bluuebunny/crossref_metadata_2025 huggingface dataset:
Mesenchymal Stem Cells Induce a Reversible Suppression of Alloimmune Reactions.
Authors: Tschiedel Sabine , Bartsch Kristina , Reinhardt Annette , Chiara Gentilini , Dietger Niederwieser
Published: April 2020

Objective Bone marrow contains pluripotent mesenchymal stem cells (MSC) that form bone, cartilage, adipose tissue and muscle. These cells are not immunogenic and escape recognition by alloreactive T cells and natural killer cells. MSC can be readily isolated from bone marrow and expanded ex vivo without modification in phenotype or loss of function. They are capable of differentiating along multiple mesenchymal lineages, play a crucial role in the bone marrow microenvironment and have profound immunosuppressive properties. In the present investigation we analysed the antiproliferative capacity of MSC using primary and secondary immune responses in vitro.

Methods MSC were isolated from heparinized bone marrow of healthy donors (n=5). Bone marrow aspirates were layered onto a Percoll cushion (density 1,073g/ml) and the MSC-enriched mononuclear fraction was collected, washed and resuspended in Dulbecco modified Eagle medium with low glucose and 10% fetal bovine serum. The cells were plated at 2x105/cm2 and maintained at 37°C in a humidified atmosphere and subcultured prior to confluency.

MSC stained positive for specific markers as SH2, SH3 and SH4 but were negative for CD14, CD34 and CD45. Immunsuppressive effects were assessed by adding third party MSC to primary mixed lymphocyte reactions (MLR) in decreasing concentrations (10%, 1%, 0,1%) on days 0–5. Cell proliferation was measured on day 6 by means of an 18-hour pulse with 3H-thymidine (3H-TdR). Secondary MLRs were performed by restimulating MSC co-cultured MLRs with the primary PBMC on day 10. In addition secondary MLRs were also tested in presence of MSC and cultured for 1, 2, 3 and 6 days in 96-well plates and proliferation was detected by 3H-TdR uptake.

Results Primary MLRs show significant inhibition of proliferation with 10% MSC added on day 0 (85% inhibition), 1 (69% inhibition) or 2 (70% inhibition) (p<0,01). Similar effects were observed by adding 1% MSC on day 0 (42% inhibition) and 1 (10% inhibition). Co-cultivation with MSC over 10 days lead to delayed proliferation in secondary MLRs with peak on day 3. Secondary MLRs were also inhibited by the addition of 1% (47% inhibition) and 10% (56% inhibition) MSC independent from co-cultivation with MSC in the primary culture (11% and 44% inhibition respectively). Restimulation with third party PBMC on day 10 lead to a primary response with a proliferation peak after 6 days.

Conclusions Our findings emphasize the immunosuppressive effects of MSC. We observed that MSC-induced inhibition of primary MLRs was reversible in secondary MLRs. This clearly demonstrates that MSC do not induce tolerance and could be used in a clinical setting due to their immunomodulatory properties.
Flow Cytometric Detection and Phenotype of Circulating CD34+ Cells in Patients Affected by Chronic Lymphocytic Leukemia.
Authors: Stagno Fabio , Parrinello Nunziata Laura , Fargione Giovannella , Triolo Anna , Privitera Antonella , Seria Elisa , Mannino Francesca , Di Raimondo Francesco , Guglielmo Patrizia , Giustolisi Rosario
Published: April 2020

Flow cytometric determination of peripheral blood CD34+ cells provides reliable measurements of circulating hemopoietic progenitors. Since the detection of the absolute number of circulating CD34+ cells has been found of clinical utility in the setting of chronic myeloproliferative disorders, we investigated whether peripheral CD34+ cells could play any role in the clinical work-up of B-cell chronic lymphocytic leukemia (B-CLL). In this view, we determined by flow cytometry the absolute number of circulating CD34+ cells in the peripheral blood of 28 patients (16 males and 12 females, median age 67 years) affected by typical B-CLL (Matutes score 5,4,3) and in different Rai stages of the disease (19 early stage: Rai 0, I, II; 9 advanced stage: Rai III, IV). Conventional and multiparameter flow cytometric analysis was performed utilizing a FACSCalibur cytometer (Becton Dickinson). Our data showed a significant increase in the number of circulating CD34+ cells in the peripheral blood of patients with B-CLL (median CD34+ cells:7.8mL) as compared to controls (median CD34+ cells 0.1mL) (p=0.008). No statistical difference between B-CLL patients in early versus advanced stage (p=0.5) and between untreated versus treated (p=0.7) was found, as well as there was no correlation with some of the clinical characteristics of B-CLL (WBC-count, LDH levels, Beta-2M). In 10 out of 28 B-CLL affected patients, circulating CD34+ cells were correlated with ZAP-70 and CD38 antigen but no correlation was found. In addition, we detected in the peripheral blood of 22 out of 28 patients small numbers of circulating CD34+ cells displaying the CD19+/CD5+ phenotype (median CD34+/CD19+/CD5+ cells:5.7mL) whereas these cells were absent in normal controls. This unexpected finding, whose significance remains to be clarified and still restricted to a small number of cases, could be directly correlated to the underlying lymphproliferative disease and might represent a pool of leukemic stem cells. However, further studies are warranted.
Deletion of p53 as Detected by Fluorescence In Situ Hybridization Is Associated with Significant Shorter Event-Free Survival in Patients with Multiple Myeloma Who Are Receiving Allogeneic Blood Stem Cell Transplantation.
Authors: Schilling Georgia , Hansen Timon , Perez-Simon Jose Antonio , Schwerdtfeger Rainer , Bornhaeuser Martin , Otterstetter Svenja , Seeger Doris , Dierlamm Judith , Ayuk Francis , Kurt Hossfeld Dieter , Carsten Bokemeyer , Axel Zander R. , Nicolaus Kroeger
Published: April 2020

