bluuebunny/crossref_metadata_2025 huggingface dataset:
Cycloxygenase-2 (COX-2) Expression Is More in Clonal Plasma Cells.
Authors: Paneesha S. , Leung E. , Borg Anton , Rose Peter , Morris Alan G.
Published: April 2020

Multiple myeloma is characterised by deregulated cytokine network with secretion of inflammatory cytokines. Recent studies have showed the independent poor prognostic value of COX-2 expression in myeloma. We have here studied the COX-2 expression in plasma cells from patients with myeloma, monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom’s Macroglobulinemia (WM) and compared with the COX-2 expression by normal plasma cells.

Methods:

Our study included bone marrow samples from 34 patients (Lymphoma: 15; myeloma: 10; MGUS: 7; WM: 1; Amyloidosis: 1). Mononuclear cells were harvested from the bone marrow samples by density gradient sedimentation using Lymphoprep TM from Axis-Shield, Norway. Mononuclear cells were stained with PE conjugated CD 38 (BD Bioscience) and APC conjugated CD138 (BD Bioscience). FITC conjugated IgG against human COX-2 (Cayman Chemical) was used to study the expression of cycloxygenase-2 following permeabilization with fix and perm kit (Caltag). Flow cytometry was performed on a Becton Dickinson FACS vantage with appropriate isotype controls. The colorectal cell line HT- 29 and human myeloma cell line RPMI 8226 were used as positive controls and the flow results are validated by western blotting and Prostaglandin E2 EIA assay (Cayman Chemical).

Results:

HT-29 cell line showed a peak fluorescence of 58 for COX-2, where as RPMI 8226 cell line revealed peak fluorescence of 141. Median plasma cell count in bone marrow of patients with MGUS was 2 %( range 2–6%). Plasma cells from MGUS patients revealed median peak fluorescence for COX-2 was 37(range: 9–74). Median plasma cell count in bone marrow of patients with myeloma was 25% (range: 10–59%). Median peak fluorescence for COX-2 in plasma cells from patients with myeloma was 24(range: 10–85). Median plasma cell count in bone marrow of patients with lymphoma was 1 %( range1–4%). Median peak fluorescence for COX-2 in plasma cells from patients with lymphoma was 11(range: 2–38).

Conclusions:

Our study reveals COX-2 expression is more in clonal plasma cells as compared to normal plasma cells. Studies are needed to ascertain the role of COX-2 in oncogenesis in myeloma and to discover how COX-2 expression leads to poor outcome in patients with myeloma. This information may lead to the therapeutic role of selective COX-2 inhibitors in the therapy of myeloma.
Synergistic Inhibition of Myeloma Cell Growth by Combinations of Dexamethasone with Other Anti-Myeloma Agents.
Authors: Bakker Frank , Ott Andrea , Richter Kathrin , Ahrens Tanja , Guenther Andreas , Burger Renate , Gramatzki Martin
Published: April 2020

During the past few years, several new agents with anti-myeloma activity have entered the clinic and improved disease outcome. In addition, inhibitors of key signal transduction pathways have shown promising results in preclinical studies. Combining novel and existing therapeutics might be essential to further improve treatment efficacy, while at the same time reducing adverse side effects. In this study, we systematically tested all possible two-fold combinations out of a repertoire of 8 anti-myeloma agents on five human maligant plasma cell lines. We included the conventional therapeutics doxorubicin and dexamethasone, the new therapeutics bortezomib and zoledronate, and the signal transduction inhibitors rapamycin (mTOR), AG490 (JAK/STAT), U0126 (MEK/MAPK) and L744.852 (farnesyltransferase). The anti-myeloma efficacy of each compound combination was tested in an MTS-based cell growth assay. Interestingly, the majority of compound combinations which blocked myeloma cell growth in a synergistic fashion included dexamethasone. The synergistic effect being mainly due to an increased sensitivity of the cell lines towards dexamethasone. In both the L363 and JK-6L cell lines, strong synergistic effects were observed if dexamethasone was combined with the mTOR inhbitor rapamycin, the MEK inhibitor U0126, the farnesyltransferase inhibitor L744.852 or, to a lesser extent, the JAK inhibitor AG490. In the INA-6 cell line, synergistic cell growth inhibition was observed if dexamethasone was combined with the MEK inhibitor U0126. No strong synergisms were found for the U266 and RPMI8226 cell lines. In the L363 cell line, we also observed strong synergistic effects between L744.852 and rapamycin, and between L744.852 and U0126, indicating that a three-fold combination of dexamethasone, L744.852 and rapamycin or U0126 could result in even more potent myeloma cell growth inhibition. Similarly, synergistic cell growth inhibition was seen in the JK-6L cell line when combining rapamycin and U0126, again suggesting that a three-fold combination including these two compounds and dexamethasone could be highly effective. In conclusion, our results offer important insights into the efficacy of combinations of anti-myeloma agents. Systematic in vitro testing could provide a first step towards finding useful combinations of agents to improve clinical outcome.
Stimulation of Erythropoiesis by Thalidomide in Multiple Myeloma Patients: Its Influence on FasL, TRAIL and Their Receptors on Erythroblasts and Plasma Cells.
Authors: Grzasko Norbert , Hus Marek , Soroka-Wojtaszko Maria , Klimek Piotr , Goracy Aneta , Dmoszynska Anna
Published: April 2020

