bluuebunny/crossref_metadata_2025 huggingface dataset:
Mcl-1 Is Overexpressed in Multiple Myeloma and Correlates with Disease Severity.
Authors: Wuilleme-Toumi Soraya , Robillard Nelly , Gomez-Bougie Patricia , Moreau Philippe , Le Gouill Steven , Avet-Loiseau Herve , Harousseau Jean-Luc , Amiot Martine , Bataille Regis
Published: April 2020

Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (>20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.
Non-Myeloablative Allogeneic Hematopoietic Transplantation in Relapsed/Primary Refractory Follicular Lymphoma.
Authors: Fabre Claire , Recher Christian , Huynh Anne , Rigal-Huguet Françoise , Abbal Michel , Dastugue Nicole , Saati Talal Al , Brousset Pierre , Attal Michel
Published: April 2020

Allogeneic stem cell transplantation (SCT) can induce long-term disease-free survival in refractory follicular lymphomas (FL) but is impaired by high rate of transplant-related mortality (TRM). Reduced conditioning followed by allogeneic SCT is a promising concept in FL, allowing a graft-versus-lymphoma (GVL) effect while reducing the TRM. We piloted a clinical trial using this approach in relapsed/primary refractory low-grade lymphomas. From 1998 to 2003, 31 patients (pts) with an HLA-identical sibling have been enrolled. Main pts characteristics were : age = 49 years (range, 32–61 y); grade : I/II FL = 26; FL with histologic transformation = 5; gender = 16 male and 15 female; ≥ 2 prior therapies before transplantation = 23; primary refractory = 8; stage IV = 22; Bulky disease = 16; mean LDH rate before transplantation = 335 UI/l (range, 205–858); residual disease at transplantation (partial response (PR) + progression) = 20. Previous treatments were as follow : anthracyclins = 26, alkylants = 21, anti-CD20 = 15, purine analogs = 7, autologous SCT = 6, radiotherapy = 5. The conditioning regimen was IV fludarabine 30 mg/m²/day (from day −5 to −2); PO busulfan 2 mg/kg/day (from day −7 to −6); antithymocyte globulin (ATG) 2.5 mg/kg/day (from day −4 to −3). 14/31 (45%) and 17/31 (55%) pts received peripheral blood and bone marrow stem cells respectively with a median number of CD34+ cells of 5 x 106/kg (range, 1.8–11.9). Graft-versus-host-disease (GVHD) prophylaxis consisted in cyclosporine A alone. All patients achieved complete donor (> 95%) chimerism after allogeneic SCT. Median duration of neutropenia (< 0.5 G/L) and thrombocytopenia (< 25 G/L) were 3 days (range, 0–16) and 3 days (range, 0–29) respectively. 8/31 pts (26%) had CMV reactivation but no CMV disease. 3/31 (10%) developed grade II–IV acute GVHD. 10/31 (32%) developed chronic GVHD (5 moderate and 5 extensive). The median follow-up time was 20 months. The TRM was 19% (6/31). Causes of death were aGVHD = 1, auto-immune hepatitis = 1, bacteraemia = 2, pulmonary toxoplasmosis = 1 and Lyell syndrome = 1. Overall response rate was 97% (21 CR + 9 PR) at day 100 and at 1 year (24 CR + 6 PR). The estimated 2-year overall survival (OS), event-free survival (EFS) and relapse-free survival (RFS) rate were 75%, 65% and 85% respectively. 2 relapses occurred. Both were treated by anti-CD20, achieved CR and are still alive. Donor age, Bulky disease and LDH were significant for EFS. Altogether, these data suggest that non-myeloablative SCT can induce a high rate of durable remissions with reduced toxicity in this poor risk population.
Reduced-Intensity Conditioning Regimen with BEAM/Rituximab for Patients with Refractory Non-Hodgkin’s Lymphomas.
Authors: Khouri Issa F. , Saliba Rima M. , Okoroji Grace-Julia , Acholonu Sandra , Maadani Farzaneh , Hosing Chitra , Champlin Richard E.
Published: April 2020

Non-myeloablative allogeneic stem cell transplantation using the combination of fludarabine/cyclophosphamide/rituximab as a conditioning regimen has been successful in treating patients with relapsed, chemosensitive non-Hodgkin’s lymphoma (Blood98:3595, 2001; J Clin Oncol21:4407, 2004). A more intense regimen however is required to have an early disease control for patients with refractory disease or in kinetic failure. We used the BEAM regimen for that purpose. Because of potential synergy and without overlapping toxicity, Rituximab (375 mg/m2 on days −6, −1, +7 and +14) was added to the preparative regimen. Transplantation was performed on day 0. Fourteen consecutive patients with refractory lymphoma or in kinetic failure were studied. The study group included 11 males and three females of median age of 46 (range, 19–60) yrs. Underlying histologies include diffuse large cell lymphoma (n= 9), follicular (n=1), mantle cell (n=1), lymphoplasmacytic (n=1), and CLL in Richter transformation to diffuse large cell (n=2). Median # of prior chemoregimens received was 3 (range, 1–8). Twelve patients had a matched sibling donor, one a mismatched sibling, and one a matched unrelated donor. The median # of CD34+ cells infused was 5 x 106 CD34 + cells/kg. Twelve patients received blood and 2 received marrow as the source of the hematopoietic graft. Tacrolimus and methotrexate were used for GVHD prophylaxis. The day-100 mortality was 7%. Eleven patients (79%) had a complete, and 2 (14%) had a partial remission. One patient (7%) had no response. All patients engrafted donor cells (median of 100% at day 30) post transplantation. Median time to ANC > 500 was 13 (11–15) days and median time to platelets > 20K was 14 (9–25) days. The actuarial risk of Gr 2–4 GVHD was 57%, Gr 3–4 GVHD 28%. Eleven of the 13 patients who were alive at day 100 developed chronic GVHD. With a median follow-up time among survivors of 34 (8–48) months, overall survival and disease-free survival were 63% (95% CI, 33–83) and 57% (95%CI, 28–78), respectively.