Deletion of chromosome band 17p13 (P53) is known to be an adverse prognostic factor for time to progression (TTP) and overall survival (OS) in myeloma patients receiving conventional or high-dose chemotherapy. In this retrospective multicenter study, we investigated the impact of P53 deletion as detected by fluorescence in situ hybridization combined with immunofluorescent cytoplasm immunoglobulin staining (cIg-FISH) on outcome after allogeneic blood stem cell transplantation (SCT) in 50 patients (pts) with advanced or relapsed multiple myeloma (MM). Median age was 51 years (34 – 67 ys.), 32 patients were male, 18 female. Thirty pts received a stem cell graft from an unrelated donor, and 20 pts from a HLA-identical sibling. P53 gene deletion was found in 5 out of 49 pts (10,2 %). Deletion 13 detected by cIg-FISH was found in 20 out of 47 pts (42%). There was a strong correlation between P53 deletion and deletion 13: four out of the five patients with P53 deletion also showed deletion in chromosome band 13q14. The estimated event-free (EFS) and overall survival (OS) at three years for the entire study population was 43% and 65%, respectively. For patients with del 13 only a trend for a worse 3 year EFS was seen (38% vs 42%, p=0.2), while pts with P53 deletion had a significant reduced EFS (0% vs 46%, p=0.0001). Three out of five pts with P53 deletion relapsed very early after allogeneic SCT (day 81, 108 and 287 respectively). These data suggest that P53 is a risk factor for patients with MM treated with allogeneic SCT which can not be overcome by this treatment strategy.
Gene Expression Profiling of MLL-PTD in AML Identifies Differentially Regulated Members of the HOX Gene Family.
Authors: Austin Stephen J. , Gilkes Mandy F. , Mills Ken I. , Burnett Alan K.
Published: April 2020

Partial tandem duplications (PTD) of the mixed lineage leukaemia (MLL) gene occur in approximately 7% of acute myeloid leukaemia (AML) patients. MLL-PTD has a frequency of approximately 10% in patients with normal karyotype and is reported to be associated with an adverse prognosis. Recent papers have reported that MLL-PTD may be useful as a marker for minimal residual disease (MRD) in AML patients with no other markers available due to normal cytogenetics. Diagnostic RNA from 133 AML patients was analysed for the presence of the MLL-PTD, twelve samples were positive and three of the twelve were also positive for FLT3 ITD. The MLL-PTD gene dosage was verified using quantitative polymerase chain reaction (QPCR). In order to identify the molecular disruption caused by MLL PTD mutations, fifty-four of the 133 patients were profiled using the Affymetrix U133A chip containing 22,283 probe sets. Four of the 54 samples analysed were MLL-PTD positive and three of these contained FLT3 mutations. Groups of genes were identified that had significance to MLL and showed more than 2-fold change in regulation in the MLL-PTD compared with wild type. Up-regulated genes found common to the two groups include HOXA9, HOX2B, HOX2C, HOX1 and CREBBP/EP 300. Whilst significant genes down-regulated more than 2-fold in MLL-PTD include MONDOA, a transcriptional activator. To identify whether MLL-PTD and FLT3 mutations used a common pathway, a comparison was made between genes with significance to either MLL-PTD or FLT3 mutations and no common genes were found. Hierarchical cluster analysis on the most significant genes, using Genespring software, created 4 main groups and distinguishes non-leukaemic samples, AML patients with MLL-PTD and those with a wild type MLL gene. This gene signature showed that MLL- PTD is a unique entity and could be identified amongst other AML subtypes. Moreover, over expression of Hox genes plays a role in MLL PTD positive AMLs and may provide an insight into the mechanism of the MLL-PTD with potential clinical implications.
Successful Use of Rituximab To Treat Central Nervous System Lymphoma in a Patient with B-Cell Post-Transplant Lymphoproliferative Disorder (PTLD).
Authors: Riggs Shirley A. , Van Buren Charles , Kahan Barry
Published: April 2020

A 27 year old male with end-stage renal failure secondary to IgA nephropathy received a living related renal transplant in 10/2002, complicated by severe cellular and humoral rejection. Immunosuppression included steroids, OKT3, cyclosporine A, rapamycin, thymoglobulin, antithymocyte globulin, and plasmapheresis. He developed multiple complications including severe pancytopenia, pleural effusions, Pseudomonas sepsis, and Vancomycin resistant enterococcus cystitis. Transplant nephrectomy was required 2/2003 for intractable rejection with blood clots in the urine, revealing large nodules of monomorphous B-cell PTLD throughout the renal parenchyma, along with type 2 acute cellular rejection. The B-cells were CD20 positive, and in situ hybridization for EBER was positive in the majority of the cells. PTLD staging was negative in other sites including brain and bone marrow. All immunosuppression was discontinued shortly following the nephrectomy, and he was placed on acyclovir for 3 months until he developed progressive cytopenias. In 8/2003 he underwent open brain biopsy following 2 grand mal seizures and an MRI showing multiple bilateral nonenhancing cortical and subcortical lesions up to several centimeters, with enhancing right temporal and parietal lesions with surrounding vasogenic edema and regional mass effect. Right temporal lobe biospy showed extensive high grade monomorphous B-cell PTLD with necrosis and vascular involvement. Lymphoma staging including lumbar puncture was otherwise negative and LDH was normal. He received a brief course of steroids and valacyclovir in the perioperative period. His performance status remained extremely poor, on hemodialysis, and thus he was not considered a good candidate for systemic chemotherapy, brain radiotherapy, or intrathecal chemotherapy. We elected to give a total of 8 courses of rituximab at 375 mg/m2 intravenously weekly. Although systemic rituximab does not generally penetrate the central nervous system very well, it was presumed that the blood brain barrier in this patient was broken down due to extensive lymphomatous cerebral disease. By 9/2003 there was improvement in the brain parenchymal lesions, and since 11/2003 there has been complete stabilization of residual involuted nonenhancing lesions. He remains in complete clinical remission, off rituximab for 22 months. This excellent response suggests that systemic rituximab can be an effective treatment in selected cases of PTLD in which extensive brain disease is present.
Rituximab and Yttrium-90 Ibritumomab Tiuxetan Radioimmunotherapy May Be Feasible Options in Patients with Low-Grade Lymphoma of the Orbit.
Authors: Esmaeli Bita , Pro Barbara , Saville M. Wayne , McLaughlin Peter
Published: April 2020