Proteins belonging to the TNF-family, i.e. Fas ligand (FasL), Fas, TRAIL, TRAIL receptor 1 (TRAIL-R1) and TRAIL receptor 2 (TRAIL-R2), are involved in the pathogenesis of anaemia in multiple myeloma patients. Thalidomide (THAL) is successfully used in the treatment of multiple myeloma (MM) and response is usually accompanied by an increase in haemoglobin concentration. In our previous study comprising 234 MM patients treated with THAL we observed that in the group of non-responders more than 50% of patients had improved hemoglobin level after 3 months of treatment. In the present study 29 patients with refractory/relapsed MM were treated with THAL in an initial dose of 100 mg daily, increased to 400 mg. We measured the expression of the erythropoietin receptor (EPOR) and proteins belonging to the TNF-family on erythroblasts as well as the expression of the TNF-family proteins on monoclonal plasmocytes using flow cytometry before and after 3 months of treatment. Erythroblasts and plasma cells were also investigated in short-term cultures of bone marrow mononuclear cells of MM patients. A clinical response was observed in 17 patients (58.6%), but haemoglobin concentration increased in 20 patients (75.9%). An increase in haemoglobin concentration was observed in 12 patients responding to THAL and in 10 patients not responding. The expression of FasL, Fas, TRAIL and TRAIL-R1 decreased significantly during THAL treatment, whereas the expression of EPOR and TRAIL-R2 did not change significantly. In vitro studies revealed that the expression of FasL, TRAIL, TRAIL-R1 and TRAIL-R2 was lower in THAL cultures than in control cultures. The expressions of TNF-like receptors/ligands on monoclonal plasmocytes did not differ significantly between cultures with and without THAL. Altogether our results suggest that THAL may stimulate erythropoiesis and increase haemoglobin level in MM patients and the way in which THAL acts may be connected with a decrease in the expression of TNF-like ligands/receptors on erythroblasts.
Is Survivin a Possible Target Antigen for Cellular Immunotherapy in Multiple Myeloma?.
Authors: Grube Matthias , Moritz Stephanie , Obermann Ellen , Rezvani Katayoun , Mackensen Andreas , Andreesen Reinhard , Holler Ernst
Published: April 2020

Survivin is a member of the inhibitors of apoptosis family and is expressed in different types of malignancies including multiple myeloma. Cytotoxic T cells recognizing survivin epitopes can be elicited in vitro and by vaccination in human individuals. We performed this study to investigate whether survivin peptide specific CD8 T cells occur in patients with multiple myeloma (MM) and in healthy individuals. To detect antigen specific T-cells, we incubated PBMC from HLA-A2.1 positive patients and healthy individuals with antigen-presenting-cells (C1R-A2) pulsed or not pulsed with a recently described, HLA-A2.1 binding survivin derived peptide (Survpep). We used quantitative real time PCR to measure IFN-gamma mRNA expression for the detection of peptide specific CTL in 23 patients and 21 healthy individuals. A peptide stimulation index (SI, test peptide reactivity / no peptide reactivity) of 2.0 or greater was considered a positive response. CD8 T cells recognizing Survpep could be detected in vitro in 9/23 (39%) patients with MM (SI ranging from 2.9 to 83) and 1/21 (5%) healthy individuals (SI 7). Positive responses were confirmed by 4-colour flow cytometry for intracellular IFN-gamma production. T cells producing IFN-gamma were further analyzed for expression of CD45RA and CCR7 to determine phenotypic characteristics in two patients (SI 83 and 63) identifying them as terminally differentiated effector T cells (CD8+, CD45RA+, CCR7−). Additionally, positive expression of survivin antigen in tumor cells was confirmed by imminohistochemical analysis of patients bone marrow specimen. In conclusion we provide for the first time evidence for T cell reactivity against Survivin antigen in patients with MM. Our data suggest (1) the immunogenicity of survivin antigen in multiple myeloma and (2) that survivin may be a useful candidate antigen for cellular immunotherapy of multiple myeloma.

CD8 T cell response (SI) to Survivin Peptide in patients with multiple myeloma (MM, n=23) and healthy individuals (HI, n=21) CD8 T cell response (SI) to Survivin Peptide in patients with multiple myeloma (MM, n=23) and healthy individuals (HI, n=21)
Thrombosis Is Uncommon in Chinese Patients Receiving Thalidomide- Experience in a Regional Hospital in Hong Kong.
Authors: Chan Man-Hong Helen , Lau Sze-Man June , Chen Yi-Tin , Chan Chi-Chung , Chan Cheuk-Hung , Lam Yim-Kwan
Published: April 2020

OBJECTIVE: To determine the incidence of venous thromboembolism and other side effects of thalidomide in Chinese population

Setting: Queen Elizabeth Hospital, Hong Kong SAR.

Subjects and Methods: Patients who had received thalidomide from 2001 to 2005 were evaluated in this retrospective study. Thalidomide was given either as a single agent at a dose of 100–400mg/day (median dose: 200mg; mean dose: 257.3mg) or in a combination with dexamethasone at a dose of 20– 40mg (oral) on days 1–4. A minimum of 4 weeks of duration of thalidomide was recruited for assessment. Toxicities were graded according to WHO criteria.

Results: Of the 55 patients recruited, 41 patients were evaluable for assessing side effects. The diagnosis of our patients included myelodysplastic syndrome (1/41, 2.4%) refractory mantle cell lymphoma (1/41, 2.4%); myelofibrosis (6/41, 14.6%); and multiple myeloma (33/41, 80.5%). Nine (22%) patients received thalidomide as first line therapy. Thirty-two (78%) patients received thalidomide as 2nd line therapy. Thirty-four (82.9%) patients used thalidomide as single agent. Seven (17.1%) patients used thalidomide as combination therapy. Duration of thalidomide treatment ranged from 4 weeks to 219.3 weeks (Mean 29.4 weeks, median: 20 weeks ). The most commonly observed toxicities were constipation (51.2%), somolence (24.4%), peripheral neuropathy (22%). Only one (2.4%) patient who suffered from myeloma complicated by Miller Fisher syndrome and tetraplegia developed deep vein thrombosis after 4 weeks treatment with thalidomide (300mg/day) plus oral dexamethasone.

