bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-Myeloma Activity of Two Novel N-Substituted and Tetraflourinated Thalidomide Analogues.
Authors: Kumar Shaji , Raje Noopur , Hideshima Teru , Ishitsuka Kenji , Roccaro Aldo , Shiraishi Norihiko , Hamasaki Makoto , Yasui Hiroshi , Munshi Nikhil C. , Figg William D. , Anderson Kenneth C.
Published: April 2020

The importance of bone marrow angiogenesis in multiple myeloma (MM) provided the initial rationale for using thalidomide in this disease, given its anti-angiogenic properties. Thalidomide (α-N-[phthalimido] glutarimide, C 13 H 10 N 2 O 4), a glutamic acid derivative, has proved useful in the treatment of both newly diagnosed and relapsed/refractory MM, however, its potential for teratogenicity lays a huge burden on its use as a therapeutic agent. Ongoing efforts have been directed towards development of analogues, which retain anti-MM activity without teratogenic effects. A product of cytochrome P450 2C19 isozyme biotransformation of thalidomide, 5′-OH-thalidomide, has been shown to be partly responsible for the anti-angiogenic effect of thalidomide. Based on the structure of this metabolite several N-substituted and tetraflourinated thalidomide analogues have been synthesized as potential anti-angiogenic agents and have shown pre-clinical activity in prostate cancer. In this study, we demonstrate potent anti-MM activity and delineate potential mechanisms of two such compounds, CPS11 (an N-substituted thalidomide) and CPS49 (a tetraflourinated thalidomide). Both CPS11 and CPS49 showed inhibition of proliferation of several MM cell lines as well as primary tumor cells from relapsed/refractory MM patients. These drugs had significant activity in myeloma cell lines resistant to conventional chemotherapy agents (dexamethasone, adriamycin, mitoxantrone and melphalan). Both the drugs were able to overcome the protective effects of growth factors like IL-6, IGF-1, and VEGF, and co-culture with bone marrow stromal cells (BMSCs), demonstrating their activity against myeloma cells in conditions simulating the natural microenvironment. In most of the cytotoxicity experiments, CPS49 was more potent compared to CPS11. An additive cytotoxic effect was noted when these agents were combined with dexamethasone and melphalan. Apoptosis was noted in MM1.S cells as evidenced by annexin and PI staining and a time- and dose-dependent increase in cleavage of poly ADP-ribosepolymerase (PARP) and caspase-8. Additionally, Z-VAD-FMK partially blocked these effects. Importantly, apoptosis triggered by these drugs was associated with down-regulation of anti-apoptotic proteins like Mcl1 and XIAP. In matrigel based angiogenesis assays both drugs demonstrated anti-angiogenic activity, CPS49 being more potent than CPS11. To further delineate their molecular mechanism of actions, we evaluated the effect of these drugs on various intracellular signaling pathways known to be important in myeloma. Both the drugs were able inhibit the PI3K/Akt and JAK/STAT pathways in MM1.S myeloma cells line. These encouraging in vitro data will provide the platform for testing these analogues initially in animal models and subsequently in the clinic.
A Phase I Trial of Oblimersen Sodium (G3139) for Relapsed or Refractory Waldenstrom Macroglobulinemia.
Authors: Gertz Morie Abraham , Geyer Susan M. , Kahl Brad S. , Badros Ashraf , Reeder Craig , Erlichman Charles
Published: April 2020

Introduction

Antisense oligonucleotides are used to inhibit messenger RNA function and inhibit protein translation. Bcl-2 expression proteins have been implicated in mediating resistance to apoptotic cell death in Waldenstrom Macroglobulinemia (WM). Oblimersen sodium is a specific inhibitor of Bcl-2 expression and has shown activity in multiple myeloma and in Waldenstrom cell lines. The in vitro data led to the development of a phase I study for WM patients with relapsed or refractory disease.

Materials and Methods

Eligible patients had symptomatic WM who had failed prior cytotoxic chemotherapy. All patients had to have one of the following: a hemoglobin <11 g/dL, a platelet count <100,000/uL, bulky lymphadenopathy, or hyperviscosity syndrome. As a phase I study, the primary end point was toxicity assessment, but all patients were assessed for response, using monoclonal protein levels. Patients were enrolled and evaluated in cohorts of three. Drug was administered as a 24-hour continuous infusion for 7 days every 21 days, with intrapatient dose escalation each subsequent cycle beginning at 3 mg/kg/day up to a maximum of 7 mg/kg/day days 1–7 for a maximum of 8 cycles.

Results

To date 9 patients have been accrued, six at dose level one and three patients at dose level two. Data is available for the first six entered. Five women and 1 man have been accrued with a median age of 74.5 years, ranging from 58 to 81. Four patients had relapsed from prior therapy, 2 were refractory. Three had a history of transfusions. The median number of prior regimens was 4 (range 2–7). One patient had a dose-limiting toxicity on the first cycle of therapy, which was grade 3 fatigue and anorexia, and the dose for this patient was reduced. Two patients had non-hematologic grade 3 toxicity, possibly related to therapy. Five out of six patients on dose level one had grade 3 or greater hematologic toxicity. These toxicities occurred after cycle 1 was completed, during the toxicity observation period and were not considered dose limiting toxicities influencing dose escalation. Three patients required dose reductions in subsequent cycles. To date one patient has had a partial response with her serum M spike falling from 3.6 to 1.1 g/dL. Her quantitative IgM fell from 6,000 mg/dL to 1,000 mg/dL(figure).