Our results indicate that a pronounced graft-versus-lymphoma occurs after BEAM/Rituximab and allogeneic transplantation for patients with refractory lymphoma or in kinetic failure. This treatment has been associated with a lower than expected treatment-related mortality and a higher chance of durable remission.
Mesenchymal Stem Cell Deficiencies in Myeloma Patients.
Authors: Garderet Laurent , Doucet Christelle , Holy Xavier , David Catherine , Ernou Isabelle , Lopez Manuel , Lataillade Jean Jacques
Published: April 2020

Progression of Multiple Myeloma (MM) is associated with disrupted bone remodelling resulting from increased osteoclast activity and reduced osteoblast number in involved bones. The aim of this study was to investigate Mesenchymal Stem Cells (MSCs) properties in MM patients to find out whether disorders exist within the Bone Marrow (BM) microenvironment and during disease progression. We examined MSCs derived from 6 MM patients at diagnosis, 3 patients with Bone Lesions (BL-MSCs) and 3 patients Without Lesions (WL-MSCs), and 3 Normal Donors (ND-MSCs). We focused on mesenchymal phenotype, clonogenic and proliferative capacities, Growth Factor Receptors (GF-R) expression, as well as osteogenic differentiation. BM adherent mononuclear cells were used to initiate MSC cultures and 2 passages were performed when confluency was reached. Adherent MSCs were numbered after each passage and CFU-F was counted (at 11 day culture). Analysis of MSC immunophenotype and GF-R expression were performed by flow cytometry. Osteoblastic differentiation assay was performed in inducible specific medium by analysing alkaline phosphatase activity and matrix mineralisation deposition (Von Kossa method). MSCs represented 0.1 to 1% of BM nuclear cells from all sources. The characteristic MSC phenotype (CD45−/CD90+/CD73+/CD105+) was observed in all sources of cells (MM- and ND-MSCs). The clonogenic capacity of BL-MSCs was 2 fold lower than that of WL-MSCs (44 ± 21 vs 89 ± 23, p = 0.05) and their proliferative capacity was 7.5 fold decreased (16,5 x 106 ± 15 vs 124 x 106 ± 68, p = 0.02, total cells after 2 passages). Expression level of PDGFa & b, IGF-1, EGF and NGF Receptors was lower in MM-MSCs than in ND-MSCs (185 ± 39 vs 413 ± 50 UA, p = 0.0004, 212 ± 63 vs 383 ± 143 UA, p = 0.01, 227 ± 50 vs 534 ± 67 UA, p = 0.0007, 229 ± 34 vs 535 ± 84 UA, p = 0.0001 and 179 ± 26 vs 441 ± 98 UA, p = 0.001, respectively), while the percentage of FGF receptor-expressing cells was dramatically decreased in MM as compared to ND (59 ± 31.5 vs 90 ± 6 %, p = 0.01). Interestingly, the osteogenic differentiation capacity of MSCs was maintained in MM-MSCs whatever the progression status of the disease. This study demonstrates that impaired bone formation in MM could be related to a deficient proliferation capacity of MSCs associated with a low growth factor receptors expression. The lack of expansion capacity increases with disease progression. Therapeutic approaches using normal expanded MSCs grafts may improve bone reconstruction in myeloma patients.
The OPG/TRAIL Interaction in an In Vitro Osteoclastogenesis Model Derived from Human Multiple Myeloma-Bone Disease.
Authors: Molica Stefano
Published: April 2020

The development of multiple myeloma (MM)-bone disease is mediated by increased recruitment and activity of osteoclasts (OCs). Osteoclastogenesis is regulated by a complex signaling system, that involves receptor activator of nuclear factor-kappa B (RANK), receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG), all belonging to the Tumor Necrosis Factor (TNF) family. The aims of our study were to investigate: the MM T cell involvement in osteoclastogenesis, and the expression of the major osteoclastogenic mediators. Unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs) isolated from 32 MM patients with or without osteolytic bone lesions, and parallel T cell-depleted cultures were used as in vitro osteoclastogenesis models. In addition, unstimulated and unfractionated PBMC cultures from 32 controls with nonneoplastic disease without any skeletal involvement were also established. Our results showed that the OCs derived from MM-bone disease PBMCs spontaneously developed and displayed a longer survival in a T cell-dependent way. Differently in T cell-depleted MM PBMC cultures, the addition of macrophage-colony stimulating factor (M-CSF) and RANKL was necessary to promote the formation of OCs, that however did not exibit a longer survival. MM-bone disease T cells overexpressed RANKL, OPG and TNF-related apoptosis inducing ligand (TRAIL), also detected in large amounts in the culture media. Despite high OPG levels, the persistence of osteoclastogenesis in our system can be related to the interaction between OPG and TRAIL, that were coimmunoprecipitated by a monoclonal antibody (mAb) against TRAIL. The evidence that TRAIL binds to OPG blocking OPG anti-osteoclastogenic effect is also supported by the addition of different concentrations of functional anti-TRAIL mAb, significantly decreasing the OC formation. The OCs developed from MM-bone disease PBMCs expressed a T cell-modulated balance of death and decoy TRAIL receptors. In particular, we found these OCs overexpressed TRAIL decoy receptor DcR2 in the presence of T cells, and death receptor DR4 in the T cell-depleted cultures. In conclusion, our results highlight that MM-bone disease T cells support the spontaneous OC formation with longer survival, involving the OPG/TRAIL interaction and the unbalanced OC expression of TRAIL death and decoy receptors.
Increased Bone Marrow Erythroid Precursors Is Associated with Shorter Survival in Low-Grade Myelodysplastic Syndromes.
Authors: Amin Hesham M. , Pierce Sherry A. , Kantarjian Hagop M. , Keating Michael J. , Freireich Emil J. , Koller Charles A. , Beran Miloslav , Albitar Maher , O’Brien Susan M. , Estey Elihu H.
Published: April 2020

