bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Studies of Bortezomib with Daunorubicin and Cytarabine:Sequence of Administration Affects Leukemia Cell Chemosensitivity.
Authors: Attar Eyal C. , Learner Emily , Amrein Philip C.
Published: April 2020

Induction chemotherapy with cytarabine and an anthracycline has been a standard treatment of newly diagnosed AML for the past 20 years. While the complete remission rate is high, only 20–40% of patients have prolonged leukemia-free survival and thus novel treatments are required. Bortezomib represents a first-in class proteasome inhibitor with activity in a variety of hematologic malignancies. Recent evidence has demonstrated synergistic cytotoxicity between the proteasome inhibitor, MG-132, and the anthracycline, idarubicin, in primitive leukemia cells.(Guzman et al. Proc Natl Acad Sci U S A 99 2002 16220-5) In order to determine if adding bortezomib to the conventional antileukemic therapies daunorubicin and cytarabine enhances cellular cytotoxicity, we conducted in vitro studies on the acute myeloid leukemia cell line, KG-1. Proliferation assays demonstrated that single-agent daunorubicin or single-agent cytarabine impaired proliferation to a greater extent than when either agent was simultaneously combined with bortezomib. Reasoning that treatment with bortezomib resulted in cell cycle arrest and protection from cycle-dependent agents, we treated cells with bortezomib and observed an accumulation of cells in the G2/M phase of the cell cycle, supporting our hypothesis. We then sought to determine whether sequential administration of bortezomib and chemotherapy affected leukemia cell proliferation. Cells were treated with bortezomib either 24 hours before or after idarubicin or cytarabine. Surprisingly, pretreatment of cells with bortezomib followed by chemotherapy resulted in increased proliferation relative to chemotherapy alone while initial treatment of cells with chemotherapy followed by bortezomib resulted in enhanced cytotoxicity. In conclusion, these data indicate that in in vitro leukemia cell cultures the timing of administration of bortezomib relative to conventional anti-leukemic agents critically affects cytotoxicity.
Signaling Mechanisms of Anti-Proliferation by Sphingosine 1-Phoshate(S1P) on Acute Promyelocytic Leukemia(APL) Cell Lines.
Authors: Mun Yeung-Chul , Hwang Eui-Soo , Lee Kyoung-Eun , Kwon Jung Mi , Nam Seung-Hyun , Yoo Eun Sun , Bae Yun-Kyung , Lee Seung-Eun , Ahn Jee-Young , Hyun Myung Soo , Kwon Ki Young , Lee Soon Nam , Seong Chu-Myong
Published: April 2020

There is abundant evidence that S1P can function as a second messenger important for regulation of calcium homeostasis, cell growth, and suppression of apoptosis. In many cases, the intracellular level of S1P and ceramide, another important sphingolipid metabolite associated with cell death and cell growth arrest, coordinately determine cell fate. N, N-dimethylsphingosine(DMS) potentiates TNF- and FasL- induced apoptosis in the human acute leukemia jurkat, U937 and HL-60 cell line, which suggests that combining DMS with cytotoxic drugs might be a useful chemotherapeutic approach. In this study, we investigated the role of S1P on proliferation, survival and its signaling mechanisms on the NB4 cell, the human acute promyelocytic leukemia cell line, which has chromosomal abnormality, t(15:17). NB4 cells were exposed to S1P at various concentrations (1, 5, 10 μM) for 48 hours, and then the cell growth was determined by MTT assay. We studied whether NB4 cells have S1P receptors or not. To investigate the signaling mechanisms of S1P on the NB4 cell, we used MAP kinase inhibitors and a PI3K inhibitor including PD98059, U0126 (specific ERK pathway inhibitor), SB203580 (specific p38 pathway inhibitor), and LY294002 (specific PI3K pathway inhibitor). We also examined the effect of pertussis toxin (PTX), a Gi protein inhibitor, on the proliferation of NB4 cells after S1P treatment.

RT-PCR analysis revealed a putative S1P receptor, Edg-3 mRNA expressed in NB4 cells. In the MTT assay, S1P inhibited cell proliferation in a dose-dependent manner. At 10 μM, NB4 cell proliferation was most inhibited. In addition, we found that the inhibitory effect of S1P on the NB4 cell proliferation was inhibited by PD98059, U0126, and PTX. However, SB203580 and LY294002 had not caused an inhibitory effect of S1P on the NB4 cell proliferation.

In contrast to previous knowledge that S1P increases the survival of leukemic cells because apoptosis is prevented by caspase inactivation, our data suggests that S1P inhibits NB4 cell proliferation, through ERK pathway activation, not p38 nor JNK pathways. In addition, the pertussis toxin(PTX)-sensitive GTP-binding protein-coupled receptor, Edg-3, seems to be involved in the antiproliferative signaling of S1P to the NB4 cell.

Understanding the signaling mechanisms of anti-proliferation by S1P can uncover new therapeutic targets for APL and the results of this study warrant further investigation on synergism of S1P with known therapeutic agent, ATRA and Arsenic trioxide on APL cells.
IL-8 and MCP-1 Regulate the Transmigration of Acute Promyelocytic Leukemia toward Alveolar Type II Cells.
Authors: Hsu Hui-Chi , Tsai Wen-Hui , Ho Chi Kuan , Tzeng Cheng-Hwai , Kou Yu Ru
Published: April 2020