Conclusion

Oblimersen was well tolerated at the first dose level in patients with WM. Hematologic toxicity is frequent in this cohort with heavily involved bone marrows when the dose was escalated during successive cycles. Dose reductions with subsequent cycles were frequently required. Evaluation of the maximum tolerated dose continues. Data on response for this dose level is promising, where efficacy will be fully evaluated in the phase II portion of this trial.

Figure Figure
IL-4 Affects Proliferation, Chemosensitivity-and Rituximab Sensitivity of Germinal Center B-Cell like (GCB) and Activated B-Cell like (ABC) Diffuse Large B-Cell Lymphoma Differently.
Authors: Nechushtan Hovav , Rosenblatt Joseph D. , Lossos Izidore S.
Published: April 2020

Diffuse Large B-cell Lymphoma (DLBCL) represent a diverse group of lymphoid neoplasms with heterogeneous clinical, histological, immunophenotypic, cytogenetic and molecular genetic features. Approximately 50% of DLBCL patients are not cured by the standard combination chemotherapy regimens. DLBCL can be subclassified into GCB-like DLBCL which are characterized by expression of genes normally expressed in germinal center B cells, and having a significantly better overall survival (OS) than the ABC-like DLBCL, which are characterized by expression of genes induced during in vitro activation of normal B cells. At least two markers of the GCB-phenotype - BCL6 and HGAL - are IL-4 target genes, increased expression of which independently predicts better OS. These observations suggest that endogenous or exogenously administered IL-4 may influence behavior of DLBCL. IL-4 mRNA was detected at low levels in 5 of 7 GCB-like and in all 4 ABC-like DLBCL tumor specimens. Two of 7 GCB-like tumors showed high expression levels of IL-4 as determined by real-time RT-PCR.

Examination of the effects of IL-4 on proliferation of GCB-like (SUDHL6, SUDHL4 and OCILY19) and ABC-like (OCILY10 and OCILY3) DLBCL cell lines showed that IL-4 mildly increased DNA synthesis, as assessed by thymidine incorporation, in all the GCB-like DLBCL. Conversely, IL-4 markedly decreased proliferation in the ABC-like DLBCL cell lines by inducing G1 arrest. IL-4 also differently affected the sensitivity of GCB-like and ABC-like DLBCL to doxorubicin. IL-4 reduced doxorubicin-induced cell death of ABC-like cell lines (20–50% reduction) while it markedly increased the killing of the GCB-like cells (40–80% induction). IL-4 also prevented serum starvation-induced cell death of the ABC-like DLBCL, but it increased cell death of the GCB-like DLBCL cell lines.

Recently, Rituximab was shown to improve survival of DLBCL patients when added to the CHOP regimen. The precise mechanisms of its action are unknown; however present data suggest that it may affect lymphoma cells either by activation of complement lysis or by mediating ADCC. IL-4 reduced the complement mediated Rituximab cell lysis of the ABC-like cell lines, while it increased the complement mediated Rituximab cell lysis of the GCB-like DLBCL cell lines. Expression levels of surface markers that modulate complement cell lysis (CD46, CD55 and CD59) were not affected by IL-4 exposure. In contrast, IL-4 did not affect killing of GCB-like and ABC-like cells by ADCC. These observations suggest that DLBCL subtypes may respond differently to the in vivo cytokine milieu of the tumor. Different responsiveness to IL-4 may modulate tumor sensitivity to the current therapeutic modalities and can potentially be explored to augment response to chemotherapy and Rituximab.
Novel Unconventional Antigen and Conventional Antigens Elicit Anti-Tumor Humoral Immune Reactions in Response to Interferon-a Therapy.
Authors: Yang Xiao-Feng , Xiong Zeyu , Liu Enli , Yan Yan , Yang Yu , Verstovsek Srdan , Prchal Josef , Wang Hong
Published: April 2020

Therapeutic responses to interferon-a (IFN-a) therapy in patients with malignancies are at least in part from the effects of IFN-a on the enhancement of anti-tumor immune responses. Antigens responsible for immune responses enhanced by IFN-a remain largely unknown. Polycythemia vera (PV) is a myeloproliferative disease (MPD) resulting from the clonal expansion of a pluripotential hematopoietic precursor cell. IFN-a therapy induces a complete remission in ~50% of PV patients, thus making PV a good model to study the molecular mechanism of the cytokine-enhanced immune responses against tumors. We hypothesized that IFN-a enhances anti-PV immune responses by modulating the expression of non-mutated self-antigens. We identified three novel MPD antigens by applying a technique, termed serological identification of tumor antigens by expression cDNA cloning (SEREX), to screen a human testis cDNA library with sera from three PV patients who responded to IFN-a therapy. Of these three novel SEREX antigens, MPD5 belongs to the newly identified group of unconventional cryptic antigens. These unconventional antigens are encoded by either the introns of genes, or the untranslated regions of mRNAs. MPD5 is the antigen encoded by the complementary strand of the intron 1 region of NEK-6 gene. Two additional MPD antigens, MPD13 and MPD67, are conventional antigens, which are encoded by the genes with conventional exon/intron structures. Our results showed that these novel MPD antigens elicit potent IgG antibody immune responses in the sera of patients with PV, as well as patients with chronic myelogenous leukemia who responded to IFN-a treatment and in patients with prostate cancer, suggesting that these antigens are broadly immunogenic. Previous reports showed that unconventional antigens can elicit cellular T cell mediated responses. Our data demonstrated that these antigens also elicit humoral IgG antibody responses, which can be further enhanced by IFN-a therapy. These findings provide new insights into the molecular mechanism underlying the regulation of the self-antigen repertoire including both conventional antigens and unconventional antigens, in eliciting anti-tumor immune responses in responses to IFN-a therapy, and suggest their therapeutic potential as the targets of novel immunotherapy for MPD.
HMGA2 Dysregulation in Myelofibrosis with Myeloïd Metaplasia (MMM) Versus Other Phi-Negative Chronic Myeloproliferative Disorders and Myelodysplasia.
Authors: Bilhou-Nabera Chrystèle , Andrieux Joris R. , Pierre-Louis Olivier , Le Bousse-Kerdilès Marie-Caroline , Dupriez Brigitte , Grardel Nathalie , Charpentier Agnès , Praloran Vincent , Bauters Francis , Laï Jean Luc , Demory Jean Loup
Published: April 2020