According to the FAB and WHO classifications, the diagnosis of acute erythroid leukemia is based on the numbers of nucleated red blood cells and myeloid blasts in the bone marrow. The WHO classification recognizes two types of acute erythroid leukemia; M6A with 51–80% erythroid precursors and with 20% or more of the non-erythroid precursors being myeloid blasts; and M6B with more than 80% of the nucleated cells in the bone marrow consisting of erythroid precursors, regardless of the percentage of the myeloid blasts. Previous studies have shown that many cases of acute erythroid leukemia arise in patients with a history of myelodysplastic syndrome and in other cases acute erythroid leukemia is associated with significant dysplastic features. The significance of the number of erythroid precursors is not well known in the myelodysplastic syndromes. In the present study, we included 617 consecutive patients with low-grade myelodysplasia (482 patients with refractory anemia [RA] and 135 patients with refractory anemia with ringed sideroblasts [RARS]). Among this group, 82 patients with 50% or more of erythroid precursors had shorter survival compared with 535 patients with less than 50% erythroid precursors (P < .01; Figure 1). The shorter survival in those with 50% or more of erythroid precursors may reflect the tendency of these patients to have worse International Prognostic Scoring System (IPSS) scores. Thus, among the patients with less than 50% erythroid precursors and primary MDS, 35% were IPSS low, 52% IPSS intermediate 1, and 13% IPSS intermediate 2. For the patients with 50% or more of erythroid precursors, the corresponding proportions were 14%, 57%, and 29%, respectively (P < .001). As a result of the association between IPSS and the percentage of erythroid precursors, the percentage of erythroid precursors had no effect on survival within individual IPSS groups. Similarly, the percentage of erythroid precursors had no prognostic significance in patients with refractory anemia with excess blasts (RAEB) or chronic myelomonocytic leukemia (CMML). Our findings demonstrate that in low-grade dysplasia (RA and RARS) the number of erythroid precursors may represent an important prognostic marker. These findings implicate that the percentage of erythroid precursors should be considered in the classification of the low-grade myelodysplastic syndromes. A multivariate analysis will be performed to ascertain the relative effects of IPSS score and the percentage of erythroid precursors on prognosis in patients with low-grade myelodysplasia.

Figure Figure
Phenotypic Aberrations of Myeloid CD34+ Cells in Patients with Myelodysplastic Syndrome (MDS): Clinical Significance and Correlation with Cytogenetics Abnormalities.
Authors: Subira Dolores , Font Patricia , Arranz Eva , Soraya Ramiro B , Castanon Susana , Renedo Mónica , Blas Carlos , Garcia B Maria F. , Gonzalez B Olga , Tomas José F. , Fernandez-Rañada José M.
Published: April 2020

Expression of abnormal markers in myeloid CD34+ cells of patients with MDS is common, but few immunophenotypic data have been compiled so far. Based on the statement that CD7 and TdT are markers associated with a bad prognosis in acute myeloid leukemia, we intended to describe the incidence of their aberrant expression in CD34+ cells and its role helping to establish the diagnosis of MDS.

OBJECTIVES: To explore the aberrant expression of TdT and CD7 in myeloid CD34+ cells of MDS patients and to describe their possible correlation with cytogenetic features.

DESIGN AND METHODS: Bone marrow specimens from 45 patients with MDS were included in this study (17 RA, 12 RARS, 5 CMML, 9 RAEB, 2 RAEB-t). In addition, we analyzed 28 samples of bone marrow from patients with cytopenias, but no diagnosis of MDS, as a cohort control. Immunophenotyping was performed with the following combination of monoclonal antibodies: TdT FITC / CD7 PE/ CD34 PCy-5. A case was regarded as positive for any of these markers when their expression was described in at least, 25% of myeloid CD34+ cells. Besides, adequate cytogenetic data and FISH analysis of chromosomes 5, 7 and 8 were obtained from 42 out of the 45 MDS samples.

RESULTS: The percentage of CD34+ myeloid cells in MDS samples was fewer than 2% in 25 cases, ranged from 2–5% in 6 cases and was equal or greater than 5% in 14 cases. Aberrant expression of CD7 and/or TdT was observed in 28 cases: 20 were positive for CD7, 5 cases were positive for TdT and co-expression of both antigens was described in 3 cases. Prevalence of these abnormal markers was much higher in patients with MDS (28/45; 62%) than in the cohort control (2/28; 7%) . Besides, we identified at least one abnormal marker in 14 of the 16 patients with high risk MDS (9 RAEB, 5 CMML, 2 RAEB.t). According to the IPSS, karyotypes were divided into subgroups of favorable, intermediate or unfavorable, being classified 29 patients with favorable karyotypes and 13 patients with no favorable cytogenetics. In the first group, the abnormal expression of CD7 and/or TdT was detected in 13/29 patients (44.8%) and in 10/13 patients in the non-favourable group (76.9%).

CONCLUSIONS: CD7 and/or TdT expression in myeloid CD34 + cells may be helpful in establishing the diagnosis of MDS. Prevalence of these aberrancies seems to be higher in cases with no favorable karyotypes, but a larger number of patients will be required to state significant differences. Possible correlation between high risk MDS patients and immunophenotypic aberrancies suggests the convenience of following the outcome of those low risk MDS patients who have expression of any of these abnormal markers.
Hematopoietic Progenitors in Myelodysplastic Syndrome (MDS) Demonstrate Cell Cycle Arrest, Mitotic Failure, and DNA Damage.
Authors: Head David R. , Jacobberger James W. , Jagasia Madan H. , Goodman Stacey A. , Flye Leanne , McClintock-Treep Sara A. , Boulos Fouad I. , Jiang Fen , Stelzer Greg , Briggs Robert C. , Shults Keith
Published: April 2020

To assess biologic characteristics of marrow progenitors in MDS, we studied a consecutive series of MDS patients receiving no current treatment.