Objective: To report a cohort of patients with lymphoid tumors of the orbit, including 1 with benign lymphoid hyperplasia of the orbit whose disease responded to immunotherapy with rituximab alone or rituximab followed by Zevalin (yttrium-90 ibritumomab tiuxetan) radioimmunotherapy.

Methods: Between October 2002 and January 2005, 8 patients with low-grade non-Hodgkin’s lymphoma and 1 with benign lymphoid hyperplasia of the orbit were treated with monoclonal antibodies against CD20. Five patients with low-grade lymphoma of the orbit (3 with follicular B cell or 2 with MALT) received rituximab followed by yttrium-90 ibritumomab tiuxetan, 2 patients with low-grade orbital lymphoma (1 with follicular B-cell and the other with small lymphocytic lymphoma) and 1 patient with benign lymphoid hyperplasia received rituximab alone. Three out of the 5 patients who received yttrium-90 ibritumomab tiuxetan were part of a prospective trial evaluating the efficacy of yttrium-90 ibritumomab tiuxetan for low-grade B-cell lymphoma of the orbit; the other 2 had been treated in other open label trials at M. D. Anderson Cancer Center. For each patient, clinical records and imaging studies were reviewed to establish the diagnosis and document response.

Results: Three men and 5 women in this cohort were between the age of 23 and 83 years old (median age, 63 years). Of the 7 patients with orbital lymphoma, 4 had stage IE and the other 3 had stage IVE, and 6 had previously untreated disease. All 8 patients experienced resolution of the orbital tumor in response to treatment with monoclonal antibodies against CD20. Follow-up time ranged from 6 months to 32 months (mean, 12 months) after completion of immunotherapy. There were no serious systemic or ocular side effects during the study period. The most commonly experienced side effect was mild fatigue. All 5 patients treated with yttrium-90 ibritumomab tiuxetan had transient pancytopenia, which normalized as expected within 3 months after treatment. One patient who received yttrium-90 ibritumomab tiuxetan developed shingles 6 weeks after treatment. There were no sequelae from this infection, which resolved with the usual course of oral antiviral therapy.

Conclusions: Rituximab and yttrium-90 ibritumomab tiuxetan may be considered as alternative treatment modalities to external beam radiation therapy for low-grade B-cell follicular non-Hodgkin’s lymphoma or MALT of the orbit. Systemic targeted immunotherapy may potentially have the advantage of lower rate of distant (out-of-field) relapse and less ocular toxicity compared with external beam radiotherapy; these potential advantages would have to be verified in long-term studies and in larger numbers of patients.
Promising Activity of Bortezomib, Rituximab, and Dexamethason (BORID) in Patients with Relapsed Mantle Cell Lymphoma.
Authors: Drach Johannes , Seidl Sonja , Pichelmayer Oskar , Kaufmann Hannes , Zielinski Christoph , Raderer Markus
Published: April 2020

Bortezomib (B) belongs to a new class of anti-cancer agents, the proteasome inhibitors, and has documented activity in multiple myeloma. Moreover, recent data indicate that B is also effective in other B-cell malignancies, most notably in mantle cell lymphoma (MCL). Phase II studies revealed remission rates between 40 and 55% of patients with relapsed or refractory MCL. Preclinical studies suggest that B has synergistic activity with rituximab (R), which provides a rationale for the exploration of such a treatment combination. We have therefore initiated a phase II study in relapsed/chemotherapy refractory MCL in order to evaluate the activity and safety of B in combination with R and dexamethasone (BORID). A treatment cycle consists of B at 1.3 mg/m2 administered on days 1, 4, 8, and 11, R at 375 mg/m2 administered on day 1, and dexamethasone 40 mg orally on days 1 to 4. Cycles are repeated every 3 weeks for a total of 6 treatment cycles. Patients with progressive MCL after at least one prior line of therapy (including CHOP or a CHOP-like regimen) are eligible. Up to now, we have enrolled 7 patients at a median age of 65 years (range, 52 to 74 years); they had received one to 6 lines of prior therapy including R in 5 of them and high-dose chemotherapy followed by autologous stem cell transplantation in 2 patients. Severe adverse events (> grade II) included 3 infections (herpes zoster, bacterial pneumonia, pneumonia of potential viral origin), grade III peripheral neuropathy in one patient, and nodular skin infiltrates in one patient. All adverse events were managable by standard means of supportive care and prolongation of the treatment interval between cycles. No severe hematological toxicity has yet been encountered. Of 6 patients evaluable for efficacy, 5 have achieved a response (1 CR, 4 PR), and 1 patient experienced stable disease. The patient in CR (previously treated by R-CHOP, autologous transplantation, and R plus thalidomide) was also negative for disease activity by PET scanning. None of the patients has yet progressed at 4 to 9 months after initiation of BORID. Recruitment of patients is ongoing, and updated results will be presented. Data obtained thus far indicate that BORID has promising activitiy and managable toxicity in patients with relapsed MCL, which warrants further investigation not only in MCL but also in other B-cell lymphomas.
Apigenin Induces Apoptosis in Primary Effusion Lymphoma.
Authors: Uddin Shahab , Al-Kuraya Khawla S.
Published: April 2020

