bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Polymorphisms Correlated with Outcome in Multiple Myeloma Patients Receiving High Dose Melphalan.
Authors: Dumontet Charles , Landi Stefano , Reiman Tony , Plesa Adriana , Bellini Ilaria , Barale Roberto , Pilarski Linda M. , Tavtigian Sean , Perry Troy , Gemignani Federica
Published: October 2019

High dose melphalan is an essential component in the treatment of patients with advanced multiple myeloma. However few data are available regarding genetic polymorphisms possibly correlated with patient outcome or toxicity in this setting. To identify polymorphisms predictive of survival and/or toxicity we performed a retrospective single nucleotide polymorphism (SNP) analysis using APEX technology in 169 patients having received high dose melphalan for multiple myeloma. All patients had received one or two courses of high dose melphalan with stem cell reinfusion. DNA was obtained from peripheral blood mononuclear cells and analyzed for 230 SNPs in genes selected for their role in drug metabolism, DNA repair, cell cycling or apoptosis. There were 108 males and 61 females and the median age was 55 years. The average number of VAD courses received before high dose melphalan was 4 (3–6). 58 patients (34%) received two high dose melphalan courses. A complete/very good response was observed in 43 of 103 evaluable patients (42%). Forty patients among 107 evaluable (37%) experienced high grade (III–IV) mucositis after high dose melphalan. The median freedom from progression was 45 months and the overall survival was 91 months. SNPs in SULT1A1, MDR, ERCC5, GSTP1, NQO1, CYP1A2, CCND1, PARP, RAD51, DRD4, P53 or associated proteins were found to be correlated with response to VAD therapy. SNPs in GRPR, ERCC5, GSTM3, GSTP1, XRCC1, ERCC4, CYP1B1, BARD1, MDR1, P53BP1, TERT, DRD4 were found to be correlated with response to high dose melphalan. Polymorphisms in CYP2E1, CYP2C9, CYP1A1, CYP1B1, TMPT, OGG1, PCNA, DRD2, XPC were correlated with disease progression while polymorphisms in ERCC1, DRD4, CYP2D6, CYP2C9, CYP1A1, CYP1A2, CYP1B1, ALDH, BARD1, BRCA1, MYH, TP53BP1 were correlated with death. Polymorphisms in XRCC1, SNP103, CYP2C9, CYP2C19, BRCA1, BRCA2, NBS1, CDKN1A were correlated with severe mucositis. These results suggest that SNPs of genes involved in drug metabolism or repair could be used to distinguish patients subgroups with different toxicity/efficacy profiles after high dose melphalan.
Elevated Normal Immunoglobulins and Stable M-Protein Levels in Multiple Myeloma Patients on Erythropoietin Treatment.
Authors: Gadassi Noa , Sagiv Sari Prutchi , Oster Howard S. , Newmann Drorit , Mittelman Moshe
Published: October 2019

Recombinant human erythropoietin (rHuEPO) is a well-known treatment for anemia in multiple myeloma (MM) patients. We have previously reported that rHuEPO treatment was associated with prolonged survival of several patients suffering from advanced disease (Mittelman et al., 1997). Recently we have demonstrated that treatment of MM patients with rHuEPO is associated with significant improvements of certain immunological parameters and functions (Prutchi-Sagiv et al., 2006), mainly related to the cellular compartment. The objective of the present retrospective study was to determine whether rHuEPO treatment, in addition to its effects on the cellular immune compartment, also modulates the humoral arm of the immune system in MM patients. Medical charts of eighteen consecutive IgG and IgA MM patients were analyzed and the levels of normal immunoglobulins (Ig) and M-protein before and during rHuEPO treatment were recorded. We have found a significant increase in the levels of normal Ig (IgG, IgA and IgM) in response to rHuEPO, during the 3–9 months fromtreatment initiation. Importantly, the levels of M-protein remained stable for a period of 10–12 months from treatment initiation. These results are in line with previous studies, including our study in murine models (Katz et al., 2007), demonstrating that EPO improves humoral immune responses. The current study highlights the concept that EPO’s immunomodulatory actions on MM patients might also involve the humoral compartment of the immune system.
MN1 Overexpression Promotes Proliferation and Self-Renewal of Primitive Normal Human Hematopoietic Cells
Authors: Imren Suzan , Heuser Michael , Chen Ling-Yi , Edin Glen , Eaves Connie J. , Humphries R. Keith
Published: October 2019

Overexpression of MN1 is a negative prognostic factor in patients with acute myeloid leukemia (AML) with normal cytogenetics. We have previously demonstrated that overexpression of MN1 is sufficient as a single genetic event to induce AML in mice with very short latency (Blood110:1639, 2007). To investigate the effects of MN1 on the functional activity of primitive human hematopoietic cells, we transduced CD34+ human cord blood cells with an oncoretrovirus (3 experiments) or lentivirus (3 experiments) encoding GFP ± MN1. Immediately post-transduction primary colony-forming cell (CFC) assays showed no significant differences in the number or type of colonies generated from the MN1 and control-transduced cells. However, the number of CFCs detectable in secondary assays of the MN1-transduced cells was 17-fold higher than from the GFP-transduced cells and this difference was found to increase a further 234-fold as detected by tertiary CFC assays. To assess the effect of MN1 on cells that can be propagated for more prolonged periods on mouse fibroblast feeders engineered to produce human Steel factor, IL-3 and G-CSF, 104 unsorted cord blood cells were plated into such cultures and the number and types of cells present was then determined 8 weeks later. The remarkable potency of MN1 in stimulating the output of primitive cells under these long-term culture (LTC) conditions was evident from the 1700 greater increase in total cell output and 277-fold higher output of CFCs as compared to control cultures and an accompanying large expansion of CD34+ cells (representing up to 18 % of the final population) whereas CD34+ cells were no longer detectable in the control cultures. Further analysis by limit dilution assay (2 experiments) showed that the frequency of cells able to produce CFCs for at least 6 weeks in these stromal based cultures was increased 10-fold (1/7656 control cells vs 1/718 MN1-transduced cells). In addition the CFC output of each of these was enhanced, on average, 6-fold. Remarkably, these effects of MN1 were sustained for a further 16 weeks as shown by the analysis of the cells they produced in secondary and tertiary LTCs. Overall this resulted in a cumulative 6250-fold increase in total cells over 24 weeks, whereas control cells declined to undetectable levels within 16 weeks. Cells harvested from the secondary and tertiary LTCs initiated with MN1-transduced cells also showed a continuing output of 878,967 ± 332,300 and 264,458 ± 120,600 CFCs after 16 and 24 weeks, respectively. This study sets the stage for further investigation of the effect of MN1 on human NOD/SCID/γcnull mouse repopulating cells and the molecular mechanisms by which MN1 blocks the differentiation fate of primitive human hematopoietic cells and how this may be related to its contribution to the genesis of poor prognosis AML.
Ott1 (Rbm15) Is Required for the Large to Small Pre B-Cell Transition
Authors: Kharas Michael , Manis John , Gilliland D. Gary , Raffel Glen D.
Published: October 2019

