bluuebunny/crossref_metadata_2025 huggingface dataset:
Microarray Analysis of PU.1-Induced Growth Arrest and Apoptosis of Myeloma Cell Lines.
Authors: Ueno Shikiko , Tatetsu Hiro , Harada Naoko , Hata Hiroyuki , Iino Tadafumi , Niiro Hiroaki , Akashi Koichi , Tenen Daniel G. , Mitsuya Hiroaki , Okuno Yutaka
Published: April 2020

PU.1 is an Ets family transcription factor, which is important for differentiation of granulocytes, monocytes/macrophages, and B cells. In the Friend leukemia model, it is reported that the failure of PU.1 down-regulation in erythroblasts reportedly results in differentiation arrest that leads to erythroleukemia. In conditional knockout mice of the 3.5 kb length of enhancer region located in14 kb 5′ of the PU.1 gene, PU.1 is down-regulated in myeloid cells and B cells down to 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like disease. In addition, a deletion of the 3.5 kb enhancer region, which also contains the suppressor region for PU.1 in T cells, results in ectopic expression of PU.1 in T cells, which leads to T cell lymphoma in those mice. Taken together, the failure of up-regulation or down-regulation of PU.1 in certain differentiation stages for each lineage appears to cause differentiation arrest and hematological malignancies. We recently reported that PU.1 is down-regulated in a majority of myeloma cell lines through the methylation of the promoter and enhancer region located in17 kb 5′ of human PU.1 gene which is homologous to that in14 kb 5′ of murine PU.1 gene. Conditionally expressed PU.1 induced cell growth arrest and apoptosis of those PU.1 low-negative myeloma cell lines, U266 and KMS12PE, suggesting that down-regulation of PU.1 is necessary for myeloma cell growth. In addition, we reported that PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of some myeloma patients. Myeloma patients with low-to-negative PU.1 expression (lower 25th percentile of PU.1 expression level distribution among 30 patients we examined) may have poor prognosis compared to those with high PU.1 expression, although more patient samples have to be examined to define the significance of the relationship of PU.1 expression levels and prognosis. To elucidate the mechanisms of PU.1 induced cell growth arrest and apoptosis of myeloma cells, we next performed DNA microarray analysis to compare gene expression levels before and after PU.1 induction. We utilized Illumina Sentrix® Human-6 Expression BeadChip. Of 47296 genes, 479 genes were up-regulated (>2fold) and 1697 genes down-regulated (<0.5 fold) either day 1 or 3 after PU.1 induction in U266 cells. Among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. Stably expressed siRNA for TRAIL partially inhibited apoptosis of U266 cells expressing PU.1, suggesting that TRAIL is related to PU.1 induced cell death of U266 cells. Among cell-cycle related genes, p21WAF1/CIP1 was found up-regulated in U266 cells, which was confirmed with protein levels. We are now examining the roles of the observed up-regulated genes in both U266 and KMS12PE myeloma cell lines.
Role of GSK3 in Multiple Myeloma Cell Growth and Survival.
Authors: Piazza Francesco A. , Gurrieri Carmela , Chioetto Gino , Colpo Anna , Bonanni Luca , Montini Barbara , Tubi Laura Quotti , Gnoato Marianna , Carraro Samuela , Cabrelle Anna , Trentin Livio , Zambello Renato , Semenzato Gianpietro
Published: April 2020

The serine-threonine GSK3 displays a crucial role in different cancer-pathogenetic pathways, including the PI3K/AKT, Wnt β-catenin and NF-κB signaling cascades, either promoting or counteracting cell survival. The aim of this study was to investigate the role of GSK3 in multiple myeloma (MM) cell growth. GSK3α and β total and phosphorylated protein levels were found differentially expressed in malignant plasma cells as compared to normal resting B-lymphocytes and to normal in vitro generated plasmablasts. Intriguingly, in freshly isolated malignant plasmacells from patients, most of GSK3 was found colocalized with Wnt receptor LRP6 and casein kinase I next to the cell membrane as compared to normal plasmacells or B-cells from other malignancies, wher it was distributed in the cytosol and in the nucleus, thus suggesting a peculiar role of this kinase in these cells. Upon stimulation of MM cells with IL-6 and IGF-I, GSK3 enzymatic activity was hampered, while stimulation with TNFα did not affect GSK3 function nor the early events in NF-κB activation. Basal and IL-6 and IGF-I driven proliferation of MM cells was slightly impaired by GSK3 blockade. Interestingly, GSK3β−/− mouse embryo fibroblasts (MEFs) proliferated slightly slower as compared to GSK3β+/+ cells; however, GSK3α inhibition and IL-6 and IGF-I stimulation, resulted in much higher proliferation of GSK3β −/− cells. Intriguingly, GSK3 inhibition with specific compounds (SB216763 and SB415286) caused a significant rescue from cell death of growth factor-deprived MM cells while resulted in reduced cell proliferation and apoptosis of MM cells grown with serum or growth factors. When GSK3 inhibitors were added to MM cell cultured with bone marrow stromal cells (BMSC), MM cells survival increased and NF-κB and β-catenin-mediated gene transcription (of IAPs and cyclinD1, respectively) was deregulated. GSK3 activity inhibition did not modify the rate of proteasome inhibitor-induced cell death in co-colture experiments with BMSC, suggesting that the sensitivity to bortezomib-induced MM cell apoptosis is independent on GSK3. Altogether, our data indicate thatGSK3 localizes on the cell membrane in primary MM cells;GSK3 is differently regulated downstream from growth factors or TNFα-induced signaling pathways in MM cells;a peculiar role of GSK3 in malignant MM cells as compared to normal MEFs with regard to cell proliferation; anda critical role of this kinase in regulating the MM microenvironment.
Pre-Clinical Validation of Polo-Like Kinase 1 as a Therapeutic Target in Multiple Myeloma with the Selective Inhibitor BI2536.
Authors: Sidhu Roger , Steffler Tara , Hipperson Gail , Jung Michelle , Fung Joyce , Pilarski Linda M. , Belch Andrew R. , Reiman Tony
Published: April 2020