The mechanisms that regulate induction of the antiapoptotic state and mitogenic signals in primary effusion lymphoma (PEL) are not well known. In efforts to identify novel approaches to block the proliferation of PEL cells, we found that apigenin (4′,5,7,-trihydroxyflavone), a flavonoid, induces apoptosis in a dose dependent manner in several PEL cell lines. Such effects of apigenin appear to result from suppression of the constitutively active AKT, FOXO transcription factor and GSK3. Our data also demonstrate that apigenin induces loss of mitochondrial membrane potential with subsequent release of cytochrome c and activation of caspase-3, followed by polyadenosin-5′-diphosphate-ribose polymerase (PARP) cleavage. Altogether, our findings suggest a novel function for apigenin, acting as a suppressor of AKT/PKB pathway in PEL cells, leading to the induction of caspase-dependent apoptosis. Therefore, apigenin may have a future therapeutic role in PEL and possibly other malignancies with constitutive activation of AKT/PKB pathway.
Role of Phosphatidylinositol 3- Kinase/AKT Pathway in Diffuse Large B-Cell Lymphoma Survival.
Authors: Uddin Shahab , Hussain Azhar R. , Bavi Prahant , Siraj Abdul K. , Al-Kuraya Khawla S.
Published: April 2020

Phosphatidylinositol 3-kinase (PI3-kinase) is a key player in cell growth signaling in a number of lymphoid malignancies including myeloma and primary effusion lymphoma. However, its role in diffuse large B-cell lymphoma (DLBCL) has not been elucidated. Therefore, we have studied the PI3-kinase pathway and apoptosis in a panel of DLBCL cell lines (SUDHL4, SUDHL8, SUDHL10 and OCI-LY19). Our data show that inhibition of PI3-kinase by a specific inhibitor, LY294002, induced apoptosis as detected by Annexin V/Propidium Iodide dual staining in the majority of DLBCL cell lines. We then dissected the PI3-kinase pathway by analyzing the downstream targets of phosphorylation by Western blot. We found that AKT/PKB was constitutively phosphorylated, and thus activated, in all DLBCL cell lines. The downstream elements of AKT, ForkHead (FKHR) and GSK3 were also constitutively phosphorylated in all DLBCL cell lines. Similarly, treatment with LY294002 prevented this phenomenon in all the cell lines regardless of their final apoptotic endpoint. Inhibition of PI3-kinase activity further downstream induced cleavage of Bid in all DLBCL cells and subsequently loss of mitochondrial membrane potential and release of cytochrome c from mitochondria in all DLBCL cell lines. The release of cytochrome C led to activation of Caspases 9 and 3 and cleavage of PARP. Finally expression of the inhibitor of apoptosis, XIAP, which is also a downstream target of AKT, was compromised in the all cell lines following LY294002 treatment. Our data demonstrate that the PI3-kinase pathway plays a major role in the survival and growth of DLBCL cells. Altogether, these results suggest that blocking the PI3-kinase pathway may be a potential target for therapeutic intervention in diffuse large B-cell lymphoma.
Loss of Expression of the Pro-Apoptotic Bcl-2 Family Proteins Bak and Bax in Rituximab- and Chemotherapy-Resistant Non-Hodgkin’s Lymphoma Cells.
Authors: Olejniczak Scott H. , Hernandez-Ilizaliturri Francisco J. , Clements James L. , Czuczman Myron S.
Published: April 2020

Sustained remissions following standard chemotherapy are achieved in less than half of patients diagnosed with non-Hodgkin’s lymphoma (NHL). A major case of treatment failure is the development of resistance to current therapies. Early studies demonstrated that rituximab was a safe and effective monotherapy for patients with indolent B-cell lymphoma refractory or relapsed following prior chemotherapy. In one study, Davis et al. demonstrated a loss of responsiveness upon rituximab re-treatment in 60% of patients suggesting the development of antibody resistance. To study mechanisms by which cells become resistant to rituximab we induced rituximab resistance in several well characterized B-cell lymphoma cell lines (Raji, RL, SU-DHL-4) by exposing them to increasing concentrations of rituximab or rituximab plus human serum as a source of complement. Individual clones were than generated from rituximab-resistant cell lines (RRCL) by limiting dilution. Characterization of the cell lines and clones generated by repeated exposure to rituximab revealed that in addition to gaining a rituximab-resistant phenotype they also developed concurrent chemotherapy resistance. In rituximab-sensitive parental cell lines, chemotherapy induced apoptosis via the intrinsic pathway. However, apoptotic cell death was completely blocked in resistant cell lines and clones. Upon investigation of potential mediators of both rituximab and chemotherapy resistance, we observed a significant down-regulation of the pro-apoptotic Bcl-2 family proteins Bax and Bak. We therefore hypothesize that resistance to chemotherapy- and rituximab-induced apoptosis is due to the down-regulation of Bax and Bak observed in all rituximab-resistant cell lines and clones characterized to date. We are currently investigating the mechanism(s) underlying the down-regulation of Bax and Bak protein in RRCL. Preliminary data suggests that wild-type Bax and Bak mRNA is expressed at a comparable level in RRCL and parental cells. This strongly suggests that Bax and Bak protein expression may be differentially controlled via post-transcriptional mechanisms in resistant cells. Currently, we are evaluating expression of Bax and Bak in archived lymphoma biopsy specimens and their correlation to treatment response or resistance. If a clinical correlation is identified, therapies aimed at restoring Bak and/or Bax expression may someday prove useful in circumventing clinical resistance to currently used immuno +/− chemotherapy-based regimens.
Mechanism of Acadesine-Induced Apoptosis in B-Cell Neoplasms.
Authors: Santidrián Antonio F. , Campàs Clara , Coll-Mulet Llorenç , Iglesias-Serret Daniel , Cosialls Ana M. , Defrias Mercé , Domindo Alicia , de Sevilla Alberto Fernandez , Pons Gabriel , Gil Joan
Published: April 2020

