bluuebunny/crossref_metadata_2025 huggingface dataset:
Constitutive Nuclear p65 NF-kB Expression Predicts for Spontaneous Apoptosis and In Vitro Sensitivity to Fludarabine in CLL Cells.
Authors: Hewamana Saman , Rowntree Clare , Lin Thet Thet , Pepper Chris , Brennan Paul , Fegan Chris
Published: April 2020

B-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B-cells in the blood, secondary lymphoid tissues, and bone marrow. The leukemia cells primarily are arrested in the G0/G1 phase of the cell cycle and appear resistant to programmed cell death. Despite their apparent longevity in vivo, CLL cells typically undergo spontaneous apoptosis in vitro although there is considerable heterogeneity in the timing and extent of this process. In this study we investigated the degree of this apoptosis in vitro, and its association with constitutive nuclear p65 NF-kB expression (n = 25). Apoptosis was measured by annexin V / propidium iodide labeling using flow cytometry and nuclear NF-kB expression was evaluated using electrophoretic mobility shift assay and quantified using a p65 ELISA. In contrast to previous reports, we found constitutive NF-kB activity varied in our patient cohort with a trend towards higher expression in samples with unmutated VH genes, high CD38 expression and high ZAP-70 expression. Importantly, we demonstrated an inverse correlation between the percentage of Annexin V-positive cells and nuclear p65 NF-kB expression (r2 = − 0.21; P = 0.02) suggesting that high levels of p65 NF-kB are cytoprotective in CLL cells. We then investigated the relationship between p65 NF-kB and in vitro response to fludarabine. Sensitivity to fludarabine was evaluated using LD50 values calculated from sigmoidal dose-response curves. These LD50 values positively correlated with nuclear p65 NF-kB activity (r2 = 0.25; P = 0.006) indicating that in vitro drug resistance to fludarabine may be mediated, at least in part, through the transcriptional effects of NF-kB. Taken together, our data indicate that NF-kB is a key regulator of both spontaneous and drug induced apoptosis in CLL cells and is therefore a promising therapeutic target for the treatment of this incurable condition.
Individualized Dosing of Fludarabine (FLU) for Low Grade Lymphoproliferative Disorders Results in Administration of Less Medication with Fewer Infections and Equivalent Survival.
Authors: Pasquale Donald , Ramanathan Nalini , Scarpace Sarah L. , Nguyen Hung , Jackson Amy I. , Ramanarayanan Jeyanthi
Published: April 2020

When used in the treatment of low-grade lymphoproliferative disorders, common practice is to administer 6 monthly cycles of FLU 25-mg/m2 daily × 5. This practice is reported to cause infections (including opportunistic such as PCP and Listeria) in over 50% of patients. It has been our practice to individualize the number of monthly cycles of FLU per treatment course based on response in general administering 1 cycle of therapy when rapid and substantial response is obtained, or 1 cycle past best response. Our objective is to describe our experience with this dosing schema - incidence of infections in relation to the number of cycles of FLU administered per treatment course and overall survival. We conducted a retrospective chart review of medical records of all patients who received FLU at the Stratton VA Medical Center. We defined one treatment course as the number of five day monthly FLU doses received. Any time interval of 3 or more months between FLU was considered to indicate a new treatment course. All infectious episodes of NCI grades 3 or higher that occurred within 6 months of the last FLU treatment course were attributed to that prior treatment course. Survival was estimated by Kaplan-Meier analysis from date of diagnosis to death or last known alive. There were 15 patients treated for CLL, 9 for low-grade lymphoma, and 4 for macroglobulinemia. Three individuals treated with FLAG for ANLL were excluded. These 28 patients received a total of 44 treatment courses (each treatment course with 1 to 6 monthly cycles) of FLU. Fifty-four (54) percent of the treatment courses were with ≤2 cycles. Mean ± SD age of the patients was 65 ± 11 years.

Number of FLU cycles per treatment course Number of treatment courses with infection Number of treatment courses without infection % treatment courses with infection 1 3 12 20 2 1 8 11 3 6 3 67 4 3 1 75 5 3 1 75 6 2 1 67

The overall incidence of NCI grade 3 or higher infections was significantly increased with increased numbers of FLU cycles per treatment course (complex Chi-square = 0.0193). Treatment cycles with ≤2 compared with 3 or more cycles per treatment course (Chi-square with Yate’s correction = 0.0008) had significantly fewer infections. Median overall survival was 158 months with 78 ± 44 months of follow-up. Our data indicates that individualized dosing of FLU based on response results in using lower numbers of monthly cycles per treatment course, lower incidence of severe infections, and equivalent (based on historical controls) overall survival.
A Multi-Center, Open-Label Study To Evaluate the Safety and Efficacy of Pentostatin, Cytoxan, and Rituxan (PCR) in the Treatment of Previously Untreated or Treated, Stage II, III or IV CLL.
Authors: Mena Raul R. , Robles Robert , Auerbach Michael , Moezi Mehdi M. , Headley David
Published: April 2020