BACKGROUND: The centrosome is the cellular organelle that nucleates the mitotic spindle. Polo-like kinase 1 (Plk1), a centrosome-associated serine/threonine kinase, serves as a key regulator of multiple steps in mitosis. Plk1 is overexpressed in a broad spectrum of tumor types, and its expression often correlates with poor patient prognosis. We have previously observed centrosome amplification in myeloma, and have validated other components of the myeloma centrosome and mitotic apparatus as therapeutic targets. The present study explored the expression of Plk1 in myeloma and the effect of BI2536, a potent and selective inhibitor of Plk1, on myeloma cells in the pre-clinical setting.

METHODS: Plk1 expression was assayed in a panel of myeloma cell lines (MMCL) and AutoMACS-purified CD138+ patient bone marrow plasma cells (BMPC). The anti-myeloma effects of BI2536, alone or in combination with conventional agents, were assayed on myeloma cells with proliferation (MTS) and apoptosis (Annexin V/propidium iodide) assays. The phenotype of treated cells was examined with DNA content analysis and immunofluorescence microscopy. The efficacy of BI2536 monotherapy was evaluated in NOD/SCID mice bearing RPMI8226 xenografts.

RESULTS: Plk1 is ubiquitously expressed in myeloma to varying degrees in both MMCL and BMPC. BI2536 inhibited the proliferation of MMCL (RPMI 8226, U266, LP-1 and KMS-11) and patient BMPC at nanomolar concentrations. The addition of BI2536 was able to overcome resistance to dexamethasone. Bortezomib in combination with BI2536 had significantly increased anti-myeloma effects compared to the use of either agent alone. BI2536-treated MMCL accumulated 4N DNA content prior to undergoing apoptosis. The phenotype of BI2536-treated cells is consistent with inhibition of Plk1, showing prometaphase arrest and monopolar mitotic spindles in a dose-dependent fashion. BI2536 induces regression of human myeloma xenografts in NOD/SCID mice. Taken together, BI2536 is a promising new agent for the treatment of multiple myeloma. This work provides further evidence that Plk1 and the amplified myeloma centrosome are targets for therapy.
Cucurbitacin I (JSI-124) Has Potent Anti-Myeloma Effects Independent of Its Inhibition of JAK2-Induced STAT3 Activation.
Authors: Ma Huihui , Ziegler Judith , Feng Ren-Tien , Lentzsch Suzanne , Mapara Markus
Published: April 2020

Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. JSI-124 is a plant natural product identified previously as Cucurbitacin I, isolated from various plant families such as the Cucurbitaceae and Cruciferae and has been recently described as a specific inhibitor of Janus Kinase-2 (JAK-2)/signal transducer and activator of transcription-3 (STAT3). Based on the critical role of the IL-6/STAT3 pathway in MM we studied the effects of JSI-124 on different MM cells in vitro. Different human myeloma cell lines including MM1.S, IM9, OPM-2, RPMI-8226, ARH77 in addition to the murine 5TGM myeloma cell lines were incubated with increasing concentrations of JSI-124. The impact of JSI-124 on cell proliferation, cell cycle and induction of apoptosis was studied using [3H]-Thymidine incorporation, cell counting, flow cytometry and Annexin/PI staining and caspase 3, 8 and 9 activation. JSI-124 was able to inhibit proliferation and induce apoptosis in several MM cell lines, including MM1.S, IM9, OPM-2, RPMI-8226, ARH77, U266 and 5TGM in a dose- and time-dependent manner. JSI-124 lead to activation of caspase 3, 8 and 9 indicating involvement of both extrinsic and intrinsic apoptotic pathways. JSI-124-mediated induction of apoptosis was independent of JAK2/STAT3 inhibition as MM1.S and 5TGM cells, which lack constitutive STAT3 (Tyr705) activation were equally sensitive to JSI-124 compared to U266 cells which show a constitutive STAT3 Tyr705 phosphorylation. However, JSI-124 treatment was able to abrogate IL-6 and bone marrow stroma (BMSC)-induced STAT3 (Tyr705) activation in MM1.S cells. In addition we were able to observe JSI-124 dependent inhibition of constitutive STAT3 (Ser727) activation in MM cell lines. To further delineate the mechanism underlying its anti-myeloma effects we studied the impact of JSI-124 treatment on NF-κB, MAPK and PI3K pathways. Indeed, JSI-124 treatment resulted in inhibition of p-p65, p-MEK1,2 and p-Akt underscoring the effect of JSI-124 on STAT3-independent signaling. Our results indicate that JSI-124 is a powerful direct inhibitor of myeloma cells blocking constitutive and IL-6/BMSC-dependent STAT3 activation in addition to STAT3 independent signaling pathways. JSI-124 might therefore serve as a potent novel anti-myeloma agent targeting both myeloma cells and its bone marrow microenvironment. Further studies are warranted to evaluate the in vivo efficacy of JSI-124 and identify the STAT3 independent pathways contributing to myeloma cell growth and induction of apoptosis.
Concordance of Assays Designed for the Quantitation of JAK2 1849G>T (V617F): A Multi-Centre Study.
Authors: Lippert Eric , Girodon François , Hammond Emma , Carillo Serge , Richard Céline , Fehse Boris , Hermans Mirjam , James Ian , Jelinek Jaroslav , Marzac Christophe , Migeon Marina , Pietra Daniela , Prchal Josef T. , Reading N. Scott , Sobas Marta , Ugo Valérie , Skoda Radek C. , Hermouet Sylvie
Published: April 2020