The OTT1 gene is fused with the MAL gene in t(1;22) infant-associated acute megakaryocytic leukemia generating a chimeric protein, OTT1-MAL. OTT1 is a transcriptional activator/repressor related to the spen/SHARP/Mint family. Mint was shown to regulate follicular versus marginal zone B-cell development in the spleen; however, Ott1’s physiologic role in B cell development is not fully understood. Recent data utilizing conditional deletion of a targeted Ott1 allele in mice delineated multiple regulatory roles for Ott1 in myeloid and lymphoid differentiation. Previous work by our laboratory showed that loss of Ott1 in addition to causing a myeloid and megakaryocytic expansion, results in a block in B development prior to the B220+CD43-IgM+ stage. We have characterized Ott1-deficient B-cell progenitors to identify stage- and process-specific requirements for Ott1 in pre B development. Ott1-deleted bone marrow and fetal liver could not generate pre B colonies in methylcellulose, however, we were able to establish IL-7-dependent pro B cell lines in vitro and observed no significant differences in proliferation, apoptosis or the ability to form v-Abl-transformed cell lines. Activated Ras or overexpression of Bcl2 failed to rescue pre B colony formation. In vivo, Ott1 null fetal liver pre B-cells expressed Ig heavy chain but failed to express the B-cell receptor (BCR) on their surface even though kappa rearrangement was detectable in vitro. In comparison to wildtype cells, B220+CD43-IgH+ Ott1 null cells were larger in size, had lower levels of IL-7R, but proliferated at higher levels and with an associated increase in apoptosis. Moreover, these cells had normal pre-BCR proximal signaling as judged by phospho-Blnk and phospho-Erk, but increased phosphorylation of S6 after IL7 and Ig stimulation. Ott1 null large pre B-cells had normal expression of Myc, but higher levels of expression of Cyclin D1. Taken together, these data indicate that loss of Ott1 results in enhanced proliferation and apoptosis in the pre B-cell compartment causing a developmental block at the large to small pre B-cell transition. Differentiation blocks at the pro and pre B stage through mutations in B cell regulatory genes, such as PAX5, BTK and BLNK, have been recently demonstrated in acute lymphocytic leukemias. It is plausible that mutations in OTT1, given its position in the tightly regulated process of B cell development, may likewise contribute to pre B-leukemogenesis.
Pre-Transplant Evaluation with Both CT and PET Following Second-Line Therapy Is Essential for Predicting Outcome in Patients with Transplant- Eligible Relapsed and Primary Refractory Hodgkin Lymphoma
Authors: Moskowitz Alison J. , Nimer Stephen D. , Zelenetz Andrew D. , Kewalramani Tarun , Vanak Jill M , Maragulia Jocelyn c , Yahalom Joachim , Moskowitz Craig
Published: October 2019

The standard treatment for relapsed and primary refractory (rel/ref) Hodgkin lymphoma (HL) for patients (pts) who demonstrate chemosensitivity to second-line chemotherapy (ST) is high dose chemotherapy followed by autologous stem cell transplantation (HDT-ASCT). Since 1994, four studies at our institution evaluated ICE (ifosfamide, carboplatin, and etoposide) based salvage therapy followed by HDT-ASCT in rel/ref HL. Chemosensitivity is a requirement for pts to proceed to HDT-ASCT, however the definition of chemosensitivity is broad. We use both functional imaging (gallium or PET) and CT to assess response to ICE; pts with chemosensitive disease fall into three groups: normalization of CT and FI (NRM), residual mass on CT but with negative FI (RM, FI-), and FI positivity regardless of CT finding (FI+). Here we report the outcome for pts who responded to ICE ST and proceeded to HDT-ASCT.

Between October 1994 and February 2008, 198 pts received ICE on 1 of 4 consecutive protocols (169 pts) or as per protocol (29 pts) and were deemed transplant-eligible. The median follow-up for surviving patients is 7.4 years. There were 99 male pts and 99 female pts; the median age was 31. With respect to the 3 pre-salvage chemotherapy risk factors (RF) (relapse within 1 year of primary treatment, presence of extranodal disease, and B symptoms): 44 pts had 0 RF, 64 had 1 RF, 75 had 2 RF, and 8 had 3 RF. Bulky disease defined as greater than 5cm or 10cm was present in 60 (30%) and 15 (7.5%) pts respectively.