All-trans retinoic acid (ATRA) can induces complete remission in patients with acute promyelocytic leukemia (APL), and can also induce retinoic acid (RA) syndrome in upto 30 % of patients. Massive infiltration of APL cells can be found in the alveolar spaces in the patients with RA syndrome, indicating that ATRA-treated APL cells can migrate from blood vessels into the alveolar space and cause a cascade of reactions which results a clinical picture similar to adult respiratory distress syndrome. In this study, we determine the role of IL-8 and monocytes chemotactic protein-1 (MCP-1) in the cross-talk between APL cells and alveolar cells, and their role in the pathophysiology of RA syndrome. In the transmigration assay, ATRA-treated APL cells (NB4 cells) can transmigrate toward alveolar type II cells (A549 cells) in a dose dependent manner (ATRA: 0.01 to 1 mM), which is also in parallel with increased granulocytic differentiation. Chemoattractant activity can be found in the conditioning medium (CM) of A549 cells in the time dependent manner (1-3 days). When exposed to either IL-8 or MCP-1, chemotactic effect can also be found in the ATRA -treated NB4 cells, but not in untreated NB4 cells. We further determined the concentration of IL-8 or MCP-1 in the CM by ELISA. Our data show that the levels of both cytokines were progressively increased in the CM of A549 cells. Synergistic effect was found when A549 cells were co-cultured with NB4 cells. In the presence of ATRA, the levels of IL-8 and MCP-1 were further increased in the CM of co-culture of NB4 cells and A549 cells (p<0.05 & p<0.05; respectively). The chemotactic activity in CM of A549 cells was partially inhibited when the CM was neutralized with anti-IL-8 or anti-MCP-1 antibodies, respectively. In binding assay by flowcytometry, we further demonstrated that both IL-8 and MCP-1 were bound by the ATRA-treated NB4 cells, but not in un-treated NB4 cells. We conclude that both IL-8 and MCP can induce ATRA-treated APL transmigrating toward alveolar space and causing cascade of reactions leading to RA syndrome.
Hsp90 Expression in Acute Myeloid Leukaemia.
Authors: Walsby Elisabeth J. , Gilkes Amanda F. , Walsh Valerie , Mills Kenneth I. , Burnett Alan K.
Published: April 2020

Heat shock protein inhibitors are a potential new class of anti-leukaemic agents with FLT-3 inhibitory activity. 17-allylamino-17-demethoxygeldanamycin (17-AAG) targets Hsp90 which functions as a molecular chaperone protein that promotes folding of nascent and stress denatured proteins helping them to achieve the correct conformation or targeting them for degradation. Hsp90 conformation is altered by binding ATP to a hydrophobic N-terminal pocket leading to hydrolysis which allows the protein to interact with a co-chaperone complex. 17-AAG fills the ATP binding pocket, displacing the nucleotide and inhibiting the function of Hsp90 as a chaperone. 17-AAG may affect signal transduction resulting from client proteins such as p53, c-Raf-1, Akt, Bcr-Abl and FLT-3. The level of expression of Hsp90 in AML is unknown. To examine the expression 252 AML cases and 9 normal samples were assessed by U133A GeneChips with the data analysed by MAS5 and GeneSpring 6.2.1 to give normalised gene expression of Hsp90α and β, a subset of which were then validated by real-time quantitative RT-PCR. The normalised expression range of Hspα was 0.132 to 2.727 with a mean of 0.982 (n=252) which was not significantly different from normal (0.876: n=9). Hsp90α expression was not significantly different in FAB groups, cytogenetic risk group, FLT-3 status overall or within cytogenetic risk groups. The expression of Hsp90β in the AML samples (n=252) (0.959: range 0.171 to 3.397) was not different from normal samples (0.944: range 0.535 to 1.095). However the APL subgroup (M3) (n=31) had a lower expression (0.722) than the other FAB groups (M0, M1, M2, M4, M5, M6, M7) p=0.029. There was no difference within other FAB subgroups. Apart from the influence of the t(15;17) subgroup expression levels did not vary significantly in the cytogenetic risk groups, or with the presence of a FLT-3 mutation overall or within cytogenetic risk groups. Conclusion: Hsp90 isoform expression in AML is variable over a 21 fold range. Expression does not correlate with FAB, cytogenetic risk group or FLT-3 mutation status. The relationship to clinical outcome is currently being analysed.
Impaired Interferon-Gamma Production as a Consequence of Profound STAT4 Deficiency in Lymphocytes of Lymphoma Patients after Autologous Hematopoietic Stem Cell Transplantation.
Authors: Robertson Michael J. , Chang Hua-Chen , Pelloso David , Kaplan Mark H.
Published: April 2020

Production of IFN-gamma has been found to be critical for optimal antitumor immunotherapy in several preclinical animal models. IL-12-induced IFN-gamma production is markedly defective in patients with lymphoma who have undergone autologous hematopoietic stem cell transplantation (AHSCT). We have investigated the mechanism of defective IFN-gamma production after AHSCT. Increasing the number of CD4 T cells in IFN-gamma assays did not substantially improve IFN-gamma production by post-transplant patient peripheral blood mononuclear cells (PBMCs). Thus, the quantitative deficiency in CD4 T cells observed for up to a year after AHSCT was not the primary mechanism of defective IFN-gamma production. Post-transplant patient T cells and NK cells expressed the IL-12 receptor beta1 subunit at significantly higher levels than did control PBMCs. Moreover, IL-12 receptor beta2 was expressed by a higher proportion of NK cells from post-transplant patients as compared to control subjects. Thus, down-regulation of IL-12 receptor subunits did not account for defective IFN-gamma production by post-transplant patient PBMCs. Levels of Jak2 and Tyk2 were comparable in control and post-transplant patient PBMCs. In contrast, IL-12-induced tyrosine phosphorylation of STAT4 was undetectable or barely detectable in post-transplant patient PBMCs, whereas it was readily detectable in control PBMCs. The total levels of STAT4 protein were also markedly decreased (P<0.027) in post-transplant patient PBMCs (3275+/−1692; mean+/−SE by densitometric analysis) as compared to control PBMCs (8644+/−732). This profound STAT4 deficiency persists for at least 6 months following AHSCT. Incubation of post-transplant patient PBMCs with IL-12 and IL-18 in combination partially reversed the defective IFN-gamma production seen after stimulation with IL-12 alone. IFN-gamma production in response to IL-12 plus IL-18 was not associated with increased expression of STAT4 but was dependent on the activity of p38 MAP kinase. These results indicate that defective IFN-gamma production is due to an intrinsic deficiency in STAT4 expression by post-transplant patient lymphocytes and suggest strategies for circumventing this deficiency in post-transplant cancer immunotherapy.
Interleukin 12 in Combination with Tumor Cell Vaccines Elicits Anti-Leukemic Immune Responses in a Murine Model of Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia.
Authors: Gruber Tanja A. , Skelton Dianne C. , Kohn Donald B.
Published: April 2020

