bluuebunny/crossref_metadata_2025 huggingface dataset:
Circulating CD52 and CD20 Levels Predict for Progression and Survival in Patients with CLL Treated with Fludarabine (F), Cyclophosphamide (C), and Rituximab (FCR).
Authors: Alatrash Gheath , Keating Michael J. , O’Brien Susan , Wang Xuemei , Manshouri Taghi , Faderl Stefan , Ferrajoli Alessadra , Kantarjian Hagop M. , Lerner Susan , Wierda William G.
Published: October 2019

The CD52 and CD20 antigens are ubiquitously expressed on CLL B cells and are important therapeutic targets for the monoclonal antibodies alemtuzumab and rituximab, respectively. Circulating soluble CD52 (sCD52) and CD20 (sCD20) have been shown to have prognostic value in CLL, non-Hodgkin’s lymphoma and Hodgkin’s disease (Blood101:2507, 2003; Br J Haem123:850, 2003). The pharmacokinetics and therapeutic efficacy of these mAbs may be adversely affected by circulating sCD52 and sCD20. Using the previously described ELISAs, we measured levels of sCD52 and sCD20 at various time points in blood (PB) and bone marrow (BM) of chemotherapy-naïve patients treated with FCR. Treatment consisted of fludarabine 25 mg/m2 d1-3, cyclophosphamide 250mg/m2 d1-3, and rituximab 375–500mg/m2 d1 as previously described (JCO23:4079, 2005). Courses were repeated every 4 weeks for a total of 6 courses. Univariate and multivariate Cox proportional hazards models were fit to evaluate the correlations of sCD52 and sCD20 and baseline characteristics with progression free survival (PFS) and overall survival (OS). A total of 291 patients were included, pretreatment characteristics [median (range)] were as follows: age=57yrs (17–86); WBC=76.9K/μL(2.1–619.5); ALC=66.7K/μL(0.8–558); HGB=12.4g/dL(6.1–18.7); PLT=155K/μL(8–406); ß2M= 3.7mg/L(1.6–16.4); LDH=550 IU/L(103–1828). Patients with Rai stage 0=8; I–II=186; and III–IV=97. The median follow-up time for all patients is 56 months. Of the 278 responding patients, 88 (31.7%) have progressed; the median time to progression has not been reached. To date, the median survival time for the 291 patients has not been reached; 57 (19.6%) patients have died. Pretreatment characteristics analyzed included age, gender, PS, WBC, ALC, HGB, PLT, ß2M, and RAI stage; post-treatment factors included PCR for IgVH in BM, and sCD52 and sCD20 (BM and PB) levels at response. Univariate analyses identified the following predictors for PFS (p < .05): age, HGB, ß2M, PCR for IgVH at response, and PB sCD20 at response. Independent predictors for PFS identified in multivariate analysis included PCR for IgVH and PB sCD20 level at response. For OS, significant (p < .05) factors in univariate analyses included: age, HGB, ß2M, PCR for IgVH at response, and BM sCD52 at response. Independent predictors for OS identified in multivariate analysis included age and BM sCD52 level at response. Residual sCD52 at response may reflect residual disease, therefore correlating with shorter OS. We are currently evaluating sCD52 and sCD20 as prognostic factors for clinical outcomes in previously treated patients that received FCR as salvage therapy.
A Study of the Binding Mode and the In Vitro Activity of the Protein Tyrosine Kinase Inhibitor SKI-606 in the BCR-ABL Positive Cells.
Authors: Grafone Tiziana , Soverini Simona , Tasco Gianluca , Mancini Manuela , Boschelli Frank , Golas Jennifer , Frauling David , Colarossi Sabrina , Ottaviani Emanuela , Gnani Alessandra , Panagiota Giannoulia , Castagnetti Fausto , Casadio Rita , Rosti Gianantonio , Martinelli Giovanni , Baccarani Michele
Published: October 2019

Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) is often caused by the selection of leukemic clones harboring mutations that destabilize the inactive conformation of BCR-ABL, to which IM preferentially binds, shifting the equilibrium toward the active kinase conformation. Hence the need for second -generation kinase inhibitors with a greater flexibility in binding to different BCR-ABL conformations. The 4-anilino-3 quinolinecarbonitrile SKI-606 is a novel Src and ABL kinase inhibitor. We report here that SKI-606 is a potent and antiproliferative agent when tested in K562 cell line and CD34+ cells from patients with chronic myeloid leukemia (CML) blast crisis. In K562 cells, SKI-606 treatment caused a dose-dependent decrease in cell viability accompanied by accumulation in subG1 phase, an effect not observed in BCR-ABL-negative HL-60 cells. Treatment of K562 with 100 nM SKI-606 for 24 hours caused a complete dephosphorylation of Lyn, Stat5 and Bcl-xl, while AKT and Bad phosphorylation was only diminished. Unexpectedly, the phosphorylation of BCR-ABL was less affected. SKI-606 treatment caused a shift to the subG1 phase also of CD34+ cells isolated from both IM-sensitive and resistant patients, the latter harboring the BCR-ABL mutations F359V, Y253H, E255V and E255K. The inhibitory concentration 50% (IC50) was 100 nM SKI-606 for Y253H, E255V, E255K and 1000nM for F359V. Cytofluorimetric analysis of cells from IM- sensitive CML patients indicated an accumulation in subG1 phase following treatment with SKI-606 alone or in combination with IM. Because the crystal structure of the BCR-ABL kinase domain in complex with SKI-606 has not yet been determined and the mode of binding of this inhibitor is unknown, we first used a molecular docking approach to determine SKI-606 binding mode to wild type (wt) form of the BCR-ABL kinase. We found that the interaction between SKI-606 and BCR-ABL was more stable when the activation loop was in the inactive conformation. Moreover, we found that SKI-606 retained the ability of efficiently binding all the above mentioned BCR-ABL variants, but not the T315I. Finally, we identified six BCR-ABL residues located around SKI-606 that, if mutated, could potentially be able to interfere with the SKI-606/BCR-ABL interaction: the charged residues K271, D381 and H361; the hydrophobic/aliphatic residues V299, A380 and M318.