Methods: Patients’ (n=17) clinical histories, lab data, cytogenetics, and morphology were reviewed for confirmation of diagnosis and blinded to study results. Patients lacking primary marrow disease (n=9) were used as controls. Marrow samples were studied at 2-5 hours after procurement (mean 3.1 hours), and re-assessed at 24 hours. A small number of marrow samples were fixed within minutes of procurement to validate findings. Each sample was assessed for CD45 antigen density, log side scatter, CD34, CD71, DRAQ 5 (DNA content), MPM-2, p-H2A.X (serine 139), and Annexin V with an FC500 (Beckman-Coulter) flow cytometer. Analysis was performed using Winlist 5.0 software (Verity Software) with DDE links to ModfitLT3.0 using modifications of published methods along with Esoterix Center for Innovation generated algorithms. Analyses were blinded to clinical results. Results: Most MDS patients demonstrated increased S-phase cell cycle fractions. Despite this, neither myeloid nor erythroid progenitors in MDS demonstrated an increased mean mitotic index vs. normals (0.315/0.359 and 1.33/1.48 respectively), and 7 MDS patients demonstrated no mitotic events in myeloid precursors (never observed in the normal dataset). DNA damage (H2A.X binding) was increased in myeloid and erythroid progenitors in MDS vs. normals (19/4% and 9/1.6% respectively). Annexin V staining was modestly elevated in maturing progenitors in MDS patients vs. normals (16.4% vs. 10.9%), and increased 4% in each group at 24 hours. Stem cells (CD34+) and lymphocytes were negative for both H2A.X and Annexin V binding. Discussion: These results indicate abnormalities in MDS marrow biology that may be useful as diagnostic tests for MDS. They suggest a model of MDS characterized by DNA damage in maturing hematopoietic progenitors in MDS, with late cell cycle arrest, accumulation of cells in S or G2, and decreased cell division (mitotic events) despite cellular marrow. In this model, DNA damage may initiate apoptosis in cells, but the findings are inconsistent with massive successful programmed cell death as a central event in MDS pathogenesis. The model has therapeutic implications for MDS.
Atiprimod (N-N-diethl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) Inhibits Myeloma in Vivo.
Authors: Hamasaki Makoto , Hideshima Teru , Tassone Pierfrancesco , Neli Paola , Ishitsuka Kenji , Akiyama Masaharu , Hayashi Toshiaki , Yasui Hiroshi , Shiraishi Norihiko , Chauhan Dharminder , Mitsiades Constantine , Podar Klaus , Gupta Deepak , Picker Donald H. , Jacob Gary S. , Richardson Paul G. , Munshi Nikhil C. , Anderson Kenneth C.
Published: April 2020

Atiprimod (N-N-diethl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) is an orally-bioavailable cationic amphiphilic compound which significantly inhibits inflammation in rat arthritis and multiple sclerosis models. It acts, at least in part, by significantly inhibiting production of interleukin (IL)-6. Since IL-6 mediates MM cell growth, survival and drug resistance in the bone marrow (BM) microenvironment, we in this study characterized the effect of Atiprimod on human MM cells. We first demonstrated that Atiprimod significantly inhibited growth (p<0.05) in MM.1S, U266, and RPMI8226 MM cell lines in a time- and dose-dependent fashion, with IC50s of 0.5–1.25 mM. It also induced cytotoxicity in dexamethasone (Dex)-, doxorubicin (Dox)-, and melphalan (Mel)-resistant MM cell lines; as well as in patient MM cells, but not in normal PBMCs. Atiprimod triggered MM cell apoptosis via activation of caspase-8 and caspase-3, followed by PARP cleavage. Importantly, neither IL-6 nor insulin-like growth factor (IGF)-1, which completely abrogate Dex-induced apoptosis, protects against Atiprimod-induced apoptosis in MM.1S cells. In combination treatment studies, both conventional (Dex, Dox, Mel) and novel (As2O3) agents augment MM cell apoptosis induced by Atiprimod. Atiprimod inhibits STAT3 and Akt, but not ERK1/2, phosphorylation triggered by IL-6, BAX, Bcl-xl, and Mcl-1 in MM.1S cells. Importantly Atiprimod inhibits both IL-6 and vascular endothelial growth factor (VEGF) secretion in BM stromal cells (BMSCs) triggered by MM cell adherence to BMSCs, as well as associated MM cell growth. Finally, Atiprimod inhibits tumor OPM1 MM cell growth in vivo and prolongs survival in a SCID mouse model. Our data therefore demonstrate that Atiprimod both induces MM cell apoptosis and inhibits cytokine secretion in the BM milieu, providing the framework for ongoing clinical trials of this agent to improve patient outcome in MM.
A Phase II Study of Velcade (V), Doxil (D) in Combination with Low-Dose Thalidomide (T) as Salvage Therapy for Patients (pts) with Relapsed (rel) or Refractory (ref) Multiple Myeloma (MM) and Waldenstorm Macroglobulinemia (WM): Encouraging Preliminary Results.
Authors: Chanan-Khan Asher Alban , Miller Kena C. , McCarthy Philip , DiMiceli Laurie A. , Yu Jihnee , Bernstein Zale P. , Czuczman Myron S.
Published: April 2020