Background: Dysregulation and overexpression of the tumorigenic gene HMGA2 has been demonstrated in the mononuclear cells of 12 consecutive MMM patients (pts) and thus appears a hallmark of the disease [Andrieux & al. Genes, Chromosomes and Cancer 2004]. Apart from pathogenetic considerations, the consistency of this finding raises the question of its specificity and usefulness as a differential diagnosis tool able to distinguish proliferative forms of MMM from other chronic Phi negative myeloproliferative diseases and on the other hand cytopenic forms of MMM from myelodysplasia.

Material and methods: We assessed the level of HMGA2 expression in 30 MMM pts (the former series and 18 additional pts), 15 healthy volunteers as controls, 20 pts with Polycythemia vera (PV), 5 having evolved to post-polycythemic myelofibrosis (PPMF), 12 pts with Essential Thrombocytemia (ET), 3 of whom having developed fibrosis (PTEMF), 1 pt with a MMM-like picture due to systemic mastocytosis and 15 patients presenting with various types of MDS, 3 of whom having marked medullary fibrosis. RNA was extracted from peripheral blood leukocytes separated by standard Ficoll centrifugation and QT-PCR was performed on cDNA with TaqMan technology.

Results: While controls were all negative, a substantial expression of HMGA2 was confirmed in 28/30 MMM pts, significantly higher than in any other situation; PV and ET patients were negative except for those having developed myelofibrosis; the pt with myelofibrosis related to mastocytosis was also negative. In the short and heterogeneous series of MDS pts, a weak expression was occasionnaly found with no obvious correlation with myelofibrosis or the subtype of MDS.

Conclusions: dysregulation and overexpression on HMGA2 characterize MMM and allow a definite discrimination from other Phi-negative MPD; in PV or ET, the gene is not expressed during the chronic proliferative phase but becomes activated in the case of an evolution towards myelofibrosis. These findings, together with the presence of transcripts in MDS, suggest that the re-activation of HMGA2 is not directly linked to the myeloproliferation nor to the myelofibrosis itself but rather associated with dysplastic hematopoïeisis either primitive or subsequent to myelofibrosis.
Fusion of Platelet-Derived Growth Factor β to a Novel Gene KIAA1509 on Chromosome 14 in an Atypical Myeloproliferative Disorder Associated with Eosinophilia.
Authors: Browett Peter J. , O’Grady Anna , Oei Paul , Holdsworth Duncan , Van de Water Neil S.
Published: April 2020

Platelet-derived growth factor β (PDGFRβ), a tyrosine kinase receptor, has recently been shown to be constitutively activated by fusion to different gene partners in atypical, bcr-abl negative, myeloproliferative disorders. As a consequence, the activated PDGFRβ gene is a potential target for the tyrosine kinase inhibitor Imatinib mesylate. The objective of this study was to further characterise a t(5;9;14) translocation found in a 22 year old female patient with an atypical myeloproliferative characterised by eosinophilia, a severe paraneoplastic lichenoid eruption involving her skin and oral mucosa, and a poor response to conventional chemotherapy. G banded cytogenetic analysis of her bone marrow at diagnosis revealed an abnromal karyotype t(5;9;14)(q33;q22;q32) in 38 of 40 metaphases examined. There was no evidence of the bcr-abl fusion gene trancript by RT-PCR. Fluorescence in situ hybridisation using a CSF1-R probe suggested a breakpoint involving the PDGFRβ gene locus on chromosome 5q33 with an unknown partner gene on chromosome 14. 5′ RACE-PCR studies using a PDGFRβ primer generated a product of 220bp that on sequencing revealed a novel gene KIAA1509 fused in frame to the 3′ end of PDGFRβ in exon 11. KIAA1509 has previously been mapped to chromosome 14 and encodes a protein of 1935 amino acids that shows homology to HOOK proteins and contains coiled coiled domains. The resulting fusion protein preserves the tyrosine kinase domains of PDGFRβ, but lacks the transmembrane domain, and would be predicted to result in constitutive activation of the tyrosine kinase receptor. In view of these molecular findings, the patient was commenced on Imatinib mesylate, which resulted in a rapid haematologic response, including resolution of the mucosal and cutaneous manifestations, and a complete cytogenetic response after 3 months therapy. These findings highlight the importance of investigating the molecular basis of the myeloproliferative disorders to identify potential novel targets for Imatinib and similar molecules.
High Peripheral Blood CD34 Count in Patients with Idiopathic Myelofibrosis (IF) Is Not Due to Fibrosis and Altered Marrow Microenvironment.
Authors: Popat Uday , Frost Adaani , Liu Enli , May Romelia , Reddy Vishnu , Durett April , Prchal Josef
Published: April 2020