These data help refining the use of SKI-606 for treatment of BCR-ABL positive leukemias.
Bcr-Abl and Wilms Tumor Gene (WT1) Kinetics in Nilotinib (AMN107) Treated CML Patients.
Authors: Schwarz Michaela , Baskaynak Gokben , Turkmen Seval , Schuld Peter , Dorken Bernd , Daniel Peter , Philipp le Coutre
Published: October 2019

Background: In normal hemopoesis WT1 expression is restricted to early progenitors and is rapidly down-regulated in maturing blood cells. WT1 transcript quantification therefore may be an adjunct diagnostic method in CML patients (pts) that remain Bcr-Abl positive under treatment. Especially, in advanced CML pts WT1 transcript kinetics may precede a change in Bcr-Abl kinetics and may therefore be helpful to indicate early response to treatment. Nilotinib is a potent, highly selective, second generation Bcr-Abl inhibitor which in vitro is 30-fold more potent than imatinib and is currently studied in imatinib resistant or intolerant pts.

Aim: The aim of this study was to compare kinetics of Bcr-Abl and WT1 transcripts in 33 CML pts with late chronic phase (CP) or advanced disease under treatment with nilotinib.

Methods: All pts were treated as part of an ongoing phaseI/II clinical trial with nilotinib at a standard dose of 400 mg BID. 20 pts were in chronic phase (CP), 4 in accelerated phase (AP) and 9 in blast crisis (BC). Among the CP pts 4/20 were enrolled because of toxicity to prior imatinib treatment. WT1 and Bcr-Abl kinetics were expressed in relation to Abl expression. Data points were taken at baseline and every 4 weeks thereafter. Quantitative PCR was carried out by Taqman-analysis.

Results: The median duration of nilotinib treatment was 8 months in CP pts, 4 in AP pts and 5.5 in BC pts. The follow up of all pts ranged between 3 and 13 months. All pts received imatinib prior to nilotinib and were Bcr-Abl positive at study entry. In CP samples WT1/Abl ratios (median:1.24x10−3) were 38 fold lower than Bcr-Abl/Abl (median:4.67x10−2) ratios at baseline (p≤0.014, t-test). However, in AP and BC pts no relevant difference of WT1/Abl and Bcr-Abl/Abl baseline levels were detectable. In BC patients a more rapid decline of WT1/Abl ratios compared to Bcr-Abl/Abl ratios was observed within the first four months of nilotinib therapy. In addition, in BC pts elevation of WT1 levels preceded hematologic relapses and was detectable earlier than elevation of Bcr-Abl/Abl ratios.

Conclusions:In contrast to AP and BC pts in CP pts WT1/Abl ratios were significantly lower than Bcr-Abl/Abl ratios confirming the restriction of WT1 expression to early progenitors.In BC pts under nilotinib therapy WT1/Abl ratios may be an early marker of response as compared to Bcr-Abl/Abl ratios.A stable correlation of WT1/Abl and Bcr-Abl/Abl ratios was observed in AP pts under treatment.
Kinase Profiling as a Tool in Molecular Dissection of Imatinib Resistant Chronic Myeloid Leukemia (CML).
Authors: Zhao Weiqiang , Cortes Jorge , Hannah Christina , Kantarjian Hagop M. , Jones Dan
Published: October 2019

Imatinib (Gleevec) resistance (IR) in CML is a multifactorial phenomenon related to ineffective blockage of the bcr-abl kinase through point mutation and/or gene amplification, or alternatively kinase bypass through clonal evolution or presently unknown mechanisms. The newer generation of bcr-abl small molecule inhibitors (dasatinib, nilotinib) may be useful in overcoming ineffective bcr-abl blockage in IR-CML but may not be effective in other mechanisms of resistance. As a tool in classifying the pattern of disease resistance, we profiled kinase levels in IR-CML following switch to new kinase inhibitors or with imatinib dose escalation. In Ficoll-purified blood and bone marrow samples, we examined levels of BCR-ABL autophosphorylation (Tyr245), and phosphorylation of two bcr-abl targets STAT5 (Tyr694/699) and CRKL (Tyr207) by Western blot. Kinase profiling was compared to hematologic responses, cytogenetic analysis, FISH for the bcr-abl locus using dual fusion probes and quantitative(q)PCR for bcr-abl transcript. We profiled 26 patients with IR-CML undergoing therapy shifts as well as serial samples from 10 patients receiving frontline dasatinib therapy for newly diagnosed CML. In all but 1 of 68 analyzed samples, detection of phospho(p)-CRKL in unsorted white blood cells was associated with bcr-abl/abl qPCR ratios above 1%. Among all 68 samples, pSTAT5 and pBCR-ABL levels declined in parallel to pCRKL but were absent in more samples than pCRKL. Persistence of detectable pCRKL despite switch to nilotinib, dasatinib or high dose imatinib was seen in 5 patients with bcr-abl kinase domain (KD) mutations including 2 with G250E and 1 each with L248V, F311L and T315I, 2 patients with bcr-abl gene amplification and 3 patients without KD mutations including 2 with cytogenetic clonal evolution. Among these 10 patients with IR-CML and persistent pCRKL, complete hematologic responses were noted in 4 and complete cytogenetic response in 3. Response to therapy switch and absence of pCRKL was seen in tumors with E355G, L387M and E459K KD mutations and in 12 patients with no mutation or genomic amplification. Among the patients receiving frontline therapy with dasatinib, effective hematologic responses correlated well with loss of detectable pCRKL and pSTAT5. However, detectable pCRKL did not disappear until at least 8 weeks following initiation of therapy, and lagged hematologic response. The 2 patients treated with frontline dasatinib therapy who had persistent pCRKL after 20 weeks of treatment also showed amplification of the bcr-abl locus in tumor cells by FISH. Kinase profiling, particularly for pCRKL, is useful for monitoring molecular response in the therapy switch period in IR-CML and correlates well with the predicted pattern of response in tumors with KD mutations. The value of monitoring kinase levels frequently following initiation of frontline kinase inhibitor therapy in newly diagnosed CML remains to be established.
FLT3 Mutations in a Large Series of Patients with Acute Promyelocytic Leukemia Treated with All-Trans Retinoic Acid and Anthracycline Monochemotherapy.
Authors: Montesinos Pau , Gonzalez Marcos , Calasanz Maria , Bolufer Pascual , Colomer Dolors , Roman Jose , Gomez Maria Teresa , Martinez Joaquin , Amutio Elena , Sanz Miguel
Published: October 2019