Conclusion: Chinese patients in our center tolerated thalidomide well. The low incidence of venous thrombosis observed suggests that anticoagulant prophylaxis may not be warranted in Chinese patiens.
Immunomodulatory Drug Lenalidomide (CC-5013, IMiD3) Augments Anti-CD40 SGN-40-Induced Cytotoxicity in Human Multiple Myeloma: Clinical Implications.
Authors: Tai Yu-Tzu , Catley Laurence , Li Xian-Feng , Coffey Rory , Breitkreutz Iris , Bae Jooeun , Podar Klaus , Hideshima Teru , Chauhan Dharminder , Schlossman Robert , Richardson Paul , Treon Steven P. , Grewal2 Iqbal S. , Munshi Nikhil C. , Anderson Kenneth C.
Published: April 2020

SGN-40, a humanized immoglobulin G1 (IgG1) anti-CD40 monoclonal antibody, mediates cytotoxicity against human multiple myeloma (MM) cells via suppression of IL-6-induced proliferative and antiapoptotic effects, as well as antibody-dependent cell-mediated cytotoxicity (ADCC). We here studied the clinical significance of an immunomodulatory drug lenalidomide on SGN-40-induced cytotoxicity against CD138+CD40+ MM lines and patient MM cells. Pretreatment with lenalidomide sensitized MM cells to SGN-40-induced cell death. Combined lenalidomide and SGN-40 significantly induced MM apoptosis, evidenced by enhanced cleavage of caspase 3/8/PARP and increased subG0 cells, compared with either single agent at the same doses. Pretreatment of effector cells with lenalidomide augmented SGN-40-induced MM cell lysis, associated with an increased number of CD56+CD3− NK cells expressing CD16 and LFA-1. Importantly, pretreatment with lenalidomide or lenalidomide and SGN-40 markedly enhanced NK-cell-mediated lysis of autologous patient MM cells by SGN-40. Lenalidomide also upregulated CD40L on CD56+CD3− NK cells, facilitating IL-2-mediated activation of NK cells. In addition, lenalidomide induced the CD56dim NK subset, which are more potent mediators of ADCC against target MM cells than the CD56bright NK subset. Finally, pretreatment of both effector and target MM cells with lenalidomide markedly enhanced SGN-40-mediated ADCC against CD40-expressing MM cells. These studies therefore demonstrate that the addition of lenalidomide to SGN-40 enhances cytotoxicity against MM cells, providing the framework for combined lenalidomide and SGN-40 in a new treatment paradigm to both target MM cells directly and induce immune effectors against MM.
Didox Induced Apoptosis Occurs by Inhibiting DNA Synthesis and Repair Via Down-Regulation of Ribonucleotide Reductase M1 in Multiple Myeloma (MM).
Authors: Raje Noopur , Kumar Shaji , Hideshima Teru , Ishitsuka Kenji , Yasui Hiroshi , Shiraishi Norihiko , Kiziltepe Tanyel , Chhetri Shweta , Munshi Nikhil C. , Anderson Kenneth C.
Published: April 2020

Ribonucleotide reductase is the rate-limiting enzyme of deoxynucleoside triphosphate synthesis and is therefore an excellent target for cancer chemotherapy. Inhibition of ribonucleotide reductase results in inhibition of DNA synthesis and has anti-neoplastic effects. Several ribonucleotide reductase inhibitors (RRIs) are already in clinical practice, including Hydroxyurea and Gemcitabine. Here we examine the anti-MM activity of a novel RRI, Didox. Our data shows that Didox has potent cytotoxicity against MM cells in 48-hour cultures. Apoptosis induced in MM cell lines by Didox (200mmol/l), evidenced by increased sub-G1 cell fraction, is caspase dependent, confirmed by blocking experiments with Z-VAD-FMK. Unlike other RRIs which mainly target the pyrimidine metabolism pathway (hydroxyurea and gemcitabine), didox targets both the purine and pyrimidine metabolism pathways in MM, as demonstrated by transcriptional profiling using the Affymetrix U133A 2.0 gene chip. Specifically, our data shows a ≥ 2 fold downregulation of genes in these pathways as early as 12 hours after exposure to Didox. Importantly, ribonucleotide reductase (RR) M1 component transcript was downregulated, associated with downregulation of this enzyme by Western Blot analysis, and inhibition of DNA synthesis. Furthermore, genes involved in DNA repair mechanisms were also downregulated after exposure to Didox, demonstrated by heirachial clustering of gene chip data and further validation by western blotting. Specifically RAD 51 homologue, which is involved in nucleotide excision repair and recombination repair, was downregulated in MM cells both transcriptionally as well as at the protein level. This was accompanied by downregulation of the BCL family protein including Bcl-2, Bclxl, and XIAP both at the transcriptional and post transcriptional levels. Since Didox acts on MM cells by inhibiting DNA synthesis and repair, combination studies with melphalan, an alkylating agent commonly used in MM, were next performed. Our data shows a strong in vitro synergism, with combination indices of <0.7 by the Chou Talahay method. When combined with DNA damaging agents like doxorubicin, additive cytotoxicity was noted. These studies therefore provide the preclinical rationale for evaluation of Didox, alone and in combination with alkylating agents and anthracyclines, to improve patient outcome in MM.
Engaging the NKG2D Receptor with a Novel Bispecific Protein (ULBP2-antiCD138) Activates NK Cells and Has Potent Antitumor Activity Against Human Multiple Myeloma.
Authors: von Strandmann Elke Pogge , Hallek Michael , Engert Andreas
Published: April 2020

