bluuebunny/crossref_metadata_2025 huggingface dataset:
Downregulation of PU.1 Is Required for Subtype of Multiple Myeloma.
Authors: Tatetsu Hiro , Hata Hiroyuki , Yamada Yasuhiro , Mitsuya Hiroaki , Okuno Yutaka
Published: April 2020

There are several recent reports that certain mutations in critical transcription factors could be associated with leukemogenesis; however, it remains unclear whether such mutations could be linked to the pathogenesis of multiple myeloma (MM). In the present study, we attempted to determine whether mutations in transcription factors could be found in the patients with MM, utilizing MM cell lines and freshly isolated MM cells, purified with CD138 antibody and magnetic beads. When we looked at the expression of Blimp-1, XBP-1, NF-kB (p65 and p50), and PU.1 using semi-quantitative RT-PCR, all except PU.1 were found expressed in all MM cell lines and freshly isolated MM cells examined. All the PCR products were sequenced, but no significant mutations were identified. As examined with RT-PCR, PU.1 was found completely downregulated in 6 of 8 MM cell lines, while only faint to no expression of PU.1 was seen in 12 of 41 freshly isolated MM cells. As PU.1 message was sequenced, no mutations were found in 29 PU.1-positive MM cells or 2 MM cell lines that had relatively high PU.1 expression. Considering that PU.1 is essential for myeloid and lymphoid differentiation and that mice with null mutation in the PU.1 gene have no granulocytes, monocytes/macrophages or B lymphocytes, together with a recent report by others that AML developed in mice with PU.1 downregulation by 20%, we asked whether PU.1 downregulation might be associated with the oncogenesis or maintenance of MM. To examine the effects of PU.1 expression on MM cells, we generated tet-off conditional PU.1 expression model of PU.1-negative U266 MM cell line. Surprisingly the induction of PU.1 expression resulted in cell cycle arrest and apoptosis of those cells. Cell cycle analysis using flow cytometry revealed that PU.1-expressing U266 cells were in the G1 arrest. May-Giemsa staining revealed that such cells were swollen, enlarged, and vacuolous. These changes were completely reverted when the expression of PU.1 was silenced by tetracycline addition. There was no change in expression levels of cell cycle-related genes (p27, cyclin D1, cyclin E) and apoptosis-related genes (Bcl-2, Bcl-XL, and A1), while caspase3 cleavage was observed by 72 hr following PU.1 induction. We are currently examining the mechanism(s) of PU.1 downregulation in MM cells and a downstream cascade of PU.1 expression during cell death.
Pathogen-Associated Molecular Patterns Are Growth and Survival Factors for Human Myeloma Cells through Toll-Like Receptors.
Authors: Jego Gaetan , Pellat-Deceunynck Catherine , David Catherine , Regis Bataille
Published: April 2020

Multiple Myeloma (MM) patients are strongly sensitive to bacterial, fungal and viral infections. Furthermore, MM is often newly diagnosed in the context of such infections. Given those recurrent infections remain a major cause of death of MM patients; we surmised that MM plasma-cells could be activated by microorganisms, therefore contributing to the initiation and progression of the disease. Microorganisms share highly conserved structures called pathogen-associated molecular patterns. They are recognized by a family of receptors that act as sensor for the innate immune system and are called the Toll-like receptor family (TLRs). We first measured the expression of the 10 known human TLRs on 16 human MM plasma-cells lines (HMCLs) and freshly purified Plasma-cells from 13 patients. The expression pattern is heterogeneous among HMCLs and does not correlate with the one of B cells. Indeed TLR-3, 4, and 8 are aberrantly expressed. On the contrary, the expression of TLR-2 and 10 is lost on most of the HMCLs. TLR-1, 7 and 9 are the most frequently expressed (15+/16, 12+/16 and 11+/16 respectively). Primary MM plasma-cells also express TLR-7 (9+/13) and TLR-9 (8+/13). Accordingly to this expression profile, culture with TLR-7 ligand (loxoribine) or TLR-9 ligand (hypomethylated DNA from bacteria) increased 1,7 to 6 times (mean 3 ± 1,4 n= 8) the proliferation of IL-6-dependent and independent HMCLs. Furthermore, we observed a resistance to serum-deprivation as well as to dexamethasone-induced apoptosis. Those effects were IL-6-mediated in IL-6-dependent HMCLs since a blocking antibody to IL-6 neutralized the growth and survival induced by TLR triggering. In conclusion, human MM Plasma-cells express a broad range of TLRs and the triggering of TLR-7 and 9 induces tumor cell growth and prevent chemotherapy-induced apoptosis. These effects are mediated by the induction of an autocrine loop of growth factors as IL-6, and others currently under identification in the case of IL-6 independent HMCLs. Thus, MM plasma-cells take advantage of infections to expand and escape to usual therapies.
Transdifferentiation of Human Myeloma Cells and Their Dedifferentiation by IL-6.
Authors: Kawano Michio M. , Liu Shangqin , Otsuyama Kenichiro , Asaoku Hideki
Published: April 2020

