bluuebunny/crossref_metadata_2025 huggingface dataset:
Orbital Non Hodgkin’s Lymphoma (NHL): A Large Monocentric Study of Initial Characteristics, Natural History and Prognostic Factors.
Authors: Jerome Meunier , Livia Lumbroso , Remy Dendale , Anne Vincent-Salomon , Bernard Asselain , Fariba Nemati , Driss Chaoui , Alain Fourquet , Laurence Desjardins , Corine Plancher , Didier Decaudin
Published: April 2020

Orbital NHL (ocular and/or adnexal involvement) represents less than 1% of all lymphomas but about half of malignant tumors of the eye and/or ocular adnexae. We therefore reviewed all patients treated at the Institut Curie between 1970 and 2003 for a NHL exhibiting initial orbital localization to define their initial characteristics, natural history and prognostic factors. Among 172 patients, 145 cases with completed datas were selected for the study. Pathological review according to the WHO classification showed 52 cases of MALT-type lymphoma (36%), 32 lymphoplasmocytic lymphomas (22%), 14 patients with lymphocytic lymphoma, 7 cases of follicular lymphoma, 13 unspecified low grade lymphomas [namely, 118 cases (82%) of low grade NHL], 22 patients with diffuse large B-cell lymphoma (15%), 2 cases of mantle cell lymphoma, 2 Burkitt’s lymphomas and one T-lymphoblastic lymphoma [namely, 27 cases (18%) of high-grade NHL]. Initial characteristics were: median age 66 years (range 3–96), sex ratio M/F 0.6, B symptoms 6%, PS≥2 in 4% of patients, stages III–IV 31.7%, bone marrow involvement 12%, elevated LDH in 18% and IPI 0-1/2/3/4-5 in 92/28/13 and 1 cases, respectively. Anatomic localizations were intra-orbital in 39 patients (27%), conjunctival in 38 (26%), eyelid in 9 cases, lachrymal in 8 and other in 8 cases. Treatment of selected patients consisted of abstention in 2 cases, surgical complete resection in 5 cases, mono or polychemotherapy alone (CT) in 4 cases, and radiotherapy alone in 98 cases (68%) or with CT in 36 cases (25%). With a median follow-up of 90 months (range 3–314), the 5-year relapse-free (RFS) and overall (OS) survivals were 64% and 79% for the low-grade NHL, and 43% and 50% for the high-grade NHL. Prognostic factors were determined for the 118 low-grade patients. In univariate analysis, age greater than 59 years, elevated IPI score and elevated LDH level were prognostic for lower RFS and OS. In multivariate analysis, age greater than 59 years was the only prognostic factor for both lower RFS and OS (Figure 1). In conclusion, with a median follow-up of 7.5 years, our large monocentric cohort of patients represents one of the most important series that defines the initial characteristics, natural history and prognostic factors of NHL with initial orbital localization.

Figure Figure
Long Term Results of the GELA Study, R-CHOP vs. CHOP in Elderly Patients with Diffuse Large B-Cell Lymphoma.
Authors: Coiffier Bertrand , Feugier Pierre , Sebban Catherine , Bouabdallah Reda , Delwail Vincent , Tilly Hervé , Gisselbrecht Christian , Bosly André , Salles Gilles , Reyes Felix
Published: April 2020

In 1998, the GELA (Groupe d’Etude des Lymphomes de l’Adulte) ran a randomized study to evaluate the benefit of the addition of rituximab to CHOP chemotherapy in elderly patients (60 to 80 years old) with diffuse large B-cell lymphoma. Early results were presented at ASH 2000 and published in NEJM (2002;346:235) with a median follow-up of 1 and 2 years, respectively. An update of these results is presented with a median follow-up of 5 years. 399 patients were included, 197 in the CHOP group and 202 in the R-CHOP group, 60% had a high risk disease according to the IPI score. Characteristics of the patients did not differ between both groups. Rituximab was given at 375 mg/m² the same day of CHOP for 8 cycles. Primary endpoint was event-free survival with events defined as progressive disease (PD) during or after treatment, relapse, institution of a new treatment, and death whatever the cause. Secondary endpoints were overall survival (OS) and progression-free survival (PFS) with progression defined as PD, relapse, or death from lymphoma or lymphoma treatment. Response rates for R-CHOP and CHOP were: CR/CRu 75% and 63%, PD 9% and 22%, and death during treatment 6% and 6%, respectively (P=.005), as previously published. With a median follow-up of 5 years, 106 events (52.5% of the patients) were observed in R-CHOP arm and 142 (72%) in CHOP arm: 19% and 31% PD or death during treatment, 5.5% and 4.5% institution of a new treatment, 20% and 34% relapses, 8% and 2.5% death in CR for R-CHOP and CHOP, respectively. Survival outcomes are presented in the table.