The decision to treat indolent CLL is often based on progressive bulky disease, worsening symptoms, and increasing hematological derangement. When treatment is indicated, these lymphoproliferative disorders are sensitive to combination immunochemotherapies utilizing pentostatin (P), a purine analog, cyclophosphamide (C), a DNA alkylator, and rituximab (R), an anti-CD20 monoclonal antibody. P+C+R represents a promising new approach in the treatment of patients with CLL. Eligibility criteria allow both treated and treatment-naïve patients diagnosed with Stage II/III/IV CLL (modified Rai classification) to be enrolled. Treatment consisted of intravenous infusions of P (4 mg/m2), C (600 mg/m2), and R (375 mg/m2) on day 1 of a 21-day cycle for a total of up to 10 cycles. Clinical evaluation (including CT scan) was performed after cycles 2, 4, 6, 8 and 10 (if necessary). The intent-to-treat (ITT) population consisted of 70 CLL patients (median age 64, range 35–83) who received a total of 286 cycles (median 4.5 per patient). ECOG status at enrollment was 0 (68%), 1 (28%) and 2 (4%). The overall response rate (CR+Cru+PR) of the 50 evaluable patients was 52% (CR 8%, Cru 14%, PR 30%, SD 46% and PD or RD 2%). 2 grade 4 neutropenia and 1 grade 4 respiratory distress was documented. 4 deaths have been recorded, all in elderly patients (>80 years old), due to acute respiratory failure, myocardial infarction, pulmonary edema and sepsis. This immunochemotherapeutic regimen is active in Stage II/III/IV CLL and the incidence of significant toxicities was low with deaths occurring only in elderly (>80 years old) patients. The study results will be updated at the meeting.
Methyl Jasmonate: A New Treatment for B-CLL?.
Authors: Berrebi Alain , Bassous Lucette , Borenshtain Rinat , Flescher Eliezer
Published: April 2020

Jasmonates are a group of plant stress hormones, which play a role in activation of apoptosis and defense mechanisms. In vitro, it has been found that methyl jasmonate MJ induces death of lymphoma and leukemia cells by independent mechanisms involving mitochondrial damage at the level of the oxidative phosphorilation.

Since CLL is a suitable model to study the effect of MJ on circulating leukemic cells, we analyzed the in vitro apoptosis and necrosis of B-CLL cells treated with MJ by PI/ Annexin V analysis and cytotoxicity by tetrazolium-based assay (XTT test). In comparison normal lymphocytes from healthy donors were also analyzed.

Interestingly, normal lymphocytes were not affected al all by methyl jasmonate. Blood samples from 28 CLL patients in advanced stages not treated or at least 4 weeks after cessation of treatment were selected and incubated with MJ. The proportion of cells undergoing apoptosis or necrosis following 4 hour of incubation was measured by flow cytometry and considered as positive if >35% cells death occurred. Following 24 hours incubation cells vialability was measured using XTT Cell Proliferation kit assay by Elisa. Cytotoxicity (%) was calculated by the ratio of optical density of control cells - drug-treated cells/ control cells ×100 (IC50). The titration of drug showed that 1 mM of MJ was the most effective to induce apoptosis of CLL cells and has no effect on normal lymphocytes. In 90% of samples the specific cells death tested by Annexin V was higher than 35%. A significant cytotoxicity was observed in 60% of the cases with an optimal titration of the drug for XTT test of 0.5 mM. Correlation between the two methods was found in 80% of the cases analyzed.

Previous experiments showed that MJ induces cytochrome C release selectively in mitochondria isolated from CLL cells and high glucose levels inhibit MJ-induced ATP depletion. Furthermore MJ induces mitochondrial membrane depolarization in CLL cells. Altogether these experiments showed that MJ kills B-CLL cells through an original pathway inducing bioenergitic cell collapse. The cytotoxic effect and specific cell death tested in the present analysis support the concept that MJ is a potential drug to treat B-CLL. A clinical trial is currently carried out in our Institution.
Early Estimates of Safety for Alemtuzumab Combined with Fludarabine for the Treatment of Relapsed/Refractory B-Cell Chronic Lymphocytic Leukemia: Phase II Multicenter Study.
Authors: Brown Jennifer R. , Stock Wendy , Katzen Harvey I. , Suarez Agustin J. , Lakhanpal Shailendra , Leis Jose F. , Flowers Christopher ,   on Behalf of ECO-1 Study Investigators
Published: April 2020

Background. Alemtuzumab (CAMPATH) is the most effective single-agent therapy available for the treatment of chronic lymphocytic leukemia (CLL). Previous pre-clinical and clinical studies suggest possible synergistic activity between alemtuzumab and purine analogues. A recent phase 2 study reported an overall response rate of 83% (complete response 30%) in patients (pts) with relapsed and/or refractory CLL treated with intravenous (IV) alemtuzumab combined with IV fludarabine for up to 6 cycles (Elter JCO 2005). We evaluated the safety and efficacy of alemtuzumab administered in a subcutaneous (SC) manner combined with IV fludarabine in pts with previously treated CLL. Safety data are presented based on interim analysis from this study.

Patients and Methods: Pts with active CLL who received at least 1 prior therapy were eligible to receive combination treatment with SC alemtuzumab 30 mg days 1–5 and IV fludarabine 25 mg/m2 days 1–5 every 28 days for up to 6 cycles. All pts received prophylaxis with trimethoprim-sulfamethoxazole and famciclovir (or equivalent) from initiation of therapy until ≥2 months following therapy discontinuation. Pts were monitored for cytomegalovirus (CMV) reactivation using polymerase chain reaction (PCR) assays every 2 weeks, and preemptive therapy with oral valganciclovir 900 mg (or equivalent) was administered in cases of PCR-positivity (≥100 copies/mL). Adverse events (AEs) were graded using the NCI Common Toxicity Criteria (version 3.0).

Results. To date, 45 pts of a target 60 pts, have been accrued and safety evaluations are available for 40 pts (median age 63 years; range 45–86) of whom 13 completed 4 cycles, 7 completed 5 cycles and 4 completed 6 cycles of therapy. Most pts had ECOG PS 0 or 1 (82%), 56% had Rai stage ≥3. Ninety percent of pts had received a prior therapeutic regimen that included fludarabine. The most common (≥ 10%) grade 3 or 4 AEs were hematological toxicities including thrombocytopenia in 11 pts (27.5 %), and anemia in 7 pts (17.5 %). Although neutropenia was documented in 13 pts (32.5 %), grade 3 febrile neutropenia was only observed in 1 patient (2.5%). Grade 3 or 4 infections occurred in 6 pts (15 %), which included 1 case of grade 3 fungal sinusitis. CMV reactivation (by PCR) was documented in 42% of patients at a median of 4 weeks (range: 2–8 weeks) after start of therapy. Other commonly observed AEs were infusion or administration-related, and were primarily grade 1 or 2 in severity. One patient died from presumed aspiration pneumonia. No other deaths due to AEs occurred.

