve as prognostic marker particularly in MCL and FL.
Detecting Cryptic Neoplastic B-Cells: Flow Cytometric Analysis of B-Cell Lymphoma Concealed in Polyclonal B-Cells and Other Intense Reactive Components, with Emphasis on Large B-Cell Lymphoma in Fine Needle Aspirate (FNA)/ Body Fluid (BF).
Authors: Wang Endi , Etzell Joan E.
Published: April 2020

Flow cytometry is widely used in the diagnosis of lymphoma. In B-cell lymphoma, light chain restriction (LCR) is especially useful for distinguishing B-cell malignancy from a reactive lymphoid proliferation. Usually the neoplastic B-cell population predominates, and monoclonality is conspicuous as demonstrated by distinct LCR. However, if the specimen contains a large reactive component, especially polyclonal B-cells, a small number of neoplastic B-cells will be buried in the background, thus creating a diagnostic challenge. We retrospectively analyzed three/four-color flow cytometry by examining 12 cases of B-cell lymphoma with a small proportion of neoplastic B-cells hidden in polyclonal B-cells and other intense reactive components. These 12 cases (9 FNA, 1 BF, 2 tissues) comprised 7% of specimens diagnostic or suspicious for lymphoma by flow cytometry at our institution. Eight cases were diffuse large B-cell lymphoma (DLBCL) (18.6% of DLBCL and 27.6% of DLBCL in FNA/BF) while four were lymphoma of other types (3% of non-DLBCL). In all the cases, LCR was obscured by polyclonal B-cells (8/12) or absence of surface immunoglobulin (sIg) in neoplastic B-cells (4/12), and morphology resembled a reactive picture. The clues by flow cytometry prompting further analysis included increased forward angle light scatter(FSC)/side angle light scatter(SSC) (12/12), brighter CD20 (6/12), dim CD20 (1/12), absence of sIg (4/12), and a distinct population of B-cells co-expressing CD10 (2/12), CD5 (1/12), CD2 (1/12), CD23 (4/12) or CD38 (1/12). By backgating/regating suspected subpopulations, the concealed neoplastic B-cells were demonstrated by enriched LCR (5 to10 fold) or tight clustering on FSC/SSC. In conclusion, cryptic neoplastic B-cells, especially diffuse large B-cell lymphoma in FNA/BF, can be extracted, according to their altered immunophenotypic features, from background polyclonal B-cells and reactive T-cells via manipulation of gating strategies.
First-Line Treatment with R-Chop/21 for Patients with Disseminated Diffuse Large B-Cell Lymphoma (DLCL). Results of a Prospective Series of 128 Patients.
Authors: Gonzalez-Barca E. , Domingo-Domenech E. , Ribera J. M. , Briones J. , Salar A. , Gallur L. , Estany C. , Escoda L. , Gardella S. , Hernandez J. A. , Rodriguez J. , Ramila E. , Ramon O. , Garcia-Pinto M. , Español I. , Vivancos P. , Asensio A. , Encuentra M. , de Sevilla A. Fernandez
Published: April 2020

Purpose: to analize the response rate and survival of a series of patients with disseminated large B-cell lymphoma treated with R-CHOP/21 x 8 cycles and to evaluate the prognostic factors and the prognostic value of gallium-67 imaging.

Patients and Methods: 128 patients with DLCL have been prospectively treated with R-CHOP/21 from March 2002 to March 2004, ages between 18 and 85 years. Survival curves were expressed as Kaplan-Meier plots. Prognostic factors were evaluated by the Chi-square test. Survival curves were compared by the Log-rank test.

Results: the median age was 65 years (limits: 27–83), 31% older than 70 years. 59 (54%) patients were males. Gallium imaging was positive at diagnosis in 91%. Clinical presentation of DLCL was: bulky disease: 40%, extra-nodal disease: 66% (³ 2 sites: 33%), B symptoms: 41%, ECOG ³ 2: 53%, LDH>N: 67%, IPI ³ 3: 60%. Chemotherapy cycles received were: <6: 20%; 6: 34%; 8: 46%. The intention-to-treat response was: CR 72%, PR 11%, failures 11%, not evaluable: 6%. Survival, with a median follow-up of 13 months, was: OS: 74%, and EFS: 70%. There are no differences between patients who have been treated with 6 or 8 cycles. EFS of patients with normal gallium imaging at the end of treatment was 90% (p=0.03). EFS was not different between patients older or younger than 70 years. EFS of patients with age adjusted IPI 0 was 100%, IPI 1:70%; IPI 3: 82% and IPI 4: 48%. Three (5%) patients relapsed. Prognostic factors for OS and EFS were: LDH > Normal (p=0.01 y p=0.005 respectively) and ECOG ³ 2 (p=0.0008 y p=0.02).