NK cells, a component of the innate immune system, attack virus-infected and malignant cells without prior antigen stimulation, mediate cellular cytotoxicity and produce cytokines such as interferon gamma (IFN-gamma) upon stimulation. There is growing evidence that NK cells also participate directly in adaptive immune responses, mainly by cross-talk with dendritic cells. One key factor responsible for the activation of innate and adaptive immune responses is NKG2D, a stimulatory receptor expressed on natural killer cells that binds to cellular ligands on malignant cells. Therefore we designed a recombinant NK receptor ligand (ULBP2) fused to an antibody (BB4) detecting the tumor antigen CD138 which is overexpressed on a variety of malignancies including multiple myeloma (MM). The major findings were that (1) ULBP2-BB4 bound both NK cells and tumor cells, (2) triggered NK-mediated cell lysis of CD138+ malignant cell lines and primary MM cells in the allogenic and autologous setting, (3) activated IFN-gamma secretion of NK cells exposed to immobilized protein, and (4) the co-therapy with ULBP-BB4 and human peripheral blood lymphocytes abrogated the tumor growth in a nude mouse model with subcutaneously growing MM cells. This is the first report on the design, expression, purification and functional pre-clinical investigation of a recombinant NKG2D ligand. The results suggest not only a potential clinical use of this novel construct in patients with MM, but might also offer an innovative therapy approach which is based on NKG2D engagement transferable to other malignancies.
Treatment Situation for Newly Diagnosed Multiple Myeloma (MM) Patients in Germany (GER)- First Results from a Representative Multicentre Treatment Survey.
Authors: Freund Mathias R.F. , Knauf W. , Goldschmidt H. , Einsele H. , Nowrousian M. R. , Berdel W. E. , Poenisch W. , Straka C. , Kellermann L. , Bammer S. , Angermund R.
Published: April 2020

Background: Over the past decade new treatment options and drugs significantly altered the treatment paradigm and treatment guidelines for patients with MM have been established (e.g. ASCO). However, it was not evaluated how these options were integrated into clinical practice. We performed a representative multicentric treatment survey to elucidate the current GER treatment reality.

Method: GER centres (ctr) likely to treat MM (university hospitals (UH), non-university hospitals with (SH) and without (NSH) specialized departments, and office based hematologists (OBH) were asked to provide feedback on current treatment modalities and patient no. Among the feedback received, statistical analyses were performed to calculate sample size to ensure representative data. Ctr detected were asked to participate in a treatment registry for all patients with treatment decision (start, change or end of treatment) in a current quarter of the year (Q3/04). Data were verified by monitoring anonymized patient’s source data.

Results: 889 ctr were contacted. 163 ctr (18%) responded with 981 patients, which represents 11% of the MM prevalence in GER. We selected 59 ctr (9 UH, 16 SH, 11 NSH and 23 OBH), sample size 500. All ctr participated. 278 male and 222 female patients were enrolled, median age 64 yrs. Therapy was watchful waiting (3%), primary therapy (42.2%), 2nd line therapy (29.8%), further therapy (21.2%) and maintenance therapy (3%). MM was diagnosed Durie & Salmon stage I, II and III in 18%, 20% and 62%, 77.2% A and 22.8% B. Diagnose was mainly made in SH (33%), followed by OBH (25%), UH (23%) and NSH (18%). Primary treatment was performed mainly at the site of diagnosis with very few forwarding to higher specialized ctr, though the patients were ≤ 60 yrs. old. The mean value of treatment lines performed prior to actual treatment decision was 1.90, differing by treating ctr with a mean value of 2.09 in UH, 1.94, 2.12 and 1.97 in SH, NSH and OBH. Autologous SCT was only performed in UH and in SH and in a total of 43% of patients below age 65. In other ctr main primary treatment option were Melphalan combinations (39%) followed by VAD/VID (27%) and Bendamustine combinations (8%). Participation in a clinical trial was 24% in UH, 15% in SH, 0% in NSH and 8% in OBH. A multivariate analysis revealed site of treatment, age, stage of disease, participation in a clinical trial and status of insurance relevant for treatment decision.

Conclusion: Treatment of MM in GER is highly decentralized resulting in a variability of primary treatment. This representative survey shows treatment decisions for primary treatment of an individual patient is dependend on site of treatment, age, stage of disease, participation in a clinical trial and status of insurance.
Poor Survival of IgD Multiple Myeloma Following High-Dose Melphalan and Autologous Stem Cell Transplantation.
Authors: Jeong Yong Pil , Kim Shin , Ko Ock Bae , Koo Ja Eun , Lee Danbi , Suh Cheolwon
Published: April 2020

Background: IgD multiple myeloma(MM) accounts for 2% of MM subtypes in western countries and has been reported to have poorer prognosis than other subtypes.

Aims: The purpose of this study was to compare the survival following high-dose melphalan and autologous stem cell transplantation (ASCT) between IgD MM and other subtypes.

Methods: Between November 1996 and January 2005, a total of 77 patients with MM who were treated by ASCT at Asan Medical Center were available for analysis.

Results: A total of 9 patients with IgD MM were identified, accounting for 11% of all MM patients. There were 36 patients(47%) with IgG MM, 17 patients(22%) with IgA and 16 patients(20%) with free light chain. Each MM subtype had similar distribution of SWOG stage at ASCT (Table 1). With median follow up of 17 months, median overall survival(OS) was 39 months. IgD MM had the lowest survival among the MM subtypes (p<0.01). Median OS of IgD MM, IgG MM, IgA MM, and free light chain MM were 12 months, 62 months, 40 months, and 55 months, respectively. Kaplan-Meier survival curve according to MM subtype was as Fig 1.

Summary/conclusion: In this small-sized single center study from Korea, IgD MM had a poorer survival than other subtype after ASCT.