Studies of myeloproliferative disorders (MPDs) aiming to evaluate the fraction of the clone bearing the JAK2 1849T mutation occasionally report discordant findings. One reason could be different sensitivity and accuracy of the various assays designed for the detection and quantitation of JAK2 1849G>T. We studied the concordance of 10 published JAK2 1849G>T assays. 29 samples of genomic DNA were distributed to 14 laboratories in France, USA, Australia, Germany, Holland, Italy and Switzerland for blinded assessment of JAK2 1849T levels. DNA was extracted from granulocytes of patients diagnosed with MPD, eosinophilia or secondary polycythemia. The 10 assays tested included 5 TaqMan assays with specificity based on primers (4) or competing probes (1) and allele-specific PCR (AS-PCR) (3), pyrosequencing (1) and FRET/melting curve (1) assays. Standards used for calibration were dilutions of DNA from plasmids (3 sets), cell lines or patient granulocytes. Results were expressed as %1849T allele/total JAK2 (13 centres) or as %1849T allele/control gene (1 centre). One centre had one false negative result; there were no false positive results. PCR equipment did not significantly affect the quantitation of 1849T: after adaptation of the technique, one centre tested one AS-PCR assay on 2 apparatus, 5 other centres tested one TaqMan assay on 3 apparatus; comparable results were obtained in the 6 centres. For 6 assays (10 centres), quantitation in the 26 positive samples, ranging from 1% to 96%, did not differ significantly. Overall variation was 30%; concordance improved with increasing mutational load (18% variation for samples with >8% 1849T). Three TaqMan and 1 AS-PCR assays gave significantly different results, 2 with overall low quantitation. For 3 assays, discordance was explained by an incorrect estimation of 1849T content in the standards. For the 4th discordant assay, expressing results as % 1849T/control gene, values tended to be higher proportional to the consensus. Interestingly, results were consistent with the presence of >2 copies of JAK2 per cell in 4 samples. The study underlined the importance of using defined standards when analysing JAK2 1849T levels. After adaptation to the equipment and with the use of correct standards, all assays gave comparable quantitation of JAK2 1849T, with a sensitivity <1%. Finally, quantitation of a second gene, in order to detect additional copies of JAK2 (>2/cell), should be considered.
JAK2 Mutations at Exon 12 and 14 in Polycythemia Vera and Idiopathic Erythrocytosis: Incidence and Correlation with Clinical Characteristics.
Authors: Besses Carlos , Martínez-Avilés Luz , Alvarez-Larrán Alberto , Cervantes Francisco , Hernández-Boluda Juan Carlos , Colomer Dolors , Florensa Lourdes , Serrano Sergi , Bellosillo Beatriz
Published: April 2020

Introduction. Exon 12 mutations of the JAK2 gene have been described in polycythemia vera (PV) and idiopathic erythrocytosis (IE) patients. These patients display a clinical phenotype different to that observed in V617F-positive PV patients, but no information is available on their clinical outcome.

Patients and methods. Twenty JAK2 V617F-negative PV or IE patients and 86 V617F-positive PV patients were assessed for exon 12 and exon 14 mutations. Analysis of cell lineage mutational status was assessed following cell sorting. Aim. To analyze the presence of JAK2 mutations at exon 12 and 14 in a cohort of V617F-negative PV and IE patients and to correlate them with the patient clinicohematological and evolutive data.

Results. Exon 12 mutations were detected in 4 (20%) V617F-negative patients (K539L, two cases, N542_E543del and H538_K539delinsL, one case each). In 16 patients, no mutations were found in either exon 12 or exon 14. Additional mutations in exon 14 were found in two V617F-positive patients. Two patients with exon 12 mutations developed thrombosis after diagnosis, with the probability of thrombosis-free survival being 75% at 5 years. Another patient with exon 12 mutation developed myelofibrosis at 20 years of PV diagnosis. JAK2 mutations were present in granulocytes, platelets and monocytes, but not in lymphocytes or NK cells.

Conclusions. Patients harboring exon 12 mutations represent a subset of JAK2 V617F-negative PV or IE patients. These patients have initial hematological data different from V617F-positive patients, but they do not differ with regard to thrombosis development and myelofibrotic transformation.

Table 1: JAK2 exon 12 and exon 14 mutations in blood cell subpopulations. Mutation Granulocytes Platelets CD14+ CD3+ CD19+ CD56+ * exon 12; # exon 14; + indicates the presence of the mutation; − indicates no detectable mutation ; ND not determined Patient 1* K539L (AAA →TTA) + + − − − − Patient 2* K539L (AAA →CTA) + + + − − − Patient 3* H538_K539delinsL + + ND − − ND Patient 4* N542_E543del + + + − − − Patient 5# V617F (GTC →TTT) + + − − − ND C618R (TGT →CGT) + + − − − ND Patient 6# V617F (GTC →TTC) + + + − − ND C616C (TGT →TGC) + + + − − ND
mTOR Inhibition for Relapsed or Refractory Hodgkin Lymphoma: Promising Single Agent Activity with Everolimus (RAD001).
Authors: Johnston Patrick B. , Ansell Steven M. , Colgan Joseph P. , Habermann Thomas M. , Inwards David J. , Micallef Ivana N.M. , Porrata Luis F. , Reeder Craig B. , Roy Vivek , LaPlant Betsy R. , Witzig Thomas E.
Published: April 2020

Background: mTOR inhibition has produced responses in mantle cell lymphoma as well as other non-Hodgkin lymphomas. This phase II study tested the oral mTOR inhibitor everolimus (RAD001, Novartis Pharmaceuticals) in simultaneous two-stage phase II lymphoma studies, including one arm involving Hodgkin lymphoma. The goals were to learn the toxicity profile and to assess the anti-tumor response. A total of 17 patients with Hodgkin lymphoma were enrolled in the uncommon arm at completion of enrollment.