Conclusion: Rituximab associated with CHOP improves the results of historical series treated with CHOP for patients of all ages, but in patients with age-adjusted IPI=3 results have to be improved with other therapeutic strategies. Normalization of gallium-67 imaging at the end of treatment is predictive of a significantly better EFS. Age is not a prognostic factor in patients who receive the same treatment. For patients with disseminated disease, LDH and ECOG are the only significant prognostic factors.
Decrease of Bone Marrow Fibrosis in Hairy Cell Leukemia (HCL) after 2-CdA Therapy.
Authors: Zak Pavel , Dedic Karel
Published: April 2020

Different degree of bone marrow fibrosis is commonly described in patients with HCL. Hairy cells produce and assemble an insoluble matrix of fibronectin, which is responsible for the bone marrow fibrosis. Production of fibronectin is related to activity of the disease. The aim of our study was to evaluate the change of bone marrow fibrosis before and after 2-CdA therapy.

Patients and methods: 18 patients, who were treated with 2-CdA and achieved complete remission in 6 months, were included in this analyses. Trephin bone marrow biopsy were obtained before therapy and after therapy in 12 and 24 moths. The 3μm thick section were stained with Gomori reticulin stain. Grading of reticulin fibrosis (scale of 0 to IV): grade 0 - reticulin fibers (RF) absent, grade I - scattered RF or foci of fine reticular network (RN), grade II - diffuse fine RN, grade III - diffuse fine RN and scattered thick fibers, grade IV - diffuse thick reticular fibers and presence of collagen fibers.

Results: Before 2-CdA therapy: reticulin fibrosis was grade 0 in 0 p., I in 3p., II in 4p, III in 11p., IV in 0p.. Decrease of reticulin fibrosis in 12 months after 2-CdA was demonstrated in 51% of patients (grade 0 in 1, I in 8p., II in 5p., III in 4p., IV in 0p.), in 24 moths 77% of patients (grade 0 in 2, I in 10p., II in 4p., III in 2p., IV in 0p). Bone marrow fibrosis was increased by one grade in 3p. in 24 months after therapy.

Conclusion: Successful 2-CdA therapy in patient with 2-CdA leads to significant reduction of bone marrow fibrosis.
Poor Survival in ATL Patients Treated with CHOP Chemotherapy.
Authors: Shapira Iuliana , Feldman Joseph , Solomon William B.
Published: April 2020

The survival rate of Adult T-cell leukemia-lymphoma (ATL) patients, especially those who develop the acute leukemic or lymphomas forms, is very poor, and such clonal malignant CD4 expansion remains one of the most severe lymphoproliferations. We conducted a retrospective analysis of 31 consecutive patients diagnosed with HTLV-1 adult T-cell lymphoma-leukemia (ATL), treated at a single institution between 1995–2004. We used analysis of variance examining the mean survival of the patients. The patients were stratified according to the treatment conventional CHOP chemotherapy versus non-CHOP chemotherapy (CVP, no therapy or AZT and interferon). Twenty-two patients have received CHOP chemotherapy and 9 patients have not received CHOP chemotherapy. The mean survival for the entire group of patients was 22.7 weeks. The median survival was 16 weeks (range from 1–74 weeks). Most of the patients were at high IPI risk. (22 patients IPI=4, 8 patients IPI 5 and one patient IPI 3). If the patients presented without hypercalcemia their mean survival was significantly higher than the patients with hypercalcemia, 22.7 weeks versus 16 weeks p<0.05.

The treatment with CHOP chemotherapy significantly improved survival mean 29.3+4.6 weeks versus 8.7 +2.5 weeks, p=0.01. The treatment with CHOP consistently improved survival in patients without hypercalcemia and in those with hypercalcemia 38.8+ 5.4 weeks versus 5.5+ 12.1 and in those with hypercalcemia 20.7+5.2 weeks versus 9.5+6 weeks. CHOP chemotherapy helped both groups of patients those who presented with hypercalcemia and those without hypercalcemia although there is a suggestion that it is less effective in patients with hypercalcemia. In this small retrospective study, no significant association was found between survival and presence or absence of pleural effusion, ascites, extensive liver or skin involvement. Hypercalcemia should be considered a poor prognostic factor in ATL patients.