Distribution of SWOG stage vs Ig type Subtype/SWOG stage I II III IV IgD MM 2 5 1 0 IgG MM 14 18 3 1 IgA MM 6 9 2 0 Free light chain MM 1 10 5 0 Fig 1 Cumulative Survival Rate Fig 1. Cumulative Survival Rate
Circulation Plays an Essential Role in Distributing Mammalian Yolk Sac Definitive Hematopoietic Progenitor Cells to the Embryo Proper; Using the Ncx1 Knockout Mouse Model To Prevent Circulation.
Authors: Lux Christopher , McGrath Kathleen , Conway Simon , Palis James , Yoder Mervin C.
Published: April 2020

The yolk sac is the lone site of primitive hematopoiesis. The role of the yolk sac in generating definitive hematopoietic progenitors, however, has remained controversial. One complicating factor preventing an accurate investigation of this subject has been the onset of early circulation which alters the localization of hematopoietic progenitors. An Ncx1 knockout mouse (which fails to initiate a heartbeat) is used here to investigate the temporal and spatial distribution of definitive hematopoietic progenitor cells (HPCs: adult type BFU-E, CFU-GM, CFU-GEMM) in an environment lacking circulation. Embryos were harvested from timed pregnancies from Ncx1 heterozygote crosses beginning at the onset of circulation 8.5 days post conception (E8.5) and ending 36 hours later (E10). Developmental age was determined by somite pair number. All embryos were carefully separated from their yolk sacs and both samples were digested and plated in methylcellulose using a previously published definitive hematopoietic progenitor colony assay. Colonies were counted at seven days and then collected for genotyping. In embryos of all genotypes at E8.5, definitive HPCs were enriched more than 28 fold in the yolk sac compared to the embryo proper (EP), but following redistribution by a functional circulation in E10 wild type and heterozygous embryos, the ratio drops to a 3 fold enrichment in the yolk sac (similar to the 3–5 fold enrichment previously reported at this age; KM & JP, Blood 2003). Ncx1 null embryos lacking circulation produced few HPCs in the EP and never redistribute the HPCs as late as E10 resulting in a 72 fold enrichment in the yolk sac even at E10. Whole mount ζ-hemoglobin mRNA staining was carried out to visualize the distribution of blood cells in the yolk sac and embryo proper in the presence and absence of circulation. The staining pattern in null embryos confirms that cells from the yolk sac blood band remain in the yolk sac and are not found in the EP. Our findings support a model in which the primitive and definitive hematopoietic progenitors are generated in the yolk sac and are only redistributed to the EP upon the onset of circulation. Not only is the yolk sac an important source of definitive HPCs, it is the developing embryo’s primary source through E10.
Up-Front Therapy for Multiple Myeloma: A Survey of Practice Patterns in the USA.
Authors: Natarajan Kavita , Lyman Gary H. , Ballester Oscar F.
Published: April 2020

Introduction: Several treatment programs are available for the initial management of patients with multiple myeloma, with no clear documented advantage(s) of one regimen over the others in terms of time to progression (TTP) or overall survival (OS).

Materials and Methods: A questionnaire was mailed to 540 randomly selected members of ASH during early 2005. Practitioners were asked their choice of therapy for newly diagnosed myeloma patients during 2004, based on 2005 NCCN guidelines, including: 1) melphalan/prednisone (MP), 2) vincristine / adriamycin / decadron (VAD), 3) high-dose decadron (HD), 4) thalidomide / decadron (Thal/Dex), 5) doxyl / vincristine / decadron (DVD); the options of a clinical trial (CT) or “other” were also included. Physicians were asked about factors influencing their choice of therapy for individual patients and their recommendations for autologous stem cell transplantation as part of the initial treatment schema.

Results: Surveys were returned by 123 physicians(19.2%), of which 93 contained evaluable data. Among responders, 52% were in private practice and 47% in academic institutions and 74% respondants reporting having been in practice for more than 10 years. A large majority of physicians (74%) utilized 3 or more different regimens, only 10.7% of responders used a single regimen for all of their patients. Thal/Dex was used by 87% of responders, with 47% of them recommending this regimen in ≥ 50% of their patients. MP, HD and VAD were used by 67.7%, 49% and 44% of responders, but only 10.7%, 4% and 3% respectively, recommended them to ≥ 50% of their patients. DVD was used by 25% of physicians. Of respondants, 64.5% did not accrued patients to clinical trials and only 7.5% of physicians accrued ≥ 50% of their patients to clinical trials. No significant differences in the choice of regimen were apparent based on years of practice. Physicians in academic centers tended to use HD (p =.002) and accrue patients to CT (p=. 001) more often than those in private practice. Factors identified as important in selecting initial therapy for individual patients included: age (92%); performance status (95%); prognostic factors, such as β2-microglobulin and cytogenetics (75%); and candidacy for stem cell transplantation (93%). Respondants consider autologous stem cell transplantation as part of the initial therapy for all eligible patients (47%), only those with responsive disease (42%) and normal renal function (30%); only in selected cases (76%).