In MM, tumor microenvironment (ME) plays an important role in disease progression, dissemination, and development of resistance to therapy. Pts with rel/ref MM have limited treatment options. Therefore, targeting the ME simultaneously with malignant cells may be an effective way to overcome resistance in pts with rel/ref MM. Orlowski et al recently reported synergistic activity of V+D in patients with hematologic malignancies. A phase II trial initiated at our institute is exploring this approach, targeting the MM cell as well as its ME, using a combination of Velcade (V), Doxil (D) and low-dose thalidomide (T) as salvage therapy for pts with rel/ref MM. Pts with rel/ref disease are eligible for this study. V is given at 1.3mg/m2 (D1,4,15,18) and D at 20mg/m2 (D1,15) every 4 weeks with daily T (200 mg) for 4–6 cycles. SWOG criterion was used to evaluate response. Low-dose coumadin (1–2 mg po qd) was used for prevention of venous thromboembolism (VTE). Eighteen pts (7M, 11F; median age 56, range 44–80 yrs; 16MM, 2 WM) have been enrolled to date. All pts had Stage III disease, median b2M of 4.8 (nl range: 0–2.15) and median prior therapies 2 (range 1–7). Prior therapies included stem cell transplant in (46%), T (54%), adriamycin(A) (70%) and steroids (92%). Thirteen pts have completed at least 1 cyc and are available for toxicity and response evaluation. One pt died of sepsis prior to completing 1 cyc and 1 pt with PR was taken of study for non-compliance after 1 cyc. ORR was 100% (13/13) with 5PR and 8MR. All pts are currently continued on therapy. Toxicity: 2 pts developed Gr. II plantar-palmar erythrodysthesia (PPE) and 1 had Gr. III cellulitis. No VTE was noted. VDT is a highly active salvage regimen in pts with rel/refr MM. Responses were noted despite prior failure of steroids, T or A. It is well tolerated without any significant non-hematologic Gr. III/IV toxicity. VTE does not appear to be a problem. Updated results of this first cohort of pts will be presented at the annual ASH meeting.
Discriminative HMGA2 Isoform Expression in CD15+ Granulocytic Cells in Myeloid Metaplasia with Myelofibrosis (MMM).
Authors: Pierre-Louis Olivier , Andrieux Joris , Desterke Christophe , Lippert Eric , Praloran Vincent , Demory Jean-Loup , Le Bousse-Kerdiles Marie-Caroline , Bilhou-Nabera Chrystele
Published: April 2020

MMM is a myeloproliferative disorder characterized by extramedullary hematopoiesis and reactive myelofibrosis. Recently, HMGA2 dysregulation has been demonstrated in 2 MMM patients showing 12q15 rearrangement and confirmed in 25 consecutive MMM patients without cytogenetic abnormalities (Andrieux, 2004). HMGA2 proteins belong to the high mobility group A (HMGA) family of architectural transcription factors regulating the expression of several genes. As MMM is a clonal disorder of CD34+ hematopoietic progenitors, we analyzed HMGA2 expression in peripheral blood sub-populations of 5 MMM patients and 7 healthy donors to determine in which sub-population HMGA2 was dysregulated.

RNA was extracted from peripheral blood mononuclear cells (PBMC) and CD15+ granulocytic cells (PBCD15+) separated through Ficoll centrifugation or from immunomagnetically selected circulating CD34+ cells (PBCD34+). Real-time quantitative PCR (RQ-PCR) using Taqman technology was performed on cDNA. As different isoforms were described in malignancies, we used two primer sets : the first one allowing the amplification of all HMGA2 isoforms (exon 1 to 3) (HMGA2 1–3), the second one allowing the amplification of the full length HMGA2 isoform (exon 1 to 5)(HMGA2 1–5).

In healthy donors and in MMM, PBMC HMGA2 expression levels were heterogeneous, depending of the cellular sub-population purity. HMGA2 1–3 or HMGA2 1–5 were both expressed in MMM and normal PBCD34+ cells, but with a higher expression level for HMGA2 1–3 as compared to HMGA2 1–5. Furthermore, both HMGA2 1–3 and HMGA2 1–5 expression levels were significantly increased in PBCD34+ MMM patients (p<10−6) compared to healthy donors. In MMM, HMGA2 expression level was significantly increased (p<10−5) in PBCD15+ as compared to PBCD34+. Moreover, PBCD15+ HMGA2 1–3 expression level was significantly higher in MMM patients compared to PBCD15+ from healthy donors (p<10−7). A persistence of HMGA2 1–5 expression was only observed in MMM PBCD15+ but was undetectable neither in normal PB neutrophils (purity>98%) nor in PB neutrophils from other myeloproliferative disorders (Polycythemia Vera and Essential Thrombocythemia).

To determine if HMGA2 level was modified during hematopoietic differentiation, we quantified HMGA2 1–3 and 1–5 isoform expression on purified healthy donor PB CD34+ and MMM CD34+ before and after culture with specific lineage growth factors (12 day-culture). Primary results showed that both HMGA2 isoform expression levels were higher during granulocytic differentiation. Our results demonstrate that HMGA2 1–5 isoform is discriminately overexpressed in MMM PBCD15+. The persistence of this HMGA2 full length expression in MMM myeloid lineage could be considered as a marker of the disease.
NFκB Activation in Primary Mediastinal Large B-Cell Lymphoma: Nuclear Localization of c-REL and Coordinate Upregulation of NFκB Target Genes.
Authors: Feuerhake Friedrich , Kutok Jeffery L. , Monti Stefano , Cattoretti Giorgio , Kurtin Paul , Pinkus Geraldine S. , de Leval Laurence , Harris Nancy L. , Savage Kerry J. , Habermann Thomas M. , Dalla-Favera Riccardo , Golub Todd R. , Aster Jon C. , Shipp Margaret A.
Published: April 2020