Current intensive chemotherapy regimens have dramatically increased survival in acute lymphoblastic leukemia (ALL) patients compared to the 1950s when single agent chemotherapy was used. Despite this success certain subsets of patients have a high rate of relapse such as those with the Philadelphia chromosome (Ph+). Because the Bcr-Abl oncogene is a novel protein product and uniquely expressed in the leukemia clone, it has the potential to generate anti-leukemic immune responses. Our lab has been studying immunotheraputic approaches for Ph+ ALL using a murine model. Previous data have demonstrated that transduction of leukemia cells with the immunomodulators CD40Ligand, CD80, and GM-CSF generate T and NK cell immune responses. When irradiated and given as a vaccine these gene-modified cells are able to protect a portion of mice from an otherwise lethal dose of leukemia. We looked at the ability to systemic IL-12 treatments to potentiate this immune response and found that IL-12 alone was able to eliminate pre-existing disease in mice. IL-12 treatments, however, did not establish immunologic memory and did not protect mice from subsequent re-challenge with a lethal dose of leukemia. IL-12 protection was primarily mediated by CD4 and CD8 T cells as demonstrated by a decrease in survival in nude mice. When CD4 or CD8 T cells were depleted individually, however, protection was maintained indicating that one cell type can compensate for the other in its absence. Depletion of NK cells from Nude mice further decreased survival indicating a role for these cells in the protection. Thus protection was mediated in part by CD4 T lymphocytes, CD8 T lymphocytes, and Natural Killer cells. The ability of IL-12 to activate three different cell types may explain the efficacy seen in this model, where other cytokines alone have failed. In combination, IL-12 and our leukemia cell vaccine are effective in eliminating pre-established aggressive Philadelphia chromosome positive leukemia and establishing long lasting immunity from subsequent lethal doses of wild type leukemia. As expected, the immunologic memory generated by vaccination with gene modified leukemia cells was mediated by CD4 T cells as indicated by depletion studies. These studies demonstrate the feasibility of immunotheraputic approaches in the treatment of Ph+ ALL.
Treatment of Philadelphia Chromosome (Ph)-Positive Acute Lymphoblastic Leukemia (ALL) with Concurrent Chemotherapy and Imatinib Mesylate.
Authors: Ribera Josep-Maria , Oriol Albert , Gonzalez Marcos , Vidriales Maria-Belen , Xicoy Blanca , Grau Javier , Esteve Jordi , Brunet Salut , Potro Eloy Del , Moreno Maria-Jose , Tormo Mar , Martin-Reina Maria-Victoria , Deben Guillermo , Parody Ricardo , Lahuerta Juan-Jose , Miguel Jesus-Fernando San
Published: April 2020

Imatinib mesylate as a single agent has modest activity in refractory/relapsed Ph-positive ALL. Use of concurrent chemotherapy and imatinib mesylate in newly diagnosed Ph-positive ALL was explored. Nineteen patients (pts) with de novo Ph-positive ALL were included in a prospective trial. Induction chemotherapy consisted of imatinib mesylate (400 mg/d, p.o.) and VCR (1.5mg/m2/wk), DNR (60 mg/m2/wk) and PDN (60mg/m2/d) for 4 weeks if adequate bone marrow response (<5% blast cells) at day +14 was observed. If not mitoxantrone (12 mg/m2, d15, 16, 17) and HD-ARAC (1,000mg/m2/12h d 18, 19) were administered. Consolidation chemotherapy (C1) included imatinib mesylate and MP, HD-MTX (1.5 g/m2), VM-26 and ARA-C. Pts with a HLA-identical family or MUD donor were submitted to SCT. IF no MUD donor was found a second consolidation cycle (C2) with imatinib mesylate, VCR, DNR, DXM and CPM was administered. Pts without family donor or with no MUD donor after 6 mo. of active search were submitted to autologous SCT. After SCT imatinib mesylate was administered from the sustained hematological recovery to the eventual relapse or at least up to 1 yr. of continuous molecular remission. Sequential MRD study was performed by cytofluorometry and quantitative RT-PCR. Up to July 2004, 19 pts (10 males, mean [SD] age 43[12] yr., range 17–62, p190bcr-abl in 75% of the cases) were included. One pt is still on treatment, 1 died in induction because of infection, 1 was refractory and the remaining 16 (89%) attained CR. Slow bone marrow response was observed in 4 out of 14 evaluable pts. Three patients have relapsed before SCT, 8 are on consolidation therapy and SCT was performed in 5 (3 from family donor and 2 MUD). 1 pt died after SCT and no relapses after SCT have been observed to date (range 1–6 months from SCT). With a median follow-up of 5 (0.2–14) mo., 3 pts have died, 2 are alive in second CR and 13 are in first CR, with a median DFS of 8 mo. and an OS probability of 66±30%. Levels of BCR-ABL/GUS x100 less than 0.05 were observed in 6/12 (50%) pts at the end of induction therapy. There was a further 1–2 log median reduction of BCR/ABL transcripts at the end of C1, (BCR-ABL/GUS x100 less than 0.05 in 5/6 cases). No additional reduction of transcripts was observed between C1 and SCT. A good correlation between molecular and cytofluorometric MRD data was observed. Use of concurrent chemotherapy and imatinib mesylate in newly diagnosed Ph-positive ALL pts is feasible and the preliminary data are promising in terms of high rate of clinical and molecular CR. Continued accrual and longer follow-up of the current cohort is needed.
Effective Treatment of Elderly Acute Myeloid Leukaemia (AML) with Continuous Combined Granulocyte Colony Stimulating Factor (G-CSF) and Single Oral Chemotherapeutic Agents.
Authors: Curnow Jennifer L. , Piccolo Francesco , Ward Christopher M. , Coyle Luke , Fay Keith , Mackinlay Naomi , Ramanathan Sundra , Arthur Christopher
Published: April 2020