Conclusions. Combination therapy with SC alemtuzumab and IV fludarabine at the dose schedule employed is feasible and well tolerated, with manageable toxicities.
A Long-Term Study of Prognosis on Monoclonal Gammopathy of Undetermined Significance.
Authors: Case Delvyn Caedren , Boyd Marjorie A.
Published: April 2020

From 1976 to 2006, 547 patients have been identified and followed for monoclonal gammopathy of undetermined significance (MGUS). Criteria of inclusion included presence of serum monoclonal protein of a concentration of 2 g per deciliter or less; no or moderate amounts of monoclonal light chains in the urine; the absence of lytic bone lesions, anemia, hypercalcemia, and renal insufficiency related to the monoclonal protein; and proportion of plasma cells in the bone marrow of 5% or less. Patients identified as MGUS were followed every 6 months with physical examination, CBC, chemistries, and paraprotein studies. Ages ranged from 32 – 100 years (median 70 years). There were 214 males and 333 females. Over the 30 years of observation, 61 patients have developed disease progression: myeloma 29, lymphoma 12, macroglobulinemia 10, chronic lymphocytic leukemia 7, and amyloid 3. Time to develop myeloma ranged from 2–24 years (median 6 years), macroglobulinemia 2–8 years (median 5 years), and amyloid 3–10 years (median 10 years). Patients developed myeloma throughout the entire period of observation: 2, 3, 3, 4, 4, 4, 4, 5, 5, 5, 6, 6, 6, 6, 6, 7, 7, 8, 8, 9, 10, 10, 11, 14, 15, 15, 18, 20, and 24 years. Survival from diagnosis of myeloma was 1+ – 14+ months (median 36 months) for myeloma and 24 – 108+ months (median 48 months) for macroglobulinemia. The only reliable method of identifying progression of disease to myeloma/macroglobulinemia/amyloid was serial determinations of paraprotein level. In this large series followed for 30 years, with a more restricted diagnosis of MGUS (paraprotein level ≤ 2 g per deciliter and 5% or less bone marrow plasma cells), 1% of all patients identified developed progression of disease. No patient developed disease progression in less than 2 years using the more restricted diagnosis. Risk of progression increased with time with patients developing myeloma even 20 years or longer of follow-up. Serial evaluations of paraprotein levels are indicated in patients identified as MGUS.
Bone Involvement in Multiple Myeloma Patients Carrying the T (4;14) Chromosomal Translocation.
Authors: Tosi Patrizia , Terragna Carolina , Nicoletta Testoni , Zamagni Elena , Renzulli Matteo , Tacchetti Paola , Montanari Elena , Perrone Giulia , Ceccolini Michela , Brioli Annamaria , Baccarani Michele , Cavo Michele
Published: April 2020

Chromosomal translocations involving the immunoglobulin heavy chain switch region (IgH) are quite common in multiple myeloma (MM), and some of them can reliably predict disease outcome. In particular, t(4;14) chromosomal abnormality is one of the most adverse prognostic factors for response duration and survival after high dose therapy and autologous stem cell transplantation. Despite the dismal prognosis, however, in this subset of patients, bone involvement, as evaluated by spine MRI, is relatively infrequent, at variance to what has been observed in other MM subtypes according to TC classification. In the present study we aimed at further testing this hypothesis by analyzing the extent of whole bone involvement in patients showing t(4;14) chromosomal translocation as compared to negative patients. For this purpose, 50 newly diagnosed MM patients (32M, 18F, median age = 54 yrs) underwent evaluation of total skeletal X-ray, whole spine MRI and, at the same time, quantification of markers of bone resorption (urinary NTX, PYR and DPYR and serum crosslaps) and bone formation (bone alkaline phosphatase-BAP and osteocalcin) was performed. Using a real-time PCR assay to detect the presence of IgH/MMSET fusion gene as a surrogate marker for t(4;14), we found 15 patients carrying this chromosomal abnormality, 7 of whom (46%) were also positive for the deletion of chromosome 13, this abnormality was detected in 11/35 (31%) patients who proved negative for IgH/MMSET hybrid transcript. The two groups of patients did not differ significantly in terms of median age, distribution of M protein isotype and light chain, beta-2 microglobulin, bone marrow plasma cell infiltration and disease stage. Spinal MRI was negative in 3/15 (20%) t(4;14) positive patients as compared to 12% t(4;14) negative patients; skeletal involvement, however, was more pronounced in t(4;14) positive patients (median skeletal score = 6.24, as compared to 3.58 in t(4;14) negative cases, p= 0.00). These data were confirmed by the evaluation of bone resorption markers; specifically, serum crosslaps were significantly increased in patients with t(4;14) abnormality compared to negative individuals (7984 pmol/L±1682SE vs 5123pmol/L±783SE p=0.04). Conversely, no difference in bone formation markers was found in the two groups of patients. Our results indicate that, despite a spinal involvement at MRI that is comparable to what is observed in negative patients, individuals who are t(4;14) positive show a more pronounced bone resorption pattern, this in contrast to what has been reported so far, but in line with the aggressive features of the disease in these patients.
Outcomes Following Syngeneic Hematopoietic Stem Cell Transplantation for Multiple Myeloma: A Matched Comparison to Autologous Transplantation.
Authors: Bashey Asad , Perez Waleska S. , Zhang Mei-Jie , Vesole David H. , Reece Donna E. , Goldschmidt Hartmut , Hari Parameswaran
Published: April 2020