Prospective multi-institution clinical trials are crucial to determine the most effective treatment regimens that will continue to improve survival rates in this aggressive disease.
Granulocyte Colony Stimulating Factor Preferentially Causes Proliferation of Pre-Existing Monosomy 7 Cells in Aplastic Anemia and Myelodysplastic Syndrome Because of Abnormal Truncated GCSF-Receptors on These Cells.
Authors: Sloand Elaine M. , Ramkissoon Shakti , Mainwaring Lori , Barrett John , Young Neal S.
Published: April 2020

Administration of granulocyte colony stimulating factor (GCSF) has been linked to development of monosomy 7 in some patients with aplastic anemia (AA) and congenital neutropenia. We assessed the effect of GCSF on monosomy 7 to determine if this chromosomal abnormality developed de novo or if GCSF simply favored expansion of a pre-existing clone. Bone marrow mononuclear cells (BMMNC) were co-cultured for 14 days with pharmacological doses of GCSF and examined by fluorescent in situ hybridization (FISH). No karyotypically healthy control, AA, or myelodysplastic syndrome (MDS) bone marrow showed development of monosomy 7. In BMMNC cultures from thirteen patients with MDS and monosomy 7, all developed substantial increases in numbers of monosomy 7 cells after prolonged exposure to GCSF. Experiments on stored AA samples obtained six months prior to the development of abnormal karyotype showed monosomy 7 on FISH ,with expansion of that clone on co-culture with GCSF. GCSFR mRNA was increased in BM from monosomy 7 patients as measured by real time PCR, and monosomy 7 cells expressed more GCSFR on the cell surface than did normal cells. As a truncated GCSFR isoform is associated with a hyperproliferative response to G-CSF and prolonged activation of signal transducer and activator of transcription (STAT) complexes, we sequenced the GCSFR DNA of monosomy 7 cells and measured the expression of the truncated GCSFR mRNA relative to full length GCSFR in patients with monosomy 7. While genomic GCSFR DNA showed no abnormalities, mRNA demonstrated increased proportions of the truncated isoform IV in all six patients tested. We examined GCSF-mediated GCSFR signal transduction of the Jak/Stat system in monosomy 7 CD34 cells. STAT-1 was increased and the STAT 5: STAT 3 rato was increased by ten-fold in all patients with monosomy 7 compared to normals and to MDS with normal cytogenetics. In conclusion, pharmacologic doses of GCSF appear to increase the proportion of monosomy 7 cells; this heightened sensitivity to GCSFmay be related to altered amounts of the truncated form of the receptor, with changes in GCSF signal transduction resulting in expansion of the monosomy 7 clone.
A Multi-Center, Open-Label, Non-Randomized Phase II Study of Elsamitrucin (SPI-28090) in Patients with Relapsed or Refractory Non-Hodgkin’s Lymphoma (NHL).
Authors: Mohrbacher Ann , Sanani Shamel , Irwin David , Espina Byron M. , Madrid Catherine , Lenaz Luigi , Rosales Rosamerlinda
Published: April 2020

Background: Elsamitrucin (SPI-28090) is a novel actinomycete fermentation product that induces single strand breaks in DNA and inhibits topoisomerase I and II. A previous Phase II trial showed evidence of promising anti-tumor activity with an acceptable toxicity profile in patients with relapsed/refractory NHL. The objectives of the current trial are to confirm and validate these earlier findings and to determine if elsamitrucin is efficacious in a particular pathologic NHL subtype(s).

Methods: Patients with relapsed or refractory NHL, indolent or aggressive, excluding HIV and Burkitt’s lymphoma were eligible for the study. Patients must have had measurable disease. All patients were given weekly Elsamitrucin 25 mg/m2 i.v. administered over 5–10 minutes x 8 weeks. All patients were treated until disease progression or toxicity. Treatment was given as an outpatient.