Primary mediastinal large B-cell lymphoma (MLBCL) is a clinically distinct entity that typically presents as localized, sclerotic disease in young, female patients. We previously characterized the transcriptional profiles of MLBCLs and identified important shared features with a clinically related disorder, classical Hodgkin lymphoma (cHL) (Blood 102:3871, 2003). Given the documented role of the NFkB survival pathway in Hodgkin Reed-Sternberg cells, we previously assessed NFkB activation in MLBCL by determining the subcellular location of the c-REL subunit of the NFkB heterodimer with a 2-color immunofluoresence assay. In a small pilot MLBCL series, c-REL was localized to the nucleus in the majority of examined cases, consistent with NFkB activation. In the current study, we evaluated c-REL subcellular localization in an additional series of MLBCLs and DLBCLs using a broadly applicable immunoperoxidase method. 100% of MLBCLs exhibited nuclear c-REL staining whereas DLBCL c-REL subcellular localization was more variable. Thereafter, we analyzed the transcription profiles of the 34 MLBCLs and 176 DLBCLs for coordinate expression of NFkB target genes, using literature-curated NFkB target gene lists from three independent sources and gene set enrichment analysis (GSEA). MLBCL signatures exhibited significant enrichment of 2 of the 3 NFkB target gene sets. In addition, 32 NFkB target genes from the combined set were significantly more abundant in MLBCLs than DLBCLs (> 30% more abundant and > 99th percentile in permutation analysis). Similar results were obtained in an independent series of MLBCLs and DLBCLs with available gene expression profiles (J. Exp. Med. 198:851, 2003). To assess the role of c-REL amplification in NFkB activation in our lymphoma series, we compared c-REL amplification, c-REL subcellular localization and coordinate expression of the identified NFkB target genes and classified the DLBCLs according to putative cell of origin. The majority of c-REL amplifications (67%) were found in DLBCLs of germinal center (GC) subtype, consistent with the observation that c-REL is part of the described GC signature. However, most (71%) of the examined GC DLBCLs had cytoplasmic c-REL expression and the GC DLBCLs did not have increased expression of NFkB target genes. Taken together with the MLBCL analyses, these studies indicate that: 1) NFkB is consistently activated in MLBCL; 2) c-REL amplification is not closely associated with NFkB activation in large cell lymphomas (LCLs); and 3) NFkB activation in LCL subtypes does not require amplification of the c-REL locus.
Expression of HMGA2 in PB Leukocytes and Purified CD34+ Cells from Normal Individuals and Patients with Myelofibrosis and Myeloid Metaplasia.
Authors: Andrieux Joris R. , Bilhou-Nabera Chrystèle , Demory Jean Loup , Pierre-Louis Olivier , Dupriez Brigitte , Grardel Nathalie , Charpentier Agnès , Praloran Vincent , Bauters Francis , Laï Jean Luc , Le Bousse-Kerdilès Marie-Caroline
Published: April 2020

Background: Myelofibrosis with myeloïd metaplasia (MMM) is a rare myeloproliferative disorder which associates clonal proliferation of multipotent hematopoietic progenitors and reactive fibrosis. Deregulation and overexpression of HMGA2 has recently been demonstrated in MMM. Using quantitative RT-PCR on blood mononuclear cells from 27 patients, we found various levels of expression. It could be hypothesized that HMGA2 expression reflected the variable increase in circulating CD34+ progenitors in MMM though we failed to establish significant correlation between the level of expression and the blood enumeration of CD34+ progenitors.

Material and Methods: We studied 24 patients diagnosed as primary MMM according to the set of criteria recently updated through an Italian Consensus Conference. Six individuals free from hematologic malignancy were used as controls. We also studied-purified CD34+ cells: 8 samples were isolated from the PB of MMM patients; given the very low concentrations of progenitors in normal PB, 2 samples pooling 8 healthy donors each were also prepared. In addition, 7 CD34+ cell samples were purified from the BM of consenting subjects collected during surgery for hip replacement.

Results: We assayed HMGA2 expression in purified CD34+ populations from MMM patients and controls: both expressed HMGA2 but markedly more the CD34+ cells from the patients. We also evidenced transcripts in their CD15+ granulocytic cells suggesting that the enhanced expression of the gene affects the whole pathological clone. HMGA2, not expressed in normal blood cells, is involved in benign solid tumors of mesenchymal origin.

Conclusion: Our data support the hypothesis that its reactivation in the clonal progenitors contribute to the pathogenesis of MMM.
HLA-Cw6 Alloreactive CTL Recognizing HLA-CW6+ Myeloma Cells but Not Non-Myeloma HLA-Cw6+ Cells Are Peptide Dependent.
Authors: Geffroy Alexandrine , Moreau-Aubry Agnes , Bataille Regis , Pellat-Deceunynck Catherine
Published: April 2020

We investigated the generation of myeloma-specific cytotoxic T lymphocytes (CTL) from unrelated normal donors non-HLA matched with the myeloma cell line SBN. The aim was to obtain alloreactive CTL specific of peptides to identify genes preferentially expressed by to myeloma cells. After coculture of SBN with PBL of unrelated donor, the T-cell line obtained was cloned and each CTL was assessed against SBN and SB-EBV (B-EBV cell line obtained by infecting B cells of SB patient with EBV) simultaneously. Only the CTL recognizing SBN but not SB-EBV were kept for further study. Among more than 200 clones screened, we isolated two different CTL CD8+ recognizing SBN only (60% of specific lysis at effector: target ratio 5). Their cytotoxicity was blocked by antibodies against HLA-I molecules and more precisely by mAb against HLA-B/Cw molecules. Both CTL recognized also other MM cell lines but only those matched with SBN for HLA-Cw0602 (XG6, BCN) suggesting an HLA-Cw6 recognition. Furthermore, antiserum directed against HLA-Cw6 abrogated the recognition of SBN, XG6 and BCN. Both CTL were not cytotoxic against BC-EBV nor against 3 other B-EBV cell lines derived from HLA-Cw0602 donors. This unreactivity was not restricted to B-EBV cell lines since PBL from 2 normal HLA-Cw0602 donors were also not recognized (cytotoxicity, TNF secretion). We directly measured cell surface HLA-Cw6 expression of both B-EBV and myeloma cells with a scFv directed against HLA-Cw6 and we showed that HLA-Cw6 was more expressed by B-EBV cells as compared to myeloma cells (2fold). So, the lack of recognition of B-EBV cells was not related to HLA-Cw6 expression level. We also looked for KIR expression by the CTL since KIR molecules could have blocked B-EBV recognition that expressed more HLA-Cw6 molecules as compared to myeloma cells. No KIR molecules (p58.1, p58.2, CD94) were found to be expressed by the CTL.