Advanced age is a poor prognostic factor in acute myeloid leukaemia (AML). There is evidence that continuous low-dose chemotherapy with oral agents can achieve partial remission in AML and may be suitable as palliative therapy in elderly patients. Such low-dose treatment is usually complicated by drug-related neutropenia and thrombocytopenia. Granulocyte colony stimulating factor (G-CSF) may lessen the associated neutropenia and some anecdotal reports suggest G-CSF alone may be able to induce remission in AML. The study aim was to assess the benefits of combining an oral chemotherapeutic agent with continuous G-CSF. We describe a retrospective audit of 12 elderly patients with AML, diagnosed between 2000 and 2002, who were deemed to be either unfit for induction chemotherapy or who had failed to respond to intravenous chemotherapy. Patients were treated with continuous oral mercaptopurine, thioguanine, or hydroxyurea with concomitant G-CSF on three to seven days/week. The median age at diagnosis was 80 years (range 70–89 years). Eight (67%) had a preceding myelodysplastic syndrome. Eleven of the twelve patients died within the study period described, with a median survival of 9 months (95%CI 4.32–13.75). Eleven patients (92%) had a response to treatment, with blast disappearance obtained within 14 days of starting treatment. All patients achieved an increase in neutrophil count >0.5x109/L. Neutrophil recovery was attained within a mean of 13 days of treatment. From the onset of treatment, patients had neutrophils greater than 0.5 x109/L for 71% of the time, despite being on continuous cytotoxic treatment. Five patients (42%) had a platelet response with a rise in count above 100x109/L. A total of nine (75%) patients experienced a period of platelet transfusion independence and four (33%) patients became red cell transfusion independent for a mean of 4.6 and 4.8 months respectively. The analysis of this small cohort suggests that G-CSF administered in combination with one of the above oral chemotherapeutic agents, may be a novel way of providing treatment to elderly patients with AML which is both tolerable and of potential survival benefit.
Comparison of FDG-PET and Ga-67 Scintigraphy in the Evaluation of Lymphoma and Its Relationship to the Pathologic Diagnosis Using WHO Classification System.
Authors: Tsukamoto Norifumi , Hideki Uchiumi , Matsushima Takafumi , Karasawa Masamitsu , Murakami Hirokazu , Kojima Masaru , Nojima Yoshihisa
Published: April 2020

In the management of lymphoma patients, 18fluoro-2-deoxyglucose positron emission tomography (FDG-PET) has emerged as an important tool. However there are conflicting results that have been reported concerning its usefulness in different histological subtypes, especially in indolent lymphomas. The aim of this study was to retrospectively assess and compare the sensitivity of FDG-PET with gallium-67 citrate scintigraphy (Ga-67) with regard to the capacity for identification of disease involvement in patients with malignant lymphoma, and to investigate the relationship with the World Health Organization’s (WHO) classification system for pathological diagnosis of such cases.

We contemporaneously performed 100 cases of FDG-PET and Ga-67 in addition to CT scans and/or MRIs prior to the treatment. Histological diagnoses included diffuse large B-cell lymphoma (DLBCL; n=38), MALT lymphoma (MALT; n=29), follicular lymphoma (FL; n=14), Hodgkin lymphoma (HL; n=8), NK lymphoma (NK; n=2), mantle cell lymphoma (MCL; n=2), Burkitt lymphoma (Burkitt; n=2), splenic marginal zone lymphoma (SMZL; n=2), and one case each with anaplastic large cell lymphoma (ALCL), peripheral T cell lymphoma (PTCL) and small lymphocytic lymphoma (SLL).

In the 100 lymphoma cases, FDG-PET was unable to detect at least one of the disease involvement sites for 2/38 in DLBCL, 3/14 in FL, 1/8 in HD, 11/29 in MALT, 2/2 in SMZL, and 1/1 in SLL. FDG-PET detected more involvement sites than Ga-67 with 11/38 in DLBCL, 6/14 in FL, 4/8 in HL, 7/29 in MALT, 2/2 in MCL, and 1/2 in NK. In contrast, Ga-67 was more informative in two cases of MALT and in one case of SMZL, in which the regions included the eyelid, pleura and spleen.

During the lymphoma imaging that was done before therapy, FDG-PET was highly sensitive in aggressive and high-grade lymphoma patients as compared to seen for Ga-67. In contrast, FDG-PET seemed to be more informative in FL with regard to indolent lymphomas, but was especially less reliable in MALT and SMZL. Anatomical imaging from CT scans and MRIs were useful in that they provided additional helpful information for the FDG-PET results in the lymphoma subgroups.