Human primary myeloma cells as well as myeloma cell lines lack the expression of Pax-5 gene and CD19 expression on their surface, and myeloma cells are well known to be heterogeneous with regard to their morphology and surface phenotype. The expression of translineage markers, such as CD33, CD7, CD56, CD4 or CD86, has been reported to be observed in most cases of multiple myeloma, although their positive % in the bone marrow mononuclear cells are variable case by case. We found that the expression of CD33 in Liu01, a subclone fromU266 cells and vitamin D3-treated ILKM3 cells, correlated to the monocytoid morphology with convoluted nuclei and the increased expression of C/EBPα. The expression of CD56 in myeloma cells is also well known, but its meaning remains to be clarified. We examined whether CD56(+) myeloma cells also express either CD7, the NK lineage marker, or neuron-specific enolase (NSE), the neuronal cell marker. The expression of NSE was found in CD56(+) myeloma cell lines (NOP-2 and Liu01 cells) as well as primary myeloma cells both by RT-PCR and immunostaining. The expression of CD7 in Liu01 and forskolin-stimulated U266 cells was observed and compatible with large granules in the cytoplasm, and showed an increase expression of perforin mRNA. However, these findings were not observed in Pax-5(+) B cell lines. Interestingly, IL-6 could downregulate the expression of CD33, CD7 or CD56 in primary myeloma cells as well as Liu01 cells. Therefore, these data suggest that Pax-5(−) myeloma cells, but not Pax-5(+) B cells, are capable of multi-lineage differentiation, and IL-6 can induce their dedifferentiation. We propose here that the myeloma cells dropping out of their lineage (B cell linage) can become stem cell-like cells; we call them “stone cells”(stem cell-like cells dropping out of the lineage).
Expression of Human Amyloidogenic Immunoglobulin Light Chains in Mice Leads to Organ Toxicity.
Authors: Ward Jennifer E. , Brenner Daniel , Cui Lei , Liao Ronglih , Connors Lawreen H. , Hoo Pam Soo , Eberhard Jeremy , Christensen Thomas , O’Hara Carl , Skinner Martha , Seldin David C.
Published: April 2020

Recent evidence from the study of different amyloidogenic proteins challenges the dogma that tissue damage is solely the result of amyloid fibril deposition. To examine whether amyloidogenic human immunoglobulin light chains (LCs) may cause acute toxic effects prior to the development of fibrillar tissue deposits in vivo, we have generated amyloidogenic LC-expressing cell lines and transplanted them into mice. A full length lambda-6 light chain was cloned from cDNA prepared from bone marrow of a patient with aggressive multi-organ AL amyloidosis. The LC was subcloned into an expression vector with a CMV promoter and transfected into SP2/0 plasmacytoma cells. Stably transfected cells were injected into syngeneic Balb/c and RAG−/− mice. Four-six weeks later, echocardiograms were performed and the mice were euthanized and serum, urine, and tissues were collected. Mice injected with LC-producing cells, but not control untransfected SP2/0 cells, had detectable circulating human LC in their serum, and 6 of 9 RAG−/− mice excreted LC and albumin in the urine. These mice had evidence of bradycardia by echocardiography, with 4 of 12 mice having heart rates lower than 500 bpm while no controls had heart rates that low, and upregulation of markers of cell stress in the heart. In the kidney, there was evidence of amorphous protein deposits and early glomerulopathy by electron microscopy in two mice examined, but no fibril deposition. Thus, short-term expression of human amyloidogenic LC in mice in vivo produces alterations in heart and kidney function prior to the development of fibrillar deposits.
Natural Killer Cells with NKDG2D Cytotoxicity but NKp30-Dull Phenotype Predominate in Myelodysplastic Syndrome (MDS).
Authors: Bai Fanqi , Painter Jeffrey S. , Alan Cantor , JianXiang Zou , Wei Sheng , Djeu Julie Y. , List Alan F. , Epling-Burnette P.K.
Published: April 2020

Natural Killer (NK) function in patients with MDS as measured by non-MHC-restricted cytotoxicity and activation-dependent cell cytotoxicity (ADCC) are reduced in patients with MDS, however, the mechanisms of the functional impairment are not known. Tumor cytolysis occurs through orchestrated control by inhibitory NK receptors (NKRs) and activating NKRs, which control signaling events that lead to polarized movement of perforin-containing granules toward the NK-tumor contact area. We found that NK cells from 23 out of 35 patients with MDS (66%) displayed reduced lysis of K562 tumor cells compared to age-matched normal controls (p<0.01). To better characterize this defect, we evaluated patient NK function against differential tumor targets including the MDS1 cell line established from an MDS patients. We found that MDS1 incited non-MHC-restricted lysis. Unactivated PBMCs, unactivated NK cells, NK cell lines (NK92 and NKL) but not purified unactivated T cells from normal donors killed MDS1 in 4-hr 51Cr-release assays. Normal NK cells and NK cell lines were also found to rapidly redistrubute perforin granules after exposure to MDS1suggesting that a perforin-dependent lytic pathway was activated. We then performed simultaneous cytolytic assays with K562, MDS1, and the 721.221 B cell lymphoma cell line as target cells. We found that NK cells from MDS patients had greater lytic activity against MDS1 (average 24% vs. average 8% at 50:1 Effector:Target ratio, respectively, p<0.01) Antibody-blocking experiments demonstrated that the NKL cell line and PBMCs from 8 out of 10 MDS patients predominantly used the NKG2D activating receptor to kill MDS1. Consistent with this finding, we showed that MDS1 cells express the major human stress-inducible endogenous proteins MICA and MICB, which are NKG2D ligands. In contast, lysis by NK92 cells and normal PBMCs was not appreciably reduced by NKG2D blocking antibodies suggesting that other unidentified NKR(s) also mediate lysis. To identify the NKRs expressed in MDS patients, we performed immunophenotyping for both the activating NKRs and inhibitory NKRs compared to age-matched normal controls. We found that two activating receptors, NKp30 and CD244 (2B4), were significantly reduced on NK cells from all MDS patients regardless of their ability to lyse NK targets. Inhibitory NKR expression and function were normal. Interestingly, NKG2D expression correlated with reduced cytolytic function. Similar to studies on normal NK cells with low NKp30 and NKp46 (NCRdull) phenotypes, these results suggest that low NKp30 expression leads to predominant NKG2D utilization for tumor cell lysis, which is reduced in MDS patients with defective NK function. Our findings provide critical information about potential importance for immunosurviellance through NKG2D-NKG2D ligands.
CpG in the Immunotherapy of B-Cell Lymphoma.
Authors: Li Jiali , Song Wenru , Levy Ronald
Published: April 2020