To investigate whether the recognition of myeloma cells was peptide dependent, we performed two experiments i.e., acid elution and cold target competition. Acid elution (glycine 0.3M, pH=2.5) abrogated myeloma recognition and cold target competition showed that B-EBV cells were not recognized by the CTL. These data suggest that the CTL recognize a peptide or a set of peptides restricted to myeloma cells or that the CTL recognition is activated by tumor cells only. Anyway, such alloreactive CTL could be helpful for GVL reaction in allotransplanted patients with myeloma.
Periocular MALT Lymphomas (Conjunctival/Orbital): Complete Responses with Antibiotic Therapy.
Authors: Coleman Morton , Leonard John P. , Decter Jules , Furman Richard R. , Niesvizky Ruben , Rollins Indira , Abramson David H.
Published: April 2020

There is mounting epidemiologic, histologic, experimental and therapeutic evidence that MALT (mucosa-associated lymphoid tissue) lymphomas may be related to chronic infections. While H. pylori infection-initiated MALT lymphomas of the stomach are now well documented, only recently has evidence for infectious sources of MALT lymphomas of the intestine (Campylobacter), skin (Borrelia), and the eye (Chlamydia) been reported. Chlamydia psittaci has been implicated in ocular MALT lymphoma in 2 reports (Ferreri et al, JNCI, 96(8):586–594, 2004 and Yeung et al, Cornea, 23:71–75, 2004). In these reports, only 2 of 4 evaluable patients treated with doxycycline were noted to have objective responses. We retrospectively reviewed data on 4 patients who have now undergone antibiotic therapy for subconjunctival MALT lymphomas, three of whom are evaluable for response to treatment. Complete evaluations for systemic disease (including CT scans of neck, chest, abdomen, pelvis, PET scans, and bone marrow examination) were all negative. Serologic tests for antibodies to Chlamydia psittaci were negative in all 4 individuals. Patients were 58, 74, 76 and 79 years of age with 2 males and 2 females. Treatment of one patient was with a PrevpacR (lansoprazole 30 mg capsule, amoxicillin 500 mg capsules, and clarithromycin 500 mg tablets BID) x 14 days, while three received doxycycline 100 mg daily for one month. Of the three patients evaluable to date for response, all demonstrated prompt and sustained responses (2 CR and 1 PR) with followup of 38 months, 12 months, and 7 months respectively. Definitive evidence for a particular infectious organism was not demonstrated, but the prompt and sustained response to antibiotics is consistent with an infectious origin. While further evaluation and followup is warranted in additional patients, we conclude that a trial of anti-microbial therapy should be considered for patients diagnosed with ocular MALT lymphoma.
A Novel N-Oxide Drug, AQ4N, Has In Vitro Activity in Lymphoma and Leukemia Cell Lines and Selectively Targets Lymphocytes and Lymphatic Tissues in Vivo.
Authors: Cleland Jeffrey L. , Wong Alvin , Alters Susan E. , Harris Peter A. , Dunk Chris R. , Curd John G. , Capizzi Robert L. , Henner William D.
Published: April 2020

An ideal treatment for lymphoma and leukemia is the use of highly selective compounds to eliminate diseased cells with minimal systemic toxicity to normal tissues (cf. imatinib mesylate; Gleevec). AQ4N (1,4 bis[[2-(dimethylamino)ethylamino}-5,8-hydroxyanthracene-9,10-dione bis N-oxide) is designed to have little or no toxicity until selectively activated by bioreduction in hypoxic cells to AQ4 (reduced AQ4N), a highly potent DNA topoisomerase II inhibitor. In a series of studies, AQ4 has been shown to have potent cytotoxicity on lymphoma and leukemia cell lines in vitro and AQ4N has selective activity in lymphatic tissues in vivo. The IC50 of AQ4, was 0.63, 12.0, 90.5 and 150 nM in Namalwa, Daudi, Ramos, and Raji human lymphoma cell lines and 1.0, 6.0, and 20 nM in HL-60, KG1a and K562 human leukemia cell lines. On several of the tumor lines the activity of AQ4 was more potent than doxorubicin (i.e. IC50 for Dox was 20.3 nM on Namalwa). AQ4N also had anti-proliferative activity at μM levels indicating a potential mechanism for activation by these cell lines. In repeat dose toxicology studies of AQ4N in pigmented rats and cynomolgus monkeys, the maximum tolerated doses (MTD; rats: 20 mg/kg/wk x 6; monkeys 6 mg/kg/wk x 6) resulted in lymphoid tissue atrophy. A decrease in lymphocyte levels and atrophy of the spleen, thymus, and mandibular and mesenteric lymph nodes were observed at terminal sacrifice of the animals. In contrast, there was an absence of myelosuppression and only mild neutropenia and minor bone marrow atrophy at the MTD. Administration of radiolabeled AQ4N (14C-benzene) to pigmented rats and cynomolgus monkeys indicated persistence of AQ4N radioactivity in lymphoid tissues for several weeks after a single dose (rats: 20 mg/kg (130–140 μCi/kg); monkeys: 10 mg/kg (135 μCi/kg)). For example, in rats the half-life of radioactive AQ4N in the spleen was 538 hrs with 0.9 μg AQ4N/g tissue (spleen) remaining one week after dosing. Monkeys demonstrated a similar effect with 76.5–86.8 μg AQ4N/g tissue observed in the spleen one week after treatment. Other tissues contained significantly less radioactive AQ4N with the exception of the liver (67.9–78.6 μg AQ4N/g tissue) and adrenal cortex (78.7–86.6 μg AQ4N/g tissue). While some hypertrophy and eosinophila was observed in the adrenal glands, liver toxicity was not observed at the MTD in the repeat dose cynomolgus monkey toxicology study. Overall, these initial findings indicate that AQ4N is active in vitro against human lymphoma and leukemia cell lines and selectively targets lymphoid tissues in vivo suggesting the potential benefit of AQ4N in the treatment of lymphoproliferative diseases.
Analysis of the Effect of the Novel Histone Deacetylase Inhibitor, Depsipeptide (FK228/FR901228) on Mantle Cell Lymphoma.
Authors: Paner Gladell P. , Alkan Serhan
Published: April 2020