Methods: Patients (pts) received 10 mg PO daily for each 28 day cycle (up to 12, with a possible extension in responders) and restaged after 2, 6, and 12 cycles. The primary endpoint is the confirmed response rate, including CR, CRu or PR.

Results: The median age of the 17 pts with Hodgkin lymphoma was 37 yrs (range: 27–68), with a median of 6 (range, 4–14) prior therapies. Fourteen pts (82.4%) had a prior stem cell transplant (SCT). Pts completed a median of 6 (range, 1–13) cycles of therapy. Fifteen of 17 patients were evaluable for response as of this analysis. The overall response rate was 47% (7/15), all partial responses. Ten patients are continuing on study while 6 have gone off due to disease progression and 1 due to other reasons. Common grade 3 adverse events (AEs) include thrombocytopenia (5 pts), anemia (5 pts) and alkaline phosphatase elevation (1 pts). 1 patient was reported to have grade 4 neutropenia.

Conclusions: Oral everolimus has promising activity with acceptable toxicity in Hodgkin lymphoma. These results provide the rationale for additional studies with this novel class of agents and to integrate mTOR inhibitors into salvage treatment regimens for Hodgkin lymphoma.
Vorinostat (Suberoylanilide Hydroxamic Acid) in Relapsed or Refractory Hodgkin Lymphoma: SWOG 0517.
Authors: Kirschbaum Mark H. , Goldman Bryan H. , Zain Jasmine M. , Cook James R. , Rimsza Lisa M. , Forman Stephen J. , Fisher Richard I.
Published: April 2020

Background: Patients with Hodgkin’s Lymphoma with relapse or refractory disease after salvage therapy and transplant face a paucity of active agents. New modalities of treatment are necessary. Vorinostat (SAHA) is an orally administered hydroxamic acid histone deacetylase inhibitor with activity against class I and II deactylases. After initial phase I data (Kelley WK, et al 2005) showed prolonged stable disease in patients with Hodgkin’s, a Phase II study of this agent was launched in patients with relapsed or refractory Hodgkin’s lymphoma.

Methods: Eligible patients had relapsed or refractory Hodgkin’s lymphoma, bidimensionally measurable disease, and performance status 0-2. Patients may have received up to five prior chemotherapy regimens; previous autologous transplant is allowed. Vorinostat was dosed at 200 mg po twice daily for 14 consecutive days on a 21 day cycle. CT scanning was performed after every three cycles, as was marrow biopsy for those with marrow involvement at time of entry into study. This was a 2-stage design with a maximum planned accrual of 35 eligible patients. Objective response was the primary endpoint.

Results: Of 27 patients accrued to the first stage of this study, 25 are eligible (14 males, 11 females). Median age is 41 (19–71) years. Toxicities attributable to drug of grade 3 or higher include fatigue, anorexia, anemia, and thrombocytopenia. One partial response was observed, for a response probability of 4% (95% CI: 0%, 20%). Although there were not adequate responses to open the second stage of accrual, four patients had stable disease lasting at least one year. Of these four, one opted for radiation at 12 months, another progressed at 14 months, whereas two continue on treatment, one with bulky disease who is stable at 12 months, one with symptomatic lung involvement and bulky disease who remains stable at 16 months. One additional patient remains on treatment with stable disease at 9 months.

Conclusions: Although the low response rate led to closure of this trial after the first stage of accrual, we observed that treatment with the oral histone deacetylase inhibitor Vorinostat can lead to protracted stable disease in patients with relapsed/refractory Hodgkin’s Lymphoma. Studies in combination with other molecular agents or chemotherapy may be warranted.
Arsenic Trioxide (As2O3) Mediated Cell Death in Hodgkin Lymphoma (HL) and Non-Hodgkin Lymphoma (NHL): Investigation of Reactive Oxygen Species (ROS), Caspase, and MAP Kinase Signaling Pathways.
Authors: Bhalla Savita , Singh Amareshwar T.K , Prachand Sheila , Haque Qudsia , O’Halloran Thomas V. , Winter Jane N. , Schumacker Paul T. , Platanias Leonidas C. , Gordon Leo I. , Evens Andrew M.
Published: April 2020