CpG is an immune stimulator that can activate both innate and adaptive immunity. By binding to its receptor, TLR9, CpG can activate dendritic cells and B cells by inducing the secretion of cytokines, and increasing the expression of costimulatory molecules, such as CD80 and CD86. In this study, CpG1826, a B type CpG was given intratumorally to BALB/c mice bearing a A20 transplantable B cell lymphoma as a s.c local tumor with accompanying metastatic disease. In this model, CpG alone delayed the growth of tumor, however, the majority of the treated tumor-bearing mice still died. We therefore tested a new therapeutic strategy, a combination of cyclophosphomide (CTX), followed by subsequent intratumoral injection of CpG. The rationale for this strategy is that the danger signals generated by chemotherapy attract the migration of immature DC to the tumor site, and these DCs are activated by subsequent injection of CpG. Our animal studies showed that the combined maneuver can completely regress well-established tumor (Fig.1). The combination of CTX with CpG could cure well-established large size (>1.2cm in diameter) tumors, and the mice remained tumor-free for 5-6 months after the initial treatment. Conversely, the majority of mice that received CTX or CpG alone died of lymphoma. We also showed that this combined effect was CpG injection site dependent, as CTX combined with intratumoral injection of CpG had a much better therapeutic effect (100% survival), as compared to injection of CpG into a distant normal site (20% survival). Our studies also indicated that this combined effect is CD8+T cell dependent, as systemic effects were lost when we repeated this experiment in a CD8+ T cell knockout mouse model. We also showed that CD4+ T-cells and B cells are not required in this system by depleting CD4+ T cells and by using a B cell knockout mouse model. Most importantly, we demonstrated the systemic effect of this combination strategy in mice bearing tumors at two different sites. As shown in Fig. 2, in combination with CTX, injection of one site with CpG controlled the growth of the second site tumor as opposed to a transient anti-tumor effect from chemotherapy or CpG alone. The systemic effect induced by injecting a single tumor site led to significant prolongation of overall survival (p<0.01). In summary, this combined chemoimmunetherapeutic strategy has showed both local and systemic anti-tumor effects, which may be a potent anti-tumor strategy in the clinic.

Fig. 1 Fig. 1. Fig. 2 Fig. 2.
Synergistic Activity of the Proteasome Inhibitor PS-341 with Non-Myeloablative 153-Sm-EDTMP in a Syngeneic, Orthotopic Model of Multiple Myeloma.
Authors: Goel Apollina , Dispenzieri Angela , Geyer Susan M. , Greiner Suzanne , Russell Stephen J.
Published: April 2020

Multiple myeloma is a highly radiosensitive malignancy but, at the present time, radionuclide-based interventions have no proven place in disease management. Bone-seeking radionuclides such as 153-Sm-EDTMP and 166-Ho-DOTMP are promising agents for systemic delivery of ionizing radiation to sites of myeloma disease activity, but they are associated with significant myelosuppression at therapeutically effective doses and have therefore been used only in the context of myeloma stem cell transplantation protocols. We previously reported that the proteasome inhibitor PS-341 potently and selectively sensitizes myeloma cell lines and primary myeloma cells to the lethal effects of ionizing X-irradiation (Goel et al, Exp Hematol. 33, 784, 2005). To determine whether PS-341 is equally effective in sensitizing myeloma cells to ionizing beta-radiation and to extend our initial observations to an in vivo model, we combined PS-341 with the bone-seeking radionuclide 153-Sm-EDTMP. In vitro clonogenic assays were performed using a panel of myeloma cell lines and demonstrated synergistic killing following co-treatment with PS-341 and 153-Sm-EDTMP. Using the orthotopic, syngeneic 5TGM1 myeloma model, the median survivals of mice treated with saline, two doses of PS-341 (0.5 mg/kg), or a single non-myeloablative dose of 153-Sm-EDTMP (22.5 MBq) were 21, 22 and 28 days, respectively. In contrast, mice treated with combination therapy comprising two doses of PS-341 (0.5 mg/kg), one day prior to and one day following 153-Sm-EDTMP (22.5 MBq) showed a greatly prolonged median survival of 49 days. Correlative studies indicated that, compared to single-agent therapy, combination treatment with PS-341 and 153-Sm-EDTMP rapidly reduced the clonogenicity of bone-marrow resident 5TGM1 cells, slowed the elevation of serum myeloma-associated paraprotein levels, and was associated with longer term preservation of bone mineral density. The myelotoxicity of single agent and combination therapies was evaluated by comparing peripheral blood cell counts in each of the treatment groups, and by clonogenicity assays of hematopoietic progenitors isolated form normal mice receiving identical treatment regimens. Treatment with 153-Sm-EDTMP led to significant, but transient, myelosuppression which did not differ between animals treated with 153-Sm-EDTMP alone versus those treated with the combination of PS-341 plus 153-Sm-EDTMP. In summary, PS-341 is a potent in vivo radiosensitizer that greatly enhances the therapeutic potency, without increasing myelotoxicity, of skeletal targeted radiotherapy in the syngeneic, orthotopic 5TGM1 myeloma model. Based on these findings, we propose to conduct a phase I clinical trial at Mayo Clinic to evaluate the combination of PS-341 plus non-myeloablative skeletal targeted radiotherapy (using 153-Sm-EDTMP) in patients with advanced or treatment-refractory multiple myeloma.
Identification of Target Antigens Recognized in MGUS for Immunotherapeutic Approaches.
Authors: Blotta Simona , Bellucci Roberto , Tassone Pierfrancesco , Prabhala Rao , Neri Paola , Shammas Masood , Batchu Ramesh R. , Bae Jooeun E. , Li Cheng , Venuta Salvatore , Ritz Jerome , Anderson Kenneth C. , Munshi Nikhil C.
Published: April 2020