Mature B-cell neoplasms include B-cell chronic lymphocytic leukemia (B-CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and splenic marginal zone B-cell lymphoma (SMZL), among others. We found that acadesine, 5-aminoimidazole-4-carboxamide (AICA) riboside, induced apoptosis in B-cell neoplasms and in normal B lymphocytes, but FL samples were resistant. T cells from these patients or from healthy donors were not affected (Campas et al. Blood101: 3674, 2003; Campas et al. Leukemia19: 292, 2005). We have studied the mechanism of acadesine-induced apoptosis. Acadesine uptake and phosphorylation to ZMP are necessary to induce apoptosis. The best known molecular target of ZMP described to date is adenosine monophosphate-activated protein kinase (AMPK). We have analyzed the expression of LKB1, an AMPKK, AMPK isoforms and related proteins in B-CLL cells. B-CLL cells express LKB1, the α1 catalytic isoform of AMPK and both the β1 and β2 isoforms of the β regulatory subunit, but do not express the α2 isoform. Moreover, the AMPK related protein Snark is expressed in B-CLL cells, while Melk and Ark5 are not. Incubation of B-CLL, MCL, SMZL cells with acadesine induces phosphorylation in Thr172 and activation of AMPK, which correlates with induction of apoptosis. Acadesine enters FL cells as it induces phosphorylation of AMPK, however FL cells are resistant to acadesine-induced apoptosis. Moreover, phenformin and metformin induce AMPK phosphorylation but not induce apoptosis in B-CLL cells. The phosphorylation of AMPK is not sufficient for induction of apoptosis by acadesine in B-cell neoplasms. In B-CLL cells, CD40L or TPA, but not IL-4 or SDF1 inhibit acadesine-induced apoptosis. Acadesine inhibit neither AKT phosphorylation induced by TPA, IL-4, SDF1 or CD40L nor basal AKT phosphorylation. Finally, acadesine induces a conformational change in Bax protein, and citochrome c release to cytosol which is not block by ZVAD.fmk. In conclusion, although further studies are necessary to elucidate the mechanism of acadesine-induced apoptosis, we propose that acadesine may provide an effective treatment for mature B-cell neoplasms such as B-CLL, MCL and SMZL.
Adoptive Immunotherapy of p53-Mutated B-CLL with a Trifunctional Antibody (Anti-CD3 x Anti-CD20) and Allogeneic Donor Lymphocyte Transfusion.
Authors: Buhmann Raymund , Stanglmaier Michael , Simoes Belinda , Yang Ting , Ruf Peter , Lindhofer Horst , Kolb Hans-Jochem
Published: April 2020

In most cases CLL patients with p53 aberrations are refractory to standard treatment approaches, even to allogeneic stem cell transplantation (ASCT). Here we report 3 patients with relapse after ASCT that were treated with the combination of the trifunctional antibody Bi20 and donor lymphocyte transfusions (DLT). The Bi20 antibody is trifunctional, it binds to CD20 and CD3 and activates phagocytosis of the leukemia cell by accessory cells via the Fc part. Bi20 was applied in escalating doses from 10 μg up to 640 μg and followed by DLT (1 x 107/kg body weight). Patient 2 and 3 received repeated courses of antibody and DLT. In all cases we observed a prompt, dose-dependent decrease of B-CLL cells in the peripheral blood, which already started hours after application of the antibody infusion. Furthermore, in all cases the reduction of the enlarged lymph nodes and spleen went along with a significant improvement of the clinical symptoms like night sweat. Clinically, side effects were restricted to fever, chills and bone pain that could be easily controlled. These effects peaked at a concentration of 80 μg and did not increase or even decreased at higher concentrations. The cytokine profile was characterized by a transient increase of IL-6, IL-12 and IL-10. With respect to the transaminases, only a transient and modest increase of γGT was observed. HAMAs (human anti mouse antibodies) were not detectable; the absence allowed repeated transfusions of the trifunctional antibodies. Moreover, graft-versus-host disease (GVHD) was not observed. Unfortunately relapse of the disease occurred in all cases. In two cases repeated application of Bi20 and T-cells induced a repeated response. The overall survival was 47, 151 and 196 days. Bi20 can induce a prompt anti tumor response in B-CLL patients with a p53 mutation. The toxicity of treatment is not severe. However the response is of short duration and further studies are necessary to improve the outcome of patients by e.g. optimizing the application schedule.
In Vitro Activity of the Humanized Anti-HLA-DR Antibodies KRN848 and Apolizumab in Non-Hodgkins Lymphoma Cell Lines.
Authors: Hill Matthew L. , Weiner George J. , Link Brian K.
Published: April 2020

Apolizumab (Hu1D10) and KRN848 are humanized IgG1 anti-HLA-DR monoclonal antibodies (mAb). Apolizumab binds a polymorphic determinant on the HLA-DR-β chain, whereas KRN848 is a pan-HLA-DR mAb. We undertook preclinical studies to assess the properties of these two mAbs with regard to antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CMC) and their direct effects on target cells. HLA-DR (+) Raji and Daudi lymphoblastic cell lines served as target cells. KRN848 is reactive with HLA-DR expressed on both cell lines, while apolizumab is reactive only with Raji.

ADCC assays were performed using 51Cr-labeled target cells and normal donor peripheral blood mononuclear cells (PBMCs) as effector cells. At an E:T ratio of 40:1 and a mAb concentration of 2 μg/ml, all mAbs mediated ADCC. Apolizumab mediated greater specific lysis of Raji cells (37%) than did KRN848 (15%) or rituximab (14%). When Daudi cells were used as targets, apolizumab induced <3% lysis, while KRN848 and rituximab both induced 20% lysis. Similar findings were seen when EBV-transformed lymphoblastoid cells served as targets, and autologous healthy PBMCs as effector cells, with the degree of lysis induced by apolizumab varying depending on the degree of reactivity of apolizumab with the lymphoblastoid cells. In CMC assays, apolizumab, KRN848 and rituximab mediated comparable lysis of Raji cells (77%, 80% and 83%) at 2 μg/ml. In Daudi cells, there was <4% specific lysis with apolizumab, 45% lysis with KRN848 and 90% with rituximab.