The five year event free survival (EFS) and overall survival (OS) were 69% and 78% respectively. Seventy six (38%) had a NRM response to ST, 73 (37%) had a RM, FI- response, and 49 (25%) were FI+. The 5 year EFS and OS for the three groups were 86% and 93%, 71% and 79%, and 41% and 51% respectively. There was a statistically significant improvement in OS and EFS for the NRM and RM, FI- groups compared to the FI+ group (p<0.0001). Surprisingly, a comparison between the NRM and RM, FI- groups also revealed a statistically significant improvement in EFS and OS for the NRM group (p=0.05 and 0.04 respectively).

Response to ICE ST followed by HDT-ASCT is associated with a prolonged EFS and OS. The quality of response to ICE ST had a significant impact on outcome. Both a residual mass with normalized FI and abnormal FI after ST predicts for an inferior outcome following HDT-ASCT. However, residual radiotracer on FI was associated with the poorest outcome in both EFS and OS.

CONCLUSION: Outcome cannot be predicted by FI alone since both RM, FI- and FI+ groups had an inferior outcome compared to NRM. The most accurate post treatment evaluation includes both CT and PET.

Figure Figure
Genome-Wide Analysis of B Cell Non-Hodgkin’s Lymphoma Disclosed Frequent Involvement of Genes in NFkB Pathway
Authors: Kato Motohiro , Nakazaki Kumi , Sato Yasuharu , Takeuchi Kengo , Sanada Masashi , Asakura Yoshitaka , Muto Satsuki , Chen Yuyan , Takita Junko , Hayashi Yasuhide , Igarashi Takashi , Watanabe Toshiki , Tobinai Kensei , Ishikawa Yuichi , Mori Shigeo , Kurokawa Mineo , Yoshino Tadashi , Kobayashi Yukio , Ogawa Seishi
Published: October 2019

B cell non-Hodgkin’s lymphoma (B-NHL) consists of different pathological entities that are frequently characterized by distinct genetic alterations. However, the knowledge on these genetic lesions in B-NHL is still limited. In order to obtain a more comprehensive view of genetic lesions in B-NHL, we performed genome-wide analysis of copy number (CN) alterations as well as allelic imbalances using Affymetrix SNP arrays in 190 B-NHL cases, including 64 samples of diffuse large B-cell lymphoma (DLBCL), 62 of follicular lymphoma (FL), 64 of mucosa-associated lymphoid tissue lymphoma (MALT-L). SNP array data were analyzed with CNAG/AsCNAR software, which enabled sensitive detection of CN alterations in allele-specific manner, and thus allelic imbalances, without depending on availability of paired normal controls. Most frequent numerical abnormalities in B-NHL were gains of chromosomes 3 and 18, although gains of chromosome 3 were less prominent in FL. Chromosomal deletions that lead to loss of heterozygosity (LOH) were commonly found in 1p, 6q and 10q. However, the more chracteristic feature of B-NHL genomes was high frequency of CN netural LOH or uniparental disomy (UPD), which was found in 35 cases of DLBCL (55%), 32 cases of FL (52%) and 18 cases of MALT-L (28%). It is widely distributed in the genome, but more frequently found in 1p, 1q, 6p, 6q and 12q. High-grade amplifications and homozygous deletions frequently provide a clue to identify relevant gene targets. In our series, 12 loci of high-grade amplifications and 14 loci of homozygous deletions were identified, and helped to specify the candidate genes. These regions included, FCGR2B amplified in 5 cases of DLBCL, RERE amplified in 2 cases of FL and CDKN2A/CDKN2B deleted in 9 cases of DLBCL. The most notable finding in the current study was, however, the identification of common genomic alterations in genes that regulate activation of NFkB pathway in more than 50% of B-NHL cases. Eight lymphoma cases harbored high-grade amplification of cREL gene, and gain including cREL was detected in 28 samples (14.7%). Fourteen cases had gains or amplification of TRAF6, and another 16 cases had deletion at 10q including PTEN. These abnormalities were supposed to cause dysregulation of NFkB. Aberrant NFkB activity has long been implicated in the pathogenesis of B-NHL, and our study confirmed that dysregulation of NFkB pathway was main mechanism of lymphomagenesis, providing further rationale that he treatment against malignant lymphoma with inhibitor of NF kappa B pathway.
Bortezomib Induces Osteoblast Differentiation Via Wnt-Independent Activation of Beta-catenin/TCF Signaling
Authors: Qiang Ya-wei , Hu Bo , Chen Yu , Zhong Ying , Barlogie Bart , Shaughnessy John D
Published: October 2019