Results: To date, 8 patients (5 males/3 females) have been enrolled on study with a median age of 70 years (range 40–83). Pathologies include diffuse large B-cell (4), diffuse mixed large B-cell (1); mantle cell (1), follicular grade 2 (1) and aggressive T-cell lymphoma (1). All patients had stage III or IV disease. The median number of prior chemotherapy regimens received was 5 (range of 2–9). A median of 4 doses (range 2–11) of Elsamitrucin have been administered to date. Myelosuppression was absent or mild in majority of the patients except for one patients that had grade 4 ANC and grade 3 thrombocytopenia. All non-hematologic toxicities were mild to moderate in severity, limited to nausea and vomiting. There were no reported liver function toxicities which was the dose limiting toxicity noted on prior Phase I trials. No drug related serious adverse events have been reported. Five patients are evaluable for response; 3 are too early to evaluate. One patient with stage IV mantle cell lymphoma who had received five prior chemotherapy regimens and radiation therapy has had a confirmed partial response after eight weeks of treatment, lasting for 8+ weeks. Another patient had unconfirmed partial response. One patient had mixed response and the other 2 had progressive disease.

Conclusions: Elsamitrucin preliminarily appears to be safe and well tolerated in this patient population. There has been some early evidence of anti-tumor activity. Accrual is ongoing and further update will be presented at the meeting.
SDX-105 (Treanda™), Active in Non-Hodgkin’s Lymphoma Cells, Induces the Mitotic Catastrophe Death Pathway.
Authors: Leoni Lorenzo M. , Niemeyer Christina , Bendall Heather , Reifert Jack , Bailey Brandi
Published: April 2020

SDX-105 (Treanda™) is an alkylating agent with a distinct mechanism of action that has been shown to be active in clinical trials in NHL and CLL patients refractory to traditional DNA-damaging agents. SDX-105 induces unique changes in gene expression in NHL cells and displays a lack of cross resistance with other 2-chloroethylamine alkylating agents. Quantitative PCR analysis confirmed that the G2/M checkpoint regulators Polo-like kinase 1 (PLK-1) and Aurora A kinase (AurkA) are down-regulated in the NHL cell line SU-DHL-1 after 8 hours of exposure to clinically relevant concentrations of SDX-105. No changes in these same genes were observed when cells were exposed to equi-toxic doses of chlorambucil or an active metabolite of cyclophosphamide. Because our previous studies demonstrated that SDX-105 treatment can activate apoptotic cell death pathways, we examined the ability of SDX-105 to induce cytotoxicity in cells unable to undergo ‘classical’ caspase-mediated apoptosis. Multi-drug resistant MCF-7/ADR cells and p53 deficient RKO-E6 colon adenocarcinoma cells were exposed for two or three days to either 50 μM SDX-105 alone or 50 μM SDX-105 and 20 μM pan-caspase inhibitor zVAD-fmk. Although zVAD-fmk was able to inhibit SDX-105 induced increases in Annexin-V-positive cells, microscopic analysis of nuclear morphology using the DNA stain DAPI in cells treated with either SDX-105 alone or in combination with zVAD-fmk showed increased incidence of micronucleation. Multi/micro-nucleation and abnormal chromatin condensation are both hallmarks of mitotic catastrophe and have been observed in tumor cells exposed to microtubule-binding drugs such as the vinca alkaloids and taxanes. Activation of mitotic catastrophe may amplify the cytotoxicity of SDX-105 and its activity in tumor cells where classical apoptotic pathways are inhibited. This may explain, at least in part, the potent anti-tumor activity of SDX-105 in tumor cells refractory to conventional 2-chloroethylamine DNA-damaging agents. Additional studies are ongoing to further elaborate the role of mitotic catastrophe in SDX-105’s mechanism of action. The capacity to induce mitotic catastrophe may explain the anti-tumor activity of SDX-105 in chemotherapy relapsed and resistant patients.
Predicting the Risk of Neutropenic Complications and Reduced Dose Intensity in Patients with Malignant Lymphoma: Results from a Prospective Study.
Authors: Dale David C. , Crawford Jeffrey , Wolff Debra A. , Poniewierski Marek S. , Culakova Eva , Lyman Gary H. ,   for the ANC Study Group
Published: April 2020

Background: Chemotherapy-induced neutropenia is frequently associated with neutropenic complications resulting in reduced relative dose intensity (RDI) potentially compromising clinical outcomes. Retrospective analyses have identified several factors but are limited by variable reporting and missing data.