Evidence for direct killing mediated by mAbs varied based on the assay used. When 51Cr-labeled Raji cells were incubated with KRN848 or apolizumab for 4–17 hours, and 51Cr in the supernatant used as a measure of cell death, there was minimal evidence (<10% specific lysis) of direct antibody-induced lysis over a range of concentrations (0.0016–2 μg/ml). In contrast, anti-HLA-DR mAbs had a significant effect on annexin-V/PI staining of target cells. Annexin-V/PI staining of Raji cells was positive after treatment with apolizumab (78%) and KRN848 (67%) at 2 μg/ml at four hours. KRN848 induced annexin-V/PI uptake by Daudi cells (39%) while apolizumab did not, demonstrating this effect was dependent on expression of the target antigen.

We conclude the anti-HLA-DR mAbs apolizumab and KRN848 can induce ADCC and CMC based on expression of target antigen. Anti-HLA-DR mAbs can directly alter the annexin-V/PI positivity of target lymphoma cells but do not induce 51Cr release, suggesting anti-HLA-DR mAbs alter cell membrane polarization and permeability but may not induce apoptosis directly.
Motexafin Gadolinium Causes Apoptosis in Lymphoma Cells by Increasing Intracellular Free Zinc and Disrupting Redox Balance.
Authors: Magda Darren , Lecane Philip , Karaman Mazen W. , Sirisawad Mint , Naumovski Louie , Miller Richard A. , Hacia Joseph G.
Published: April 2020

Motexafin gadolinium (MGd, Xcytrin®) is an anti-cancer agent that is being studied in clinical trials in various types of cancer including hematologic malignancies. MGd has been shown to generate reactive oxygen species and induce apoptosis in myeloma and lymphoma cells, and to cause an increase in intracellular levels of free zinc. We now report the effect of MGd and exogenous zinc on intracellular levels of free zinc, oxidative stress, proliferation, and cell death in human B-cell lymphoma and other hematologic cell lines. To better understand the molecular basis of these cellular responses, gene expression profiling analyses were conducted on Ramos cell cultures treated with MGd and/or zinc acetate. In cultures treated with MGd and zinc, 21% of Ramos cells were apoptotic within 8 hours of treatment as demonstrated by Annexin V staining. The apoptotic fraction increased to 30% within 12 hours and 68% by 24 hours. Analogous results were obtained using JC-1, with 38% of Ramos cells exhibiting non-aggregated (green) JC-1 fluorescence characteristic of mitochondrial dysfunction within 8 hours of combined treatment with MGd and zinc. This fraction increased to 52% by 12 hours and 74% by 24 hours. MGd and zinc similarly increased cell death in other B-cell lines (Raji, DB, DHL-4 and HF-1) tested. We also found that increased levels of oxidative stress and intracellular free zinc preceded and correlated with cell cycle arrest and apoptosis. Cultures treated with MGd or zinc acetate alone elicited transcriptional responses characterized by induction of metal response element-binding transcription factor-1 (MTF-1) and hypoxia inducible transcription factor 1 (HIF-1) regulated genes. Cultures co-treated with MGd and zinc acetate displayed further increases in the levels of MTF-1 and HIF-1 regulated transcripts and also additional transcripts regulated by NF-E2-related transcription factor 2 (NRF-2). HIF-1 activation can have negative consequences for cell growth and survival by induction of targets linked to apoptosis, whereas NRF-2 activation is characteristic of cells responding to disruption of redox balance. These data demonstrate that MGd increases intracellular free zinc and oxidative stress levels in lymphoma cells that activate adaptive survival responses but eventually lead to cell death.
Motexafin Gadolinium Enhances Rituximab-Induced Cytotoxicity Against B-Cell Lymphoma Cell Lines: Role of Elevated Intracellular Calcium.
Authors: Chen Jun , Ramos Jason , Sirisawad Mint , Miller Richard A. , Naumovski Louie
Published: April 2020

Motexafin gadolinium (MGd, Xcytrin®) is a tumor selective redox active drug that is directly cytotoxic to some hematolymphoid cell lines and chronic lymphocytic leukemia (CLL) patient samples. MGd has shown single agent activity in early phase II studies of lymphoma and CLL patients. Rituximab, an anti-CD20 antibody, is used widely in the treatment of B-cell malignancies. We evaluated the effects of MGd, rituximab and the combination in HF-1, a follicular lymphoma-derived cell line. Data analysis with CalcuSyn software revealed that the combination of MGd and rituximab showed synergistic growth inhibition and cytotoxicity compared to either agent used alone. MGd/rituximab activated a caspase-dependent apoptotic pathway as demonstrated by loss of mitochondrial membrane potential and PARP cleavage. Similar results were obtained with the combination of MGd and rituximab in DHL-4 and Ramos lymphoma cell lines. Since intracellular calcium ([Ca2+]i) levels have been implicated in rituximab cytotoxicity, we explored [Ca2+]i in rituximab and MGd/rituximab treated cells. Rituximab and MGd/rituximab treated cells have increased levels of [Ca2+]i. Elevation of [Ca2+]i with thapsigargin, an agent that releases calcium from internal stores or the ionophore A23187 that transports calcium into cells, results in synergistic cytotoxicity with rituximab or the MGd/rituximab combination. These in vitro findings suggest a role for [Ca2+]i in MGd/rituximab-induced cell death and support the combined use of MGd and rituximab in the treatment of B-cell lymphoma.
Identification of Genes That Potentially Allow Prediction of Interferon Response in Chronic Phase CML Diagnostic Samples through Gene Expression Analysis.
Authors: Barbany Gisela , Hagberg Anette , Strömberg-Olsson Ulla , Isaksson Anders , Bengtzon Mats , Höglund Martin , Simonsson Bengt
Published: April 2020