Bone loss in multiple myeloma (MM) and other conditions represents a serious clinical problem, and extensive research is being conducted to develop new drugs that target signaling cascades whose disruption is known to cause bone loss. Inhibition of Wnt/beta-catenin/TCF signaling induces proliferation of mesenchymal stem cells and/or suppresses their differentiation into osteoblasts. Osteolysis in MM and several other diseases has recently been linked to the suppression of Wnt/beta-catenin/TCF signaling. The antitumor activity of the proteasome inhibitor bortezomib in MM has been linked to its bone anabolic effects. However, the molecular basis of bortezomib’s action on bone is not well characterized. Here we show that bortezomib activates the beta-catenin/TCF pathway in murine and human osteoblast progenitors and human mesenchymal stem cells derived from bone marrow of healthy donors and patients with MM, leading to their subsequent differentiation in-vitro as assessed by matrix mineralization and calcium deposition. Pull-down assays followed by immunoblotting and immunofluorescence microscopy revealed that bortezomib induced stabilization of beta-catenin in both the cytoplasm and nucleus. Nuclear translocation of stabilized beta-catenin was associated with the activation of beta-catenin/TCF transcriptional activity as detected by luciferase activity from TOPFLASH reporter constructs. This activation was not dependent on the drug inducing Wnt ligand or receptor expression, the suppression of Wnt signaling inhibitors or change in disheveled protein levels. Synergy experiments showed that recombinant Wnt3a or LiCl-induced inactivation of GSK3beta did not enhance bortezomib-induced changes in TCF activity. Matrix mineralization was not accompanied by the induction of alkaline phosphatase or osteoprotegerin, which can be activated by Wnt3a in these cell types. Blocking the activation of beta-catenin/TCF signaling by dominant negative TCF attenuated bortezomib-induced matrix mineralization. These results add to the growing body of evidence demonstrating a critical role of the Wnt/beta-catenin pathway in bone biology and provide mechanistic insights into the recently recognized bone anabolic effects of bortezomib. As the effects of bortezomib completely bypass all known regulatory mechanisms governing Wnt signaling, these data also provide a strong rationale for the use of bortezomib in the treatment of diseases linked to suppression of Wnt/beta-catenin signaling.
Downregulation of Ribosomal Proteins Is Seen in Non 5q- MDS
Authors: Sohal Davendra P. , Pellagati Andrea , Zhou Li , Mo Yongkai , Opalinska Joanna B. , Alencar Christina , Heuck Christoph , Wickrema Amittha , Friedman Ellen , Greally John , Ebert Benjamin L. , Warner Jonathan , Boultwood Jacqueline , Verma Amit
Published: October 2019

Recent work (Ebert et al, Nature 2008, Jan 17:335-9) has shown that ribosomal protein S14 (RPS14) is underexpressed in myelodysplasia (MDS) with deletion of chromosome 5q and plays a role in its pathogenesis. The role of ribosomal proteins in other more common subtypes of MDS is unknown. We conducted a meta-analytical comparison of gene expression profiles of 60 cases of non 5q- MDS CD34+ cells with 52 normal CD34+ profiles. These datasets were obtained from seven independent studies from NCBI’s GEO database. The data was integrated based on UniGene IDs and were quantile normalized to ensure cross-study comparability. (Based on our previous approach; Sohal et al PLOS One, 2008, Zhou et al, Blood, 2008). Using significance analysis of microarrays (SAM) and a false discovery rate (FDR) of just 0.04%, we found that ribosomal protein were the class of genes that were most significantly altered in MDS. We observed that RPL35a, RPS9, RPL10, RPL22, RPS14, RPS10, RPS15a, RPS24, RPL24, RPL36, RPL21, RPL23 were strikingly downregulated in non-5q- MDS CD34+ cells. To determine if these alterations were a result of changes in DNA copy numbers of these genes, we examined 20 MDS samples by high resolution array comparative genome hybridization (aCGH) performed on Nimblegen whole genome tiling arrays. aCGH at 6kb resolution revealed deletions in RPL14, RPL22, RPL36, RPS10, RPS5 and even RPS14 in distinct selected cases of non 5q- MDS. These small deletions, which were not identifiable by traditional karyotyping methods, may be putative mechanisms of ribosomal protein downregulation and ultimately, in MDS development and/or progression. Since MDS is also characterized by aberrant epigenetic silencing of genes, we next examined the methylation status of ribosomal gene promoters by high resolution global DNA methylation profiling by using the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)). This assay uses differential methylation-specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. While there were sporadic changes in some patients, we did not observe any consistently significant changes in methylation of these ribosomal gene promoters on comparison with normal anemic controls.

In summary, we show novel widespread alterations in ribosomal protein expression in MDS, at least some of which are associated with genomic deletions. These findings illustrate the applicability of meta-analytical genomic approaches in a heterogeneous disease such as MDS. Most importantly, our data points to the dramatic role of alteration in the protein translational machinery in pathogenesis of MDS.
Cyclin D1 as a Universally-Expressed Mantle Cell Lymphoma-Associated aTumor Antigen for Immunotherapy
Authors: Wang Michael , Sun Luhong , Qian Jianfei , Han Xiaohong , Zhang Liang , Lin Pei , Yi Qing
Published: October 2019

Mantle cell lymphoma (MCL) accounts for 5% to 10% of all non-Hodgkin lymphomas and has the worst prognosis among all lymphomas. The hallmark of MCL is a t(11;14) translocation resulting in overexpression of cyclin D1 by tumor cells of virtually all patients. In this study we examined whether cyclin D1 could be an effective tumor-associated antigen for immunotherapy. We identified a cyclin D1 peptide (P101) for HLA-A*0201, created heteroclitic peptide (Py101) for better binding, and generated peptide-specific T-cell lines from HLA-A*0201+ blood donors and MCL patients. After 5 to 7 rounds of in vitro stimulation with peptide-pulsed autologous dendritic cells (DCs), T-cell lines were obtained, which contained about 45% CD4+ and 55% CD8+ T cells. As exemplified by the results of peptide-tetramer staining, the frequencies of peptide-specific CD8+ T cells increased during in vitro stimulation, from 5.7% of specific T cells at fourth stimulation to 19.7% at sixth stimulation. These cell lines proliferated in response to autologous DCs pulsed with cyclin D1 peptide Py101 (but not unpulsed), as measured by 3[H]-thymidine incorporation and CFSE-dilution assays. These results indicate that the T cells are indeed specific for cyclin D1 Py101 peptide. Moreover, the T cells efficiently lysed peptide-pulsed but not unpulsed T2 cell and autologous DCs, and cyclin D1+ and HLA-A*0201+ human MCL lines MINO, SP53, Jeko-1, and Granta 519, and more importantly, HLA-A*0201+ primary lymphoma cells from MCL patients. No killing was observed on HLA-A*0201− primary lymphoma cells or HLA-A*0201+ normal blood cells including B cells. These results indicate that these T cells are potent cytotoxic T cells and recognize cyclin D1 peptides naturally presented by patient lymphoma cells in the context of surface HLA-A*0201 molecules. Flow cytometry analysis was used to examine the expression of granzymes, perforin, and Fas ligand (FasL) by the T cells. Based on these results, T cells may kill their target cells via the perforin/granzyme pathways, because they express perforin and high levels of granzymes but not FasL. Two independent methods were used to examine the cytokine expression profiles of the T cells. Upon restimulation with DCs pulsed with Py101 peptide, but not with unpulsed DCs, 11.2% of the T cells expressed IFN-γ by intracellular cytokine staining. IL-4-expressing T cells were very few (0.3%). To detect cytokine secretion, an ELISPOT assay was used to enumerate IFN-γ-secreting cells. After restimulation with DCs pulsed with cyclin D1 peptides, or with cyclin D1+/HLA-A*0201+ SP53 and cyclin D1+/HLA-A*0201+ primary lymphoma cells from patients, large numbers of IFN-γ-secreting cells were detected. Taken together, our work identifies cyclin D1 as a potentially important antigen for immunotherapy in MCL.
Cancer Stem Cell Activity in Mantle Cell Lymphoma Is Inhibited by TLR9 Activation
Authors: Brennan Sarah , Meade Brooke , Wang Qiuju , Weiner George , Matsui William
Published: October 2019