The cytotoxic activity of As2O3 in leukemia and myeloma has been shown to be mediated in part by ROS. We determined the mechanism of cytotoxicity of As2O3 in HL and NHL cell lines. Ramos, HF-1, SUDHL4 (NHL lines), and L428 (HL line) cells were cultured with increasing clinically relevant As2O3 concentrations (1.0mm-10mm) at 24-72 hours with and without the oxidizing agent, buthionine sulfoxime (BSO, 100mm), and with and without caspase inhibitors Z-VAD-FMK and Z-IETD-FMK. Apoptosis was measured by Annexin-V/propidium iodide (PI) staining and detected by flow cytometry (FACS). ROS was measured by oxidation of 2′7′ dichlorofluorescein diacetate (H2DCFDA) to Dichlorofluorescein (DCF) and analyzed by FACS. Immunoblots were performed for bcl-2, PARP, cleaved caspases 3, 8, 9, and mitogen activated protein kinase (MAPK) pathways (ERK, JNK, and p38 activation). As2O3 alone at 10mM resulted in dose- and time-dependent apoptosis in all cell lines (HL and NHL) with >75% annexin+/PI+ between 48 and 72 hours. In Ramos and L428 cells, a combination of As2O3 (2 mM) and BSO 100mM resulted in >80% annexinV+/PI+, while either agent alone resulted in <15% apoptosis (p=0.001). A four-fold increase in ROS was seen in all four cell lines with As2O3/BSO, but not with As2O3 alone. Both ROS production and apoptosis induced by As2O3/BSO and As2O3 alone were reversible with the anti-oxidant N-acetylcysteine (NAC) (10mM). Furthermore, As2O3/BSO induced PARP cleavage and caspase-3 activation in all NHL cell lines, but not in L428. In Ramos, Z-VAD-FMK and Z-IETD-FMK blocked As2O3-induced apoptosis (suggesting a caspase-dependent mechanism), but had no effect on As2O3/BSO-induced cell death (caspase-independent). To investigate the signaling pathways involved, we performed western blot analysis for phospho-p38 (p-p38), phospho-JNK and phospho-ERK (p-ERK) in Ramos and L428 cells following As2O3 alone and As2O3/BSO. We found up-regulation of p-p38 in Ramos cells, while in L428, p-ERK was activated following As2O3 and As2O3/BSO. Inhibitors of p-38 in Ramos cells resulted in up-regulation of ERK. In summary, As2O3 induced dose- and time-dependent apoptosis in HL and NHL cell lines was significantly enhanced with the addition of BSO, and inhibited by NAC, suggesting that apoptosis was in part ROS-dependent. In Ramos, As2O3 alone resulted in caspase-dependent apoptosis, while addition of BSO resulted in caspase-independent apoptosis. As2O3 alone and combined with BSO resulted in activation of MAPK pathways in HL and NHL lines. These data provide a basis and a mechanism for As2O3-induced cell death in HL and NHL and have therapeutic implications.
Long Term Outcome of Gastric MALT Lymphoma Patients Treated with Anti-Helicobacter (Antibiotic and Proton-Pump Inhibitor) Regimens.
Authors: Chini Claudio , Pinotti Graziella , Stathis Anastasios , Proserpio Ilaria , Bertoni Francesco , Cavalli Franco , Zucca Emanuele
Published: April 2020

Background: Primary mucosa-associated lymphoid tissue (MALT) lymphoma of the stomach is often associated with Helicobacter pylori (Hp) infection and Hp eradication therapy is widely accepted as initial treatment. The aim of this study is to report the clinical characteristics and long term outcome in a large series of patients with gastric MALT lymphoma exclusively treated with Hp eradication therapy.

Methods: 105 newly diagnosed gastric MALT lymphoma patients (54 men and 51 women) with median age of 64 years (range 20–94) referred to our institutions between June 1990 and November 2006 were eligible for the study. Staging was performed according to the Lugano staging system. All patients received anti-Hp eradication therapy as initial treatment with standard regimens combining antibiotics (usually Amoxicillin, Clarithromycin and/or Metronidazole) and proton-pump inhibitors (Omeprazole in most cases). Responses, evaluated with regular endoscopic biopsies every 3–6 months were graded according to the Wotherspoon’s histological score system.

Results: 100 patients had stage I and 5 patients had stage IIE1 disease. Hp was positive in 83 patients (79.5%) and was eradicated in all positive patients but 19 patients required a second line antibiotic therapy. Symptoms disappeared or markedly diminished and endoscopic features improved in almost all patients after Hp eradication. Histological regression of the gastric lymphoma was achieved in 78 of 102 evaluable patients (76%, 95% C.I.; 67%–84%) with histological complete response (Wotherspoon’s score 0–2) in 66 and partial response (score 3) in 12 patients. Of the 78 patients who achieved a lymphoma regression, the histological remission was consistently confirmed at follow-up endoscopies in 28 patients (36%), while 33 (42%) had histological score fluctuations (from 0–4), sometimes with transient histological relapses followed by spontaneous histological remissions. Ten patients had a frank lymphoma relapse (2 with high-grade transformation) and 7 died in remission for other causes. At a median follow up time of 6.3 years, the overall survival in the entire group is 92% at 5 years (95% C.I.; 84%–96%), 83% at 10 years (95% C.I.; 70%–91%) and 78% at 15 years (95% C.I.; 62%–88%). Only one patient died for lymphoma (after histological transformation). Additional tumors were observed in 22%. A history of autoimmune disease was present in 15% of the patients and did not affect the outcome.

Conclusions: Hp eradication usually results in long term disease control, independently of the lymphoma pathological remission status. A watch and wait policy seems safe in patients with local histological relapse with no endoscopic evidence of gross disease.
Long-Term Phenotypic Correction of Hemophilia A Mice Following Intravenous Injection of miRNA-Regulated Lentiviral Vectors.
Authors: Matsui Hideto , Ozelo Margareth , Hegadorn Carol , Labelle Andrea , Burnett Erin , Tuttle Angie , Brown Brian , Naldini Luigi , Hough Christine , Lillicrap David
Published: April 2020