Background: The prognostic significance of FLT3 internal tandem duplication (ITD) and D835 mutations in acute promyelocytic leukemia (APL) remains unclear. Recent studies have revealed an association between FLT3 mutations and leukocytes > 10x109/L, M3-variant subtype and BCR3 isoform. It has been also suggested that patients with FLT3 mutations have a higher induction death rate and a shorter survival. Objectives: To analyze in patients with newly diagnosed APL treated with an ATRA plus anthracycline-based protocol if the presence of FLT3 ITD and D835 mutations influences the clinical and biological presenting features, and the disease outcome.

Material and methods: Between 1997 and 2005, 733 consecutive patients were enrolled in the PETHEMA LPA96 and LPA99 trials. Treatment consisted of Induction therapy with oral ATRA (45 mg/m2/d) and intravenous idarubicin (12 mg/m2/d x 4 days) followed by three courses of consolidation with anthracycline monochemotherapy. In the LPA99 trial, ATRA was added in each cycle of consolidation for intermediate and high risk patients (according to Sanz score). In both trials, maintenance therapy consisted of intermittent ATRA and low dose chemotherapy with methotrexate and 6-mercaptopurine. Using RT-PCR, analysis of the FLT3 ITD and D835 mutations was available in 251 and 191 patients respectively.

Results: Overall, 61 patients (24%) had a FLT3 ITD mutation and 20 patients (10%) had a FLT3 D835 mutation. Only one patient had both mutations. FLT3 ITD was associated with BCR3 isoform (78% vs 32%, p < 0.01), leukocytes > 10x109/L (64% vs 17%, p < 0.01), M3v subtype (46% vs 13%, p < 0.01), bone marrow blasts > 70% (95% vs 80%, p < 0.01) and expression of CD2 and CD34 surface antigens (41% vs 14% and 38% vs 9%, respectively, p < 0.01). FLT3 D835 was not associated with leukocytes > 10x109/L (35% vs 25%, p=0.31) or any other biological characteristic. There was a trend towards a higher induction death rate in patients with FLT3 ITD or D835 mutations, when compared to patients with wild-type FLT3 (15% vs 16% vs 6%, respectively, p=0.08). There was no significant difference on relapse-free survival (RFS) and overall survival (OS) at 7 years in patients with FLT3 ITD, when compared to those with FLT3 D835 or wild-type FLT3 (81% vs 100% vs 87% for RFS, p=0.17; and 76% vs 84% vs 81% for OS, p=0.37).

Conclusion: In a large cohort of patients with newly diagnosed APL, the incidence of FLT3 ITD and FLT3 D835 mutations was 24% and 10%. This study was only able to demonstrate an association of FLT3 ITD with leukocytes > 10x109/L, M3v subtype, BCR3 isoform, and expression of CD2 and CD34 surface antigens, but this mutation had no independent prognostic value in patients with APL receiving state-of-the-art treatment.
Acute Promyelocytic Leukemia Stem Cell Derived from Leukemic Progenitor.
Authors: Rosen Galit P. , Kogan Scott C.
Published: October 2019

Efforts to identify the leukemic stem cell (or leukemia-repopulating cell, LRC) of acute myeloid leukemia (AML) have led to considerable insights, but also to a number of unresolved questions. While the LRC is believed to arise primarily from the hematopoietic stem cell, data from chronic myelogenous leukemia blast crisis and acute promyelocytic leukemia (APL) suggest that they may arise from more committed cells.

In order to clarify the potential of committed progenitors to become LRCs in some human AMLs, we utilized the MRP8-PML/RARA transgenic murine model of APL to perform initial studies directed at the identification of the LRC in APL. Unsorted spleen cells of MRP8-PML/RARA leukemic mice injected into cohorts of sublethally irradiated mice using limiting dilutions showed that the LRC of APL is present in 1:100 to 1:300 cells. FACS analysis of six primary and serially transplanted leukemic specimens revealed that normal hematopoetic stem cells and myeloid progenitors were rare, comprising .007% and .078% of total splenic cells, respectively. These data raised the possibility that the LRC is present at higher frequency than these normal progenitors and does not belong to any of these populations.

Given the scarcity of normal progenitors, we surmised that the LRC may be part of the leukemic population. We performed FACS analysis of seven primary leukemias to elucidate potential heterogeneity within the leukemic cell population. Myeloblasts had the immunophenotype lymph−Gr1+ckit+CD16/32+CD34mod/+. A smaller population that made up 1.4–12.7% of all cells was lymph−Gr1−ckitlo/mod and had variable expression of CD16/32 and CD34. Such cells have been identified as myelomonocytic cells and represent a stage in myelopoiesis beyond the GMP. Our findings suggest a hierarchy in which Gr1−ckitlo/mod leukemia repopulating cells proliferate and differentiate to maintain the population of abnormal promyelocytes.