Patients with Mantle Cell Lymphoma (MCL) often respond to initial cytotoxic therapy, but invariably relapse. This clinical pattern suggests that standard therapies fail to eliminate a population of chemoresistant cells responsible for tumor regrowth and disease relapse. Studies in myeloid leukemias and multiple myeloma have suggested that cancer stem cells (CSC) are relatively quiescent and that this property promotes drug resistance. Therefore, the induction of CSC proliferation may increase their susceptibility to anti-cancer treatment. Unmethylated CpG DNA sequences induce the activation and differentiation of normal B cells through toll like receptor 9 (TLR9), and synthetic CpG oligonucleotides (ODN) are currently undergoing clinical trials in MCL and other B cell non-Hodgkin’s lymphomas. We initially examined the effects of TLR9 agonists on the clonogenic growth of the human MCL cell lines Granta 519, Jeko-1, and Rec-1 and found that the unmethylated CpG ODN 2006 (5ug/mL, 48 hours) significantly inhibited the ability of each of these cell lines to form colonies in methylcellulose (p<0.01). We previously demonstrated that clonogenic CSC in multiple myeloma and Hodgkin’s lymphoma may be identified based on the relative activity of the intracellular detoxification enzyme aldehydedehydrogenase (ALDH). Similarly, each of the MCL cell lines contained small populations (0.5−2.3%) of ALDH+ cells that were enriched for in vitro clonogenic growth upon primary and secondary plating in methylcellulose. Following treatment with CpG ODN 2006, the population of ALDH+ cells was significantly reduced in each cell line (p<0.05). The self-renewal potential of normal and cancer stem cells is lost through the process of differentiation, and treatment of the MCL cell lines with CpG ODN 2006 resulted in the loss of CD19 and CD20 expression and induction of the plasma cell antigen CD138 by FACS. Similarly, RT-PCR for regulators of B cell differentiation revealed decreased PAX-5 and BCL-6 expression coupled with increased transcription of BLIMP-1 and XBP-1. We hypothesized that the plasma cell differentiation induced by TLR9 agonists may improve the efficacy of anti-tumor agents with known activity against plasma cells, specifically the proteosome inhibitor bortezomib. Treatment with CpG ODN 2006 or bortezomib (10–100nM, 48 hours) alone induced apoptosis in a small percentage of cells within each MCL line, but the combination of these agents synergistically increased the proportion of Annexin V+ cells. In summary, cellular activation by CpG ODN 2006 reduces MCL clonogenicity and stem cell function through the induction of differentiation. In turn, plasma cell maturation appears to increases the sensitivity of MCL to bortezomib and suggests a novel mechanism for the anti-tumor activity of CpG ODN.
Epoetin Alfa Reduces the Need for Red Blood Cell Transfusions and Does Not Affect Remission Rate or Survival When Given to Patients with Hematologic Malignancies Receiving Hyper-CVAD Therapy.
Authors: Mattiuzzi Gloria , Kantarjian Hagop , Cabanillas Maria , Foudray Maria , Garris Rebecca , O’Brien Susan , Thomas Deborah A. , Cortes Jorge
Published: October 2019

Background: Recent reports suggest that the use of erythropoietic stimulating agents (ESA) may adversely affect survival when used in the management of patients with certain solid tumors. Anemia is common in patients with acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LL), and Burkitt’s lymphoma (BL). The use of erythropoietin (EPO) in this group of patients may be beneficial; however, the impact of EPO on the survival of patients with hematologic malignancies is unknown.

Objective: To evaluate the effects of EPO on rate of transfusions, survival and remission rates in patients with ALL, LL and BL treated with Hyper-CVAD.

Methods: We evaluated 109 pts with newly diagnosed acute lymphocytic leukemia (ALL), lymphoblastic lymphoma (LL), and Burkitt’s lymphoma (BL) who participated in a randomized trail comparing EPO (40,000 units SQ weekly within 14 days of starting chemotherapy) vs. standard of care. Both arms received blood transfusions as per institutional guidelines.