Hemophilia A is an excellent candidate disorder for the use of gene therapy as a treatment modality. To date, although lentiviral delivery of the factor VIII (FVIII) transgene has the potential to provide sustained therapeutic correction of the hemophilia A phenotype, this has not been achieved in adult animals because of the anti-FVIII immune response. We have used lentiviral vectors to deliver the canine FVIII transgene to hemophilia A neonates and although no anti-FVIII immune response occurred, and indeed the treated mice displayed long-term tolerance to the canine FVIII antigen, this strategy did not provide sustained therapeutic levels of plasma FVIII. To overcome these limitations, we modified our lentiviral vector and the protocol for viral delivery to enhance transduction of hepatocytes and direct transgene expression away from antigen presenting cells. We engineered lentiviral vectors that encode the B-domain deleted canine FVIII cDNA under the transcriptional control of either a non-viral ubiquitous promoter or two different liver-restricted promoters. However, no plasma FVIII was detected in any of the adult hemophilia A mice after intravenous injection of the various lentiviral vectors because of an anti-canine FVIII immune response. An alternate pseudotype (GP64) was used to enhance transduction of hepatocytes and a target sequence for a hematopoietic-specific microRNA was incorporated into the transgene to prevent FVIII expression in antigen presenting cells that may arise from promoter trapping. When hemophilia A mice received intravenous infusions of these modified vectors, where the cFVIII trangene is under the control of either of the liver-restricted promoters, all treated mice (n=4) showed sustained FVIII expression (mean FVIII levels 28.2±2.4 mU/mL) for more than 150 days (last time analyzed) without developing anti-FVIII antibodies. Moreover, temporary depletion of Kuppfer cells prior to viral administration resulted in a 3-fold elevation of levels of plasma FVIII (mean FVIII levels 83.3±2.1mU/mL; n=4). Analysis of the biodistribution of the integrated FVIII transgene and expression of canine FVIII mRNA indicate an enhanced restriction of FVIII expression in hepatocytes with the use of the modified lentiviral vectors. These results demonstrate, for the first time, the long-term therapeutic potential of modified lentiviral vectors for treating adult pre-clinical animal models of hemophilia A.
In Vitro and In Vivo Selection of Genetically Modified Cells Using shRNA Against HPRT and Treatment with 6-thioguanine.
Authors: Porter Christopher C. , DeGregori James
Published: April 2020

Inefficient transduction, poor long term expression, and engraftment failure of ex vivo manipulated cells have slowed the practical advancement of gene therapy trials. Thus, the ability to select for or amplify a population of cells that has been modified to express a gene of interest might enhance the effectiveness of gene therapy. Strategies for in vivo expansion of genetically modified cells that have been studied to date have relatively high toxicity or low efficacy in selection of hematopoietic stem cells. We hypothesized that resistance to the purine analog 6-thioguanine (6TG) could be programmed via lentiviruses, and that treatment with 6TG would allow for selection of genetically modified cells in vitro and in vivo. Using short hairpin RNAs, we achieved efficient knockdown of hypoxanthine phosphoribosyl transferase (HPRTkd), the enzyme required for 6TG cytotoxicity, in the murine hematopoietic progenitor cell line FL5.12. In so doing we were able to provide Fl5.12 cells with resistance to 6TG. In the presence of 6TG, HPRTkd cells continued to proliferate for at least 30 days, whereas control transduced cells ceased proliferating after 7-10 days. 6TG treatment of mixed cultures of GFP+-HPRTkd cells and untransduced cells resulted in selective outgrowth of HPRTkd cells. Knockdown of HPRT in FL5.12 cells was found to attenuate the checkpoint activation, cell cycle arrest and apoptosis seen in control transduced cells when treated with 6TG. Knockdown of HPRT in murine primary hematopoietic cells also allowed for selection of transduced cells with 6TG ex vivo. Furthermore, and most importantly, after transduction of whole bone marrow and transplantation into sub-lethally irradiated recipient mice, a single, short course of treatment with 6TG resulted in up to 12 fold greater percentages of circulating transduced granulocytes as compared to untreated controls. These results suggest that genetically modified hematopoietic stem cells can be selected in vivo using 6TG. This strategy may be useful for therapy of a variety of hematopoietic diseases, particularly those that affect hematopoietic progenitors. The benefits of this strategy include the following: 1) the use of a lentivirus with a self inactivating long terminal repeat, 2) a very short cassette encoding drug resistance, making the vector easier to manipulate, and 3) a very well tolerated and relatively non-toxic medication for selection.
Efficient Transfer of CD40L mRNA into Mantle Cell Lymphoma for the Production of a Whole Tumor Cell Vaccine.
Authors: Brody Joshua D. , Li Linhong , Feller Stephanie , Fratantoni Joseph , Levy Ronald
Published: April 2020

Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma with the worst long-term prognosis of any NHL subtype. Current therapeutic options are unsatisfactory. MCL patients’ malignant B cells are ineffective antigen-presenting cells (APCs), perhaps resulting from low level expression of the immune co-stimulatory molecules that are essential to activate T cells upon interaction with the T-cell receptor. The MCL cells can be engineered to be effective APCs and thereby function as a therapeutic cellular vaccine in combination with chemotherapy and/or stem cell transplantation to eradicate residual disease. However, primary MCL cells are difficult targets for gene transfer by both viral and non-viral methodologies. Ligation of CD40 resulting from co-culturing with hCD40L expressing murine fibroblasts was shown to be superior to a panel of other immune stimulants and cytokines in upregulating co-stimulatory markers and inducing anti-tumor T cell responses (Hoogendoorn et al. 2005). We now report on a technology platform, based on electroporation of mRNA for CD40L, for the introduction of CD40L protein expression and subsequent induction of immune co-stimulatory molecules by MCL tumor cells. Primary MCL malignant B cells were obtained from patients’ lymph node biopsies by mechanical dissociation, placed in single cell suspension and cryopreserved prior to modification. Full-length 5′-end capped hCD40L mRNA transcript was generated by in vitro transcription with a commercially available T7 polymerase kit. The transfected MCL cells were immunostained with fluorophore-conjugated monoclonal antibodies against hCD40L, hCD80 and 86 then analyzed by FACS. Data showed hCD40L could be detected in ≥ 80% of the transfected MCL cells as early as 2 hrs post transfection. At 3 days post manipulation, hDC40L expression could be detected on approximately 30% of the transfected MCL cells. Cell viability remained at approximately 80% during the 3 day in vitro culturing. FACS analysis of the immune co-stimulatory molecules revealed that forced expression of hCD40L caused an up-regulation of CD80/86, which was increased approximately 10 fold compared to the expression levels in naïve, non modified cells. The increased expression level of CD80/86 was maintained for 3 days. Furthermore, when the hCD40L modified MCL cells were mixed with allogeneic PBMC, they stimulated IFN-γ production at a level 4 fold higher than was observed with naïve, non modified MCL cells mixed with allogeneic PBMC. This provides proof-of-concept that MCL cells modified by mRNA-hCD40L transfection have the potential to be used as a cellular vaccine. Such transduced cells function to protect animals from tumor challenge. The process can be scaled up to produce >2×1010 modified tumor cells. This simple, non-viral cell manipulation system is practical and will be a useful tool for immunotherapy of human hematopoietic malignancies such as MCL.
Targeted Killing of Glioblastoma Multiforme In Vivo by IL-13 Zetakine Redirected CTLs Made Glucocorticoid Resistant with Zinc Finger Nucleases.
Authors: Reik Andreas , Holmes Michael C. , Zhou Yuanyue , Mendel Matthew , Liu Pei-Qi , Lee Gary , Paschon David , Rebar Edward , Ando Dale , DiGiusto David , Gregory Philip D. , Jensen Michael C.
Published: April 2020