Interferon (INF) was the first drug to significantly prolong survival in chronic myeloid (CML) and has been extensively used to treat CML patients over the past years. Today, in the Imatinib era INF still has a role as additional agent in patients with a suboptimal response to Imatinib. However, not all patients will respond to INF and the reasons for this heterogeneity are largely unknown. The aim of the study was to identify biomarkers that allow prediction of future response to INF in CML diagnostic samples. Microarray-based gene expression analysis enables the study of thousands of genes in one single experiment and has been shown to be a useful tool to identify biomarkers that can assign samples to specific categories. Gene expression profiles in diagnostic samples from 15 CML chronic phase patients were analyzed using cDNA microarrays with 7458 genes. Blood samples were collected at diagnosis and all patients were treated with alpha-interferon. Seven patients were responders, defined as patients achieving a complete or major cytogenetic response at 12 months or earlier after initiation of interferon. Eight patients that had no cytogenetic response at 6–18 months were regarded as non responders. A list of 61 genes differentially expressed in both sample groups was generated by sorting those genes that showed the largest difference in median expression between the groups. The top 20 genes were subsequently used in an “all pair” selection procedure to identify those genes that are highly predictive of response to INF. The genes selected by this procedure were LTF, PRG2, JARID1A, NRGN, RNASE2 and DEFA4. The accuracy of these genes in predicting response to INF was determined in a “leave one out cross validation” procedure and the estimated error rate was found to be 0,13. The expression level of these six genes was verified with real-time PCR. The data generated by real-time PCR were used in a principal component analysis that separated the samples in two clusters.We conclude that it might be possible to use microarray-based gene expression analysis to predict future treatment outcome in CML diagnostic samples.
In Vitro and In Vivo Antileukemic Effects of the mTOR-Targeting Drug Rapamycin in Patients with Imatinib-Resistant CML.
Authors: Mayerhofer Matthias , Boehm Alexandra , Aichberger Karl J. , Esterbauer Harald , Derdak Sophia , Pickl Winfried F. , Sillaber Christian , Valent Peter
Published: April 2020

The mammalian target of rapamycin (mTOR) has been described to be activated in BCR/ABL-transformed cells and to mediate rapamycin-induced inhibition of cell growth. We have recently shown that rapamycin downregulates expression of vascular endothelial growth factor (VEGF), a mediator of leukemia-associated angiogenesis, in primary CML cells. In the current study, we analyzed growth-inhibitory in vitro- and in vivo effects of rapamycin in leukemic cells in patients with imatinib-resistant CML, and asked, whether the rapamycin-induced suppression of VEGF in these cells is related to growth-inhibition. Rapamycin dose-dependently inhibited the in vitro growth of primary CML cells in all pts with imatinib-resistant CML tested (rapamycin, 10 nM: 50±17% of control; p<0.05). Moreover, rapamycin was found to downregulate growth of Ba/F3 cells exhibiting wild type (wt) BCR/ABL or various imatinib-resistant mutants of BCR/ABL including the T315I-mutation, which renders BCR/ABL resistant against tyrosine kinase inhibitors. We also found that rapamycin and imatinib exert synergistic inhibitory effects on growth of Ba/F3p210WT cells as well as on Ba/F3 cells containing imatinib-resistant mutants of BCR/ABL. In all cells tested including imatinib-resistant primary cells, rapamycin was found to downregulate expression of VEGF mRNA and secretion of the VEGF protein. However, exogenously added VEGF did not counteract the rapamycin-induced decrease in proliferation suggesting that these two effects of rapamycin are separable. In a consecutive phase of the project, we treated 7 pts with imatinib-resistant CML with rapamycin at 2 mg per os daily for 14 consecutive days, with dose-adjustment allowed to reach a target serum rapamycin concentration of 10–20 ng/ml. A major leukocyte response with decrease from >20,000 to normal values (<10,000/μL) was obtained in 3 pts, and a minor transient response in 2 other pts, whereas 2 pts did not respond. No molecular or cytogenetic responses were seen. No severe side effects of rapamycin were recorded. In aggregate, rapamycin may be an interesting targeted drug in imatinib-resistant CML. However, combinations with other targeted (or conventional) drugs may be required to obtain sufficient antileukemic activity in vivo.
Monitoring of Chronic Myeloid Leukemia (CML) Imatinib Response by Fluorescence-In-Situ-Hybridization (FISH).
Authors: Yip Sze Fai , Wan Thomas Shek Kong , Lie Albert Kwok Wai , Liu Herman Sung Yu , Chan Li Chong
Published: April 2020