Relapse is the main cause of treatment failure following autologous hematopoietic cell transplantation (HCT) for multiple myeloma (MM). Syngeneic HCT offers the advantage of a myeloma-free-graft. However, a potential disadvantage is the lack of a graft versus myeloma effect (GVM). We compared the probabilities of treatment-related mortality (TRM), disease progression, progression-free survival (PFS) and overall survival (OS) after syngeneic versus autologous HCT for MM done between 1988 and 2003. Median follow up was >70 months in both groups. 43 syngeneic HCT recipients were matched to 170 autologous HCT recipients using a propensity score. A numerical propensity score for each syngeneic HCT recipient was calculated using the variables of age, Durie-Salmon stage at diagnosis, sensitivity to pretransplant therapy, time from diagnosis to HCT and year of HCT. Propensity scores ranged from 0.004–0.286. Syngeneic HCT recipients (cases) were matched in random order to autologous transplant (control) recipients with similar propensity scores. Patients who underwent tandem transplants were excluded. Median age (range) was 53 and 52 years in cases and controls. Most patients in both groups (60% of cases, 64% of controls) were transplanted within 12 months of diagnosis. Except for a higher proportion of patients with IgG myeloma (59% vs. 39%, p<0.01) and PBSC grafts (92% vs. 51%, p<0.01) in the control group there were no statistically significant differences in baseline characteristics of the two groups. 5-year outcomes are summarized in the table.

5-year outcome, probability (95% CI) Syngeneic Autologous Treatment-related mortatlity 14 (5–26) 10 (6–15) Disease progression 42 (26–58) 71 (64–78) Progression-free survival 44 (28–60) 19 (13–26) Overall survival 59 (43–74) 40 (32–48) Medican follow up survivors, months 71 (23–161) 85 (3–145)

In multivariate analysis, risks of progression and treatment failure were significantly lower after syngeneic than autologous HCT [disease progression RR= 0.43 (95%CI, 0.23–0.78, p=0.004); treatment failure RR= 0.59 (95%CI 0.35–0.98, p=0.04)]. TRM at 1 year was 14% (5–26) in the syngeneic group and 9% (5–13%) in controls (p=0.33). The 5-year risk of mortality was lower in the syngeneic group but the difference was not statistically significant (RR= 0.61, 95%CI 0.36–1.05, p=0.07). Disease recurrence accounted for 79% of deaths in the autologous and 47% in the syngeneic cohort. We conclude that syngeneic HCT for MM results in superior PFS and lower progression rates compared to autologous HCT, confirming previous smaller analyses and emphasizing the importance of a disease-free graft. Interestingly, these data suggest that relapse rates similar to those observed after nonmyeloablative allogeneic transplantation – another source of tumor free grafts – can occur in the absence of clinical graft versus host disease.
Expression and Clinical Significance of β-Catenin in Multiple Myeloma.
Authors: Li Juan , Zhang Dian-Bao , Luo Shao-kai , Zhao Ying , Huang Bei-Hui , Gu Jing-Li
Published: April 2020

OBJECTIVE: To detect the expression of β-catenin in mononuclears of bone marrow of healthy people, primary and relapsed/refractory multiple myeloma(MM), and analysis the clinical date and the curative effect of the MM patient, to open out the clinical significance of the expression of the β-catenin in MM.

METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Wes tern blot were used to detect mRNA and protein expression of β-catenin in bone marrow samples of 12 primary MM patients, 14 relapsed/refractory MM patients, and 11 healthy people.

RESULTS: The positive rates and the expression levels of β-catenin mRNA were significantly lower in healthy people than in MM patients (27.3% vs. 88.5%, 0.22±0.09 vs. 0.80±0.15, P <0.01), the expression level of β-catenin mRNA(β-catenin/β-actin) was significantly lower in newly diagnosed patients than in MM patients (0.7196±0.11 vs. 0.8517±0.16, P <0.05). β-catenin protein were not detected in all of the healthy people; while the positive rates of β-catenin was 69.2% in MM patients (P <0.01), and its expression levels was significantly higher in relapsed/refractory patients than in primary patients (0.3231±0.11 vs. 0.2065±0.08, P<0.05). In 10 primary MM patients which can be evaluated the curative effects, the expression rate in no response patients was significant lower than in response patients (14.3% vs. 100%, P <0.05). To stage the patients, the statistics show the expression of β-catenin protein in Durie/Salmon stage III was significant higher than stageII(87.5% vs. 40%, P <0.05) and in ISS stage III was significant higher than lor IIstage(100% vs. 45.5% or 33.3%).

CONCLUSION: To analysis the relationship between β-catenin and β2-MG or serum LDH, we found the β-catenin protein was positive correlative with the expression level of β2-MG (r=0.688, P<0.01). and serum LDH(r=0.502, P<0.05).
IL-10 Promoter Region -592A/C Genotype Increases the Risk of Multiple Myeloma in Japanese Patients.
Authors: Saitoh Takayuki , Kasamatsu Tetsuhiro , Matsuoka Hitomi , Endoh Miyuki , Inoue Madoka , Yamane Arito , Irisawa Hiroyuki , Handa Hiroshi , Matsushima Takafumi , Tsukamoto Norifumi , Karasawa Masamitsu , Ogawara Hatsue , Nojima Yoshihisa , Murakami Hirokazu
Published: April 2020

Background: Interleukin 10 (IL-10), a cytokine that regulates inflammation, is involved in multiple myeloma (MM) cell proliferation and survival. However, it is unclear whether IL-10 polymorphisms alter the susceptibility to MM. We examined the single nucleotide polymorphism located within the promoter region of IL-10 genes in Japanese patients with MM and monoclonal gammopathy of undetermined significance (MGUS).

Patients and Methods: Fifty-eight patients with MM [age range, 34–87 years; stage I (n=11), stage II (n=12), stage III (n=35)], 31 patients with MGUS (age range, 43–85 years), and 185 healthy controls were included. The MM group consisted of 34 M-component type IgG, 12 IgA, 1 IgM, 9 Bence-Jones, and 2 non-secretory. Genotyping was determined by the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique. Genotype and allele frequencies were compared between the study groups by χ2 test. Odds ratios (OR) and 95% confidence intervals were calculated.