Background: Mantle cell lymphoma (MCL) comprises approximately 3–5% of the non-Hodgkin’s lymphomas and the vast majority of these patients are incurable. Histone deacetylases (HDAC) inhibitors are a new class of chemotherapeutic drugs that have been shown to inhibit cell proliferation and induce apoptosis in a variety of malignancies. Depsipeptide (FK228/FR901228) is a novel inhibitor of class I HDAC and was shown to be effective in patients with cutaneous T-cell lymphoma.

Design: A MCL cell line, JeKo-1 was treated with varying concentrations of depsipeptide and analyzed for evidence of apoptosis. Apoptosis was analyzed by flow cytometry (Annexin and 7AAD staining) and by microscopic examination. The effect on the status of H3 histone acetylation and changes in the expression of the Bcl-2 family proteins (Bcl-2, Bcl-xl and Mcl-1) following treatment with depsipeptide were investigated by flow cytometric evaluation. Requirement for de novo protein synthesis and activation of JNK in depsipeptide-induced apoptosis was analyzed by treatment with cycloheximide (CHX) and JNK inhibitor (SP600125) prior to addition of depsipeptide respectively. The effect of depsipeptide in 5 primary MCL patient samples was then analyzed for morphologic evidence of apoptosis.

Results: Depsipeptide (0.25 mM/L; 80%apoptosis) induced marked apoptosis in JeKo-1 cells and this was shown to be a dose dependent fashion following 12 hours incubation. There was increase in H3 acetylation, after treatment with depsipeptide. Bcl-2, Bcl-xl and Mcl-1 showed decreased expression in the depsipeptide treated samples. Treatment with CHX (100ng/ml and 500ng/ml) and SP600125 (20ml/L) had no effect to the ability of depsipeptide-induced apoptosis. Primary MCL cells of 5 patients also showed marked apoptosis with depsipeptide treatment.

Conclusion: Depsipeptide causes increased H3 acetylation and induces apoptosis in MCL cell line and primary MCL cells. The mitochondrial apoptotic pathway is possibly altered by the depsipeptide treatment causing apoptosis, by down-regulation of Bcl-2, Bcl-xl and Mcl-1 proteins. Based on the recent phase I clinical trial demonstrating clinical response in cutaneous T cell lymphoma, our study supports the potential clinical utilization of depsipeptide in patients with MCL.
Immunotargetting of the Wilms’ Tumor WT1 Antigen for Dendritic Cell and B-Cell-Based Vaccination of Leukemia.
Authors: Berneman Zwi N. , Van Driessche Ann , Ponsaerts Peter , Gao Liquan , Stauss Hans J. , Schroyens Wilfried A. , Van de Velde Ann , Gadisseur Alain , Lenjou Marc , Nijs Griet , Van Bockstaele Dirk R. , Van Tendeloo Viggo F.
Published: April 2020

The Wilms’ tumor antigen (WT1) is overexpressed in almost all leukemias and in several solid tumors. Overexpression of WT1 blocks the normal differentiation and enhances proliferation of hematopoietic progenitor cells. WT1 is also used in the detection of minimal residual disease. Using WT1-specific MHC class I tetramers, we were able to detect ex vivo low numbers of WT1-specific CD8+ T cells in the peripheral blood or bone marrow of leukemia patients, but not of healthy donors. In one particular donor we could detect up to 24% WT1 tetramer positive cells at the time of diagnosis. WT1 tetramer positive cells were present in all types of leukemia, except for CLL, and also in patients with MDS. Because WT1 plays an important role in leukemogenesis, it could serve as an antigenic target for dendritic cell-based immunotherapy. We used the mRNA electroporation strategy that allows presentation of multiple WT1 epitopes by MHC class I molecules, irrespective of the HLA haplotype. Monocyte-derived DC (Mo-DC) were electroporated with in vitro transcribed WT1 mRNA. RT-PCR and Western blot analysis showed that WT1 RNA and protein, respectively, was present for up to 5 days in WT1-electroporated DC, but not in mock- or EGFP mRNA-electroporated Mo-DC. Importantly, using a CD8+ T cell clone that secretes IFN-gamma upon recognizing the HLA-A2 immunodominant WT1126–134 epitope, we showed that WT1 mRNA-electroporated Mo-DC processed the WT1 protein via the MHC class I pathway and presented the WT1 epitope to the T cells in an HLA- and antigen-specific manner. Since Mo-DCs are a non-expandable source of antigen-presenting cells, we also used proliferating CD40-activated B (CD40-B) cells as inducers for WT1-specific T cell immunity. CD40-B cells were expanded to high numbers from a limited amount of peripheral blood and subsequently electroporated with WT1 mRNA. In T cell clone activation experiments, WT1 mRNA-electroporated CD40-B cells were as efficient as Mo-DC in presenting the WT1 epitope in a MHC class I-restricted manner. Based on these results, we are currently focusing on the in vitro (re)activation of autologous WT1-specific cytotoxic T cells of leukemia patients using WT1-loaded autologous Mo-DC or CD40-B cells and on the immunological parameters to break immune tolerance against the WT1 tumor self antigen.