Results: From September 2003 to July 2008, 109 pts were included in the study, 5 patients were excluded from the survival analysis because they did not receive study drug (1) or for violation of the study (4), randomized to no EPO but received EPO. The median age of patients was 41 years (range: 15–84), the majority had ALL (82 pts, 79%). Median baseline Hgb was 8.8 g/dl (range 6–12.6). There was no difference in the complete remission rate or survival between the 2 groups (Table 1). Seventy-six patients (38 in each arm) who remained in the study for at least 5 months were evaluable for number of transfusions during that period of time. Patients randomized to EPO received significantly less number of transfusions than patients that did not receive EPO [N=, 16 units (range 6–32) versus 19 units (range 11–40)], p = 0.026 (Mann-Whitney U test). Five patients (deep vein thrombosis = 3; pulmonary embolism =1; and CVA = 1) in the EPO group and 2 patients (deep vein thrombosis =2) in the NO EPO group developed possible related adverse events over 6 months of evaluation (p= 0.285)

EPO (N= 54) No EPO (N= 50) P value Complete remission, n (%) 51 (94) 48 (96) 0.873 Survival at 1 year (%) 80 81 NS Survival at 2 years (%) 75 70 Survival at 3 years (%) 72 70

Conclusions: The use of EPO appears not to have a negative impact on remission rate or survival in patients with ALL, LL or BL receiving HyperCVAD treatment, while EPO significantly decreased the number of transfusions required. There was no significant increase in the risk of thrombotic events associated with EPO.
Phosphoinositide 3-Kinase p110δ Is Dispensable for HoxA9/Meis1 and MLL-AF9 Mediated Leukemogenesis.
Authors: Gibbs Kenneth , Nolan Garry P.
Published: October 2019

The Phosphoinositide 3-kinase(PI3K)/PTEN pathway has been implicated in the pathogenesis of a number of hematopoietic malignancies and solid tumors. The only PI3K subunit invariably expressed in AML blasts is the catalytic p110δ. Recent reports have demonstrated that impairment of p110δ activity prevents growth of AML blasts in culture and increases their sensitivity to apoptosis inducing agents. However, a definitive requirement for p110δ in AML pathogenesis remains undefined. To identify the requirement of p110δ in AML initiation, the transforming potential of two retroviral models of AML (HoxA9/Meis1 and MLL-AF9) was compared in hematopoietic progenitors from wildtype (WT) and p110δD910A/D910A mice (homozygous knock-in for a catalytically inactive form of p110δ). Mice transplanted with p110δD910A/D910A progenitors expressing either HoxA9/Meis1 or MLL-AF9 oncogenes developed a phenotypically similar AML at rates comparable to those transplanted with transformed WT progenitors. Moreover, tumors derived from p110δD910A/D910A progenitors were transplantable to secondary recipients. These results indicate that the catalytic activity of p110δ is not required for HoxA9/Meis1 or MLL-AF9 driven leukemogenesis, suggesting that there are compensatory mechanisms that require further investigation.
Natural Killer (NK) or NK/T Cell Lineage Large Granular Lymphocytosis Associated with Dasatinib Therapy for Philadelphia Chromosome Positive Leukemia.
Authors: Kim Dong Hwan , Kamel-Reid Suzanne , Chang Hong , Sutherland Robert , Jung Chul Won , Kim Hyeoung Joon , Lee Je-Jung , Lipton Jeffrey H.
Published: October 2019

Dasatinib, a dual tyrosine kinase inhibitor, is known to modulate or suppress T-cell activation and proliferation. We report a series of patients of chronic peripheral lymphocytosis development, identified as natural killer (NK) cells or NK/T-cells based on their large granular lymphocyte (LGL) morphologies and CD16+CD56+CD3− or CD3+ immunophenotypic profiles during dasatinib therapy. All cases that developed LGL lymphocytosis achieved optimal molecular response (8/8 in LGL+ patients vs 3/10 in LGL− patients, p=0.002). A 51Cr release assay demonstrated that NK cell cytotoxicity has been enhanced in a case of LGL lymphocytosis compared to normal healthy donors (Figure 1), and that NK cell cytotoxicity in dasatinib-responders was superior to that in non-responders (Figure 2). In summary, the present study suggests that NK or NK/T cell lineage LGL lymphocytosis develops associated with dasatinib therapy and that LGL might have a therapeutic effect on Ph+ leukemic cells.

Figure 1. Cytotoxicity of NK cells isolated from the patients developing large granular lymphocytosis following dasatinib therapy as assessed by 51Cr release assays using target cells as K562 (A) and T2 cell line (B) as target cells. Figure 1. Cytotoxicity of NK cells isolated from the patients developing large granular lymphocytosis following dasatinib therapy as assessed by 51Cr release assays using target cells as K562 (A) and T2 cell line (B) as target cells. Figure 2. The result of 51Cr release assays comparing cytotoxicity of NK cells isolated from patients responding to dasatinib therapy (responder) and not responding (non-responder) following dasatinib therapy using K562 cell line as target cells. Figure 2. The result of 51Cr release assays comparing cytotoxicity of NK cells isolated from patients responding to dasatinib therapy (responder) and not responding (non-responder) following dasatinib therapy using K562 cell line as target cells.
Disruption of the NHR4 Domain Structure in AML1-ETO Abrogates Its Interaction with SON and Promotes AML1-ETO-Associated Leukemogenesis.
Authors: Ahn Eun-Young , Yan Ming , Malakhova Oxana A , Boyapati Anita , Ommen Hans Beier , Hines Robert , Hokland Peter , Zhang Dong-Er
Published: October 2019