Monoclonal gammopathy of undetermined significance (MGUS) is a benign condition with 30% of patient developing symptomatic multiple myeloma (MM). The events leading to the transformation from MGUS to MM remain unclear, however several evidences have supported the idea that the immune system, in patients with MGUS, may play a role in controlling the progression to MM; and the identification of antigenic targets could open the way for future immunotherapeutic approaches to delay or prevent such progression. To identify target antigens that are recognized by the immune system in MGUS, we have screened a cDNA expression library, produced from purified CD138+ bone marrow myeloma cells, with high dilution (1:500) serum from 3 MGUS patients with stable disease for 1 to 4 years. Using this approach we were able to identify a panel of 11 gene products to which an antibody response was observed in serum from individuals with MGUS. Responses appeared to be directed against intracellular proteins involved in variety of cellular functions such as apoptosis (SON, Hip1), DNA and RNA binding (KIAA0530, GPATC4), signal transduction regulators (AKAP11), transcriptional co-repressors (IRF2BP2), developmental proteins (OFD1) and proteins involved in the ubiquitin-proteosome pathway (PSMC1). Interestingly PSMC1, the 26S subunit of proteosome, has recently emerged as a potential target for antimyeloma therapies and AKAP11 located on the 13q14.11, a locus frequently involved in the pathogenesis of aggressive MM. Importantly, two of these identified genes with antibody response have been previously identified by SEREX screenings; KIAA0530 has been reported to induce a strong antibody response in solid tumors such as head and neck, testicular, breast and colorectal cancers and SON, a protein involved in apoptosis with similarities to oncoproteins such as myc and mos, has been previously reported by us to induce an antibody response associated with a complete response after donor lymphocyte infusion in patients with MM. Our studies are now focused on testing these proteins in a series of MGUS serum at different stages of disease as well as in early stage myeloma. Although preliminary, these data open the possibility to identify target antigens that are important in the disease process of MGUS and may allow us to design future vaccines and immunotherapeutic approaches targeting these antigens in MGUS as well as in MM.
DKK-1 Is a Widely Expressed, Potent Tumor-Associated Antigen in Multiple Myeloma Recognized by Cytotoxic T Lymphocytes.
Authors: Yi Qing , Qian Jianfei , Yang Jing , Wang Siqing , Zhan Fenghuang , Shaughnessy John D. , Wezeman Michele
Published: April 2020

Idiotype proteins have been used for immunotherapy in multiple myeloma (MM); and the results thus far have been disappointing. Therefore, the identification and use of novel and more potent tumor-associated antigens are urgently needed to improve the efficacy of the treatment. Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by the tumor cells of most myeloma patients and may be responsible for suppressed osteoblast formation. Based on these data and the fact that DKK1 is not expressed in normal tissues except placenta, prostate, and mesenchymal stem cells, we hypothesized that DKK1 is a novel tumor-associated antigen in MM. In this study we examined the capacity of DKK1 to trigger the induction of antimyeloma cytotoxic T lymphocyte (CTL) responses. Using DKK-1 peptide-pulsed dendritic cells (DCs), we successfully generated DKK-1 peptide (DKK120-29 and DKK166-74V)-specific CTL lines and clones from HLA-A2+ MM patients and healthy individuals. In our experiments, mature DCs obtained from cultures of blood monocytes were pulsed with HLA-A2-restricted DKK1 peptides. Autologous T cells were then stimulated weekly with these DCs, and cytotoxicity was examined against DKK1 peptide-pulsed T2 cells, myeloma cell lines, and primary myeloma cells isolated from patients. After 4 cycles of in vitro stimulation and subsequent T cell cloning and expansion, specific CD8+ CTL lines and clones were obtained. These CTLs not only had cytolytic activity against DKK1 peptide-pulsed T2 cells, but also significantly lysed HLA-A2+ MM cell line U266 and primary tumor cells in vitro. No killing was observed against HLA-A2+ normal lymphocytes including B cells or HLA-A2− myeloma cell lines or primary myeloma cells from patients. The CTL response could be inhibited by anti-HLA-A2 antibody indicating that the response was indeed restricted by HLA-A2 molecules. IFN-γ, but not IL-4, was secreted by the T cells indicating that the effector cells were type-1 CD8+ T cells. Further functional studies are underway. Thus, these results demonstrate that DKK1-specific CTLs are able to lyse myeloma tumor cells including primary myeloma cells from patients, and identify DKK1 as a potentially important antigenic target for antimyeloma immunotherapeutic strategies
Anti-CD20 Monoclonal Antibody (mAb) with Enhanced Affinity for CD16 Activates NK Cells at Lower Concentrations and More Effectively Than Rituximab ®.
Authors: Weiner George J. , Bowles Julie A. , Link Brian K. , Campbell Mary A. , Wooldridge James E. , Breitmeyer James B.
Published: April 2020

Growing evidence indicates the affinity of mAb for CD16 (FcgRIII) plays a central role in the ability of the mAb to mediate anti-tumor activity. Both polymorphisms in CD16 and structure of mAb Fc can impact on the affinity between CD16 and mAb. Little is known about how affinity between mAb and CD16 impacts on the ability of mAb to activate NK cells. We evaluated how CD16 polymorphisms, and mAb with modified affinity for CD16, impact on NK cell phenotype and cytokine production. MAb consisted of R, anti-CD20 with enhanced affinity for CD20 (AME-133), and AME-133 with Fc engineered to have enhanced affinity for both CD20 and CD16 (Fc-eng-133). Peripheral blood mononuclear cells were obtained from normal subjects that were homozygous for high affinity CD16 (VV at position 158), homozygous low affinity (FF at 158), and heterozygotes and were mixed with mAb and Raji lymphoma cells at an effector:target ratio of 1:1. Higher concentrations of mAb were needed to induce CD16 modulation, CD54 upregulation, and IFNg production on NK cells from subjects with the lower affinity CD16 polymorphism. The dose of mAb needed to induce NK activation was lower with Fc-eng-133 irrespective of CD16 polymorphism.