Conclusions: Thal/Dex appears to be currently the most commonly recommended up-front therapy for multiple myeloma in the USA, in spite of the lack of published data documenting patient benefit in terms of TTP and OS. A sizable proportion of physicians do not recommend autologous stem cell transplantation as part of the initial therapy of newly diagnosed myeloma patients despite confirmed randomized clinical trials documenting benefit.
Modified Thalidomide, Cyclophosphamide and Dexamethasone (TCD) to Patients with Multiple Myeloma as Primary Therapy Prior to Peripheral Blood Stem Cell Collection.
Authors: Yang Deok-Hwan , Lee Je-Jung , Kim Yeo-Kyeoung , Baek Jin-Ho , Sohn Sang-Kyun , Jin Choi-Young , Shin Ho-Jin , Kim Hyeoung Joon
Published: April 2020

In the treatment of multiple myeloma (MM), autologous peripheral blood stem cell (PBSC)-supported high-dose melphalan is now considered standard therapy, especially for younger patients. Before PBSC, a new immunomodulatory drug, thalidomide, has replaced VAD regimen as the induction therapy of choice prior to hematopoietic stem cell procurement. Thalidomide has alternative mechanisms of action and usually combines with dexamethasone or alkylating agent, but the optimal doses and the intervals of chemotherapy has been evaluated. We reported preliminary data that the patients with previously untreated multiple myeloma were treated by combined different dose-scheduled thalidomide, cyclophosphamide, and dexamethasone as primary therapy and performed autologous stem cell collection. A total of 52 patients with MM were initially treated thalidomide-based chemotherapy from June 2003. Thalidomide was given with the three different method: 400mg/day on D1–5, D15–19 (arm A), 50mg/day daily (arm B) combined with cyclophosphamide 150mg/m2 P.O. on D1–4 and dexamethasone 20mg/m2 P.O. or I.V. on D1–5, D15–19, or thalidomide 200mg/day daily combined with dexamethasone 20mg/m2 on D1–4, 9–12, 17–20 in odd cycles and on D1–4 in even cycles (arm C), repeated every 28 days. Low-dose aspirin or warfarin was taken as prophylaxis for thrombosis. Thirty-nine (arm A: 17, arm B: 12, arm C: 10) of the 52 patients who received at least 4 cycles or more were evaluated for response and toxicity. Median age was 65 (range: 39–80) years and the total number of 158 cycles of toxicities were evaluated. After median 4 months of time to response followed, the overall response rate was 71.8% (76.5% v 75.0% v 60.0%), including 23% (17.5% v 23% v 28%) of complete or near complete response. Two patients (5.1%) died due to infection during treatment. When the toxicity of therapy was evaluated with more Grade III, two patients (5.1%) showed neurotoxicity, six patients (15.4%) showed neutropenia, and two patients (5.1%) had deep-vein thrombosis. Thirteen patients who achieved more than partial response proceeded to PBSC collection and yielded a median number of 3.78 x 106 CD 34+ cells/kg. This low or intermediate dose or periodic thalidomide combination showed positive responses, reducing toxicities, and adequate numbers of blood stem cells in patients eligible for subsequent high-dose chemotherapy.
Thymoglobulin (Polyclonal Rabbit Anti Thymocyte Globulin) Has In Vitro Activity in Multiple Myeloma.
Authors: Kumar Shaji , Timm Michael , Kimlinger Terry , Kline Michael , Haug Jessica L. , Rajkumar S. Vincent , Greipp Philip R.
Published: April 2020

Background: Multiple myeloma is a plasma cell malignancy that remains incurable with current approaches and newer therapies are needed to improve the outcome of patients with MM. While monoclonal antibody base therapies have been successful in some of the hematological malignancies, especially lymphoma, such approaches have not been very useful in the setting of myeloma. Targeting of antigens like CD138 on the myeloma cell surface has been hampered by the ubiquitous nature of this protein in the body. Thymoglobulin (polyclonal rabbit antithymocyte globulin, Genzyme) has been extensively evaluated in the setting of allogeneic blood and marrow transplantation and is currently in clinical use. Given the polyclonal nature of this product, it has antibodies against several B cell antigens and forms the rationale for its evaluation in B cell malignancies like myeloma.

Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity following drug treatment was measured using the MTT viability assay. Drug induced apoptosis in the cell lines was measured by flow cytometry after staining with Annexin V-FITC and propidium iodide (PI). Apoptosis in primary patient derived plasma cells following treatment was measured after staining for Apo 2.7.

Results: rATG was cytotoxic in vitro to several MM cell lines (RPMI 8226, U266, OPM1, OPM2) including the IL-6 dependent cell line Kas6/1. The LC50 in most of the cytotoxicity assays was around 1 mg/mL. Additionally, rATG was cytotoxic MM cell lines resistant to conventional agents such as doxorubicin (Dox40), melphalan (LR5) and dexamethasone (MM1R). The drug retained its cytotoxicity when myeloma cells were grown in the presence of various cytokines like IL-6, IGF-1 and VEGF. rATG treatment resulted in a time and dose dependent induction of apoptosis in MM cell lines. rATG was also able to induce apoptosis of freshly isolated myeloma cells from patient marrows. When tested in combination with other anti-myeloma agents an additive effect was seen with doxorubicin, PS341 and melphalan.

Conclusions: Thymoglobulin appear to have in vitro activity against various myeloma cell lines as well as patient derived primary myeloma cells. Ability of the drug to overcome resistance to conventional drugs as well as the effect of combining rATG with these agents points towards non-overlapping mechanisms of action. Ongoing studies are trying to identify the particular B-cell antigens that are targeted by correlating response to expression of various B cell antigens expressed by these cell lines. These studies will provide the rational for future clinical development of this agent in myeloma alone or in combination with other agents.
Dose Reduced Conditioning and CD3/CD19 Depletion for Haploidentical Allogeneic Hematopoietic Cell Transplantation in Adults: Fast Engraftment and Low Toxicity.
Authors: Bethge Wolfgang A. , Faul Christoph , Bornhaeuser Martin , Lang Peter , Handgretinger Rupert , Kanz Lothar
Published: April 2020