Table 1. 5-year survivals

R-CHOP CHOP P value Median event-free survival 3.8 y 1.1 y =0.00002 5-year event-free survival 47% 29% Median progression-free survival Not reached 1 y <0.00001 5-year progression-free survival 54% 30% Median overall survival Not reached 3.1 y =0.0073 5-year overall survival 58% 45%

Progression-free survival is shown in the figure; PFS does not included 19 patients who died in CR from causes not related to lymphoma or its treatment, 5 in CHOP arm and 14 in R-CHOP arm. It better reflects the long term effect of treatment on the disease. No severe late toxicity was observed in R-CHOP treated patients and deaths not related to lymphoma did not show any pattern.

In conclusion, long term results continue to show a major benefit for the addition of rituximab to CHOP in the treatment of patients with DLBCL. This improvement increases with time.
Rituximab/Chemotherapy Induction Treatment Followed by High-Dose Therapy and Autologous Transplantation in Patients with Mantle Cell Lymphoma.
Authors: Thieblemont C. , Antal D. , Lacotte-Thierry L. , Delwail V. , Bouafia F. , Tartas S. , Michallet A.S. , Espinouse D. , Giraud C. , Salles G. , Guilhot F. , Coiffier B.
Published: April 2020

Between September 1998 and September 2003, 25 patients with Mantle Cell Lymphoma (MCL) were treated in our centres with a treatment combining rituximab + chemotherapy at induction with subsequent consolidation high-dose chemo/radiotherapy (HDT) and autologous PBSC-transplantation. Twenty-two (80%) patients received this treatment in first line therapy. Median age of patients was 53 (range: 41–65). All patients had a disseminated disease with a bone marrow (BM) involvement in 23 (92%) patients. Elevated LDH was present in 11 patients. All patients except one had a good performance status. IPI score was low in 5 patients, low-intermediate in 10 patients, high-intermediate in 7 patients and high in 3 patients. Chemotherapy induction regimens were: R-CHOP (n=7 pts), R-ACVB (n=4 pts), R-ESHAP (n=1 pts), R-DHAP (n=1pts), or sequential CHOP+DHAP/ESHAP+Rituximab (n=12pts) regimens. Rituximab was giving simultaneously with the chemotherapy in 13 patients or just before harvest in 12 patients. Number of rituximab injections was four in all patients except one that received 5 injections. PBSC harvest was done after cyclophosphamide or cyclophosphmide + etoposide followed by G-CSF and was successful in all patients with a median number of CD34+ cells at 6.44 106/kg. HDT included TBI in 21 patients.

After induction therapy, all patients were in response: 16 (64%) reached a CR and 9 (36%) a PR because of persistence of a weak (<5%) BM involvement. Evaluation three months after transplant showed a CR in 17 (68%) patients and a PR in 7 (24%) patients because of weak BM involvement. At 3 years, 80% (20/25) of the patients are still alive (Figure). With a median follow-up at 4 years, median time to progression was 3.3 years. Four patients died from progressive disease. Transplantation toxicities were similar to those observed with regular induction treatment without rituximab with a median time of achievement of a PN count > 0.5GI/L at 10 days. One patient died before neutrophil recovery because of undocumented pulmonary infection. No patient presented late neutropenia. In conclusion, combined induction chemotherapy with rituximab followed by HDT dramatically improved the overall survival and the progression free survival in MCL patients, without adding hematological toxicities and infectious complications.