Measure of activation - % of NK cells CD54 bright: EC50 ng/ml (mean moAb concentration +/− SEM): N=4 per polymorphism

R AME-133 Fc-eng-133 p<0.01 - R vs AME-133; R vs Fc-eng-133; AME-133 vs Fc-eng-133 for each polymorphism VV 6.2 +/− 1.0 3.9 +/− 0.5 1.0 +/− 0.3 VF 11.6 +/− 1.7 5.7 +/− 0.6 1.4 +/− 0.4 FF 29.9 +/− 10.6 11.5 +/− 2.5 2.8 +/− 0.3 p value VV vs FF <0.05 <0.05 <0.05

At saturating mAb concentrations, peak NK activation was greater for Fc engineered AME-133 when compared to R mAb for all samples studied

Ratio of Fc-eng-133 to R % NK cells CD54 bright 1.35 +/− 0.07 IFNg production 2.64 +/− 0.43

These data indicate tumor cells coated with mAb with enhanced affinity for CD16 are more effective at activating NK cells at both low and saturating mAb concentrations irrespective of CD16 polymorphism, and provide further evidence for the clinical development of such mAb with the goal of improving clinical response to mAb.
Correlation of Suppression of FDG PET Uptake with Serum Free Light Chain Levels - Both FDG PET-CT and Serum Clonal Free Light Chain Response Precede and Predict the Likelihood of Subsequent Complete Remission in Newly Diagnosed Multiple Myeloma.
Authors: Walker Ronald , Rasmussen Erik , Cavallo Federica , Jones-Jackson Laurie , Anaissie Elias , Alpe Terri , Epstein Joshua , Van Rhee Frits , Zangari Maurizio , Tricot Guido , Shaughnessy John , Barlogie Bart
Published: April 2020

18F-labeled Fluorodeoxyglucose Positron Emission Tomography - computed tomography (PET-CT) imaging results from newly diagnosed patients with multiple myeloma (MM) were correlated with serum free light chain (FLC) results from Total Therapy 3 (TT3). Patients had baseline PET-CT scans and serum kappa and lamda FLC analysis at diagnosis and at 2, 14 and 30 days post first cycle of VDTPACE and at pre-transplant 1 of two planned autologous stem cell transplants (TX1), correlating PET-CT response, clonal and nonclonal FCL response, and likelihood of subsequent complete clinical response (CR). Patients were selected with serum clonal FLC levels above the normal range that had been enrolled for sufficient time for a complete response (CR) to be recorded. Correlations between the serum clonal and nonclonal FLC levels and PET-CT response defined by both reduction in number of PET-defined focal lesions (PET-FL) (5 mm or larger areas of circumscribed uptake visually definable above background activity) and reduction in the maximum standardized uptake value normalized to the patients’ lean body mass (max SUV) were performed.

Results: Reduction in FLC and PET-FL and max SUV are correlated, as was normalization of clonal FLC levels with likelihood of subsequent CR (Fig 1). The Spearman’s correlation coefficient between percent reduction in clonal FLC and percent reduction in max SUV was 0.04 (n=49, p=0.79) at 2 days post-VDTPACE, 0.32 (n=48, p=0.028) at 30 days post-VDTPACE, and 0.52 (n=33, p=0.002) prior to TX1. The greatest reduction in max SUV occurred by 2 days post-VDTPACE while the maximum reduction in clonal FLC occurred at 30 days post-VDTPACE, indicating that the PET response lead and predicted the FLC response in near “real time.” While normalization of the clonal FLC levels at 30 days was strongly predictive of subsequent CR (p<0.001, n=125), normalization of the non-clonal FLC levels was not (p=0.41, n=124).

Conclusion: We conclude that normalization of serum clonal FLC correlates both with improvement in FDG PET-CT scanning in both max SUV and number of PET-defined focal lesions and with likelihood of subsequent CR, though it “lags” behind FDG PET response by nearly a month, with FDG PET following the clinical course in near real time.

Figure 1: Kaplan-Meier Analysis Correlating Normalization of Clonal FLC Levels by 30 Days Post Initiation of Treatment with Likelihood of Subsequent Clinical Remission (p=0.0001, n=125). Figure 1:. Kaplan-Meier Analysis Correlating Normalization of Clonal FLC Levels by 30 Days Post Initiation of Treatment with Likelihood of Subsequent Clinical Remission (p=0.0001, n=125).
Mabthera (Rituximab) Plus CVP Chemotherapy for First-Line Treatment of Stage III/IV Follicular Non-Hodgkin’s Lymphoma (NHL): Confirmed Efficacy with Longer Follow-Up.
Authors: Solal-Celigny Philippe , Imrie Kevin , Belch Andrew , Robinson Katherine Sue , Cunningham David , Rueda Antonio , Catalano John , Offner Fritz , Walewski Jan , Raposo João , Smith Paul , Marcus Robert
Published: April 2020

Design/Methods: We recently demonstrated in a phase III trial that the addition of rituximab to each of 8 cycles of CVP (R-CVP) chemotherapy significantly improves the clinical outcome of previously untreated patients with stage III/IV CD20 positive follicular NHL when compared to CVP alone (Marcus et al., Blood2005; 105: 1417–23). A multivariate Cox regression analysis of time to progression or death (TTP) showed a treatment benefit in all patient subgroups according to baseline risk factors, except for patients with a baseline hemoglobin level below normal. We now present an updated analysis of all major trial endpoints with 42 months follow-up (FU).

Results: A total of 321 patients (median age 53 years) were recruited (159 CVP, 162 R-CVP). Approximately half of the patients had high-risk disease according to the Follicular Lymphoma International Prognostic Index (FLIPI, score 3–5). The median TTP was more than doubled for patients receiving R-CVP compared to CVP alone (33.6 months vs 14.5 months, p<0.0001). Median time to new lymphoma treatment or death (TNLT) was 12.3 months in the CVP group and nearly quadrupled to 46.3 months in the R-CVP group (p<0.0001). Superior response rates for R-CVP were confirmed (CR+CRu rate 41% vs 11%, p<0.0001) with a median response duration (DR) of 13.5 months in the CVP arm versus 37.7 months in the R-CVP arm. Median disease free survival (DFS) in complete responders was 44.8 months for patients receiving R-CVP and 20.5 months in patients receiving CVP alone (p=0.0005). Thirty-five patients in the CVP arm and 23 patients in the R-CVP arm have died. Kaplan-Meier estimates of 3-year OS rates were 81% in the CVP arm and 89% in the R-CVP (p=0.07). Importantly, significantly more patients receiving CVP died due to lymphoma progression compared to patients receiving R-CVP (25 vs 12 deaths, p=0.02). Subgroup analysis for TTP, ORR, DR and OS according to risk factors at baseline are ongoing and will be presented.