Results: The IL-10-592A/C genotype, which shows higher production of IL-10, was associated with an increased risk of MM (OR, 2.15; 95% confidence interval [95% CI], 1.17–3.93; P=0.012). The IL-10-592A/A genotype, which shows lower production of IL-10, was associated with a decreased risk of MM (OR 0.52; 95% CI, 0.27–1.00; P=0.047). Interestingly, 6 of 7 patients (86%) with transformation of MGUS to MM showed the IL-10-592A/C genotype. There was no association between the development of MGUS and the genotype of IL-10-592 polymorphism (Fig. 1). There was a trend to better overall survival of IL-10-592 A/A compared to IL-10-592 A/C or IL-10-592 C/C, although not significantly (Fig. 2). In addition, we noted that the ethnic distribution of IL-10-C592A was significantly different. The high producer IL-10-592 C/C genotype is present in only 10% of the Japanese population in our study compared to 35% of the Caucasian population in previous studies.

Conclusion: These results suggested that the IL-10-592 A/C genotype contributes to the susceptibility of MM development and prognosis in Japanese patients. Genetic variations of IL-10-592 polymorphism may also influence ethnic differences in myeloma incidence.

Figure Figure
Frequent Expression of Cancer/Testis Antigens CT7 and LAGE-1 in Advanced Stage Multiple Myeloma: Are They Possible Targets for Immunotherapy?.
Authors: Andrade Valeria Cristina da Costa , Colleoni Gisele Wally Braga , Vettore Andre L. , Felix Roberta Spetic , Almeida Manuella S.S. , Carvallho Fabricio , Yamamoto Mihoko , Zago Marco Antonio
Published: April 2020

Introduction: Recently, Jungbluth and collaborators (Blood2005;106(1):167) demonstrated that CT7 and MAGE-A3/6 are frequently expressed in advanced stage MM patients and that higher levels of CT7 and MAGE-A3/6 proteins also correlate with elevated plasma-cell proliferation index. These findings suggest a possible pathogenic role of such proteins in MM and also show that they could be attractive targets for immunotherapy.

Objectives: To analyze global expression of 13 CTs (MAGE-A1, MAGE-A2, MAGE-A3/6, MAGE-A4, MAGE-A10, MAGE-A12, BAGE-1, CT7, GAGE family, LAGE-1, PRAME, SP17 and SSX-1) in a panel of normal tissues and monoclonal gammopathies.

Material and Method: We studied 13 normal tissues (skeletal muscle, bladder, lung, spleen, heart, brain, thymus, uterus, stomach, mammary gland, pancreas, prostate and colon, Clontech), six normal bone marrow aspirates (donors for allogeneic stem-cell transplants), one pool of 10 normal bone marrow samples (Clontech), three monoclonal gammophaties of undertermined significance (MGUS), five solitary plasmacytomas, 39 multiple myeloma samples (two Durie-Salmon stage II and 37 stage III) and MM cell line U266. Normal testis was used as positive control. Total RNA was extracted using Trizol reagent (Invitrogen). After cDNA synthesis, the expression of CTs was evaluated by RT-PCR and 2% agarose gel electrophoresis.

Results: SP17 was positive in all seven normal bone marrow samples and in the 13 normal tissues tested. Thus, it was excluded from further analyses. CT7 was positive in one MGUS and in one plasmacytoma. U266 cell line was positive for all CTs, except SSX-1. The frequency of CTs expression in MM patients was: CT7 = 30/39 (77%); LAGE-1 = 18/39 (46%); MAGE-A3/6 = 16/39 (41%); MAGE-A2 = 14/39 (36%); GAGE family = 13/39 (33%); BAGE-1 = 11/39 (28%); MAGE-A1 = 10/39 (26%); PRAME = 9/39 (23%); SSX-1 = 10/39 (26%); MAGE-A12 = 8/39 (20,5%); MAGE-A4 and MAGE-A10 = 0%. It is important to note that from the 18 cases with less CT’s positivity (0, 1 or 2 positive-CTs), three were negative for all of them. Twelve of the remaining 15 cases (80%) were positive for CT7. We did not find association between International Scoring System and percentage of expressed CTs in 36 analyzed MM cases.

Conclusion: Our results showed high frequency of expression of CT7 in advanced stage MM patients and support its importance as a target for immunotherapy, because it has a high incidence of positivity even in cases without expression of other CTs. As far we know, this is the second study showing high frequency (~ 50%) of LAGE-1 expression in MM (van Baren et al, 1999), also highlighting its importance as therapeutic target.
p38MAPK Inhibitor LSN2322600 Modulates the Bone Marrow Microenvironment and Inhibits Osteoclastogenesis in Multiple Myeloma.
Authors: Ishitsuka Kenji , Hideshima Teru , Neri Paola , Vallet Sonia , Shiraishi Norihiko , Raje Noopur , Shen Zhenxin , Kiziltepe Tanyel , Ocio Enrique M. , Podar Klaus , Chauhan Dharminder , Munshi Nikhil , Campbell Robert M. , De Dios Alfonso , Shih Chuan , Starling James J. , Tamura Kazuo , Anderson Kenneth C.
Published: April 2020

The interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment plays a crucial role not only in proliferation and survival of MM cells, but also in osteoclastogenesis. In this study, we examined diverse potential of novel p38MAPK inhibitor LSN2322600 (LSN) for MM therapy in vitro and in vivo. The cytotoxic activity of LSN against MM cell lines was modest; however, LSN significantly enhances the cytotoxicity of Bortezomib by down-regulating Bortezomib-induced heat shock protein (HSP) 27 phosphorylation. We next examined the effects of LSN on cytokine secretion in MM cells, bone marrow stromal cells and osteoclast precursor cells. LSN inhibited IL-6 secretion from long-term cultured-bone marrow stromal cells (LT-BMSCs) and bone marrow mononuclear cells (BMMNCs) from MM patients in remission. LSN also inhibited MIP-1 α secretion by fresh tumor cells, BMMNCs and CD14 positive cells. Since these cytokines mediate osteoclastogenesis, we further examined whether LSN could inhibit osteoclastogenesis. Importantly, LSN inhibited in vitro osteoclastogenesis induced by macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of nuclear factor- κ B ligand (sRANKL), as well as osteoclastogenesis in the severe combined immunodeficiency (SCID)-Hu mouse model of human MM. These results suggest that LSN represents a promising novel targeted strategy to reduce skeletal complications as well as to sensitize or overcome resistance to Bortezomib.
S-Dimethylarsinoglutathione (SGLU/ZIO101) Is a Novel Active Organic Arsenical That Displays a Unique Gene Expression Profile in Myeloma Cells.
Authors: Boise Lawrence H. , Morales Alejo A. , Croutch Claire R. , Gutman Delia , Gale Robert Peter , Lee Kelvin P.
Published: April 2020

Arsenic Trioxide (ATO) is highly active in acute promyelocytic leukemia (APL) and has activity in several other diseases including multiple myeloma. Since arsenicals are active and it is known that organic arsenicals are less toxic than ATO, the testing of new organic arsenicals is warranted. One such compound, ZIO-101 is in phase I/II studies. Therefore we previously compared the ability of ZIO-101 and ATO to kill four myeloma cell lines (RPMI 8226, U266, KMS11, MM.1s) that display differential sensitivity to ATO. Sensitivity to ATO and ZIO-101 did not correlate, as the most ATO resistant line (RPMI 8226) was highly sensitive to ZIO-101. We and others have reported that glutathione (GSH) is a critical regulator of ATO-induced cell death and we have utilized ascorbic acid (AA) as a GSH depleting agent both in vitro as well as clinically. We therefore also tested the effects of GSH depletion on ZIO-101-induced cell death in MM cell lines. BSO was much more effective at sensitizing cells to ATO than to ZIO-101. Moreover while AA could sensitize cells to ATO, it actually protected cells from cell death induced by ZIO-101. Taken together these data suggest ZIO-101 is active against myeloma cells although factors that determine the potency of this compound are different than those for ATO. To better characterize these differences gene expression profiling of the cellular response to ZIO-101 was performed. RNA was isolated from the 4 cell lines treated with ZIO-101 for 0, 6 and 24 h and profiling performed using Affymetrix Hu133 plus 2 arrays. We initially focused on genes that demonstrated similar changes in all four cell lines. 320 probes demonstrated an increase of 1.5 or greater at 6 h while only 58 increased at 24 h. Additionally 265 genes were decreased by at least 1.5 fold at 6 h while only 12 genes were down regulated 1.5 or greater at 24 h. Interestingly the pattern of gene expression was distinct from that observed in similar experiments with ATO. Most notably genes associated with metal responses (MT-1, ZnT-1) and oxidative stress responses (increased expression of HO-1, NQO-1, malic enzyme, GSH synthesis pathway, ferritin) were either absent or only transiently increased. In contrast there was increased expression of the pro-apoptotic gene Noxa compared to ATO treatment in the 4 cell lines. Taken together these data indicate the cellular response to ZIO-101 does not include the up regulation of protective pathways and suggest that ZIO-101 does not initiate cell death through the induction of oxidative stress. This may reflect differences in either metabolism or mechanism of action. Thus resistance to one form of arsenic does not preclude use of another. A phase I/II study of ZIO-101 in myeloma is underway.
Striking Coincidence of IgD M-Protein and Translocation t(11;14) in Hungarian Myeloma Patients.
Authors: Mikala Gabor , Adam Emma , Kozma Andras , Kapas Balazs , Nahajevszky Sarolta , Masszi Tamas
Published: April 2020

Multiple myeloma exhibiting production of IgD M-protein is a rare subset (appr. 1 %) of all patients. Translocation t(11;14) is, on the other hand, the most common primary translocation in myeloma that involves the IgH locus and cyclin D1, approximately 16–22% of all patients harbor this genetic rearrangement. This cytogenetic abnormality has been previously associated with nonsecretory behavior and also with production of IgE M-protein. Out of 317 myeloma patients treated in our department in 2004–2006, five patients had IgD M-protein (four belonged to IgD-lambda and one to IgD kappa subtype). Cytogenetic analysis by interphase fluorescent in situ hybridization (FISH) revealed the presence of the IgH/Cyclin D1 abnormality in all five of them (100%), while the prevalence of this translocation was 17% in the entire myeloma population seen by our ward and tested by FISH (132 patients). One IgD patient had a complex IgH/MYC/CyclinD1 translocation, also confirmed by conventional cytogenetic analysis of metaphases, while two of five patients had additional copies of IgH and Cyclin D1 genes, possibly indicating atypical translocations and/or hyperdiploidy. Although the number of patients with this rare disease in our report is low (5), we find the uniform presence of translocation t(11;14) a striking occurrence that may explain some of the unusual biological properties of this myeloma subtype.
Ex Vivo Anti-Tumor Activity of Rosiglitazone on Multiple Myeloma Cells.
Authors: Huang Haiwen , Wu De Pei , Leung Anskar Y.H. , Liang Raymond , Lie Albert K.W.
Published: April 2020

Peroxisome proliferation activated receptor-g (PPAR-g) belongs to the family of nuclear hormone receptors (NHRs), it is normally expressed in adipocytes, adrenal gland, spleen and liver. Recently it was reported that PPAR-g can also be found in tumor tissues. Activation of PPAR-g by its ligands has potential anti-neoplastic effects in a variety of human malignancies, including leukemia through inhibition of cell proliferation, induction of apoptosis and terminal differentiation. The ligands of PPAR-g may represent a promising, novel therapeutic approach for certain human malignancies.