Figure Figure
Myelodysplasia and Acute Myeloid Leukemia Following Therapy for Indolent Lymphoma with Fludarabine, Mitoxantrone, Dexamethasone (FND) Plus Rituximab and Interferon Alfa.
Authors: McLaughlin Peter , Estey Elihu , Glassman Armand , Romaguera Jorge , Samaniego Felipe , Ayala Ana , Hayes Kimberly , Maddox Anne Marie , Preti H. Alejandro , Hagemeister Fredrick B.
Published: April 2020

Treatment-related myelodysplasia (t-MDS) can occur following chemotherapy, especially with DNA-damaging agents such as alkylators or topoisomerase II inhibitors. Antimetabolites such as the nucleoside analogs have only infrequently been associated with post therapy MDS (t-MDS). In a prospective chemo-immunotherapy trial between 1997–2003 in patients with stage IV indolent lymphoma, 202 patients were treated and 8 have developed MDS between 2–5 years after therapy, including 4 who received only fludarabine, mitoxantrone, and dexamethasone (FND) for 6–8 courses, with or without rituximab. The other 4 MDS patients received other agents including alkylators, either as salvage chemotherapy or as part of the initial regimen. Complex cytogentic abnormalities were present in all patients. Abnormalities of chromosome 7 were present in six of the eight, three of whom received FND ± rituximab therapy only. The abnormalities of chromosome 7 were monosomy 7 in four (one of which had add 7p in the remaining chromosome), one del 7q, and one der 7. MDS with features classically associated with DNA damaging agents can occur following therapy with the FND combination, with or without rituximab.
A Small-Molecule Bcl-2 Inhibitor Amplifies NKT-Cell Killing of U937 Lymphoma Cells.
Authors: Lickliter Jason , Cox Joanne , Nieda Mie , Lin Henry , Nicol Andrew
Published: April 2020

Invariant Vα24+ Vβ11+ NKT cells regulate immune responses and are capable of direct cytotoxic activity against tumour-cell targets mediated via the perforin/granzyme-B, Fas ligand and TRAIL pathways. A potential mechanism for tumours to escape immune killing is to block downstream apoptotic pathways by overexpression of the Bcl-2 oncoprotein. We therefore evaluated whether the Bcl-2-expressing U937 lymphoma line could be sensitized to NKT-cell cytotoxicity by coexposure to the HA14-1 small-molecule Bcl-2 inhibitor. Vα24+ NKT cells were purified from donor blood samples using magnetic beads and activated by exposure to KRN7000 and IL-2. U937 cells were labelled with the PKH26 membrane dye and then cocultured with activated NKT cells in a 10:1 or 20:1 effector: target ratio in U-bottomed 96-well plates. Cultures were performed in the presence or absence of HA14-1. After 4 hours, the cells were stained with annexin V-FITC and analysed by flow cytometry. Apoptotic and viable target cells were quantified as annexin-V positive and negative cells, respectively, in the PKH26-positive gate. Results of experiments on NKT cells derived from 3 donors are shown in the figure. Bars represent the mean percentage of viable U937 cells in triplicate assays and error bars indicate the SD. At the concentration of HA14-1 used, there was only minor single-agent cytotoxicity. However, a significant increase in U937 apoptosis was observed with the combination of HA14-1 and NKT cells compared to NKT cells alone. We conclude that the addition of the HA14-1 Bcl-2 inhibitor significantly increases the cytotoxicity of NKT cells against U937 lymphoma targets. This provides preliminary data supporting the possibility that the use of a combination of a small molecule inhibitor with immune therapy may increase the clinical efficacy of immune therapies that work via cytotoxic killing of malignant targets.