Idiopathic myelofibrosis (IF) is due to an acquired somatic mutation(s) of a single hematopoietic multipotent progenitor and thus circulating myeloid, erythroid, megakaryocyte, B and T cells (Reeder et al Blood. 2003;101: 1981–1983) are clonal. It has been suggested that fibrosis may occur secondary to cytokines secreted by the malignant clone. Although marked elevation of circulating CD34 cells occurs in patients with IF, it is not known whether it is a consequence of the primary defect of the hematopoietic stem cell, or secondary to fibrosis and impaired marrow microenvironment. To answer this question, we studied CD34 count in peripheral blood of patients with IF and secondary myelofibrosis due to pulmonary hypertension. Nineteen patients with either primary or secondary pulmonary hypertension due to connective tissue disease and 11 patients with IF were studied. Bone marrow biopsy revealed fibrosis in all 19 patients with PH: 11 severe, 3 moderate, and 5 mild. All eleven patients with IF had severe fibrosis. Clonality studies in 13/14 informative patients with PH showed polyclonal platelets and granulocytes in contrast to monoclonal platelets and granulocytes in patients with IF. Peripheral blood was assayed for CD34+ cells using a three-color direct immunofluorescent staining method. Mean absolute CD34 count was significantly (P=0.02) lower - 6/ul (range: 1–18/ul) in patients with PH compared to 142 /ul (range 4–833/ul) in patients with IF. We therefore conclude that high CD34 count is not seen in patients with PH with secondary myelofibrosis; furthermore, elevated CD34 in patients with IF is not due to marrow fibrosis and altered microenvironment.
Lineage Analysis of the FIPL1-PDGFRα Fusion Gene in Myeloproliferative Hypereosinophilic Syndrome.
Authors: Lemery Steven J. , Robyn Jamie A. , McCoy J. Philip , Kubofcik Joseph , Kim YaeJean , Nutman Thomas B. , Dunbar Cynthia E. , Klion Amy D.
Published: April 2020

Hypereosinophilic syndrome is a rare disorder characterized by hypereosinophilia and eosinophil-mediated tissue injury. An imatinib sensitive myeloproliferative variant (MHES) has been described which has a male predominance, and is associated with elevated serum tryptase levels, tissue fibrosis, increased atypical mast cells, and the presence of the fusion oncogene FIP1L1-PDGFRα which has tyrosine kinase activity. The FIP1L1-PDGFRα mutation has been detected in peripheral blood mononuclear cells, however, the hypercellular bone marrow and elevated serum tryptase levels suggest that multiple lineages might be involved in the clonal process. We analyzed peripheral blood from eight patients with the FIP1L1-PDGFRα mutation. Individual patient samples were sorted by flow cytometry to collect greater than 95% pure populations of CD3, CD14, and CD19 cells. Density gradient centrifugation followed by negative selection for CD16, CD3, CD14, and CD19 using an immunomagnetic bead column was used to purify eosinophils to > 99% purity. Bone marrow from one patient was obtained, and mast cells were cultured from CD34 positive cells. Three techniques were used to assay for the presence of the FIPL1-PDGFRα fusion gene: nested RT-PCR, TaqMan quantitative PCR, and FISH. Eosinophils were positive for the fusion gene in all patient samples that were analyzed. Monocytes were also positive in all but one instance. Surprisingly some patients showed positivity in lymphoid lineages as well. The bone marrow derived pure mast cell culture was positive for the mutation, consistent with the elevation of serum tryptase and atypical appearance of mast cells in MHES. In conclusion, although MHES seems to have a multilineage predilection, specific lineages involved may vary between patients. This may reflect differences in the progenitor stage at which the mutation occurs. Whether the pattern of lineage involvement has any relation to the phenotypic expression of the disease remains to be elucidated.
DHEA Can Inhibit the Proliferation of Myeloma Cells and the IL-6 Production of Bone Marrow Mononuclear Cells from the Myeloma Patients.
Authors: Otsuyama Ken-ichiro , Ma Zi , Liu Shangqin , Li Fu-Jun , Abroun Saeid , Zheng Xu , Maki Yasuko , Tsuyama Naohiro , Obata Masanori , Ishikawa Hideaki , Kawano Michio M.
Published: April 2020

Growth expansion of monoclonal plasma cells (myeloma cells) in the bone marrow (BM) is triggered by increase in serum interleukin-6 (IL-6). Indeed, high levels of IL-6 have been found in the serum of patients with progressive multiple myeloma (MM), and a reduction of the tumor cell mass has been reported by inhibition of myeloma cell proliferation with anti-IL-6 antibodies. On the other hand, IL-6 is considered to increase physiologically in its serum level along with age, and the age-related increase in serum IL-6 could be related to the age-dependent diseases such as rheumatoid arthritis, atherosclerosis, Alzheimer’s disease and B cell malignancies including myelomas. Recent reports also show that the age-associated decline in the serum level of an adrenal sex hormone, dehydroepiandrosterone sulfate (DHEA-S), is closely related to the increase in serum IL-6. Here, we show that DHEA-S is decreased significantly more than that by physiological decline with age in overt myeloma, but not in other lymphoid malignancies such as malignant lymphoma or macroglobulinemia. DHEA-S as well as DHEA suppressed IL-6 production from a BM stromal cell line, KM-102, and bone marrow mononuclear cells from patients with MM. Furthermore, DHEA inhibited in vitro growth of the U-266 cell line and primary myeloma cells from the patients, and also in vivo growth of U-266 cells intraperitoneally implanted in SCID-hIL6 transgenic mice. DHEA upregulated the expression of peroxisome proliferator activated receptor (PPAR)β and IκBα gene in myeloma cells and BM stromal cells, which could explain the suppressive effect of DHEA on IL-6 production through the downregulation of NF-κB activity. PPAR agonists such as troglitazone, carbacyclin and WY14643, also inhibited the growth of myeloma cell lines and primary myeloma cells. Therefore, these findings suggest that DHEA has a capability of inhibiting the growth of myeloma cells and IL-6 production of bone marrow mononuclear cells through activation of PPAR and/or suppression of NFκB activity, and inversely a decreased level of DHEA and DHEA-S could upregulate IL-6 activity, which could be responsible for progression of myeloma cells.
CD8+ T Cells Specific for Cancer-Testis Antigens Are Found in Many Patients with Multiple Myeloma and Correlate with Disease Burden.
Authors: Goodyear Oliver C. , Piper Karen , Arrazi Julie , Khan Naeem , Mahendra Premini , Pratt Guy , Moss Paul
Published: April 2020