Conclusion: With longer FU, the combination of 8 cycles of rituximab with CVP chemotherapy continues to provide a major benefit as first line treatment for patients with advanced stage follicular NHL.

Kaplan Meier plot of time to death due to disease progression Kaplan Meier plot of time to death due to disease progression
Toxicity Profiles in Patients Using Bortezomib for Multiple Myeloma: Korean Multicenter Analysis.
Authors: Bang Soo-Mee , Lee Jae Hoon , Yoon Sung-Soo , Park Seonyang , Min Chang-Ki , Suh Cheolwon , Sohn Sang Kyun , Min Yoo-Hong , Lee Je-Jung , Kim Kihyun , Seong Chu-Myong , Yoon Hwi-Joong , Jo Deog-Yeon , Lee Kyung Hee , Lee Na-Ri
Published: April 2020

Introduction: The proteasome inhibitor bortezomib has demonstrated clinical activity in pts with MM. Its adverse effects including thrombocytopenia and peripheral neuropathy affected 30% to 60% of patients overall, and interrupted the therapy in 10% to 20%. There were no toxicity data in Asian pts using bortezomib for MM.

Methods: We reviewed the clinical records of MM pts from 24 centers in Korea using NCI Common Toxicity Criteria version 3.0. They were treated with bortezomib alone or in combination with other agents including thalidomide.

Results: 92 pts with MM were treated with a median age of 60yrs (range: 42–77). Median number of previous treatments was 3 (range: 1–10), 73% had been treated with three or more of the major classes of agents including thalidomide (67%) and autologous stem cell transplantation (51%). Regimens were bortezomib only in 36 (39%), bortezomib plus dexamethasone in 33 (36%), and bortezomib plus thalidomide-containing regimen in 23 (25%) pts. Analysis of response revealed CR +nCR in 31 (34%) and PR in 27 (30%), for an ORR of 64% in 90 evaluable pts. The most commonly reported adverse events were thrombocytopenia, sensory neuropathy, infection and fatigue (figure 1). Six therapy-related mortality (7%) were reported within 20 days after the last dose of bortezomib. Causes of death were infection in 3, disease progression in 2 and suicide in 1 patient. Twelve pts (13%) ceased the therapy due to toxicity; neuropathy in 7, infection in 4 and diarrhea in 1 pt. Neuropathy more than grade 2 were more frequent in pts who received thalidomide (11/23) when compared with those without thalidomide (17/69) (p=0.036). But preexisting neuropathy and number of prior regimens did not affect the neuropathy.

Conclusion: The incidence of thrombocytopenia and neurotoxicity were similar but the gastrointestinal toxicities were relatively low in Korean pts compared to that in western studies. Significant neuropathy was associated with the combination of thalidomide to bortezomib. This finding will give a useful message for the development of safe salvage regimen including bortezomib.

Figure Figure
Methylation of Progesterone Receptor Promoter-Associated CpG Island in Polycythemia Vera and Related Myeloproliferative Disorders.
Authors: Jelinek Jaroslav , Verstovsek Srdan , Bueso-Ramos Carlos E. , Prchal Josef T. , Issa Jean-Pierre J.
Published: April 2020

Polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF) are clonal myeloproliferative disorders (MPD). A recently discovered activating mutation of JAK2 tyrosine kinase has been found in most patients with polycythemia vera (PV), in about half of those with essential thrombocythemia (ET) and myelofibrosis (MF), and in 10–20% patients with chronic myelomonocytic leukemia, Philadelphia-negative CML, atypical or unclassified MPD and megakaryocytic leukemia. It is not known what other factors determine the disease phenotype of PV, MF, and other MPD, and what factors other than JAK2 lead to disease progression. Very little is known about epigenetic changes in PV. DNA methylation of promoter-associated CpG islands is a well-recognized mechanism of epigenetic silencing used by tumors for evasion from regulatory mechanisms, and it is an alternative to genetic lesions in cancer causation. Using a genome-wide screen for differentially methylated CpG islands, we found methylation of progesterone receptor promoter region (PGR) in PV granulocytes. We then developed pyrosequencing assays for quantitative detection of PGR methylation in bisulfite-treated PCR-amplified DNA. The PGR methylation above normal control levels was observed in ET (2/12 patients, 17%), PV (10/22 patients, 45%), MF (8/12 patients, 67%), and patients with acute myeloid leukemia and antecedent PV (6/7 patients, 86%). We compared the levels of PGR methylation in MPD with the mutation status of JAK2. The 1849G>T JAK2 mutation was present in 16/27 (59%) MPD patients with unmethylated PGR and 21/26 (80%) patients with methylated PGR; the difference not statistically significant; p=0.135. The role of progesterone receptor signaling in hematopoiesis is not known. Using real time quantitative RT-PCR assay for progesterone receptor expression we found detectable levels in granulocytes from 4/5 normal individuals while the expression in granulocytes from 5/5 PV patients was not detectable. To assess the functional significance of progesterone receptor silencing, we explored the effect of mifepristone, a progesterone receptor antagonist, on the response of BFU-E progenitors to erythropoietin. Mifepristone increased the sensitivity of BFU-E progenitors from normal blood to low concentrations of erythropoietin (60–250 mU/ml) suggesting that disabling of progesterone receptor may increase the response of hematopoietic cells to proliferative stimuli. In conclusion, our data show that PGR methylation is present in half of PV patients and it is even more frequent in MF and PV transformed to AML. Silencing of progesterone receptor by methylation may be an epigenetic change contributing to MPD phenotype and transformation to leukemia.
The Jak2V617F Oncogene Associated with Polycythemia Vera Regulates G1/S-Phase Transition.
Authors: Sattler Martin , Walz Christoph , Crowley Brian J. , Gramlich Jessica L. , King Kendra L. , Podar Klaus , Griffin James D.
Published: April 2020