Figure Figure
Clinical Characteristics and Outcome of a Large Series of Patients with Chronic Lymphocytic Leukemia (CLL) According to ZAP-70 Expression.
Authors: Bosch Francesc , Crespo Marta , Villamor Neus , Muntañola Ana , Ferrer Ana , Domingo Abel , Rozman Maria , Rozman Ciril , Lopez-Guillermo Armando , Montserrat Emili
Published: April 2020

The prognosis of patients with CLL has been traditionally assessed by using clinical stages. Clinical stages, however, are a mere reflection of the biological diversity of CLL. In this regard, we and others have recently shown that ZAP-70 expression separates CLL into two clinical forms with different outcome. The aim of this study was to analyze the distribution of age, gender, and different prognostic parameters according to ZAP-70 expression in 222 pts with CLL (median age, 62 yrs; female/male: 89/133). ZAP-70 was determined by flow cytometry, a high expression being defined as >20% ZAP-70 positive CLL cells. Median follow-up of the series was 7.1 years. Clinical stages at diagnosis were: Binet stage A, 187 pts; B, 21 pts; and C, 14 pts. Rai Stage 0, 123 pts; I, 53 pts; II, 28 pts; III, 7 pts; and IV, 11 pts. Overall median survival was 13 years. Parameters associated with a high ZAP-70 expression were lymphocyte doubling time >12 mos., atypical morphology, bone marrow infiltration > 60%, increased serum LDH and timidin kinase, CD38 expression >20%, del(13q14), unmutated status of IgVH, and development of Richer syndrome. Interestingly, ZAP-70 expression distribution correlated with Rai’s stage (0 vs. 1 to 4). Also, CD38 expression was able to separate two subpopulations with different survival among CLL cases with low ZAP-70 expression (median survival 18 yrs vs. 13 yrs), but not in pts. with ZAP-70>20%. About 50% of pts in Binet A progressed during the follow-up. Median time to progression for patients with high ZAP-70 expression was 3.3 years whereas it was not reached for patients with low ZAP-70 expression. Median survival of pts with low ZAP-70 expression was 16.3 years, whereas it was of 8.5 years in those with high ZAP-70 expression. In conclusion, ZAP-70 expression identifies a subgroup of patients with CLL with unfavorable prognostic factors related to the proliferation and progression of the disease.

Prognostic Parameters according to ZAP-70 expression

Variable ZAP-70 >20% ZAP-70 < 20% p= Median age 60 yrs 63 yrs Gender (female) 39% 41% LDT < 12 mos 42% 23% 0.009 Atypical morphology 54% 12% 0.000 BM infiltration >60% 50% 33% 0.03 Increased LDH 13% 4% 0.028 Increased TK 66% 28% 0.000 CD38 > 20% 67% 32% 0.000 Del(13q14) 38% 55% 0.027 Unmutated IgVH 88% 11% 0.000 Richter Syndrome 8% 2% 0.039 Rai Stage > 0 53% 37% 0.039 TTP (Binet A) 3.3 yrs NR 0.000 Median Survival 16.3 yrs 8.5 yrs 0.000
Anti-CD20 Antibody Rituximab and Anti-CD23 Antibody IDEC-152 Induce Apoptosis of Malignant B-Cells in Combination with Chemical Antagonists of XIAP.
Authors: Mangiola Massimo , Welsh Kate , Kitada Shinichi , Pedersen Irene M. , Pathan Nuzhat , Pinilla Clementia , Kipps Thomas J. , Reed John C.
Published: April 2020