Result of FDG-PET and Ga scintigraphy

CT positive CT positive Histology No. of cases PET negative Ga negative ALCL 1 0 0 Burkitt 2 0 1 DLBCL 38 2 13 FL 14 3 8 HL 8 1 5 MALT 29 11 14 MCL 2 0 2 NK 2 0 1 peripheral T 1 0 0 SLL 1 1 1 SMZL 2 2 1
Prognostic Value of ZAP-70 Expression Detected by Immunohistochemistry on Bone Marrow Biopsies in Early Phase Chronic Lymphocytic Leukaemia.
Authors: Ambrosetti Achille , Zanotti Roberta , Chilosi Marco , Zanetti Flavia , Lestani Maurizio , Pattaro Cristian , Remo Andrea , Perbellini Omar , Pizzolo Giovanni
Published: April 2020

New biological prognostic factors have been developed over the last years with the aim of predicting at presentation the heterogeneous clinical course of B chronic lymphocytic leukaemia (B-CLL) and of planning a risk-adapted treatment strategy. Among them ZAP-70 expression on leukemic cells as evaluated by molecular analysis or flow cytometry, initially proposed as surrogate of IgVH mutational status, appears a strong prognostic marker in B-CLL. However the optimal methodological approach to immunophenotipical demonstration of ZAP-70 expression as still matter of debate. We evaluated the cytoplasmic expression of ZAP-70 protein in leukemic cells by immunohistochemical method on bone marrow trephine biopsies taken at diagnosis of 84 patients with B-CLL. We used a mouse anti-human monoclonal antibody to ZAP-70 (clone 2F3.2, 1/200, Upstate, Lake Placid NY) with a polymeric labelling two-step method (Dako cytomation EnVision+, HRP). The results were correlated with age, sex, Binet stage, pattern of bone marrow infiltration, survival and clinical outcome. They were 54 males (64%), aged 34 to 80 years (median 62.5). At presentation 69 (82%) were Binet stage A, 9 (11%) stage B and 6 (7%) stage C. Among the 60 survivors, the median follow-up period from diagnosis was 78.5 months (range 22 – 236 months) Thirty-nine cases (46%) of B-CLL patients had cytoplasmic expression of ZAP-70. This group of patients presented higher percentage of advanced Binet stage (B–C) (p= 0.001). The ZAP-70 positivity was significantly related to inferior OS (55% vs 90% at 7 years)(HR 4.67 CI 1.95–11.14) and PFS (15% vs 57% at 7 years) (HR 5.52 CI 2.57–11.86), confirmed in multivariate analysis. ZAP-70 expression was correlated to poorer outcome also when we evaluated only the 69 stage A patients and the 56 cases with non-diffuse bone marrow infiltration, whereas in patients with diffuse pattern ZAP-70 expression had no prognostic significance. In conclusion, immunohistological detection of ZAP-70 on bone marrow samples at diagnosis appears a useful methodological approach to identify patients with different prognosis in B-CLL.
High Doses of CD34+ Cells Chemomobilized with G-CSF PBPC Collection Had Greater CD34(+) Cells Collected Than G-CSF Alone and Trended toward Reduced Progression Rate after Autologous PBPC Transplantation for Breast Cancer in a Phase III Trial.
Authors: Gajewski James L. , Saliba Rima , Rondon Gabriela , Ueno Naoto T. , Donato Michele L. , Anderlini Paolo , Korbling Martin , McMannis John D. , Giralt Sergio A. , Khouri Issa F. , Mehra Rakesh , Champlin Richard E.
Published: April 2020

We evaluated intensive chemotherapy prior to PBSC mobilization vs. G-CSF alone for PBPC mobilization in a randomized controlled trial of 143 patients who received high dose chemotherapy with cyclophosphamide 6 g/m2, thiotepa 750 mg/m2 and BCNU 450 mg/m2 over 3 days with PBSC support for treatment of breast cancer. 74 patients were randomized to chemomobilization with cisplatin 135 mg/m2, etoposide 1200 mg/m2, and cyclophosphamide 4.5 g/m2 over 3 days plus G-CSF (6mcg/kg q12h)(CVP-G), while 69 patients received G-CSF alone with the collection goal >4 x 106 CD34(+) cells/kg. 77 patients were stage II–III, 66 were stage IV. The CVP-G group had improved mobilization with a collection of 18 (range 3–141) x 106 CD34(+) cells/kg compared with 4(2–13) x 106/kg for the G-CSF group (p<0.001). Granulocyte and platelet recovery post-transplant was 9/9 days for the CVP-G mobilized group, 10/11 days for the G-CSF group (p<0.001). Actuarial risk of progression at 6 years in patients mobilized with CVP-G was 59±7% vs. 66±6% for G-CSF alone (p=0.2). Rate of progression post-transplant was evaluated according to quantities of collected CD34(+) cell dose [for CVP-G, first quartile ≤ 9.0, second ≤17, third ≤40, and fourth >40]. Only one patient in the G-CSF alone group collected >9 x 106 CD34(+) cells. In the CVP-G group progression trended toward being reduced in patients with CD34(+) cell doses collected of >9 x 106/kg (57±9% vs. 65±11%, p=0.06) This effect was consistent for all stages. Overall survival was improved in the CVP-G patients collecting ≥9 x 106 CD34(+)/kg 62±7% vs. 27±11% those collecting less. For patients receiving CD34(+) doses ≤9 x 106/kg, the progression rate was 65±11% in the CVP-G group and 68±6% in the G-CSF group (p=NS). Higher doses of CD34(+) cells collected by chemomobilization than G-CSF alone and those patients with the higher doses of CD34(+) collected trended toward lower disease progression after high dose chemotherapy.
Localization of Binding Sites for Low-Density Lipoprotein Receptor in Coagulation Factor VIII.
Authors: Makogonenko Evgeny M. , Sarafanov Andrey G. , Ananyeva Natalya M. , Strickland Dudley K. , Saenko Evgueni L.
Published: April 2020