The V617F activating point mutation in Jak2 has recently been detected in a high proportion of patients with the myeloproliferative disorders polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. Using the Jak2V617F-mutant erythroid leukemia cell line HEL as a model, potential mechanisms that contribute to transformation were investigated. Inhibition of Jak2V617F with a small molecule kinase inhibitor reduced cell growth of HEL cells in a dose dependent manner with an IC50 of 300 nM. This inhibition of growth was associated with a G1 cell cycle arrest, with minimal or delayed apoptosis. The major Jak2 target in normal hematopoietic cells, STAT5, was found to be activated by Jak2V617F. Treatment of the cells with either a Jak2 kinase inhibitor, or with a Jak2-targeted siRNA, decreased STAT5 activation, and also resulted in decreased expression of cyclin D2 and increased expression of p27Kip. Of interest, we found that Jak2V617F induced high levels of reactive oxygen species (ROS), an activity associated with several other tyrosine kinase oncogenes. Expression of a constitutively active form of STAT5 by itself was capable reducing expression of p27Kip and increasing production of ROS, suggesting that each of these signaling events are downstream of STAT5. Additionally, treatment of HEL cells with the anti-oxidant N-acetylcystein increased expression of p27Kip, suggesting that Jak2V617F regulates cell cycle progression at least in part through STAT5 activation of ROS, and ROS regulation of p27Kip. Cell growth of HEL cells was found to be blocked by anti-oxidants. Overall, our results suggest that constitutive activation of Jak2 contributes to a transforming phenotype and therefore hints at novel targets for drug development that may aid traditional therapy.
Detection of Activated STAT5 in Neoplastic Mast Cells in Patients with Systemic Mastocytosis: Role of c-kit D816V.
Authors: Sonneck Karoline , Mayerhofer Matthias , Gleixner Karoline V. , Kerenyi Marc , Krauth Maria-Theresa , Fritz Richard , Sillaber Christian , Moriggl Richard , Valent Peter
Published: April 2020

Recent data suggest that activated STAT5 contributes to growth and differentiation of mast cells (MC) and that STAT5-knock out mice are MC-deficient. We have recently shown that constitutively activated STAT5 acts as a potent oncogenic signaling molecule in hematopoietic progenitor cells (Cancer Cell2005;7:87–99). In the present study, we examined the expression of activated STAT5 in neoplastic MC in systemic mastocytosis (SM) and asked whether the SM-related oncogene c-kit D816V is involved in STAT5-activation. For the immunohistochemical detection of activated tyrosine phosphorylated STAT5 (P-Y-STAT5), we used the specific monoclonal antibody AX1 (Advantex) which does not react with inactive STAT5. In all patients with SM tested (indolent SM, n=11; smouldering SM, n=2; aggressive SM, n=1; mast cell leukemia, n=1; all exhibiting c-kit D816V), MC were found to display P-Y-STAT5. Expression of activated STAT5 was also demonstrable in the c-kit D816V-positive mast cell leukemia-derived cell line HMC-1. The reactivity of HMC-1 cells with AX1 antibody was abrogated by a STAT5-specific blocking-peptide. To define the role of c-kit D816V in STAT5-activation, Ba/F3 cells with doxycycline-inducible expression of c-kit D816V (Ton.kit) were employed. In these cells, induction of c-kit D816V was followed by a massive increase in phosphorylated STAT5 as determined by a specific DNA-binding assay, whereas the total amounts of STAT5-mRNA and of the STAT5-protein showed only a slight increase or remained unchanged. In summary, these data show that neoplastic MC in SM express activated STAT5 (P-Y-STAT5), and that the transforming c-kit mutation D816V leads to persistent activation of STAT5 in these cells.
Heme Oxygenase-1 (HO-1): A Novel KIT D816V-Dependent Target in Neoplastic Human Mast Cells (HMC-1).
Authors: Kondo Rudin , Mayerhofer Matthias , Gleixner Karoline , Vales Anja , Krauth Maria-Theresa , Derdak Sophia , Gruze Alexander , Pickl Winfried , Sillaber Christian , Greish Khaled , Maeda Hiroshi , Valent Peter
Published: April 2020

Systemic mastoyctosis is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC). Most patients express the D816V-mutated variant of c-KIT, which mediates resistance against most available tyrosine kinase inhibitors. Therefore, current research is focusing on novel targets in MC. We analyzed expression and function of the survival factor heme oxygenase-1 (HO-1) in neoplastic MC. As assessed by Northern blotting and RT-PCR, the human MC line HMC-1 that exhibits KIT D816V, was found to express HO-1 mRNA. Expression of the HO-1 protein was demonstrable by immunocytochemistry and Western blotting. To examine the role of mutated KIT in expression of HO-1, Ba/F3 cells with doxycycline-inducible expression of c-KIT D816V were employed. In these experiments, KIT D816V was found to induce HO-1 promoter activity and expression of HO-1 mRNA and HO-1 protein in these cells. Moreover, the KIT-D816V-targeting compound PKC412 was found to downregulate expression of HO-1 in HMC-1 cells. The KIT D816V-induced upregulation of HO-1 in Ba/F3 cells was completely blocked by a combination of LY294002 (PI3-kinase inhibitor) and PD89059 (MEK inhibitor), but not by single agents, suggesting involvement of multiple signaling pathways. We next examined whether targeting of HO-1 is associated with decreased survival. As assessed by 3H-thymidine uptake, the pegylated HO-1 inhibitor zinc-protoporphyrin (PEG-ZnPP) reduced proliferation of HMC-1 cells in a dose-dependent manner (IC50: 5 μM). The PEG-ZnPP-induced inhibition of growth was found to be associated with induction of apoptosis (control: 1±0.6 vs PEG-ZnPP, 5 μM: 55±5% apoptotic cells, p<0.05). The HO-1 inductor hemin (10 μM) increased the expression of HO-1 in HMC-1 cells and partly rescued these cells from PKC412-induced growth-inhibition (PKC412, 1 μM: 39±8% vs PKC412 + hemin: 23±11%). Finally, PKC412 and PEG-ZnPP were found to produce cooperative (additive) growth-inhibitory effects on HMC-1 cells. In summary, our data show that HO-1 is a novel survival factor and interesting target in neoplastic human MC exhibiting the D816V-mutated variant of KIT.
PKC412 Inhibits In Vitro Growth of Neoplastic Mast Cells Expressing the D816V-Mutated Variant of KIT: Comparison with AMN107 and Imatinib, and Evaluation of Drug-Interactions.
Authors: Gleixner Karoline V. , Mayerhofer Matthias , Aichberger Karl J. , Derdak Sophia , Sonneck Karoline , Boehm Alexandra , Samorapoompichit Puchit , Gruze Alexander , Pickl Winfried , Manley Paul , Fabbro Doriano , Sillaber Christian , Valent Peter
Published: April 2020