Methods: Results from an ongoing prospective study of cancer chemotherapy patients based at more than 100 randomly selected US centers are reported. Demographic, clinical and treatment-related variables were obtained on 345 patients with malignant lymphoma (ML). Data are presented on the first 265 patients completing at least one cycle of chemotherapy. Planned and unplanned reductions in relative dose intensity (RDI) were estimated along with the risk of neutropenia and febrile neutropenia (FN) over the first four cycles of treatment are presented.

Results: Patients with Hodgkin’s disease (HD; N=43) received five different regimens including ABVD (84%) while patients with Non-Hodgkin’s lymphoma (NHL; N=222) received 33 different regimens the most common of which was CHOP (53%). Neutrophil nadirs <1000 were reported in 54% of subjects, two-thirds of which occurred during the 1st cycle of treatment. One or more episode of FN occurred in 18% (29% HD; 16% NHL) with 60% in cycle 1. Overall average RDI was 90% with 25% receiving <85%. Differences in delivered RDI were found based on practice setting (P=.07), geographic location (P=.007), age (P=.007), ECOG performance status (P=.065) and peripheral vascular disease (P=.021). Significant reductions in planned RDI but not unplanned RDI were observed for increasing age (P=.008). Over the first three cycles of treatment, patients with HD experienced a greater risk of FN (29%) than patients with NHL (16%) (P=.042). Correspondingly, HD patients received an average RDI of 93% with two-thirds of reductions unplanned compared to NHL patients who received an RDI of 90% with two-thirds were planned reductions (P=.03). In multivariate logistic regression analysis, significant predictors of reduced RDI were age, chemotherapy regimen, peripheral vascular disease and cerebrovascular disease (P<.001; R2=.435).

Conclusions: This prospective study confirms previous retrospective surveys that showed a substantial proportion of patients with ML receive reduced RDI potentially compromising clinical outcomes. Predictive models based on the risk factors identified should enable clinicians to target supportive care efforts in a more cost-effective manner.
A New Mechanism of Communication between Stem Cells Involving Vertical Transfer of mRNA by Its Intracellular Delivery within Membrane-Derived Microvesicles.
Authors: Ratajczak Janina , Miekus Kasia , Kucia Magda , Dvorak Petr , Ratajczak Mariusz
Published: April 2020

Membrane-derived microvesicles (MV) are spherical membrane fragments that are released from eukaryoctic cells upon their activation. We reported that MV transfer cell membrane-derived receptors between cells and/or directly stimulate target cells by the ligands expressed on their surface (Blood2001:98;3143; AIDS2003:17;33). Recently we observed that embryonic stem cells (ES) shed MV into culture media and that co-culture of ES with hematopoietic stem cells enhances the latter’s expansion. Similarly, co-culture of ES with somatic cells induces their dedifferentiation. Although the mechanisms responsible for these phenomena are not clear, we hypothesized that ES-derived MV (ESMV) could play an important role. To address this we isolated ESMV from murine (ES-D3) and two human (CCTL-12 and CCTL-14) embryonic cell lines and focused on molecules that may be responsible for epigenetic changes of cells co-cultured with ESMV. We found that ESMV as compared to the ES cells from which they originated are highly enriched in mRNA. This increase in mRNA content suggested a segregation mechanism that enriches ESMV in cytoplasmatic mRNA during their shedding from ES. Using real-time RT-PCR we found that ES-MV are highly enriched (x 103−107) in mRNA for early transcription factors that regulate self-renewal of stem cells (e.g., Oct-4, Gata-4, Rex-1 and Nanog). Intrigued by these observations we hypothesized that ESMV could penetrate the cells and deliver ES-derived mRNA and that this could be a novel mechanism for reprogramming target cells. Supporting our hypothesis we found (i) by confocal microscopy that ESMV do indeed penetrate the cells (e.g., BM-derived CD34+ cells or ES themselves), and (ii) by Western blot analysis that mRNA delivered to the target cells by ESMV is not trapped in the endosomal compartment but is delivered to the cytoplasm and actively transcribed into appropriate proteins (e.g., Oct-4). Based on these data we postulate that MV may transfer mRNA between the stem cells and play a role in vertical transfer of genetic information. Our recent similar data on MV derived from other cell types (normal and malignant) lend further support to this novel hypothesis and mechanism of cell to cell signaling/communication.