The thiazolidinedione (TZD) class drug rosiglitazone (RGZ), one of synthetic ligands of PPAR-g, is currently used for the treatment of type 2 diabetes. In this study, we cultured myeloma cell line U266 with different concentration of rosiglitazone, as well as combined with dexamethasone. Cell proliferation was measured by [3H] thymidine incorporation after 48h incubation. Rosiglitazone was found to generate inhibition of cell proliferation on U266 cells in a dose-dependent manner. Cell cycle analysis by flow cytometry showed that rosiglitazone can arrested U266 cells in G0/G1 phase and the G2/M phase cells were significantly decreased compared to controls. We also studied the effect of rosiglitazone on expression of different anti-apoptosis protein FLIP and survivin by RT-PCR, the result revealed that rosiglitazone can also decrease the FLIP and survivin expression. Furthermore, exposure to rosiglitazone can induce the decreased caspase-3 activity in U266 cells, which was associated with apoptosis induction. When rosiglitazone was combined with dexamethasone, the data demonstrated that cell growth inhibition and apoptosis they exerted was much greater than they were used alone, the decreased expression of FLIP and survivin and decreased caspase-3 activity were also greater than when rosiglitazone was used alone. Based on these findings, we suppose that rosiglitazone alone and in combination with dexamethasone holds promise as novel therapy for myeloma.
Monoclonal Gammopathy as a Determinant of Adverse Outcomes in Patients with Chronic Kidney Disease.
Authors: Hutchison Colin A. , Townend John , Mead Graham P. , Chandler Katie , Harper Jo , Bradwell Arthur R. , Zakeri Rosetta , Cockwell Paul
Published: April 2020

Elevated serum free light chain (sFLC) concentrations are associated with glomerular and tubulointerstitial disease. We have analyzed a cohort of patients with chronic kidney disease (CKD) to determine the relationship between sFLC and clinical outcomes. 369 patients from the renal clinics at the Queen Elizabeth hospital in Birmingham were followed for a period of 8 years. We reviewed renal outcomes and all cause mortality over this period. sFLC levels were measured on baseline serum samples using a nephelometric immunoassay (The Binding Site Ltd.). The CKD patients had mean sFLC concentrations significantly higher than the normal population (kappa: 77 vs. 8.4mg/L p<0.0001; lambda: 94.7 vs. 13.4mg/L p<0.03). 65 patients (18%) had abnormal sFLC ratios suggesting clonal production of sFLC. There were 270 endpoints during follow-up (106 deaths and 164 end stage renal failures). By Kaplan Meier survival analysis an abnormal sFLC ratio is significantly associated with a worse composite outcome (p<0.02), with the dominant factor being the progression of renal impairment to end stage disease. This is the first study to assess if an abnormal sFLC ratio is predictive of outcomes in patients with CKD. These findings provide the basis for further work to evaluate whether monoclonal FLCs are prognostic of a worse outcome in patients with CKD.
Incidence, Clinical Presentation and Outcome of Secondary Multiple Extramedullary Plasmacytomas: Experience at Roswell Park Cancer Institute.
Authors: Manochakian Rami , Miller Kena C. , Coignet Jean-Gabriel , Padmanabhan Swami , Musial Laurie , DePaolo Dawn , Chanan-Khan Asher Alban
Published: April 2020

Introduction: Extramedullary plasmacytomas are malignant plasma cell tumors that arise outside of the bone marrow. They occur either as solitary tumors with a cure rate of over 95% with radiation therapy alone; or they may present as manifestations of advanced metastatic multiple myeloma, referred to as secondary extramedullary plasmacytomas (SEP). SEP usually present as multiple tumors, primarily in the head and neck area and often after multiple relapses in MM patient. Although the etiology of multiple SEP remains unknown, they are reflective of clinically advanced disease that no longer relies on the bone marrow microenvironment. The incidence, clinical outcome and natural history of SEP are not clearly delineated in the literature. With the recent use of novel therapies (thalidomide, lenalidomide, bortezomib) resulting in prolonged survival we have noticed an increase incidence of patients relapsing with SEP at our institute. This observation prompted us to formally evaluate SEP to characterize the clinical pattern of presentation, clinical course and correlation with use of novel therapies of SEP.

Methods: This is a single institute retrospective review. All patients seen at our institute with the diagnosis of MM were evaluable. Data was queried since 1990, which included a total of 343 patients.

Results: To date 100 patients have been reviewed and among these, 20 patients (20%) were noted to have SEP. Characteristics of these patients are outlined in details in table (1). 14 patients (70%) remain alive. However, none achieved a complete remission after the diagnosis of SEP.

Conclusion: In this study, the overall incidence of SEP was 20% on the patients so far evaluated. Majority of SEP patients have received prior novel agents. Systemic treatments seem to be effective in controlling progression of SEP though achievement of CR status has not been possible with current therapeutic interventions. Our finding are intriguing as the incidence of SEM seems to be increasing, which may reflect either unveiling of the natural history of the disease through improved survival from the use of novel agents, or this may be reflective of a change in the biology of MM.

We intend to present the complete analysis of all cases at the meeting.