In most patients with systemic mastocytosis (SM) including aggressive SM and mast cell leukemia (MCL), neoplastic cells express the oncogenic c-KIT mutation D816V. KIT-D816V is associated with constitutive tyrosine kinase (TK) activity and thus represents an attractive target of drug therapy. However, most available TK inhibitors including STI571=imatinib, fail to block TK-activity of KIT D816V at pharmacologic concentrations. We provide evidence that the novel TK-targeting drugs PKC412 and AMN107 decrease TK-activity of D816V-mutated KIT and counteract growth of Ba/F3 cells with doxycycline-induced expression of KIT D816V as well as growth of the human mast cell leukemia cell line HMC-1 expressing this c-KIT mutation. PKC412 was found to be the superior drug with IC50 values of 50–250 nM and without differences seen between HMC-1 cells exhibiting or lacking KIT D816V. By contrast, AMN107 exhibited potent effects only in the absence of KIT D816V in HMC-1 cells. Corresponding results were obtained with Ba/F3 cells exhibiting wild-type or the D816V-mutated variant of KIT. Moreover, we found that PKC412 and AMN107 inhibit growth of primary neoplastic MC in a patient with KIT D816V+ SM. The growth-inhibitory effects of PKC412 and AMN107 on HMC-1 cells were associated with TK-inhibition of KIT and with induction of apoptosis. In addition, PKC412 was found to downregulate expression of CD2 and CD63, two cell surface antigens upregulated in SM. In co-incubation experiments, PKC412 was found to synergize with AMN107, imatinib, and 2CdA in producing growth inhibition in HMC-1 cells lacking KIT D816V, whereas in KIT D816V+ HMC-1 cells, drug-interactions were additive rather than synergistic. Together, PKC412 and AMN107 alone and in combination counteract growth of neoplastic mast cells. Both drugs may therefore be considered as novel promising agents for targeted therapy in patients with aggressive SM or MCL.
Chimeric Anti-CD20 Monoclonal Antibody (Rituximab;Mabthera) in Remission Induction and Maintenance Treatment of Relapsed /Resistant Follicular Non-Hodgkin’s Lymphoma: Final Analysis of a Phase III Randomized Intergroup Clinical Trial.
Authors: Van Oers Marinus H.J. , Van Glabbeke Martine , Teodorovic Ivana , Rozewicz Cynthia , Klasa Richard , Marcus Robert E. , Wolf Max , Kimby Eva , Hagenbeek Anton
Published: April 2020

Background. Last year we reported interim results of a randomised phase III trial in patients with relapsed/refractory stage III/IV positive follicular NHL, demonstrating that the addition of rituximab (R) to CHOP chemotherapy for remission induction as well as rituximab maintenance treatment significantly improves the clinical outcome (Blood2004;104:169a). Based on these interim results, randomisation to the induction part of the trial was halted and the protocol was amended.

Study design. Patients with stages III or IV follicular lymphoma at initial diagnosis and relapsed after or resistant to a maximum of two non-anthracycline containing systemic chemotherapy regimens, were randomized to remission induction with either 6 cycles of standard CHOP (once every 3 weeks) or CHOP + R (375 mg /m2 at day 1 of each cycle of CHOP). Those with a complete or partial remission after 6 cycles of therapy underwent a second randomization to no further treatment (observation) or maintenance treatment with R (375 mg/m2 once every 3 months) until relapse or for a maximum period of two years.

Results. At the time of the preplanned second interim analysis (February 2004) 461 patients had been included. Of these, 369 could be evaluated for response (188 CHOP; 181 R-CHOP). Both treatment arms yielded similar partial response rates (CHOP: 53.7% - R-CHOP: 52.5%), but highly significant different CR rates (CHOP: 18.1%; R-CHOP: 30.4%; p=0.0004). Of 319 patients randomized for maintenance treatment, 268 were evaluable (132 observation; 136 R maintenance). A highly significant advantage was observed in progression free survival in patients randomized to R maintenance, when compared with the observation arm (median PFS 38 vs.15 months; p<0.0001). At that time there was no impact on OS: the observed difference (in favor of R maintenance) was not significant for an interim analysis. Toxicity of CHOP and R-CHOP induction was similar, and R maintenance was associated with minimal toxicity. These results of the planned second interim analysis showed that the formal criteria for stopping the trial had been met. It was concluded that this is the first trial to show that: 1) in patients with relapsed/resistant follicular lymphoma R-CHOP remission induction results in a highly significant increase in CR rate as compared to CHOP; 2) rituximab maintenance treatment significantly improves PFS in patients responding to induction treatment. The final analysis of the study results will be performed in September 2005, with an additional follow-up of at least 18 months. The final results will be presented at the meeting.