We infer from the dilution analysis data that the LRC in this MRP8-PML/RARA mouse model of acute promyelocytic leukemia cannot be a hematopoietic stem cell or a normal myeloid progenitor cell. Also, it is apparent that the entire blast population is not capable of transferring disease; otherwise, all the mice injected would have developed leukemia. While our data does indicate that while leukemogenesis in APL is a hierarchy, it originates from a newly identified leukemia progenitor cell that is Gr1−ckitlo/mod that may be more mature than a GMP. It is quite likely that the LRC lies within this population of cells. Future work will further delineate the immunophenotype of the LRC in this model and in human leukemia, as well as molecular abnormalities that are important in differentiating from the LRC into the myeloblast phenotype.
Comparison of Three Different NOD/SCID-Related Strains in Preclinical Models of Acute Leukemia.
Authors: Agliano Alice , Martin-Padura Ines , Merighetti Paola , Mancuso Patrizia , Rabascio Cristina , Shultz Leonard D. , Bertolini Francesco
Published: October 2019

Transplantation of human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) primary cells and cell lines in a variety of different strains of immunodeficient mice has led to preclinical models extensively used to investigate AML and ALL stem cells, biology, and drug sensitivity. We investigated the engraftment kinetics of two AML cell lines (HL-60 and KG-1), two ALL cell lines (MOLT-16 and 697) and AML primary cells from an AML M4 patient (QD1-EIO, described in Fusetti et al, Cancer Res 2000) in 3 different strains of NOD/LtSz-Prkdcscid (NOD/SCID, NS)-related immunodeficient mice. NS, NS/beta2 null (NSB) and NOD/SCID/IL-2Rgamma null (NSG) mice were injected ip with 10x106 AML or ALL cells. Mice were observed daily and sacrificed when leukemia-related symptoms were evident. Overall, leukemia-related symptoms were observed in 71, 84 and 86% of NS, NSB and NSG mice, respectively (n=42, p<0.01), after a median of 53, 49 and 35 days (p<0.001). Leukemia engraftment was investigated in the marrow, the blood and the spleen by means of morphology, flow cytometry and quantitative PCR for human genes. AML HL-60 and KG-1 cells accounted for 10-6, 5-1 and 7-3 % of peripheral blood cells in NS, NSB and NSG mice, respectively. ALL MOLT-16 and 697 cells accounted for 2-12, 22-27 and 1-27 % of blood cells in NS, NSB and NSG mice, respectively. AML primary cells QD1-EIO accounted for <1, <1 and 3% of blood cells in NS, NSB and NSG mice, respectively. Similar engraftment results were observed in the marrow and in the spleen. Leukemia cell-injected NSG mice, compared to NS and NSB, showed a significantly higher increase of circulating endothelial mature cells (CEC, enumerated by flow cytometry as CD45−, CD13+ VEGFR2+ cells) and progenitors (CEP, enumerated by flow cytometry as CD45−, CD13+ VEGFR2+ CD117+ cells, see Shaked et al, Cancer Cell 2005). This CEC and CEP increase paralleled leukemia engraftment. Taken together, our data indicate that the 3 different strains have significantly different leukemia engraftment behavior and kinetics. Engraftment in NSG mice is significantly faster compared to the other two strains, leukemia-related microenvironment is differently modulated, and less leukemic burden might be needed to observe leukemia-related symptoms. These data might have major implications to design future studies on leukemia-initiating stem cells, leukemia biology and preclinical leukemia treatment studies.
BAFF-R Receptor Also Functions in the Nucleus of Normal and Neoplastic Human B Lymphocytes.
Authors: Fu Lingchen , Lin-Lee Yen-Chiu , Tamayo Archito , Yoshimura Linda , Ford Richard J.
Published: October 2019

B-lymphocyte stimulator (BLyS) is a relatively newly described tumor necrosis factor (TNF) superfamily cytokine involved in cell survival and proliferation in normal and neoplastic B cells, particularly in the aggressive B cell non-Hodgkin’s lymphomas (NHL-B). BLyS binds to three receptors: BAFF-R (or BR3), BCMA and TACI. However, recent studies have shown that BLyS regulates B cell survival and proliferation predominately through BAFF-R. Mice with mutant BAFF-R show significant decrease in the peripheral blood B-lymphocyte compartment, similar to the phenotype of BLyS knockout mice. BLyS/BAFF-R signaling functions through activation of the critical transcription factor NF-kB pathways, especially the NF-kB2 pathway, which mainly involves the p52/ rel-B complex.

Our previous studies have shown that BLyS is constitutively expressed in aggressive NHL-B cells leading to increased survival and proliferation of the malignant B cells. In this study, we found by western blotting and confocal microscopy that BAFF-R, the major B cell associated cell membrane receptor for BLyS, was also present in the B cell nucleus, in addition to its location in plasma membrane and cytoplasm in both normal peripheral blood B lymphocytes and NHL-B cells. Nuclear presence can be increased by anti-IgM and soluble CD154 treatment in normal peripheral blood B lymphocytes, and is constitutively expressed in aggressive NHL-B. Inhibition of BLyS expression by specific BLyS siRNA decreases nuclear BAFF-R level and survival in LBCL cells. Immunostaining experiments show that in the cytoplasm of normal and neoplastic B cells, BAFF-R interacts with the improtin a and b which are members of the classic karypherin pathway. A candidate nuclear localization sequence (NLS) was also identified in the BAFF-R protein sequence, and mutation of this putative NLS can block BAFF-R entering nucleus, which is consistent with our hypothesis that BAFF-R undergoes nuclear translocation. To further investigate the functional role of BAFF-R in nucleus, we performed confocal immunostaining analysis and co-immunoprecipitation, that shows that BAFF-R co-localizes with some NF-kB family members such as c-rel in the B cell nucleus. We also found that nuclear BAFF-R/c-rel complex can bind to the NF-kB binding site on the promoters of NF-kB target genes such as BLyS, CD154, Bcl-xL and Bfl-1 /A1 by chromatin immnoprecipitation (ChIP) assay. Luciferase reporter assays show that BAFF-R has transactivation activity on these NF-kB target genes. Furthermore, NLS mutant BAFF-R decreases NF-kB target gene promoter activity, compared to wild type BAFF-R, and NHL-B cell proliferation. These findings indicates that in addition to activating NF-kB pathways at the plasma membrane, BAFF-R may also promote survival and proliferation of both normal and NHL-B cell in the nucleus by directly regulating transcriptional activity of key NF-kB target genes and may functions as a transcriptional co- factor with NF-kB.
TRAF6 and C-myb Show Novel Functions in the Nucleus of Lymphoma B Cells.
Authors: Pham Lan V. , Lin-Lee Yen-Chiu , Zhou Hai-Jun , Tamayo Archito T. , Yoshimura Linda C. , Ford Richard J.
Published: October 2019