We tested the effects of Rituximab (anti-CD20) and IDEC-152 (anti-CD23) on apoptosis of B-cell malignancies, using established non-Hodgkin’s B-Cell lymphoma cell lines and freshly isolated Chronic Lymphocytic Leukemia (CLL) B-cells. We used monolayers of stably transfected CHO-cells expressing FcRγIII-A to present antibody to B-cells and promote crosslinking. Established B-cell lymphomas (n = 3) were cultured in the presence of FcRγIIIA-expressing CHO monolayer with or without MAbs and apoptosis was measured by annexin V/propidium iodide staining at various times thereafter. Both antibodies induced time-dependent apoptosis of B-cell lymphoma cell lines. After 48 hrs of treatment with either Rituximab or IDEC-152, the majority of the malignant B-cells were apoptotic (remaining viable cells = 28.7% ± 0.2137% for Rituximab and 30.87% ± 0.7332% for IDEC-152). Rituximab and IDEC-152 also induced marked increases in caspase activity in B-cell lymphoma cell lines, with fold-increases above baseline control cells of 25 ± 0.9031 and 24 ± 0.3839, respectively. In contrast, neither Rituximab nor IDEC-152 induced striking effects on primary CLL B-cells (n = 6). We therefore tested the combination of Rituximab or IDEC-152 with other agents that target anti-apoptotic proteins, exploring whether more efficient induction of apoptosis can be achieved. We cultured lymphoma cell lines and primary CLL specimens with chemical antagonists of XIAP (Schimmer, et al. Cancer Cell5: 25, 2004), an anti-apoptotic protein that inhibits effector caspases. When used at concentrations where XIAP antagonists alone were non-apoptotic (approximately 2.5 μM), a significant increase in apoptosis was achieved in cultures of lymphoma and CLL cells treated with either Rituximab or IDEC-152. These findings suggest that Rituximab or IDEC-152 may more efficiently induce apoptosis of malignant B-cells when combined with an apoptosis-sensitizing agent. (Supported by CA-81534; CA-78040; and an unrestricted grant from Genentech, Inc.).
Fusion of NUP214 to ABL1 on Amplified Extrachromosomal Elements in T-ALL.
Authors: Cools Jan , Graux Carlos , Melotte Cindy , Quentmeier Hilmar , Ferrando Adolfo A. , Levine Ross , Vermeesch Joris R. , Stul Michel , Dutta Binita , Boeckx Nancy , Bosly Andre , Heimann P. , Uyttebroeck Anne , Mentens Nicole , Somers Riet , MacLeod Roderick A.F. , Drexler Hans G. , Look A. Thomas , Gilliland D. Gary , Michaux Lucienne , Vandenberghe Peter , Wlodarska Iwona , Marynen Peter , Hagemeijer Anne
Published: April 2020

The Philadelphia translocation, encoding the BCR-ABL1 (BCR-ABL) fusion gene, is typically found in chronic myeloid leukemia (CML) and precursor B-cell acute lymphoblastic leukemia (B-ALL), but is exceptionally rare in T-cell acute lymphoblastic leukemia (T-ALL). To study the potential involvement of ABL1 gene rearrangements in T-cell malignancies, we screened 90 T-ALL cases by fluorescence in situ hybridization (FISH), using BCR and ABL1 probes. No BCR-ABL1 fusion signals were observed, confirming the low frequency of this rearrangement in T-ALL, but we did observe marked amplification (> 10 signals per nucleus) ABL1 of in 5 of 90 (5.5 %) T-ALL patients.

Amplification of ABL1 occurred on small extrachromosomal elements that were not detectable by conventional cytogenetics. and hence are referred to as episomes. FISH, and array-CGH analyses delineated the amplicon as a 500 kb region from chromosome band 9q34, containing the oncogenes ABL1 and NUP214 (CAN). Molecular analysis led to the identification of a NUP214-ABL1 fusion gene, which is generated as result of the circularization of the genomic region between ABL1 and NUP214 to form the episomes. This is the first example of an oncogenic fusion gene generated by extrachromosomal amplification. The NUP214-ABL1 transcript was detected in 5 patients with ABL1 amplification, in 5 of 85 (5.8 %) additional T-ALL patients, and in 3 of 22 T-ALL cell lines. The constitutively phosphorylated tyrosine kinase NUP214-ABL1 is sensitive to the tyrosine kinase inhibitor imatinib mesylate (STI-571). The recurrent cryptic NUP214-ABL1 rearrangement is associated with increased expression TLX1 of (HOX11) or TLX3 (HOX11L2), and with deletion of CDKN2A (p16), consistent with a multi-step pathogenesis of T-ALL. Our results identify a novel mechanism for the generation of a fusion gene on extrachromosomal elements, and indicate the importance of activated tyrosine kinase signaling in the pathogenesis of T-ALL. NUP214-ABL1 expression defines a new subgroup of T-ALL patients that could benefit from imatinib treatment.
Dysregulated Apurinic/Apyrimidinic Endonucleases (Ape1 and Ape2) Lead to Genetic Instability in Multiple Myeloma.
Authors: Shammas Masood A. , Koley Hemant , Shah Sima , Batchu Ramesh B. , Tassone Pierfrancesco , Anderson Kenneth C. , Munshi Nikhil C.
Published: April 2020