Genetic modification of cytolytic T-lymphocytes (CTL) for enhancing their functional immunobiology is a promising immunotherapeutic approach for the treatment of cancer and infectious disease. CTLs modified to express a chimeric antigen receptor comprising an extracellular IL13 domain and cytoplasmic CD3 domain (IL13-zetakine) can be re-directed both in vitro and in animal models to target glioblastoma multiforme (GBM), which is characterized by high expression of IL13Ralpha2. Patient-derived IL13-zetakine/HyTK expressing CD8+ CTL clones have entered early stage clinical trials. However, their clinical application is frequently limited in this patient population by the pervasive use of dexamethasone, a potent glucocorticoid analogue employed in the management of cerebral edema. Thus iatrogenic dexamethasone-mediated T-cell functional anergy and apoptosis in these patients is a barrier to realizing the full clinical utility of this adoptive therapy strategy. We hypothesized that knocking out the expression of the glucocorticoid receptor would render therapeutic CTLs resistant to the effects of synthetic glucocorticoids, including dexamethasone. We therefore developed engineered zinc finger nucleases (ZFNs) to specifically disrupt the glucocorticoid receptor (GR) locus in the human genome. ZFNs include the cleavage domain of the restriction enzyme FokI linked to an engineered zinc finger DNA-binding domain and can be designed to cleave a predetermined site in the genome. Natural repair of such DNA breaks via the error-prone non-homologous end joining pathway results in the inactivation of the target gene at frequencies which permit the isolation of knock out clones. Employing adenovirally delivered and transiently expressed ZFNs targeting exon 3 of the human GR gene, we isolated IL13-zetakine+ CD8+T-cells containing a biallelically mutated GR locus. These cells were characterized by the absence of full length GR protein, lack of glucocorticoid hormone-induced gene regulation and resistance to glucocorticoid hormone-mediated immunosupression and apoptosis. Importantly, the ZFN-modified, glucocorticoid-resistant CTLs demonstrated zetakine re-directed cytolytic activity and tumor cell specificity in chromium release assays in vitro and in an orthotopic mouse model of GBM in vivo. These results indicate that glucocorticoid-resistant IL13-zetakine targeted CTLs should retain function in cancer patients receiving glucocorticoids. A clinical trial to test this hypothesis is currently under development.
A Prospective Study of Imatinib 400 mg vs 800 mg Frontline in High Risk Ph+ Chronic Myeloid Leukemia (CML) Patients.
Authors: Baccarani Michele , Castagnetti Fausto , Porkka Kimmo , Nielsen Johan L. , Hjorth-Hansen Henrik , Haznedaroglu Ibrahim , Nagler Arnon , Rege-Cambrin Giovanna , Specchia Giorgina , Amabile Marilina , Testoni Nicoletta , Palandri Francesca , Pane Fabrizio , Alimena Giuliana , Martinelli Giovanni , Rosti Gianantonio , Simonsson Bengt
Published: April 2020

Background: imatinib mesylate (IM) is the drug of choice for the front-line treatment of Ph+ CML, at a dose of 400 mg daily (M. Baccarani et al Blood2006;108:1809–1820). Several biological and clinical observations suggest that increasing the dose may improve the results. The cytogenetic response to IM 400 mg daily is significantly related with Sokal relative risk. High risk patients could benefit of a dose increase front-line.

Aim: to compare the effects of 400 mg and 800 mg daily in previously untreated, early chronic phase patients, high Sokal risk. The primary efficacy variable of the study is the complete cytogenetic response (CCgR) rate after 12 months, on an intention-to-treat analysis.

Patients and Methods: this is a multicentric international study running in Italy, Sweden, Denmark, Finland, Norway, Turkey and Israel, approved by the local Ethic Committees, conducted according to Helsinki Declaration and Good Clinical Practice. 215 patients with confirmed Ph+ CML previously untreated, high risk according to Sokal formulation (J. Sokal et al Blood1984;63:789–799) were enrolled over a 3-year period and were randomized (1:1) to receive IM 400 or 800 mg daily. Cytogenetic response was assessed by conventional cytogenetics and FISH analysis after 3, 6 and 12 months. Molecular response was evaluated every three months.