The tumor necrosis factor (TNF) family (TNF-R; CD40; BAFF-R) plays a key role in neoplastic as well as normal B cell growth and survival mechanisms. TNF receptor-associated factor-6 (TRAF-6) is an adapter molecule that regulates several important signaling pathways critical for cell growth and cell survival. It is a member of seven closely related TRAF proteins that serve as signaling molecules, coupling to TNF-receptor superfamily to intracellular signaling, particularly in the CD40 Signalosome. TRAF6 has shown to be over-expressed and play an important role in cell growth and cell survival through the activation of the key transcription factor NF-kB in aggressive non-Hodgkin’s lymphoma B cells (NHL-B), common B cell neoplasm that have been increasing in recent years. Although much of TRAF-6 functions have focused primarily as an adaptor molecule in signaling pathways in the cytoplasm, the role of TRAF-6 in other cellular compartments has not been investigated. Here, we demonstrate, by confocal microscopy as well as cellular fractionation studies that TRAF-6 resides not only in the cytoplasm but also in the nucleus of lymphoma B cells. Immunoprecipitation studies show that TRAF6 is auto-ubiquitinated in the cytoplasm but not in the nucleus, suggesting that nuclear TRAF6 functions differently than cytoplasmic TRAF6. Chromatin immunoprecipitation (ChiP) cloning assays using anti-TRAF6 polyclonal antibody reveal over 200 clones, one of which contains a 130 bp fragment belonging to the proximal 5′ end of the c-myb oncogene promoter. Further experiments demonstrate that nuclear TRAF6 co-localized with SUMO1 and c-myb, suggesting that TRAF-6 may enter the nucleus through SUMO1 interaction and serve as an E3 sumo ligase, in addition to its known adapter role in cytoplasmic signaling. Over-expression studies show that TRAF6 enhances c-myb sumoylation in lymphoma B cells, where this oncogene is over-expressed. C-myb correlates with TRAF6 protein and mRNA expressions in NHL-B cells, suggesting that TRAF6 may be involved in the modulation of c-myb expression through sumoylation, regulating key genes that are regulated by c-myb. Small interfering RNA (siRNA) targeting c-myb results in inhibition of lymphoma cell survival, suggesting that SUMO1/TRAF6/c-myb interactions are important in cell survival pathways in aggressive NHL-B. Such pathways could represent novel targets for the development of therapeutic agents for aggressive B cell lymphomas.
Regulation of pri-miRNA BIC Transcription and Processing in Burkitt Lymphoma.
Authors: van den Berg Anke , Kluiver Joost , de Jong Debora , Blokzijl Tjasso , Harms Geert , Jacobs Susan , Poppema Sibrand , Kroesen Bart-Jan
Published: October 2019

BIC a primary microRNA (pri-miR-155) transcript is highly expressed in the vast majority of Hodgkin lymphoma and diffuse large B cell lymphoma cases. High miR-155 levels are also observed in these B cell malignancies. In contrast, only very low or no expression of BIC and miR-155 are observed in Burkitt lymphoma cases. In the normal lymphoid system, BIC expression is observed in a proportion of the germinal center B cells and we have previously shown induction of BIC expression by BcR stimulation.

In this study we show the crucial involvement of PKC and NF-κB in the regulation of BIC expression upon BcR triggering of the Burkitt lymphoma cell line, Ramos, which is commonly used as a model system for germinal centre B cell stimulation. Surprisingly, Northern blot analysis did not reveal any miR-155 expression upon induction of BIC expression whereas other microRNAs were clearly detectable. Ectopic expression of BIC in Ramos and HEK293 cells resulted in miR-155 expression in HEK293 but not in Ramos cells suggesting a specific block of BIC to miR-155 processing in Ramos. In line with the results obtained with Ramos, lack of miR-155 expression after induction of BIC expression was also observed in other Burkitt lymphoma cell lines, indicating a generic and specific blockade in the processing of BIC in Burkitt lymphoma. In contrast, induction of BIC expression in normal tonsillar B cells resulted in very high levels of miR-155 expression and induction of BIC expression in Hodgkin lymphoma cell lines also resulted in elevated levels of miR-155. More recent reports have indicated that miRNA processing may be regulated by editing of pri-miRNA sequences by adenosine deaminases acting on RNA. ADARs can convert adenosines to inosines in stemloop sequences which may interfere with proper recognition and cleavage of pri-miRNA by the microprocessor complex. To evaluate whether ADAR editing might explain the observed differences in BIC to miR-155 processing, we analyzed the miR-155 sequence including 100nt up- and downstream of BIC RT-PCR products from stably transfected Ramos cells and PMA/Ionomycin stimulated CA-46 Burkitt lymphoma cells and compared those with sequences obtained from stably transfected HEK293 cells and L1236 cells. Analysis of 30 individual clones per cell line revealed no or very few conversions in the BIC transcripts of the four cell lines. This indicates that ADAR editing is not responsible for the observed BIC processing blockade in BL.