AML1-ETO is generated from t(8;21)(q22;q22), which is commonly associated with acute myeloid leukemia (AML), especially FAB M2 AML. While full length AML1-ETO fails to promote leukemia due to its detrimental effects on cell proliferation, an alternatively spliced isoform without its C-terminal NHR3 and NHR4 domains strongly induces leukemia. The NHR4 domain has a zinc-chelating topology and has been implicated in transcriptional repression through recruitment of the NCoR/SMRT complexes. Here, we report that disruption of the zinc-chelating structure in the NHR4 domain of AML1-ETO by replacing only one critical amino acid leads to rapid onset of leukemia, suggesting that the NHR4 domain is responsible for generating inhibitory effects on leukemia development. To identify the molecular mechanism of this leukemogenic effect, we searched for NHR4-interacting factors with yeast two hybrid screening, and identified a novel NHR4-interacting protein, SON, a potential DNA/RNA-binding protein. The N-terminal fragment of SON with 81 amino acids, which is encoded by exons 1 and 2, was sufficient to interact with the NHR4 domain of ETO. Interaction of endogenous SON and AML1-ETO was also detected in t(8;21) AML cell lines. A serine substitution of cysteine 663 which disrupts the zinc-chelating topology of the NHR4 domain completely abolished its interaction with SON, while retaining the interaction with another ETO-binding protein, N-CoR. Knockdown of SON by siRNA resulted in significant growth arrest. Disruption of the interaction between AML1-ETO and endogenous SON by overexpressing the SON N-terminal fragment rescued cells from AML1-ETO-induced growth arrest. These results suggest that SON is an indispensable factor for cell growth and the interaction between endogenous SON and AML1-ETO generates negative effects on cell growth. In t(8;21) AML patient-derived primary leukemic cells and cell lines, abnormal cytoplasmic localization of SON was detected, which may keep cells proliferating in the presence of full length AML1-ETO. Taken together, these results uncovered the crucial role of the NHR4 domain in determination of cellular fate during AML1-ETO-associated leukemogenesis and revealed the interaction of the NHR4 domain with SON, an essential protein for normal cell growth.
Autocrine IGF-1/IGF-1R Signaling Is Responsible for PI3K/Akt Constitutive Activation in Acute Myeloid Leukemia.
Authors: Chapuis Nicolas , Tamburini Jerome , Cornillet-Lefebvre Pascale , Gillot Lucile , Bardet Valérie , Willems Lise , Park Sophie , Green Alexa Samantha , Ifrah Norbert , Dreyfus Francois , Mayeux Patrick , Lacombe Catherine , Bouscary Didier
Published: October 2019

The PI3K/Akt axis is implicated in cell proliferation and survival and appears promising for target inhibition. The constitutive activation of PI3K (PI3K+) that reflects intrinsic cytokine-independent signaling properties of blast cells is detected in 50% of AML samples at diagnosis. Several cytokines and growth factors are known to stimulate the PI3K/Akt pathway, such as FLT-3, SCF or IGF-1 but the mechanisms leading to the constitutive PI3K activation are unknown. The IGF-1/IGF-1R signaling is thought to have a crucial role in the development and progression of cancers. In AML, IGF-1 promotes cell growth and survival via PI3K signaling. As we previously reported an IGF-1 autocrine production in AML leukemic cells, we investigated whether IGF-1/IGF-1R signaling could be responsible for the constitutive activity of PI3K in AML. We show here in primary AML blast cells that IGF-1R is constantly expressed and constitutively phosphorylated in PI3K+ samples. The constitutive phosphorylation of Akt is fully inhibited in 70% of PI3K+ samples by aIR3, a neutralizing anti-IGF-1R monoclonal antibody but neither ERK nor P70S6K phosphorylation are modified by aIR3. Moreover, siRNA-mediated decrease of the IGF-1 autocrine production markedly reduces Akt phosphorylation in leukemic cells. Finally, no mutation is detected within the complete sequence of IGF-1R cDNA from PI3K+ AML samples. In conclusion, the constitutive activity of PI3K/Akt proceed from the activation of IGF-1R due to an IGF-1 autocriny in 70% of PI3K+ primary AML samples. Our current data strengthen the rational of targeted therapy against IGF-1R in AML.
Intensified Induction Chemotherapy with Regimen Containing Intermediate Dose Cytarabine for De Novo Acute Myeloid Leukemia.
Authors: Wang Jianxiang , Mi Yingchang , Liu Jiazhuo , Fu Mingwei , Wang Ying , Lin Dong , Bian Shougeng
Published: October 2019

Abstract□F

Objective To analyze the complete remission (CR) rate, disease free survival (DFS) and overall survival (OS) of de novo acute myeloid leukemia (AML) patients induced with HAD (Homoharringtonine-HHT, cytosine arabinoside-AraC, daunorubicin-DNR) regimen containing intermediate dose AraC (ID-AraC) and to explore the impact of cytogenetic abnormalities on the prognosis.

Methods 87 AML patients were treated with HAD regimen containing ID-AraC as induction therapy. HAD regimen was as follow: HHT 2mg/m2.d, Day 1–7; Ara-C 100mg/m2, Day 1–4, 1–1.5g/m2/q12h, Day 5–7; DNR 40 mg/m2.d, Day 1–3. CR rate, DFS and OS were calculated.83 patients who had karyotype results were divided into 3 groups according to SWOG criteria respectively. Differences in CR rate, DFS and OS among different groups were evaluated.

Results The CR rate of the 87 cases was 80/87 (92%). Median DFS of the 80 CR patients was NR (not reach). DFS rates at 1 and 3 years were 76.3% and 63.4% respectively. The median OS of the 87 patients was 16 (range from 2 to 67) months. OS rates at 1 and 3 years were 86.0% and 58.7% respectively. According to SWOG criteria, CR rate, median DFS and OS were 100%, NR for the favorable group„dG88.9%, NR and 16 months for the intermediate group„dG83.3%, 4.5 months and 7.5 months for the adverse group. The differences among the three groups were statistically significant excepting for CR rate between adverse and intermediate groups.