Results: as of August 2007, 137 patients are evaluable for CCgR rate at 12 months (primary efficacy variable). Patients in CCgR at that time were 78/137 (57%). Treatment failures during the study (no complete hematologic response or 100% Ph+ at 6 months, or loss of response) were 24/137 (17%), patients off-treatment for protocol violations or refusal were 10/137 (7%), patients off-treatment for toxicity were 7/137 (5%).

Conclusions: the results of this preliminary analysis show that the CCgR rate at 12 months is overall 57%, in line with the results of the IRIS trial (imatinib 400 mg daily - T. Hughes et al, NEJM 2003, 349;15: 1423–1432) in the same risk category (69% all risks, 49% high Sokal risk). At the time of writing is too early to analyze the results by arm: a second analysis will be performed in November (datalock, October 31) and the results will be presented on site.
Angiogenesis Assessed in Tissue Microarrays of Diagnostic Follicular Lymphoma Correlates with Outcome.
Authors: Clear Andrew J. , Lee Abigail M. , Morris Kelly J. , Calaminici Maria , Macdougall Finlay , Proto-Siqueira Rodrigo , Goff Lindsey K. , Lister Thomas A. , Gribben John G.
Published: April 2020

Micro-vessel density (MVD) is a powerful prognostic factor in cancers but its value in haematological malignancies is more controversial. To examine its prognostic value in follicular lymphoma (FL) we assessed number and distribution of blood vessels by high-throughput tissue microarray and automated image analysis measurements in tissue microarrays (TMA) of paraffin-embedded, diagnostic lymph node biopsies taken from fifty-nine FL patients. These patients were selected from those who died from lymphoma progression less than 5 years after diagnosis (short survivor group) (n=34) and those who survived more than 15 years from diagnosis (long survivor group) (n=25). Immunohistochemistry was used in TMAs to study the number and location of vessels staining positive for the endothelial cell markers CD31 and CD34 and pericyte coverage using PDGFR. Interactive quantification using image analysis software was used to provide details of absolute numbers of vessels from each patient, as well as vessel size and their location. Image stacks of immunofluorescence stained sections were obtained using laser-scanning confocal microscopy to trace the tumour vasculature. Results demonstrated that both total vessel count and mean vessel area were significantly different between the two groups. Samples from the long survivor group were significantly more likely to have fewer (p=0.025), but larger vessels (p=0.002) than those from the short surviving group. The differences in vessel size and number were more prominent in inter-follicular vessels compared with those inside the neoplastic follicles. The smaller and more numerous blood vessels seen in the poorer prognostic sub-group likely reflects active, sprouting angiogenesis as confirmed by confocal microscopy. This study validates the use of TMAs to examine angiogenesis and demonstrates the powerful prognostic value of assessing MVD in FL. These results suggest that sprouting angiogenesis represents a therapeutic target in this disease and ongoing studies are investigating the mechanisms contributing to alteration in angiogenesis in different prognostic subgroups in FL and in transformation of this disease. The prognostic significance of MVD assessment in TMA is currently being evaluated in a validation set of FL samples.
Majority of Transformed Lymphomas Have High SUVs on PET Scanning Similar to Diffuse Large B Cell Lymphoma (DLBCL).
Authors: Noy Ariela , Weissler Matthew , Schoder Heiko , Gonen Mithat , Yeung Henry
Published: April 2020

Background: In our previous work, we correlated non-Hodgkins (NHL) histology with intensity of FDG uptake (SUVmax) on positron emission tomography in untreated patients: an SUVmax greater than 10 predicted an aggressive lymphoma with >80% certainty and an SUVmax greater than 13 predicted an aggressive lymphoma with >90% certainty. We sought to evaluate SUVmax in a series of patients with transformed lymphoma.

Methods: All PET scans for lymphoma indications at our institution 1999-5/2007 were evaluated. Patients were included if they had biopsy proven transformation. FDG-PET scans were included if they were no more than sixty days prior to or 90 days after biopsy proven transformation. Patients had no therapy for sixty days prior to PET scan unless there was documented progression of disease after the last treatment.

Results: Among the indolent diagnoses (n= 44), the following specific histologies were identified: 9 follicular lymphoma grade 1 (FL I), 9 FL grade 2 (FL 2), 7 small lymphocytic lymphoma / chronic lymphocytic leukemia (SLL/CLL), 11 marginal zone lymphoma (MZL) including one of mucosa associated lymphoid tumor (MALT) type, one mycoses fungoides, and one indolent lymphoma otherwise not specified. 5 FL 3 (FL 3), including one specified as grade 3a (FL 3a) were considered indolent if they preceded a diagnosis of DLBCL. Among the aggressive diagnoses (n= 28), the following specific histologies were identified: 37 DLBCL, one follicular lymphoma grade IIIa included as it was preceded by FLI, one large peripheral T-cell lymphoma, and 5 Large cell lymphoma(LCL), not otherwise characterized on account of biopsy size. Standard uptake values (SUV) were measured at the biopsy site when possible (n=38). In 6 patients the biopsy site was completely excised before the PET scan. The SUV of biopsy site ranged from 3-38, with a median of 10 and mean of 15. Among evaluable patients, 17/36 (47%) biopsies had an SUV above 10; and 12/36 (33%), above 13. The SUVmax for a transformed aggressive lymphoma ranged from 3.2–40, with a median of 12 and mean of 15. 27/44 (61%) patients had an SUVmax above 10; and 21/44 (48%), above 13.

Conclusions: Similar to patients with de-novo aggressive lymphoma, the majority of patients with transformed lymphomas have high SUVmax for a given pre-treatment staging study. Therefore, transformation to aggressive lymphoma should be suspected in patients with indolent lymphoma found to have high SUVs on FDG PET, and biopsies should be directed to the site of greatest PET avidity whenever feasible.