Glivec, a tyosine kinase inhibitor is the drug of choice for newly diagnosed chronic phase CML. Whilst Q-PCR is the most sensitive method to monitor response; cytogenetic analysis as well as FISH is routinely used in many laboratories. FISH has the advantage over marrow cytogenetic by allowing analysis of more cells, and cells in interphase, obviating necessity for an invasive marrow procedure. We performed 126 FISH studies using BCR-ABL dual color dual fusion translocation probe for 69 CML patients on imatinib. For each samples, 300 nucleated cells were counted. Clinical information was available from 57 patients (34 chronic-phase (CP), 7 accelerated phase (AP), 8 blastic phase (BT) and 8 post-BMT relapses). Most CP patients (94%) could achieve sustained complete hematological responses (CHR), including 14 of the 15 patients pre-treated with IFN. IFN pre-treated patients received imatinib generally late in the course of CP compared to those without (median 19M vs 0.8M, p<0.001). At 12–15 months, 61% CP patients were FISH-ve. Fewer IFN-pretreated patients achieved FISH-ve responses (41.7% vs 90%, p=0.03). With a mean follow-up of 25 months (4–55M), none of the FISH-ve patients had disease transformation while AP/BT developed in 2 FISH+ patients (0% vs 22%, p=0.14). In CML-AP, early institution of imatinib (1–10M) achieved more FISH-ve responses than late treatment (30–36M) and degree of FISH response after 6–12 months of imatinib in CML-AP appeared to predict prognosis. For patients treated in BT, 4 received imatinib upfront and 4 after remissions or reversion to CP after chemotherapy. FISH negativity in the first few months of imatinib treatment, however, did not predict better prognosis. Two patients remained FISH-ve and disease-free 52 months without transplant, suggesting imatinib may be useful as maintenance after chemotherapy induction for BT patients not eligible for transplant. The observation that imatinib after chemotherapy induction has better prognosis than imatinib upfront requires confirmation by larger studies. In post-BMT relapses (8 patients: 1 cytogenetic relapse, 4 CP relapses, 1 blastic relapse, 2 AP relapses), hematological response could be achieved in all. Half became FISH-ve at 6–9 months and remained so at latest follow-up (44–56M). Three patients (1 CP, all 2 AP) were FISH+ at 6–9 months (42–90% Ph+ cells) and remained FISH+ thereafter, with one progressed to blastic disease at 24 months. FISH response at 6–9 months, thus, appears to carry prognostic significance in post-transplant CML relapses. In summary, our initial experience suggests monitoring of imatinib response by FISH provides useful prognostic information in different stages of CML.
RANBP2/ALK Rearrangement Is a New Rare but Recurrent Fusion Gene in Childhood Myeloid Disorders.
Authors: Harbott Jochen , Bruch Jochen , Busch Kerstin , Gammerdinger Ulrike A. , Rottgers Silja , Teigler-Schlegel Andrea , Viehmann Susanne
Published: April 2020

The ALK gene which is typically rearranged in anaplastic large cell lymphoma (ALCL) and inflammatory myofibroblastic tumor (IMT) is located in the band 2p23. An involvement of this band was detected in 6 patients (0.35%) when we checked the cytogenetic database of our laboratory with approximately 1500 successfully analyzed karyotypes of childhood AML and MDS. Patient 1 had a t(2;4)(p23;q27) and patient 2 showed a del(2)(p23). In both cases, the 2p23 abnormality was part of a complex karyotype that included several non-specific alterations. Among the remaining four children, three (pts. 3–5) showed an inv(2)(p23q13) and patient 6 a t(2;2)(p23;q13). We performed a hybridization using the LSI ALK Dual Color Break Apart Rearrangement Probe (Vysis, Downers Grove, IL) onto all six patients. Whereas in the nuclei of pts 1, 2, 3 two normal fusion signals were found, a split hybridization signal showed the ALK involvement in the inv(2) and the t(2;2) of patients 4, 5 and 6. The latter case was further analyzed using chromosome arm-specific painting probes and this approach also confirmed the translocation. For the identification of the partner gene we used the primers for RANBP2/ALK which were described for IMT. In all patients with ALK involvement a PCR product was found and the sequencing identified identical breakpoints within the ALK and RANBP2 gene. In addition, a FISH and RT-PCR screening was performed in 20 children with AML or MDS, but no ALK involvement could be detected. All 3 ALK rearranged patients were originally diagnosed as myeloid disease (AML/MDS), but showed different unspecific morphological features. To the best of our knowledge this is the first description of an inv(2)(p23q13) and a t(2;2)(p23;q13) and the first report of an RANBP2/ALK rearrangement in childhood AML/MDS. Further studies will help to elucidate the role of this rearrangement in pediatric leukemia.
Bone Marrow-Derived, BCR/ABL+ and Flk1+CD31−CD34− Stem Cells from CML Patients That Are Insensitive to STI571 In Vitro.
Authors: Song Yongping , Fang Baijun , Zhang Yanli , Wei Xudong , Lu Lulu , Zhu Xinhu , Wang Ping , Li Yufu , Gao Quanli , Wang Guiye , Qiu Lugui , Zhao Chunhua
Published: April 2020

To evaluate the effect of imatinib mesylate (STI571, Gleevec) on primitive/committed malignant progenitor cells in chronic myelogenous leukemia (CML) and to further elucidate the mechanisms involved in CML relapse, bone marrow-derived, malignant, BCR/ABL+Flk1+CD31CD34− cells with hemangioblastic characteristics from CML patients were labeled with carboxy-fluorescein diacetate succinimidyl diester dye to enable high-resolution tracking of cell division. Then they were cultured in Methocult GF+ media with or without STI571. After culture, the cells were separated by fluorescence-activated cell sorting into populations of viable quiescent versus cycling cells. Then the mechanisms involved in CML stem cells that became resistant to STI571 were studied. We found that in the most sensitive cases, STI571 killed almost all dividing cells; however, a significant population of viable BCR/ABL-positive cells were recovered in the undivided peak and confirmed to be the leukemic stem cells. STI571 also appeared to exhibit antiproliferative activity on the quiescent population. We further demonstrate that apoptosis was restricted to dividing cells, whereas nonproliferating BCR/ABL+Flk1+CD31CD34− cells were resistant to apoptosis induced by imatinib, Ara-C, VP-16, ceramide, or arsenic, either alone or in combination. These studies suggested that CML primitive stem cells remain viable in the presence of STI571 and that inhibition of BCR/ABL tyrosine kinase by STI571 restores normal hematopoiesis by removing the proliferative advantage of CML committed progenitors but that elimination of all CML progenitors may not occur. So despite dramatic short-term responses in vivo, such in vitro insensitive to STI571, might translate into disease relapse after prolonged therapy.