Conclusions HAD regimen containing ID-AraC as induction chemotherapy regimen is very effective in de novo AML of adult patients, can achieve higher CR rate and longer survival time than standard dose HAD regimen. Most of the patients were able to endure therapy. Cytogenetics is the important prognostic factor for AML patients. The differences among the three groups were statistically significant.
A Phase I/II Study with CP-4055 in Patients with Haematologic Malignancies.
Authors: O’Brien Susan , Rizzieri David A. , Vey Norbert , Kantarjian Hagop M , Jacobsen Tove Flem , Nilsson Bo I , Staudacher Karin , Giles Francis
Published: October 2019

Background: CP-4055 (ELACYT™, cytarabine 5′-elaidic acid ester) is a novel cytotoxic nucleoside analogue. While CP-4055 has similar mechanisms of action to cytarabine, it is, unlike cytarabine, independent of nucleoside transporters for cellular uptake.

Aims: Determination of efficacy and safety of CP-4055 in patients (pts) with haematologic malignancies receiving CP-4055 either as monotherapy or in combination with idarubicin.

Methods: The first patient was enrolled in May 2006. Pts received IV CP-4055 over 2 hours (2 hr IV) or 24 hours (CIV, Arm B) in a day 1–5 q3w schedule.

Results: Seventy-seven pts (46 male and 31 female) with a median age of 61 years (range 21– 92) were enrolled in the first phase I part. The majority of the pts presented with refractory/ relapsed AML and most pts had received two or more previous chemotherapeutic regimens. The recommended dose for CP-4055 monotherapy was determined to be 2000 mg/m2/d. Four pts had CR/CRp among 29 relapsed/refractory AML pts treated with this regimen The starting dose for CP-4055 in combination with idarubicin was 1150 mg/ m2/d CIV. Four pts (2 male, 2 female) have been enrolled. MTD has been determined at 1150 mg/m2/d and accrual is ongoing at 1000 mg/m2/d. DLTs were typhilitis and hand-foot-syndrome. In the phase II monotherapy part, accrual of patients with AML who have failed two prior lines of therapy is ongoing. The first evaluation for safety and futility will be prepared after 20 pts.

Summary/Conclusion: CP-4055 was well tolerated when administered both as 2–4 h IV and as CIV d1–5 q3w. The recommended dose for CP-4055 monotherapy was determined to be 2000 mg/m2/d in a d1–5 q3w schedule. The starting dose for CP-4055 in combination with idarubicin was 1150 mg/m2/d. MTD has been reached and accrual is ongoing at 1000 mg/m2/d.. The study continues.
QLT0267, a Small Molecule Inhibitor of ILK and FLT3 Is Cytotoxic to Acute Myeloid Leukemia (AML) ‘Stem Cells’ Detected in Immunodeficient Mice.
Authors: Muranyi Andrew L , Dedhar Shoukat , Hogge Donna E.
Published: October 2019

The phosphatidylinositol-3-kinase (PI-3K) and FMS-like tyrosine kinase 3 (FLT3) receptor signaling pathways are constitutively active in many AML blast samples suggesting these as therapeutic targets. Integrin linked kinase (ILK) is involved in Akt and GSK3 activation, key downstream effectors of the PI-3K pathway, and participates in the regulation of apoptosis, cell cycle progression, and tumour angiogenesis in many solid tumours. ILK is also expressed ubiquitously in AML blasts. In previous experiments to explore the effect of targeting ILK in AML, QLT0267, a small molecule inhibitor of ILK, was shown to be cytotoxic to AML blasts and colony forming cells (CFC) from some patient samples. Since AML samples containing the FLT3 internal-tandem duplication (ITD) were more susceptible to QLT0267-induced cell kill than FLT3 wildtype (WT) cells we tested the possibility that QLT0267 could inhibit FLT3 as well as ILK. In vitro kinase assays from 4 AML samples showed that QLT0267 produces equivalent inhibition of ILK and FLT3 (both WT and ITD) while Western blotting of 2 AML samples cultured with QLT0267 showed a dose and time dependant decrease in both FLT3 and Akt phosphorylation. 5 AML samples (4 FLT3-ITD, 1 FLT3 WT), were cultured for 24 h ± QLT0267, and assayed for AML CFC or 6-week suspension culture initiating cells (SC-IC). The mean percents kill for 20 and 50 μM QLT0267, respectively, were 92%, and 100% for AML-CFC and 71% and 92% for SC-IC. CD34+CD38− blasts (enriched for AML cells which engraft in immunodeficient mice) from these same samples were analyzed for expression of ILK, pGSK3, and FLT3. Intracellular staining detected ILK and pGSK3 protein in CD34+CD38− cells at levels similar to those present in other AML cell populations. QRT-PCR showed FLT-3 expression in CD34+CD38− cells from all 5 AML samples with 2 of these showing higher expression in this population than in the remainder of AML blasts. To determine if simultaneous targeting of ILK and FLT3 would kill AML progenitors that engraft in mice 4 AML samples (FLT3-ITD +) were cultured for 24 h ± QLT0267, and then injected IV into sublethally irradiated NOD/SCID or NOD/SCID IL2γRnull mice. As shown in the Table, treatment with 20 μM QLT0267 significantly reduced AML engraftment for 3 of 4 samples while the 50 μM dose was effective for 2 of the 3 samples tested (p<0.05, student t-test). Thus, combined targeting of ILK and FLT3 will kill AML cells, including candidate leukemic stem cells that sustain long-term engraftment in mice. Further preclinical evaluation of the potential therapeutic usefulness of this strategy is ongoing.

QLT0267 (μM) 0 20 50 AML Sample % engraftment ± SD AML cells in mouse bone marrow  Week 16 (n=) 1 86 ± 11  (4) 53 ± 30  (6) 2 ± 3  (4) 2 46 ± 45  (5) 3 ± 5  (6) 0  (2) 3 47 ± 40  (5) 1 ± 2  (4) 0  (6) 4 90 ± 14  (3) 1 ± 1  (3) ND