Our data provide evidence for two levels of regulation for mature miR-155 expression: one at the transcriptional level involving PKC and NF-κB, and one at the processing level. Burkitt lymphoma cells not only express low levels of BIC but also prevent processing of BIC via an, as yet, unknown mechanism.
Impact of IL-10 Polymorphisms in Survival of Lymphoid Neoplasms.
Authors: Domingo-Domenech Eva , Benavente Yolanda , Gonzalez-Barca Eva , Montalban Carlos , Guma Josep , Wang Sophia S. , Whitby Denise , de Sevilla Alberto Fernandez , Rothman Nathaniel , de Sanjose Silvia
Published: October 2019

BACKGROUND

Polymorphisms in interleukin-10 (IL-10) genes can influence immune responses that may affect the outcome of lymphoid neoplasms.

METHODS

We analyzed two single-nucleotide polymorphisms (−1082 and −3575) in the IL-10 promoter region in 472 consecutive non-immunessuppresed cases diagnosed with lymphoid neoplasms. Genotypes were tested for an association with overall survival and classical prognostic factors by multivariate analysis, adjusted for clinical characteristics and other confounders. Haplotypes were reconstructed using haplo.stats package and their implications analyzed in lymphoma survival by multivariate analysis.

RESULTS

In our series, patients with IL-10-3575AA genotype had a better overall survival (p= 0.002) for all lymphoid neoplasms as well as within the non-Hodgkin lymphoma patients (p=0.04). We also found that patients carrying the IL-10-1082GG genotype presented a better median overall survival when compared to genotypes IL-10-1082AA/AG. (p=0.05). When information on both genotypes were included in a multivariate analysis, IL-10-3575AA was the only genotype identified as an independent prognostic factor for survival (HR=0.2, 95%CI 0.05–0.92).

Haplotype analysis showed that patients with A-G/A-G diplotype had a longer overall survival (p=0.0042) and it appeared to be an independent prognostic factor for survival (HR:0.26; 95%CI 0.08–0.83).

CONCLUSIONS

IL-10-3575AA and IL-10-1082GG genotypes, together with A-G/A-G diplotype were identified as markers of a favorable lymphoma survival.
Aberrant Methylation of SHP-1 (SH2-Containing Phosphatases 1) and SOCS-1 (Suppressor of Cytokine Signalling 1) Genes in Immunodeficiency-Related Lymphomas.
Authors: Rossi Davide , Deambrogi Clara , Lunghi Monia , Cerri Michaela , Garbellotto Sara , Spina Valeria , De Paoli Lorenzo , Riccomagno Paola , Gloghini Annunziata , Lucioni Marco , Capello Daniela , Paulli Marco , Carbone Antonino , Gaidano Gianluca
Published: October 2019

SHP-1 and SOCS-1 are members of a family of negative regulators of signaling that act downstream of cytokine receptors, receptor tyrosine kinases and receptor complexes of the immune and hemopoietic system. SHP-1 and SOCS-1 act through the inhibition of the JAK2/STAT pathway and their inactivation is considered to be a relevant mechanism in JAK2/STAT pathway induction. Several observations point to induction of the JAK/STAT pathway as an important mechanism of lymphomagenesis. Since DNA methylation is a mechanism of gene silencing involved in the pathogenesis of human cancers, including lymphoma, here we tested the involvement of SHP-1 and SOCS-1 methylation in immunodeficiency-related lymphoma. Tumor samples from 34 HIV-related non-Hodgkin lymphoma (HIV-NHL) and 25 post-transplant lymphoproliferative disorders (PTLD) were analyzed for SOCS-1 and SHP-1 methylation by methylation-specific polymerase chain reaction. The tumor panel included 15 HIV-diffuse large B cell lymphoma (HIV-DLBCL), 10 HIV-Burkitt lymphoma (HIV-BL), 9 HIV-primary effusion lymphoma (HIV-PEL), 10 PTLD-immunoblastic (PTLD-IB), 7 PTLD-centroblastic (PTLD-CB), 4 polymorphic PTLD (P-PTLD), 3 PTLD-BL and 1 PTLD multiple myeloma (MM). Among HIV-NHL, SHP-1 methylation occurred in 23/34 (67%) cases, including 12/15 (80%) HIV-DLBCL, 4/10 (40%) HIV-BL, and 7/9 (78%) HIV-PEL. SOCS-1 methylation occurred in 6/34 (17%) HIV-NHL, including 5/9 (55%) HIV-PEL and 1/15 (7%) HIV-DLBCL. When considering EBV status, SHP-1 was methylated in 10/11 (91%) EBV-negative HIV-NHL and in 12/22 (54%) EBV-positive HIV-NHL. Among PTLD, SHP-1 methylation was detected in 20/25 (80%) cases, including 7/10 (70%) PTLD-IB, 6/7 (86%) PTLD-CB, 3/4 (75%) P-PTLD, 2/3 (66%) PTLD-BL and 1 PTLD MM. SOCS-1 methylation was detected in 3/25 (12%) PTLD, including 1/10 (10%) PTLD-IB and 2/7 (28%) PTLD-CB. When considering EBV status, SHP-1 was methylated in 12/13 (92%) EBV-negative PTLD and in 6/11 (54%) EBV-positive PTLD. The implications of our data are threefold. First, SHP-1 methylation is involved in the majority of HIV-NHL and PTLD. Second, it is remarkable that virtually all EBV-negative HIV-NHL and PTLD carry SHP-1 methylation. In EBV-positive B-cells, EBV infection activates the STAT pathway. It is conceivable that, in EBV-negative HIV-NHL and in EBV-negative PTLD, SHP-1 inactivation through aberrant methylation may surrogate EBV infection for STAT activation. Third, similar to observations in NHL of the immunocompetent host, SOCS-1 methylation is rarely implicated in the pathogenesis of immunodeficiency-related NHL. This notion is supported by the phenotype of SOCS1-deficient mice, that die of a myeloproliferative disease but do not develop a lymphoproliferative disease.
The IMiDs® Immunomodulatory Drugs Revlimid® (Lenalidomide) and CC-4047 Induce Growth Arrest and Apoptosis in NHL Tumor Cells In Vitro.
Authors: Corral Laura G. , Zhu Dan , Wang Yuedi , Stein Bernd
Published: October 2019

IMiDs® immunomodulatory drugs are thalidomide analogues that have been developed for improved anti-cancer and anti-inflammatory properties and decreased side effects. Many IMiDs® immunomodulatory drugs have been shown to have activities in hematologic cancers and solid malignancies, as well as having profound effects on the bone marrow microenvironment. Specifically in NHL, it was shown that addition of Revlimid® or CC-4047 to Rituxan enhances anti-tumor activity in a SCID mouse lymphoma model.