Multiple myeloma (MM) is associated with significant genomic instability. Homologous recombination (HR), which is elevated in MM, is considered to be responsible for this instability. As endonucleases play an important role in mediating HR, here we have evaluated the role of endonuclease in biology and progression of MM. Gene expression profile using Affymetrix U133 array showed > 2 fold elevation of Ape1 or Ape2 or both in 5 of 6 MM cell lines and 12 of 15 patient samples. Immunocytochemistry confirmed upregulation of Ape1 protein in MM cell lines. A Plasmid degradation assay confirmed significantly elevated endonuclease activity in MM cells compared to normal plasma cells. To identify the pre-dominating endonuclease activity, the degradation assay was carried out in the presence of specific endonuclease inhibitors. Harmane and methoxyamine (MA), specific inhibitors of apurinic/apyrimidinic endonucleases effectively inhibited significant endonuclease activity, while other endonuclease inhibitors ACPD and FK506 had minimal effects, confirming predominant role of apurinic/apyrimidinic endonucleases (APE) in mediating increased endonuclease activity in MM. We investigated the role of elevated APE endonuclease activity on DNA recombination and subsequent genomic re-arrangements. Using a plasmid-based assay we have previously demonstrated significantly elevated homologous recombination (HR) in MM. Inhibition of endonuclease by methoxyamine suppressed HR activity by 85 ± 2% in MM cells. Next, we evaluated whether inhibition of HR by methoxyamine can affect the frequency of acquisition of new genetic changes in MM cells using single nucleotide polymorphism (SNP) arrays (Affymetrix) as indicator of genomic instability. In three independent experiments, methoxyamine reduced the acquisition of new loss of heterozygocity (LOH) loci by an average of 71%. These data suggest that the dysregulated APE endonucleases contribute significantly to the genomic instability, acquisition of new mutations and progression of MM and provides the rationale for targeting endonuclease activity to prevent disease progression including development of drug resistance.
Constitutively Activated FGFR3 Mutants Signal through PLCγ- Dependent and -Independent Pathways for Hematopoietic Transformation.
Authors: Chen Jing , Williams Ifor R. , Lee Benjamin H. , Duclos Nicole , Huntly Brian J.P. , Donoghue Daniel J. , Gilliland D. Gary
Published: April 2020

Ectopic expression of FGFR3 associated with t(4;14), frequently containing the activating mutation K650E (TDII), has been implicated in the pathogenesis of human multiple myeloma. To investigate the role of autophosphorylated tyrosine residues in FGFR3 signal transduction and transformation, we characterized a series of FGFR3 TDII mutants with single or multiple Y→F substitutions. Phenylalanine substitution of a non-activation loop tyrosine residue Y760, essential for PLCγ binding and activation, significantly attenuated FGFR3 TDII-mediated PLCγ activation, as well as transformation in Ba/F3 cells and a murine bone marrow transplant (BMT) leukemia model. In contrast, single substitution of other non-activation loop tyrosine residues Y577, Y724 or Y770 had minimal to moderate effects on TDII-dependent transformation. Substitution of all the four non-activation loop tyrosine residues significantly attenuated, but did not abolish FGFR3 TDII transforming activity. However, substitution of both activation loop tyrosine residues Y647/Y648 resulted in complete abrogation of FGFR3 TDII kinase activity and transforming activity in vitro and in vivo. Similar observations were obtained in the context of constitutively activated fusion TEL-FGFR3 associated with peripheral T-cell lymphomas (PTCL) with chromosomal translocation t(4;12)(p16;p13). Moreover, two independent EμSR-FGFR3 TDII transgenic mouse lines developed pro-B cell lymphoma, and PLCγ was highly activated in primary lymphoma cells as assessed by tyrosine phosphorylation, along with elevated activation of STAT3 and expression of Bcl-XL in the tumor tissues. These data indicate that engagement of multiple signaling pathways, including PLCγ-dependent and PLCγ-independent signaling pathways, are required for full hematopoietic transformation by constitutively activated FGFR3 mutants.
Allogeneic Transplant in Patients with Poor Prognosis B-Chronic Lymphocytic Leukemia (B-CLL): Comparative Study between Myeloablative(M) and Non-Myeloablative(NM) Conditioning Regimen.
Authors: Caballero M.D. , García-Marco J.A. , Martino R. , Esteve J. , Mateos M.V. , León A. , Sarrá J. , Ribera Josep Maria , Sanz G. , de la Serna J. , Díez J.L. , San Miguel11 J.F.
Published: April 2020