Proteins from the family known as ‘cancer-testis antigens’ (CTAg) are expressed in some cases of multiple myeloma and subsets of acute myeloid leukaemia. CTAg can stimulate CD8+ T cell responses in patients with melanoma but there are no reports of CTAg-specific immune response in patients with haematological malignancy. Such information is critical to assess whether or not these antigens act as targets for tumour-specific immunity or if they could be used as targets for immunotherapy. We have used twelve peptide epitopes from a range of cancer-testis antigens which have been previously defined as epitopes for CD8+ T cells. These were used to screen for tumour-specific T-cells in blood of patients with multiple myeloma at various stages of their disease. The IFNγ cytokine secretion assay was used to detect functional responses and magnetic selection was employed to increase the sensitivity of detection. FACS analysis was used to quantitate the frequency of responding cells. 37 patients were screened with an age range of between 45 and 88 years. Blood samples were taken at monthly intervals and the percentage of CD8+ T cells responding to each peptide was calculated. 13 patients responded to 1 or more of the peptides with a range between 0.01% and 0.7% of the total CD8+ T cell pool. The frequency of the tumour-specific response fluctuated during treatment in individual patients. Analysis of the CTAg-specific immune response in relation to disease course revealed that the immune response was generally correlated with tumour burden as revealed by the paraprotein level. CTAg HLA-peptide tetramers incorporating peptides from LAGE-1 and MAGE-2 were able to directly visualize CTAg-reactive T cells in PBMC. CTAg-specific CD8+ T cells may have been primed and expanded by expression of CTAg on tumour cells or following ‘cross presentation’ through dendritic cells. In conclusion, T cells specific for cancer-testis antigens are present in the blood of a subset of patients with multiple myeloma. The clinical significance of this observation is currently being addressed.
Specific Killing of Multiple Myeloma Cancer Cells by Epigallocatechin-3-Gallate Extracted from Green Tea.
Authors: Shammas Masood A. , Koley Hemanta , Batchu Ramesh B. , Neri Paola , Tassone Pierfrancesco , Prabhala Rao , Anderson Kenneth C. , Munshi Nikhil C.
Published: April 2020

Epidemiological studies have demonstrated chemopreventive effects of green tea in several types of cancers. The active ingredient present in green tea, and not black tea, is epigallocatechin-3-gallate (EGCG). EGCG has been shown to interfere with multi-step carcinogenesis and it exerts its effects in pharmacologically attainable non-toxic concentrations. We examined the effects of EGCG on growth of MM cells. We observed dose- and time-dependent induction of apoptosis in MM cells following EGCG treatment. Apoptotic cell death, as measured by annexin V labeling was 38 ± 6% and 60 ± 8% at 24h with 100 mM and 200 mM EGCG concentration, respectively. This increased to 73 ± 5% and 88 ± 2% at 72h, respectively. Importantly, no significant apoptosis was observed in normal cells exposed to 200 μM EGCG. The apoptotic cell death was predominantly caspase 9 dependent and not caspase 8 dependent as evaluated using specific caspase 8 and 9 inhibitors. The anti-tumor effects of EGCG has been demonstrated to be mediated via a metastasis-associated 67-kDa laminin receptor. Gene expression profile of MM cell lines (n=6) and patient myeloma cells (n=15) compared to normal plasma cells (n=3) showed 3.1 ± 1 and 3.9 ± 0.7-fold elevation of laminin receptor respectively. Significant upregulation of this receptor was observed in 6 of 6 MM cell lines and 14 of 15 MM patient samples. We are currently investigating possible role of laminin receptor in EGCG mediated cell death in MM and combinations that may enhance EGCG-mediated MM cell death. These data suggest EGCG as a promising natural agent with excellent therapeutic index.
FDG PET Functional Imaging in Multiple Myeloma - Clinically Important Caveats, Pitfalls, and Pearls.
Authors: Walker Ronald , Jones-Jackson L. , Miceli M. , Anaissie Elias , Alpe T. , Walker R. , Epstein Joshua , Tricot Guido , Shaughnessy John , Barlogie Bart
Published: April 2020

We have performed 2,000+ Fluorodeoxyglucose PET (PET) scans for multiple myeloma (MM) staging and restaging at our facility since October 2001. While the usefulness of the PET scan for MM is reported by us and others elsewhere, we have reviewed our list of “incidental” but important findings, some of which are unique or occur more commonly with MM patients and some of which are common to many patients, the more common of which we present below: Occult infection occurs very commonly in patients with MM due to direct tumor effect on the immune system and to medication (especially high dose dexamethasone). Of the 2000+ PET scans for MM done at our facility, 300+ infections (about one half occult) have been detected by PET. These most commonly involve central lines (septic thrombophlebitis), diskitis, lung (either bacterial or fungal), and periodontal abscesses (a source of bacteremia in these patients), though non-catheter related spontaneous septic thrombophlebitis also occurs.