Here we tested the direct effects of Revlimid® and CC-4047 on NHL tumor cells by treating Raji cells with each drug alone or with each drug in combination with anti-CD20 antibodies B1 and Rituxan. CC-4047 alone caused up to 40% inhibition of proliferation at 10 μM in Raji cells, which corresponded to G1 arrest. In combination with B1, CC-4047 showed a small additive effect at 10 μM while Revlimid® effects were minimal up to 10 μM. In combination with Rituxan, CC-4047 showed a slight additive effect at 10 μM and Revlimid® at 50 μM.

We have also developed a co-culture assay of PBMC and NHL tumor cells as an in vitro model of tumor-host immune system interaction, to further explore the anti-tumor potential of the drugs in NHL. This assay is non-radioactive and flow cytometry based. In this co-culture system using Raji and PBMC, we have shown that pre-treatment of PBMC with Revlimid® or CC-4047 can enhance the PBMC activity in inducing Raji cell apoptosis in a dose dependent manner. In addition, our data indicate that pre-treatment of Raji cells with Rituxan can further enhance the apoptosis induced by PBMC pre-treated with Revlimid® or CC-4047. Since minimal additive effect between each drug and Rituxan was observed in the Raji single tumor cell model, these studies suggest that the co-culture system is a more appropriate cellular model to assess the anti-tumor activities of certain IMiDs® immunomodulatory drugs. This system can reveal the effects of certain IMiD® immunomodulatory drugs not observable with single tumor cell proliferation models.

In summary, our data clearly demonstrate that Revlimid® and CC-4047 directly induce NHL tumor cell growth arrest and effectively enhance tumor cell apoptosis induced by PBMC. These results support clinical evaluation of Revlimid® and certain IMiDs® immunomodulatory drugs in relapsed B-cell NHL in combination with Rituxan.
Motexafin Gadolinium (Mgd), a Redox Stress Inducer, Lowers p53 Protein but Not Message in Lymphoma Cell Lines: Mechanisms of Reactive Oxygen Species (ROS)-Mediated Apoptosis in Lymphoma.
Authors: Singh Amareshwar , Prachand Sheila , Evens Andrew M. , Schumacker Paul , Gordon Leo I.
Published: October 2019

Disruption of the tumor suppressor p53 is a frequent event in malignant disorders, including lymphomas. Loss of constitutive low-level p53-dependent expression of anti-oxidant proteins could render cells more susceptable to the acute oxidant stress induced by oxidant generating agents such as MGd, bortezomib and rituximab. Alternatively, loss of p53 would render cells more resistant to apoptosis normally seen after strong activation of p53 activity in response to DNA damage. We hypothesized that the ROS generating expanded porphyrin, MGd, which we have previously shown to cause apoptosis in lymphoma and myeloma cells, results in loss of low-level, basal p53 expression, making cells more susceptable to oxidant stress. In order to test this hypothesis, we incubated Ramos, HIF-1 and SUDHL-4 lymphoma cells for 30, 60, 120 and 300 min with MGd (0.1–100 μM) with or without zinc acetate (Zn) and ascorbate. Following incubation, whole cell lysates were collected, electrophoresed on SDS-PAGE gels and blotted with p53-DO1 antibody. GAPDH was used as an internal standard. We found that at 300 min incubation, there was a dose-dependent loss of p53 by immunoblotting in the presence of Mgd and either Zn or ascorbate in Ramos and SUDHL-4 compared with control, and a dramatic loss with MGd/Zn/ascorbate in each of the cell lines. Maximum loss of p53 was observed at 100 μM MGd with both Zn 100 μM and ascorbate 100 μM. MGd, Zn or ascorbate alone had no effect. To determine if p53 message was altered, Ramos cells were treated for 300 min with the same concentrations of MGd, ascorbate and Zn and total RNA isolated by the Qiagen method and dissolved in RNase-free water. 1.0 μg RNA was used for reverse transcription using the Taq DNA polymerase system. PCR amplificatiion was performed at 94° C for 30 sec (denaturation), 58° C (annealing) and 72° C (extension) for 40 cycles. Anti-sense primers and sense primers (IDT Technologies, Coralville, IA) for p53 and GAPDH were used and DNA was identified by 1% agarose gel electrophoresis. We found that there was no loss of p53 message under these conditions in the Ramos cell line.