Clearance of coagulation factor VIII (fVIII) is mediated by a hepatic receptor low-density lipoprotein receptor-related protein (LRP), a member of low-density lipoprotein receptor (LDLR) family. It has been recently discovered that LDLR acts in concert with LRP in regulating fVIII level. FVIII has the domain structure A1-A2-B-A3-C1-C2, and to identify the portions providing the interaction with LDLR, in surface plasmon resonance-based assay we studied the binding of fVIII and its fragments to immobilized recombinant ligand-binding domain of LDLR (residues 1-292). The affinity values were determined from the families of binding signals obtained for five concentrations (10–150 nM) of each analyte. The binding signals for full-length fVIII, and its portions A3-C1–C2 (or light chain, LCh) and A1/A3-C1–C2 heterodimer (derived from activated fVIII) were best fitted to a two-site model, with equilibrium dissociation constants KD(1) ~1, 4, 14 nM and KD(2) ~14, 45 and 37 nM, respectively. Noteworthy, we did not observe any significant binding for the isolated C2 domain (at 300 nM). This suggests that the LDLR-binding site within LCh is likely located within the A3 domain, similar to that found previously for LCh-LRP interaction. The binding signals for A1-A2-B (heavy chain, HCh) were best fitted to a one-site model, with KD ~60 nM. We registered a dose-dependent, high-affinity binding of the isolated A2 domain to LDLR, with KD ~14 nM whereas the A1 domain showed no appreciable binding. This suggests that within HCh, A2 domain bears the LDLR-binding site. Von Willebrand factor did not significantly block the binding of fVIII to LDLR as compared to a 3-fold inhibition of fVIII binding to LRP. This indicates that within fVIII/vWf complex, the A2 binding site for LDLR is more available than that for LRP. Anti-A2 monoclonal antibody 413 (epitope 484–509) inhibited the A2 binding to LDLR in a dose-dependent manner, similarly to that demonstrated for fVIII-LRP interaction. A number of A2 point mutants with substitutions of the residues critical for A2 binding to LRP, megalin and VLDL were found to have significantly reduced affinity also for LDLR. The obtained data indicate that fVIII interacts with LDLR preferentially via the binding sites located within the A2 domain of HCh and within the A3 domain of LCh, and that the A2 site is likely to be universal for the interactions with four tested members of LDL receptor family.
Expression of Protein Z (PZ) and Protein Z-Dependent Protease Inhibitor (ZPI) In Situ in Human Malignant Tissues.
Authors: Sierko Ewa , Wojtukiewicz Marek , Tokajuk Piotr , Zimnoch Lech , Ramlau Rodryg , Kisiel Walter , Broze George J.
Published: April 2020

The activity of the hemostatic system components facilitates tumor growth and dissemination. There exists a mechanism of direct inhibition of factor Xa that involves protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) system. ZPI also attenuates the activity of factor XIa. Data on the presence of PZ and ZPI in malignant tissue in situ are lacking.

The aim of this study was to assess the localization of PZ and ZPI in situ in colon, breast, gastric, laryngeal, pancreatic, renal, endometrial cancer, non-small cell lung cancer (NSCLC), malignant melanoma and glial neoplasms. Studies were performed on tumor tissues obtained at surgical treatment of previously untreated patients. Immunohistochemical procedures according to the ABC method and Envision Double Staining System (Dako) employed a polyclonal antibody against PZ and a monoclonal antibody against ZPI. Protein Z was localized in cancer cell bodies in all types of malignant tumors examined. Staining intensity was more pronounced in less differentiated cancer cells of anaplastic gliomas. In contrast, more differentiated cancer cells of gastric cancer revealed stronger staining than less malignant ones. Cancer cell bodies of NSCLC and renal cancer were characterized by weak intensity of staining for PZ. In pancreatic and renal cancer, as well as in malignant melanoma, the intensity of staining for PZ in cancer cells was irregular: both high and low PZ expression was observed independently of the degree of malignancy. Protein Z was also revealed in association with macrophages in colon cancer, NSCLC and malignant melanoma. Expression of ZPI was observed in cancer cell bodies in all examined specimens. The staining intensity for ZPI was irregular: both strong and weak expression of ZPI was observed. Various percentages of ZPI-positive cancer cells were observed in different specimens of the tissues examined. In colon cancer, ZPI expression was detected in tumor infiltrating macrophages. Double staining studies revealed co-expression of both proteins in cancer cell bodies in all examined tumor types, although there were areas of tumor foci where only one protein (either ZP or ZPI) was localized. Our results suggest that PZ and ZPI, apart from being inhibitors of blood coagulation, may play an additional regulatory role in tumor progression.
Intracellular Trafficking of Factor V Subsequent to Its Endocytosis by Megakaryocytes.
Authors: Seifert Lorna W. , Wahle-Ritchie Alexa , Bouchard Beth A. , Tracy Paula B.
Published: April 2020

Recent studies by our laboratory have identified physical and functional differences between plasma- and platelet-derived factor V. Additional studies indicate that the platelet-derived molecule originates from megakaryocyte endocytosis of the plasma-derived cofactor via a receptor-mediated, clathrin-dependent mechanism, and is subsequently packaged and stored in platelet α-granules. We hypothesize that plasma-derived factor V is modified intracellularly following its endocytosis by megakarycytes to generate the unique platelet-derived cofactor molecule. Thus, the time-dependent, intracellular trafficking of fluorescently-labeled factor V by the megakaryocyte-like cell line, CMK, was determined by confocal microscopy using various organelle-specific, fluorescent markers. Previously, we had demonstrated that subsequent to its endocytosis factor V partially co-localizes with two other proteins known to be endocytosed by megakaryocytes, fibrinogen, an α -granule protein, and transferrin, an iron transport protein. In the current study, we demonstrated that subsequent to their endocytosis, factor V and transferrin partially co-localized to early endosomes as determined using an antibody directed against Rab5. Complete co-localization of anti-Rab5 with an antibody against early endosomal antigen-1 (EEA-1) confirmed the specificity of the anti-Rab5 antibody for early endosomes. Endocytosed factor V was also shown to partially co-localize with von Willebrand factor, an α -granule protein that is synthesized by megakaryocytes. Its synthesis by megakaryocytes was confirmed by partial co-localization of this antibody with anti-Golgi antibodies against GM130, a structural element of the Golgi apparatus, and p230 trans, a protein involved in vesicular transport from the trans-Golgi network. Factor V also partially co-localized with these Golgi markers, consistent with the hypothesis that factor V is modified intracellularly subsequent to its endocytosis. Co-localization studies were also performed using LysoSensor Blue, which partitions into acidic organelles with a pH ~5.1 exhibiting an increase in fluorescence intensity upon acidification. Neither factor V nor transferrin co-localized with LysoSensor Blue confirming that they are not trafficked to lysosomes subsequent to their endocytosis. In conclusion, these combined observations suggest that subsequent to its endocytosis by megakaryocytes factor V is trafficked through early endosomes and ultimately stored in the α -granule with vWF and fibrinogen. Further, these data suggest that prior to its packaging into α -granules factor V may undergo retrograde transport through and O-linked glycosylation in the trans-Golgi network, which is consistent with our previous observations that purified, platelet-derived factor V contains an N-acetyl glucosamine or galactosamine at Thr402 that is not observed in its plasma counterpart.
Different Prevalence of Drug Metabolism Gene Polymorphisms (GSTP and GSTT) in FA Patients Compared to a Normal Population.
Authors: Santos Moema Nene , Rocha Vanderson , Zanette Dalila , Porcher Raphael , Ionescu Irina , Tavela Marli , Soulier Jean , Leblanc Thierry , Fisher Gustavo , Baruchel André , Zago Marco Antonio , Gluckman Eliane
Published: April 2020