Haploidentical hematopoietic cell transplantation (HHCT) has been made feasible with the use of megadoses of CD34+ peripheral blood stem cells. Despite this progress, HHCT is still complicated by high treatment related toxicity, slow engraftment and immune reconstitution. A new regimen using graft CD3/CD19 depletion with anti-CD3- and anti-CD19-coated microbeads on a CliniMACS device and dose reduced conditioning may allow HHCT with lower toxicity and faster engraftment. CD3/CD19 depleted grafts not only contain CD34+ stem cells but also CD34 negative progenitors, natural killer-, dendritic- and graft-facilitating-cells. As dose reduced conditioning fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2) and OKT-3 (5 mg/day, day -5 to +14) without posttransplant immunosuppression was used. So far 9 consecutive adult patients have been transplanted with this regimen. Median age of the patients was 37 (range, 27–58) years and the diagnoses were AML (n=3), ALL (n=3), NHL (n=2) and multiple myeloma (n=1). All patients were high risk with relapse after preceding hematopoietic cell transplantation. The CD3/CD19 depleted haploidentical grafts contained a median of 8 x 10E6 (range, 5.2–17x10E6) CD34+ cells/kg, 6x10E7 (range, 0.02–8.6 x10E7) CD56+ cells/kg and 2x10E4 (range, 0.006–8.2x10E4) CD3+ T-cells/kg. 8 of 9 patients had a donor-recipient KIR-mismatch. The conditioning regimen was well tolerated with maximum acute toxicity being grade 2–3 mucositis and nausea. In 4/9 patients reversible peripheral neuropathy grade 2–3 occurred, combined with multifocal leucencephalopathy in one patient. Subsequently the dose of fludarabine was reduced to 150 mg/m2. Engraftment was rapid with median time to neutrophils >500/μL of 13 (range, 10–17) days, platelets >20000/μL of 11 (range, 8–14) days and full donor chimerism after two weeks in all patients. All evaluable patients had fast immune reconstitution with T-cells >100/μL by day +100. Five cases of grade 2 skin GVHD rapidly responded to steroids. Three patients with controlled fungal pneumonia at time of transplant had resolution of infiltrates with neutrophil reconstitution and antifungal therapy. Treatment related mortality in the first 100 days was low with one death due to idiopathic pneumonia syndrome. Three patients died after day 100, one each due to relapse, systemic adenoviral infection and CMV infection. Overall survival is 5/9 patients with a median follow-up of 406 days (range, 44–661). In conclusion, this regimen seems to be promising in adult high risk patients lacking a suitable donor and a prospective phase I/II study is ongoing.
Generation of a Common Progenitor Population from Human Embryonic Stem Cells That Gives Rise to Both Embryonic Erythropoiesis and Definitive Hematopoiesis.
Authors: Zambidis Elias T. , Osborne Jihan , Civin Curt I.
Published: April 2020

Human embryonic stem cells (hESC) provide a valuable new tool for dissecting the earliest developmental events of human hematopoietic-stem progenitor cell (HSPC) genesis. We have recently reported the efficient step-wise differentiation of hESC to embryonic (primitive) erythroid cells followed by definitive erythro-myeloid hematopoietic cells from human embryoid bodies (hEB). Hematopoiesis proceeds spontaneously from hEB-derived cells and appears to model the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development.

We now extend our studies to define conditions which may favor differentiation into definitive hematopoietic cells from hEB. We previously demonstrated that hEB-derived primitive hematopoiesis requires fetal calf serum, but proceeds in the absence of supplemented recombinant growth factors to developing hEB’s. We now show that supplementing hEB differentiation cultures with a broad array of hematopoietic growth factors dramatically enhances the number of primitive erythroblasts, definitive myeloid, BFU-E, CFU-E, and multi-potent mixed colonies in methylcellulose CFC assays while not affecting the kinetics of hematopoietic differentiation. The inclusion of VEGF-165 during hEB differentiation was found to have an exceptionally potent effect in increasing the multilineage generation of both primitive and definitive hEB-derived hematopoietic cells. To further define the hEB-derived population which gives rise to primitive and definitive hematopoiesis we FACS-purified a population of CD45-CD31+CD34+ hEB cells which we and others have shown are capable of both endothelial and hematogenous differentiation. Co-culture of this purified population in serum-containing OP9 stromal layers gave rise primarily to definitive-type erythro-myeloid cells including mature beta-globin-expressing erythroid cells, neutrophils, monocytes/macrophages, CD41+ megakaryocyte precursors, and CD56+ NK-like cells. Alternative culture of this purified CD45-CD31+CD34+ hEB population in serum-free, stromal-free cultures supplemented with erythropoietin and VEGF-165 produced an abundant population of embryonic (nucleated gamma, epsilon, zeta chain-hemoglobin-expressing) erythroblasts.

To isolate a clonogenic, expandable human hemangioblastic progenitor of primitive and definitive hematopoieisis as well as endothelial cells in our system, we have prepared transgenic hESC lines expressing a GFP reporter cDNA under the hemato-endothelial regulatory sequences of the human genomic SCL/TAL1 locus. Our hESC model reveals, for the first time, the putative existence of a population of human hEB progenitors capable of both embryonic-type and definitive blood cells depending on the differentiation environment.
Dendritic Cells Reconstitution after Different Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Wu De Pei , Cao Junjie , Li Caixia , Wu Xiaojin
Published: April 2020

Objective: To compare the dendritic cells reconstitution after different allogeneic hematopoietic stem cell transplantation in early time

Methods: From June 2004 to March 2005, Twenty-eight patients undergoing allo-HSCT were enrolled in this study. There were 16 patients who undergone normal HSCT, 8 patients who undergone Haploidentical HSCT and 4 patients who undergone Nonmyeloablative HSCT. Three-colour flow cytometry was applied to study the alteration of the percentage and number in circulating peripheral blood dendritic cells subsets on day 14,day 30,day 60 after transplantation among three distinct type HSCT.