While related to MM, extramedullary disease from MM (EMD) is seen more commonly with PET than MRI since PET has a wider field of view. In our series of 172 patients with both baseline PET and MRI, PET detected EMD in 11/11 patients versus MRI detection rate of 7/11. On follow-up, PET detected three times more EMD (29/31 sites) than MRI (9/31sites). Detection of EMD by PET at baseline is a profoundly negative prognostic factor in our series (12 month EFS 20% for EMD+ vs. 47% for EMD−, p = 0.002, and OS 42% for EMD+ vs. 70% for EMD−, p=0.005, n=48). Following tumor response in hypo- or non-secretory disease is difficult by standard prognostic factors (SPFs). FDG PET results were actively used for clinical management in 10 of 11 non-secretory patients at our facility.

A major pitfall of PET scanning for MM comes from the use of steroids. Treatment with chemotherapy in general and steroids (i.e. prednisone or dexamethasone) in particular can result in a false negative PET scan by producing a profound but transient suppression of tumor metabolism. In addition, the hyperglycemic effect of the steroids produces competitive inhibition of FDG (a glucose analog) uptake, also causing suppression of FDG tumor uptake that can lead to an underestimation of disease. Second primary malignancies are frequently seen the MM age group. In our population, we have found unsuspected breast cancer, breast lymphoma, thyroid cancer, melanoma, colon cancer and lung cancer. In addition, we have found several functioning thyroid adenomas and premalignant colon polyps. Being a whole body imaging device for metabolism, FDG PET is a powerful though nonspecific tool for imaging that can not only help stage and restage the malignancy under question but which can yield a plethora of additional clinically useful information if used properly and with its limitations understood.
Induction of Multiple Myeloma-Specific Cytotoxic T Lymphocytes Using HLA-A2.1-Specific CD19 and CD20 Peptides.
Authors: Bae Jooeun , Martinson Jeff A. , Klingemann Hans G. , Treon Steven , Anderson Kenneth C. , Munshi Nikhil C.
Published: April 2020

We have identified novel CD19 and CD20 antigen-derived HLA-A2.1-specific immunogenic peptides, CD19150–158 (KLMSPKLYV) and CD20188–196 (SLFLGILSV), for generating cytotoxic T lymphocytes (CTLs) against malignant B-cell diseases. Initial testing showed that the CTLs displayed antigen-specific and HLA-A2.1-restriced cytotoxic activity against both Burkitt’s lymphoma and chronic lymphoid leukemia cell lines. The observed cytotoxic activity of the CTLs was shown to be specific to the CD19150–158 or the CD20188–196 peptides. Additionally, the CTLs displayed a distinct phenotype (majority CD69+/CD45RO+) along with a significant (p<0.05) increase in cell proliferation and IFN-γ release following re-stimulation with HLA-A2.1+/CD19+/CD20+ tumor cell lines. Based on emerging information that clonogenic myeloma cells express CD19 and/or CD20, we evaluated the activity of the CD19 and CD20 peptide specific-CTLs against several multiple myeloma cell lines. Five of 10 myeloma cell lines evaluated were HLA-A2.1-positive and expressed both CD19 and CD20 antigens. CD19 peptide specific-CTLs generated from normal donors were able to specifically lyse CD19+/HLA-A2.1+ MM cell lines (30% lysis; 10:1 E:T ratio) but did not lyse CD19−/HLA-A2.1+ or CD19+/HLA-A2.1− cell lines. Similarly, the CD20-specific CTLs generated from normal donors lysed CD20+/HLA-A2.1+ MM cell lines (25% lysis; 10:1 E:T ratio), in a manner restricted to HLA-A2.1 and specific to antigens. We next showed IFN-γ production by the CTLs after exposure to CD19+/HLA-A2.1+ or CD20+/HLA-A2.1+ MM cells. Moreover, we have demonstrated the ability to expand CD20-CTLs under serum-free culture conditions while maintaining their cytotoxic activity (28–49%). In ongoing studies, we are evaluating the ability of CD19- and CD20-specific CTLs to eliminate clonogenic myeloma cells in vitro and in vivo in a SCID mouse model of myeloma. These preclinical studies strongly suggest that immunogenic CD19 and CD20 peptide-based vaccines represent a promising immunotherapeutic approach in myeloma.
PS-341 Induces Selective Radiosensitization of Multiple Myeloma Cells.
Authors: Goel Apollina , Dispenzieri Angela , Greipp Philip R. , Witzig Thomas E. , Mesa Ruben A. , Russell Stephen J.
Published: April 2020

Sublethal doses of ionizing radiation induce a complex cellular survival response, one component of which is activation of the nuclear factor kappa B (NF-kB) pathway. PS-341 is a selective inhibitor of the 26S proteasome which prevents the proteasomal degradation of I-kB and thereby inhibits the activation of NF-kB. We therefore hypothesized that PS-341 should synergistically enhance the anti-myeloma effect of ionizing radiation (IR). The individual and combined effects of PS-341 and IR were analyzed using human MM cell lines (MM1, RPMI 8226, JJN-3 and ARH-77) and bone marrow samples from myeloma patients.