Fanconi Anemia patients have an increased toxicity of drugs used in conditioning regimen (CT) for hematopoietic stem cell transplantation (HSCT), due to defective DNA repair processes. This observation has led to diminish the doses of drugs (such as cyclophosphamide and busulphan) and irradiation in the preparative regimen. We and others have reported that genetic polymorphisms, which interfere with metabolisms of drugs used in CT, are associated with toxicities after HSCT. In order to test the hypothesis that probably Fanconi patients have a difference in prevalence of pharmacogene polymorphisms, we have compared the samples of 49 Fanconi patients (median age of 6,2 years) with 107 samples of normal population of the same ethnic group. Polymerase chain reaction (PCR) and restriction-digestion protocols were used to identify most of the polymorphisms. We have selected pharmacogenes knonwn to interfere with the metabolisms of drugs used in the CT or GVHD prophylaxis. The following gene polymorphisms were analyzed: P450 cytochrome family [CYP2B6*2(C64T), *4(A785G), *5(C1459T), *6(G516T)]; glutathion-S-Transferases [GSTM1 (null), GSTT1(null), GSTP1(G313A)], Thiopurine-S-methyl transferase [TPMT *3B (G460A) and *3C (G238C)], Multiresistance-drug [MDR1 (C3435T)], Methylenetetrahydrofolate reductase [MTHFR (C677T, A1298C)], Vitamin D receptor [VDR (ApaI, TaqI and BsmI)]. All gene polymorphisms studied were found in the same frequency in FA as in normal controls, except polymorphisms of GSTP and GSTT. In fact, FA patients had a higher frequency of mutant alleles (AG or GG) of GSTP (86%; n=42) compared to 51% (n=55) in normal controls (p<0.001). GSTP polymorphisms are associated with reduced ability to detoxify chemotherapeutic agents thereby increasing the effective dose of the drug within the cell. It has also been associated with increased risk of breast, prostate, bladder and testicular cancer. We have also observed a decreased frequency of GSTT wild type (null; 8%, n=4) in FA patients compared to normal controls (25%, n=27) (p=0.01). In conclusion, this study shows that prevalence of GSTP and GSTT gene polymorphisms in FA is different from the normal population.
Human CIK Maintain Their In Vitro and In Vivo Anti-Tumor Ability after Cryopreservation.
Authors: Baker Jeanette , Sheehan Kevin , Monterola Gina , Staines Nancy , Negrin Robert S.
Published: April 2020

Adoptive cellular therapy holds promise for improving the outcome of hematopoietic cell transplantation (HCT). At present, donor lymphocyte infusion post-HCT is efficacious for only a limited number of diseases, yet can induce significant graft versus host disease (GVHD). To improve the outcome of this approach, it would be beneficial to identify populations of T cells that retain graft versus tumor (GVT) effects with reduced propensity for GVHD. We have previously described studies of murine expanded Cytokine Induced Killer (CIK) cells which are ex vivo activated and expanded T cells that express both T and NK markers. CIK cells mediate cytotoxicity both in vivo and in vitro in a non- MHC restricted NKG2D dependent manner. Human CIK cells were expanded from PBMC from 9 healthy donors, cultured with IFNg, CD3 and IL-2 and maintained in AastromRepliCell® biochambers for 21–28 days. We aimed to determine whether cryopreservation of the CIK affects viability, cytotoxicity and phenotype. Cells were cryopreserved immediately after harvest at 10x106/ml and stored in liquid nitrogen vapor phase. CIK viability was not compromised with cryopreservation and cells thawed at 1, 2, 4, 8, 10 and 28 weeks after freezing were 96% viable (range 95%–99%). Immediately upon thawing, CIK cells showed diminished cytotoxicity against the B cell lymphoma cell lines DB and SUDHL4 with 6–10% killing at the 40:1 E:T ratio. However, thawed CIK cells regained their pre-freeze cytotoxic activity against these targets within 5 hours of being placed in reactivation medium containing IL-2 at 300 IU/ml. Reactivation of the CIK cells was extended up to 48 hours but showed no further increase in cytotoxicity beyond that attained at 5 hours; nor did increasing the IL-2 concentration to 1500 IU/ml in the reactivation medium improve CIK cell activity over the same time course. Cell viability declined during reactivation, decreasing from an average 96% upon thawing to 60% over 48 hours. Thawed CIK cells placed in reactivation medium maintained their cytotoxic activity up to 14 days in vitro. The cytotoxicity of reactivated CIK cells was assessed in vivo using SCID mice inoculated IP with 1x106 human ovarian cancer UCI-101 cells expressing the firefly luciferase gene. The mice were treated weekly with 2x107 cryopreserved and thawed human CIK cells that were re-cultured for 5 hours before injection. Following each administration of CIK cells, there was a reduction of tumor signal. Weekly treatments resulted in a better survival outcome for the mice receiving CIK cells as compared to PBS control mice. This study demonstrates that human CIK cells may be reactivated after cryopreservation and regain their cytotoxic potential. These finding have important implications for the application of these cells as adoptive cellular therapy.
Interferon-gamma Expressing EBV LMP2A-Specific T Cells for T Cell Therapy Against EBV-Positive Hodgkin’s Lymphoma.
Authors: Sun Qi , Niyazova-Brewer Nargisa , Bao Lei , Chen Lipai , Lucas Kenneth G.
Published: April 2020