Characteristics of patients with secondary plasmacytoma Characteristics Sex 8 (40%) and 12 (60%) F Age Median: 66, Range(46–86) Stage 16(80%) stage III and 4(20%) < stage III Type 9(45%) IgG,6(30%) IgA, 4(20%) Free Light Chains and 1 (5%) nonsecretory Prior treatments 19(95%) had prior Thalidomide or Bortezomib,17(85%) had prior Thalidomide and12(60%) had prior Bortezomib Locations of Plasmacytomas 5(25%) Head&Neck,7(35%) Paraspinal, 2(10%) Abdomen,4(20%) Thorax and 2(10%) at other locations
Bone Marrow Fibrosis Is a Negative Predictor for Osteolytic Lesions in Patients with Myeloma.
Authors: Paneesha Shankaranarayana , Pol Raj , Chachlani N. , Pratt Guy , Rose Peter , Borg Anton
Published: April 2020

Multiple myeloma is a clonal lymphoproliferative disorder characterised by proliferation of plasma cells in marrow and is associated with an unbalanced bone remodelling leading to osteolytic lesions. Bone marrow interstitial fibrosis is common in myeloma. Correlation of marrow fibrosis with clinical parameters and survival in patients with myeloma is conflicting. We have analysed the impact of marrow fibrosis at presentation in 83 (F: 45; M: 38) patients with median age 67 yrs on osteolytic lesions and survival. Reticulin silver impregnation is employed as standard matrix stain. H & E slides were reviewed for degree and pattern of plasma cell infiltration. Marrow reticulin was quantified from Grade 0–4. 50 had IgG myeloma, 15 had Ig A myeloma, 13 had LC myeloma, 2 had Ig D myeloma and PCL and one had Ig M myeloma. Median marrow plasma cell count was 60%. 42% had increased marrow reticulin (> grade2). 33 received VAD, 16 melphalan and prednisolone, 7 dexamethasone, 6 cyclophosphamide, thalidomide, dexamethasone, 6 cyclophosphamide, 4 thalidomide, 1 dexamethasone and thalidomide combination and 10 only bisphosphonates as initial therapy.

Haemoglobin < 10 gms/dl was in 47% patients. 67% were positive for BJP. 33% had hypercalcaemia. 51% had >2 osteolytic lesions at diagnosis. 55% had β2-microglobulin >4mg/L and 22% had renal failure at diagnosis. 47% needed more than one line of therapy. Marrow reticulin was a negative predictor for osteolytic lesions (Fisher’s Exact test; p: 0.001). Marrow reticulin didn’t correlate with other laboratory parameters. Marrow reticulin, sex, BJP & number of osteolytic lesions at diagnosis did not impact on OS (Log Rank test p values: 0.6, 0.07, 0.6 & 0.4 respectively). Known poor prognostic markers Hb <10gm/dl, hypercalcaemia, renal failure and elevated β2-microglobulin were associated with poor OS (Log Rank test p values: 0.02, 0.04, 0.004 & 0.002 respectively) with median follow-up 27 months. Number of osteolytic lesions and anaemia at diagnosis had impact on progression free survival (Log Rank test; p values: 0.01 & 0.02 respectively) where as raised β2-microglobulin, marrow reticulin, hypercalcaemia and renal failure (Log Rank test; p values: 0.07, 0.7, 0.9 & 0.35 respectively) had no impact. Marrow reticulin also had no effect on requirement for more than one line of therapy (p value: 0.17).

Marrow reticulin is a negative predictor for osteolytic lesions, even though it does not appear to impact on survival in patients with myeloma. Our study also confirms the poor prognostic role of anaemia, hypercalcaemia, renal failure and raised β2-microglobulin in patients with myeloma. Further study of the hepatocyte growth factor/c-Met interaction may identify the negative correlation between marrow fibrosis and osteolytic lesions. This may enhance understanding of skeletal prophylaxis in patients with myeloma.
Combination of the Proteasome Inhibitor Bortezomib and a Histone Deacetylase Inhibitor PXD101 Results in Synergistic Inhibition of Osteoclastogenesis and Significantly Stronger Inhibition of Multiple Myeloma Growth In Vitro and In Vivo.
Authors: Feng Rentian , Oton Ana B. , Patrene Kenneth , Anderson Gulsum , Mapara Markus Y. , Belani Chandra , Roodman David , Lentzsch Suzanne
Published: April 2020

Bortezomib, a proteasome inhibitor, has been known to have in vitro and in vivo activity against multiple myeloma (MM). PXD101, a novel, low molecular weight histone deacetylase (HDAC) inhibitor recently has been recognized to induce growth arrest and apoptosis in MM cells. We hypothesized that these two agents, with different molecular targets, can be combined with a high probability of additive or synergistic effects without exerting cross-resistance.

In this study, we investigated the activity of the combination of PXD101 and bortezomib against osteoclast generation and MM cell growth. To evaluate the effects of bortezomib and PXD101 on the formation of osteoclasts from mononuclear hematopoietic precursors, we generated osteoclasts cultured from human bone marrow cells from healthy donors and MM patients using in vitro RANKL/M-CSF stimulation. Proliferation of MM cell lines treated with PXD101, bortezomib, or their combination was determined by measuring 3H-Thymidine incorporation. Annexin V-FITC/PI staining was used to determine the degree of apoptosis, and trypan blue was used to assess cell viability. We utilized a xenograft murine model to test the in vivo synergistic activity of the two agents against MM tumor formation.

We found that even though either drug alone significantly inhibited osteoclast formation at concentrations of PXD101 at 25 nM or bortezomib at 1 nM, the combination resulted in synergistic inhibition in osteoclast assays. Treatment of MM cells lines (MM.1S, RPMI-8226, OPM2) with serial dilutions (10–100 nM) of the combination also led to significantly stronger inhibition of proliferation, apoptosis and cell death than single drug treatment. In addition, the combination demonstrated synergistic induction of cell death in primary human CD138+ myeloma cells isolated from patient samples. In vivo, the combination of PXD101 and bortezomib resulted in significantly higher xenograft tumor inhibition compared to either drug treatment alone and was not more toxic.

The current study provides the rationale for clinical evaluation of this novel PXD101-bortezomib combination for patients with MM. The data generated here support the hypothesis that simultaneous targeting of different proliferative and anti-apoptotic pathways both in tumor cells and activated osteoclasts avoids cross-resistance and constitutes a dynamic approach to treat MM.