Treatment of myeloma cell lines with 10nM PS-341 produced a modest anti-proliferative effect with G2/M-phase arrest and p21cip/waf-1 induction but minimal procaspase-3 cleavage and minimal apoptotic cell death: Similar sublethal damage was observed after single exposure to IR at a dose of 6 Gy. In contrast, sequential exposure to PS-341 (10 nM) and IR (6 Gy) resulted in potent (synergistic) induction of apoptosis, assayed by both fluorescein isothiocyanate conjugated annexin V-propidium iodide assay and cell cycle analysis. Overall cell viability was greatly reduced by the combination of PS3451 and IR, but not by either modality alone, as determined by 3-(4,5 dimethylthiazol-2yl) 2–5 diphenyltetrazolium bromide (MTT) assay and analysis of polyadenosine-5′-diphosphate-ribose polymerase (PARP) cleavage -product after 72 hours. As predicted, exposure to 6 Gy IR led to potent induction of nuclear NF-kB activity and this response was almost completely inhibited by simultaneous treatment with PS-341. The combination of PS341 and IR was next evaluated on CD138+ve and CD138-ve cell fractions from bone marrow aspirates of myeloma patients. Synergistic killing of primary myeloma (CD138+) cells was observed with relative sparing of normal marrow elements (CD138−). We conclude that PS-341 is a potent radiosensitizer for myeloma cell lines and primary myeloma cells. This provides a strong rationale to combine PS-341 with conventional external beam or with targeted radionuclide therapy for effective treatment of MM patients.
Phase II Study of Oxaliplatin in Patients with Relapsed or Refractory Non-Hodgkin’s Lymphoma: Final Results.
Authors: Oki Yasuhiro , McLauhlin Peter , Pro Barbara , Hagemeister Frederick H. , Bleyer Archie , Loyer Evelyn , Younes Anas
Published: April 2020

Many patients with NHL suffer from refractory or relapsed disease, and platinum-based combination therapy is widely used for salvage therapy. Oxaliplatin, a novel platinum derivative, is a more potent cytotoxic agent than cisplatin, demonstrating greater efficacy against many tumor cell lines, including lymphoma cell lines. Clinical studies of oxaliplatin for other malignancies have shown a favorable toxicity profile. The objective of this phase II trial was to investigate the activity of oxaliplatin in patients with relapsed or refractory non-Hodgkin’s lymphoma (NHL). Patients with measurable NHL who had experienced failure of one or more prior chemotherapy regimens were considered eligible. They were required to have an ECOG performance status £ 2 and adequate organ function. Patients were excluded if they had HIV infection, CNS lymphoma, or had chemotherapy or radiotherapy within 4 weeks prior to entering the study. All patients gave their written informed consent to participate. Oxaliplatin was administered at 130 mg/m2 every 21 days for up to six cycles in the absence of progression. Thirty-one patients (23 with aggressive NHL, 8 with indolent NHL) were enrolled on this trial. Thirty eligible patients received oxaliplatin, who were all assessable for toxicity. Response and survival were also analyzed in all 30 patients based on intention to treat analysis. The median patient age was 62 years, and most of the patients had an ECOG performance status of 0 or 1. The most common toxic effect was sensory neuropathy (87%). Grade 3 and 4 toxic effects included sensory neuropathy (10%), neutropenia (17%), and thrombocytopenia (20%). Objective responses occurred in 8 (27%) of the patients: 7 with aggressive NHL, 1 with indolent NHL. The median failure-free survival duration in the entire group, aggressive NHL group and indolent NHL group was 3.0, 2.1 and 4.0 months, respectively. It is noteworthy that three of the five patients with MCL (60%) had a significant response to oxaliplatin, including two CRus, with a median FFS duration of 5 months. When patient group is divided by IPI score (0/1 and 2 /3), the median FFS duration was 3.4 months and 2.6 months, respectively. Median response duration for entire group was 3.0 months. In summary, oxaliplatin has modest activity in patients with previously treated NHL, especially those with aggressive disease. The favorable toxicity profile of oxaliplatin warrants further investigation of it either in combination chemotherapy or with targeted biologic therapy.
Sapphyrins Exhibit Tumor Selectivity and Efficacy in Animal Models of Hematologic Malignancies.
Authors: Naumovski Louie , Sirisawad Mint , Lecane Philip , Ramos Jason , Magda Darren , Wang Zhong , Thiemann Patti , Boswell Garry , Cho Dong-Gyu , Sessler Jonathan , Miller Richard A.
Published: April 2020

Sapphyrins are pentapyrrolic metal-free expanded porphyrins that localize to tumors. We have previously demonstrated that sapphyrins induce apoptosis in a variety of hematologic tumor cell lines including lymphomas (Ramos, DHL-4, HF-1), leukemias (Jurkat, HL-60), and myelomas (8226/S, 1-310, C2E3, 1-414). Through chemical modification of the parent compound, PCI-2000, a number of derivatives were generated and tested for induction of apoptosis in Ramos cells. PCI-2000 and one of the more potent apoptosis-inducing derivatives, PCI-2050, were injected into CD-1 nude mice bearing Ramos xenografts. Animals were sacrificed 48 hrs after injection and analyzed for drug uptake in the tumor, liver and spleen using flow cytometry. For PCI-2000, the relative uptake was spleen>tumor>liver. For PCI-2050 the relative uptake was tumor>spleen>liver, suggesting that PCI-2050 preferentially localizes in tumors compared to PCI-2000. Tumor cells isolated from PCI-2050 treated animals grew less well in culture and had more apoptotic cells than those derived from PCI-2000 or control animals. Uptake of PCI-2050 into xenograft tumor cells and tumor cell killing was dose dependent. PCI-2050 (10 umol/kg x 2 days in a row) was administered to Ramos xenograft bearing animals that were then monitored for tumor growth. In both minimal tumor (animals treated before tumor was palpable) and established tumor (palpable tumor) models, PCI-2050 reduced tumor growth by 60–75%. Alternative dosing strategies revealed that split dosing (allowing 1 or more days between doses) was more efficacious in tumor control than dosing 2 days in a row. At the doses used in this study, there was no myelosuppression or lymphosuppression, hepatic or renal abnormalities as assessed by complete blood count and comprehensive serum chemistry analysis, respectively. Our work demonstrates that PCI-2050 induces apoptosis in tissue culture and inhibits tumor growth in an animal tumor model while exhibiting minimal toxicity. PCI-2050 and other sapphyrin derivatives will be further evaluated as potential anti-cancer agents.
Consolidation with Alemtuzumab in First Remission Induces Pronounced MRD Reduction and Clinical Remissions - Update on a Randomized Phase III Trial of the German CLL Study Group (GCLLSG).
Authors: Ritgen Matthias , Schweighofer C. , Fingerle-Rowson Günther , Eichhorst Barbara , Busch Raimunde , Stilgenbauer Stephan , Döhner Hartmut , Kneba Michael , Hallek Michael , Wendtner Clemens M.
Published: April 2020