Hodgkin’s lymphoma (HL) accounts for approximately 67% of lymphomas. Patients with HL refractory to standard intervention have poor prognoses. LMP2A is one of the Epstein-Barr virus (EBV) oncoproteins persistently expressed in approximately 50% of HL cases, providing an ideal target for T cell therapy against EBV-positive HL. However, polyclonal EBV-specific T cells have achieved only limited responses in clinical trials, with eventual disease progression or relapse. While multiple possibilities could underlie the unsatisfactory efficacy, the most obvious shortcoming of the current generation of therapeutic T cells is the low frequency of HL-relevant effectors in the T cell preparations. We designed a strategy to enrich LMP2A-specific T cells from polyclonal EBV-specific T cell preparations. With an “Interferon (IFN) Capture” procedure, LMP2A-specific T cells were isolated from T cell cultures generated with stimulation by autologous EBV immortalized B lymphoblastoid cells based on their antigen-specific expression of IFN-gamma. The enriched LMP2A-specific T cells were subsequently expanded with the anti-CD3 antibody OKT3 to a scale applicable for clinical use. Our results showed that a ten fold enrichment can be achieved for T cell cultures containing as low as 0.5% LMP2A T cells, and 5 fold for those containing higher than 10%. Thus, 2 consecutive expansions increased the LMP2A-specific T cells to nearly 50% from 0.5% in the starting culture. Cytokine expression analysis showed that the enriched T cells retained their antigen specific production of IFN-gamma, and a subpopulation upto 10% of the IFN-gamma producing T cells were capable of expressing interleukin (IL)-2. In contrast, no cells expressing immuno-suppressive cytokines IL-4, IL10 and TGF-beta were detected. The enriched T cells were mostly CD45RO+/CD45RA-, indicating that they were proliferation competent. Chromium release assays confirmed that the enriched T cells maintained antigen-specific cytotoxicity. With a panel of four HLA A02 restricted tetramers, we could further demonstrate that the enriched T cells were polyclonal recognizing multiple LMP2A epitopes and retained their spectrum of specificity throughout the enrichment and expansion. This system provided the foundation for T cell therapy against EBV-positive HL with new generation therapeutic T cells.
Better Molecular Response (MR) to Imatinib (IM) in Early Chronic Phase (CP) Versus Late CP Chronic Myeloid Leukemia (CML) Patients (pts) in Complete Cytogenetic Response (CCR): A Comparison at 24 Months of 2 Clinical Trials of the GIMEMA Working Party on CML on Behalf of the GIMEMA Working Party on Chronic Myeloid Leukemia (GIMEMA-CML).
Authors: Poerio Angela , Amabile Marilina , Iacobucci Ilaria , Soverini Simona , Colarossi Sabrina , Ottaviani Emanuela , Terragna Carolina , Grafone Tiziana , Luatti Simona , Castagnetti Fausto , Pane Fabrizio , Saglio Giuseppe , Russo Domenico , Rosti Gianantonio , Baccarani Michele , Martinelli Giovanni
Published: April 2020

We sought to determine the differences in molecular response between early and late CP pts with CML who achieved a CCR after treatment with IM at the standard dose of 400mg/d. We studied 2 different cohorts of patients in CCR: 67/191 (35%) pts after α-Interferon (α-IFN) failure enrolled on the CML/002/STI571 protocol

53/76 (70%) pts treated front line with a combination of IM and pegilated IFN-α (PEG-IFN) enrolled on the CML/011/STI571 protocol

Cytogenetic response was monitored on bone marrow (BM) metaphases and molecular response was assessed by real time RT-PCR (TaqMan) BM and peripheral blood (PB) samples, collected at baseline, 3, 6, 9 and 12 months during the first year, and every 6 months thereafter. Molecular response was expressed as the ratio between BCR/ABL and β2-microglobulin (β2-M) x100. The lowest level of detectability of the method was 10−5. Negative results (i.e. undetectable transcript) were confirmed by nested PCR performed 4 times (sensitivity 10−6).

For the purpose of this analysis, a major molecular response (MMR) was defined as a BCR-ABL/β2M value <0.0001%, which turned out to be roughly equivalent to a 3-log reduction and a complete molecular response (CMR) was defined as negative (undetectable) BCR/ABL levels confirmed by nested PCR.

We observed a progressive decrease of the amount of BCR/ABL transcript in pts who achieved a CCR. At 24 months the median reduction in BCR/ABL transcript level was:

a 3-log reduction in late CP pts

a 4-log reduction in early CP pts

In the latter group of pts MR was assessed also at 36 months. So we observed that 36 months after the first dose of IM and PEG-IFN pts who were still in CCR had the median value of BCR/ABL transcript of 0.00001% both in BM and PB. Therefore all these pts achieved a MMR. However only 8/53 (4%) pts were in CMR (undetectable BCR/ABL at least once as assessed by nested PCR).

We conclude that front-line treatment with IM results in a better quality MR (4-log reduction in BCR/ABL transcript levels in early CP pts, as against a 3-log reduction in late CP pts).

Figure Figure