CONCLUSIONS: These results suggest that there is post-translational modification of p53 induced by the ROS-generating expanded porphyrin MGd, and that this effect depends on the presence of ascorbate or Zn, similar to the effects of MGd, ascorbate and Zn for lymphoma and myeloma cell apoptosis. These data suggest that oxidant generating agents can result in loss of low-level basal p53 protein expression in lymphoma cells and that this might be important in cellular apoptosis, perhaps through mechanisms that depend on downstream p53 targets.
Quantitative Assessment of Circulating t(14;18) Positive Cells by RQ-PCR at Diagnosis and Follow-Up Correspond to Clinical Characteristics and Predicts Time to Treatment Failure in Follicular Lymphoma.
Authors: Pott Christiane , Boettcher Sebastian , Brueggemann Monika , Raff Thorsten , Ritgen Matthias , Unterhalt Michael , Dreyling Martin , Hiddemann Wolfgang , Hoster Eva , Kneba Michael
Published: October 2019

Follicular lymphoma (FL) is characterized by the t(14;18) translocation and is incurable with standard treatments in advanced stages. However, the prognosis of FL is heterogeneous and there are long term survivor with a slow disease progression. The detection of residual t(14;18) positive cells by PCR has become an important tool in the diagnostic work-up and during the clinical follow-up in patients with FL. Therefore we evaluated the prognostic value of RQ-PCR assessment at diagnosis and follow-up in patients with advanced FL.

Patients and methods: PB and bone marrow (BM) of 80 patients with advanced stage, t(14;18)+ FL treated frontline with CHOP (n=42) or CHOP+ Rituximab (n=28) in a prospective trial of the German Low-Grade Lymphoma Study Group (GLSG) was analysed. t(14;18)+ cells were quantified by RQ-PCR (ABI PRISM 7700).

Results: Patients with stage IV disease (p=0.003), presence of B-symptoms (p=0.002) or >4 involved lymph node regions (p=0.004) had significantly higher level of PB t(14;18)+ cells, whereas the RQ-PCR levels did not correlate with sex, bulky disease, LDH and age. RQ-PCR level seemed to correlate also with the FLIPI defined risk groups (median level 0.00015 low risk, 0.00275 intermediate risk, 0.008 high risk, p=0.059). RQ-PCR values showed a significant influence on TTF (p=0.037, univariate Cox Regression). Circulating lymphoma cells in diagnostic PB above a threshold of 0.01 corresponded to the time to treatment failure (TTF), patients with a high lymphoma load (>0.01) demonstrated a significant lower TTF (median 2.8 years) compared to patients with levels <=0.01 (median not reached at a median observation time of 27 months, p=0.0396).

MRD quantification after CHOP induction demonstrated a significant reduction of about 2 logs of lymphoma cells (p=0.0021) in PB, but the majority of patients (77%) remained MRD positive. R-CHOP removed lymphoma cells more efficiently (>2 logs) inducing a molecular remission (MR) in 76% (p<0.0001). Achievement of MR after induction tended to correlate with improved PFS (PFS at 2 years MRD+ 53%, MRD neg. 90%, p=0.068).

Conclusions: Quantitative assessment of circulating t(14;18)+ in diagnostic PB as well as after treatment has demonstrated prognostic relevance and can be used as a surrogate marker for clinical outcome of patients with FL. The results provide further evidence for the value of RQ-PCR based MRD assessment and demonstrate that achievement of MR is one of the major goals in the therapy of FL.
Serum Free Light Chain (SFLC) Elevation Is Associated with High β2-Microglobulin and with a Shorter Time to Treatment in Waldenström Macroglobulinemia (WM).
Authors: Itzykson Raphael , Le Garff-Tavernier Magali , Katsahian Sandrine , Diemert Marie-Claude , Guis Laurence , Choquet Sylvain , Musset Lucile , Leblond Veronique
Published: October 2019

WM is a rare lymphoproliferative disorder caracterised by bone marrow lymphoplasmacytic infiltration with IgM monoclonal gammopathy. Assessing the tumor burden in WM is challenging and is not directly related to IgM level or bone marrow infiltration. Thus treatment criteria are based on clinical symptoms related to IgM or pancytopenia. SFLC quantification is a useful tool for the diagnosis and follow-up of gammopathies such as amyloidosis and light chain myeloma. So far its quantification has never been studied in WM. We investigated whether SFLC quantification was correlated with monoclonal component value and established prognostic factors, or was predictive of a shorter time to treatment.

We analyzed 45 untreated WM patients (IgMκ: 37; IgMλ: 8) at the time of diagnosis (n=23) or during the follow-up (n=22). Patients were assessed for physical examination, body CT scan, serum protein electrophoresis with albumin (g/l) and monoclonal component (g/L) measurements, SFLC level (The Binding Site®, cut-off: SFLCκ 19.4 mg/L and SFLCλ 26.3 mg/L), peripheral blood count and β2-microglobulin (β2-m). Cut-off values of prognostic factors were those defined by the International Prognostic Index for WM (Morel ASH 2006) ie. β2-m > 3mg/L, Hb < 115 g/L, platelets < 100 G/L, albumin < 35 g/L. Treatment was initiated at diagnosis in 7 patients or during the follow-up in 23 patients. Median follow-up for the whole population was 15 months (1 – 74).

Median SFLCκ value was 39 mg/L (1.73–399), median SFLCλ was 12.6 mg/L (1.93–19400). 75 % of patients had upper to normal SFLC value (34/45). Table 1 summarizes univariate analysis of (1) the relationship between high SFLC value (SFLC > N) and known biological prognostic factors, and (2) the impact of SFLC > N and other biological parameters on time to treatment. Our results showed that high SFLC correlated with β2-m but not with other biological prognostic factors or with monoclonal IgM value. (2) Patients with SFLC > N had a shorter time to treatment than patients with normal SFLC value. Furthermore, taking SFLC as a continuous variable in patients with SFLC > N, treatment time was strongly correlated to the SFLC quantification (κ: p=0.01; λ: p=0.01). SFLC could thus be used as a new marker of tumor burden in WM and its prognostic value must be tested in larger WM series. The correlation between SFLC variation and response to treatment is still under investigation.

Table 1 SFLC > N (1) time to treatment (2) SFLC > N NA p=0.03 monoclonal IgM value p=0.08 p=0.001 β2-m > 3 mg/L p=0.03 p=0.44 Hb < 115 g/L p=0.43 p=0.009 Platelets < 100 G/L p=1 p=0.36 Albumin < 35 g/L p=0.3 p=0.011