The monoclonal antibody alemtuzumab is known to be effective in combination or alone in patients with late stage chronic lymphocytic leukaemia. Flow cytometric studies on minimal residual disease (MRD) showed the high potency beyond clinical response criteria even in late stage chronic lymphocytic leukaemia (CLL). The aim of the phase III trial of the GCLLSG was to investigate the role of alemtuzumab as consolidation therapy in CLL patients in first clinical remission after fludarabin (F) or F plus cyclophosphamide (FC).

Patients in partial or complete clinical remission were randomized to either arm A (alemtuzumab 3 x 30 mg i.v. for up to 12 wks) or no further treatment (arm B). From 21 eligible patients 11 (1CR, 1 nPR, 9PR) were randomized to arm A. All patients received standard infection prophylaxis with famciclovir and trimethoprim/sulfamethoxazole. Before, at 6 and 12 weeks after start of consolidation therapy and regulary during follow up blood and bone marrow samples were send for molecular MRD assessment by allele specific real time PCR. The PCR allowed MRD assessment with a sensitivity of at least 10E-4 in 11 eligible cases.

Results: MRD analysis showed clear reduction of CLL content by first line treatment to a median MRD level of 2.2E-3. Short after alemtuzumab treatment all treated patients showed pronounced MRD reduction to a median MRD level of 5.0E-5. During molecular follow-up median MRD level remained below 1E-4 for one year with a tendency to increase afterwards. After a median follow-up of 31.3 months from start of initial treatment occurred one relapse in the treatment arm compared to 7 of 10 in arm B. The median progression free survival (PFS) calculated from start of consolidation therapy was significantly shorter in the control group versus the treatment group (pfs 25.2; p= 0.04). Nevertheless due to severe acute infections (CTC III and IV) this study had to be stopped prematurely.

Conlusion: The addition of alemtuzumab as a consolidation regimen after remission induction by F or FC is highly effective and leads to sustained MRD reduction below 10E-4. This translates into significant improved clinical outcome even in this small patient cohort compared to the control group. Encouraged by the molecular responses the GCLLSG initiated a new phase I/II trial as a dose finding study in CLL patients after F based induction of clinical remission.
Rituximab® and High Dose Methylprednisolone (HDMP) for the Treatment of Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia.
Authors: Castro Januario E. , Bole Jan , Prada Carlos E. , Kipps Thomas J.
Published: April 2020

High-dose gluocorticoids and the anti-CD20 mAb Rituximab each can effect partial responses in patients with chronic lymphocytic leukemia (CLL), although complete and durable responses to treatment with either of these agents have not been reported. We examined whether patients with relapsed and/or refractory CLL could respond to these agents when used in combination in a pilot clinical trial. Fourteen patients with progressive, symptomatic, and relapsed/refractory disease were treated with three four-week cycles of high-dose Methylprednisolone (HDMP) at 1gr/m2 daily for 5 days and weekly Rituximab® at 375mg/m2 for four weeks. The median age of the patients was 60 years, the male to female ratio was 4:1, the ECOG performance status was < 2, and the average number of prior treatments was 2. All patients failed or were intolerant to fludarabine and 86% had high-risk disease by the modified Rai classification. Sixty-five percent of the patients had CLL cells that expressed ZAP-70 and unmutated immunoglobulin variable region genes. Response assessment was performed at the end of each cycle, two months after completion of treatment, and each 3–6 months thereafter until the patients experienced disease progression and/or required further treatment. Objective responses were observed in all 14 patients, with 6 patients achieving a complete response (CR) and the remainder a partial response (PR) as per the NCI-working group criteria. We observed a significant decrease in peripheral white blood cell (WBC) counts, increase in hemoglobin, elevation of platelet counts and a dramatic decrease in lymphadenopathy and splenomegaly. Five of the treated patients have not required further treatment with a median follow up of 26 months. Of these 5 patients 3 have maintain a CR. The median time to progression (TTP) was 12 months. Overall, the treatment was well tolerated and all the patients, except one, completed 3 cycles of therapy. The majority of adverse events were Grade I-II (fluid retention, cough, transient hyperglycemia, fatigue). In addition, we observed 7 episodes of grade III-IV toxicity secondary to (anemia, CMV esophagitis and GI bleeding with one case each and two cases each of thrombocytopenia and neutropenia). These data suggest that treatment with the combination of Rituximab® and HDMP has increased activity over treatment with either agent alone and may produce durable complete responses in patients with refractory and / or relapsed CLL.