Results: The dendritic cells subsets number of myeloablative HSCT patients were very low. No difference was observed in the kinetics of DC1 (Lin−HLA-DR+CD11c+)and DC2 (Lin−HLA-DR+CD123+) reconstitution between the normal HSCT group and Haploidentical HSCT group patients(p<0.05). There was significant difference between myeloablative HSCT group and normal healthy individuals(p<0.01). There was significantly different between the non-myeloablative HSCT group and the myeloablative HSCT group in the kinetics of DC1 and DC2 reconstitution(p<0.05).

Conclusion: The early reconstitution of dendritic cells in Nonmyeloablative HSCT patients is earlier than the patients who undergone myeloablative HSCT. The early reconstitution of dendritic cells in normal HSCT and Haploidentical HSCT patients were later than others.
Competitive Repopulation Assay for Human Stem Cell Engraftment in NOD-scid IL-2rγnul Mice.
Authors: Giassi Lisa J. , Laning Joseph , Biber Kristen , Cuthbert Amy , Phillip Lowry A. , Shultz Leonard D. , Greiner Dale L.
Published: April 2020

Umbilical cord blood (UCB) is a source of stem cells for hematopoietic transplantation (Laughlin, et al. NEJM2004; 351:2265) but application in adults is limited by marginal stem cell numbers. Combining cord blood units has shown promise in achieving engraftment (Barker, et al. Blood2002; 100:41a [abs #142]) without crossed immunologic rejection. Short-term hematopoiesis showed contributions from both cords, but long-term engraftment derived predominantly from just one cord. To study this process we have developed an in vivo pre-clinical competitive repopulation assay using NOD-scid IL-2rγnull mice that support high levels of human UCB engraftment (Shultz, et al, J. Immunol.2005, 174:6477). NOD-scid IL-2rγnull mice were transplanted with T-cell depleted UCB containing 3x104 HLA-A2+ CD34+ cells, 3x104 HLA-A2− CD34+ cells, or a 1:1 mixture (6x104 CD34+ cells). Human hematopoietic chimerism, measured by human CD45 and HLA-A2 expression, was quantified in bone marrow 6 and 12 weeks after transplantation. Variable mixed engraftment within individual as well as between 6 separate experiments was observed. At both 6 and 12 weeks, mixed contribution from both cords was achieved although rarely in a 1:1 ratio despite the infusion of equivalent CD34+ cell numbers. HLA-A2 expression did not provide a selective advantage. The complete dominance of one cord observed in the clinical report and in animal studies (Yahata, et al. Molecular Therapy2004; 10: 882) suggests an important role for the immune activity of UCB T-cells in this competitive process, a hypothesis we are currently testing. This model has a rapid turnaround and can test multiple permutations, providing a preclinical vehicle for characterizing optimal combinations of cord cells and for testing conditions that allow one cord to ultimately prevail.

Legend: Engraftment of human CD45+ cells at 6 weeks. All bars represent single mice except for the HLA-A2+ group, which is the average (±SEM) of 3 mice. Legend: Engraftment of human CD45+ cells at 6 weeks. All bars represent single mice except for the HLA-A2+ group, which is the average (±SEM) of 3 mice.
Regulatory and Activated T Cells in Stem Cell Autografts Collected by Mobilization with High-Dose Cyclophosphamide and Granulocyte Colony Stimulating Factor.
Authors: Condomines Maud , Quittet Philippe , Lu Zhao-Yang , Nadal Laure , Latry Pascal , Lopez Ernesto , Baudard Marion , Rossi Jean-François , Tarte Karin , Klein Bernard
Published: April 2020

High-dose chemotherapy (HDC) supported by autograft of hematopoietic progenitors (HP) is a standard therapy for patients with multiple myeloma (MM). High-dose cyclophosphamide (CTX) and G-CSF are widely used to collect HP. As the number of lymphocytes in the autograft is a powerful prognostic factor in patients with MM, our purpose was to study how CTX-G-CSF treatment affects the phenotype and function of T cells, in particular regulatory T cells (Treg), in 15 patients with MM. CTX induced severe T cell immunosuppression with a slow and partial T-cell recovery (a threefold decrease) at the time of HP collection. CTX-G-CSF treatment did not affect the percentages of central memory (CD45RA−, CCR7+), effector memory (CD45RA−, CCR7−), and late effector (CD45RA+, CCR7−) CD4 or CD8 T cells but a decrease of naïve CD4 cells (CD45RA+, CCR7+) was found. The percentages of CD25+ cells increased two- to threefold in CD4 or CD8 T cells, respectively. Post-CTX treatment CD4CD25+ cells included both activated CD4CD25low cells and CD4CD25high T cells. The latter were Treg because they expressed high level of FOXP3 and membrane CTLA-4 mRNA and protein and displayed functional suppressor function. In CTX-G-CSF leukaphereses from 15 patients with MM, the mean Treg number was one fifth that of CD34 and the CD3, CD4 and CD8 numbers respectively 3 fold, 2 fold and equal that of CD34. Post-CTX-G-CSF treatment CD3 cells did not cell cycle in vivo and died in short-term culture in vitro. Adding IL-2 or IL-15 induced their survival and cell cycle, and stimulation with anti-CD3 MoAb led to efficient growth in vitro. These results suggest that following CTX-G-CSF treatment, CD3 cells are preactivated in vivo and do not cell cycle, likely due to a lack of T cell growth factors in vivo. The current data indicate that CTX-G-CSF treatment profoundly affects T cell function without eliminating Treg. The persistence of Treg could be explained by an opposite effect of CTX known to kill Treg and of G-CSF amplifying Treg. Given the major impact of lymphocyte count on patients’ survival post HDC and HP and T cell graft, the present data invite to define novel therapeutic strategies to improve T cell recovery in vivo while limiting Treg expansion.