Sustained complete remissions (CR) have been reached with allogeneic transplant in patients with poor prognosis B-CLL; however, mortality rates are high (20–50%); in order to reduced TRM, NM conditioning are widely used in haematological malignancies; however it is not clear if the use of NM regimens can maintain the efficacy reducing the toxicity. IN this report we performed a retrospective comparison between 30 patients (group A) who have received myeloablative conditioning consisted of TBI plus Cy in 23 pts (74%), TBI, Cy plus VP-16 in 6 pts (19%) and BuCy in 1 patient and 31 patients (Group B) who have received a NM transplant. Conditioning regimens in Group B included: Fludarabine plus Melphalan, 20 pts (64%), Fludarabine, Busulphan and ATG, 5 pts (16%), Fludarabine, TBI and ATG, 4 pts (13%) and Fludarabine plus TBI, 1 patient. All patients received peripheral blood stem cells from a HLA related identical donor. T-cell depletion was performed in 14 patients of the group A. Median age at transplant was significantly higher in the group B patients (53 versus 45, respectively) (p<0,005); no differences were observed in terms of status at transplant and n° of previous chemotherapy lines as well in the risk of graft versus host disease (GVHD) and transplant related mortality (TRM) (See Table, below).

With a median follow-up of 71 and 36 months for groups A and B respectively,Overall Survival and Event Free Survival are similar for both groups (53% versus 64% and 60% versus 68%, respectively). Although patients in the NM transplant group were older toxicity was similar in both groups; moreover a similar efficacy has been observed suggesting the clear role of graft versus tumour effect in B-CLL probably more important that the type ofconditining.

Table 1

GROUP A Myeloablative Group B Non-myeloablative p Number of previuous chemotherapy lines 2 (1-6) 2 (1-8) NS Acute GVHD 15/30 (48%) 20/31 (64%) NS Grade II-IV 11/30 (35%) 12 /31(38%) NS Chronic GVHD 12/26pts at risk (46%) 18/27 pts at risk (66%) NS Extense 8 pts (30%) 9 pts (33%) NS TRM 7/30 (23%) 7/31 (22%) NS
Molecular Composition of Intercellular Contacts in Human Mesenchymal Stem Cells.
Authors: Wuchter Patrick , Boda Judit , Straub Beate , Krause Ulf , Seckinger Anja , Grund Christine , Kuhn Caecilia , Gottschling Sandra , Franke Werner W. , Ho Anthony D.
Published: April 2020

The long-term fate of stem cells depends on their interaction with the niche. Mesenchymal stem cells (MSC) from human bone marrow have been demonstrated to differentiate into various tissues, such as bone, cartilage, muscle and fat. As the interaction with the microenvironment plays a major role in differentiation, we have characterized the cell-cell contact among MSC. We have demonstrated the occurrence of a novel kind of adhering junction, consisting of slender, villiform-to-vermiform cell projections (processus adhaerentes). In this study, we have systematically analyzed the molecular composition of these junctions.

A panel of antibodies specific for various components of tight junctions, gap junctions, adherens junctions and desmosomes was used. The expression of these antigens was verified by light and electron microscopy and by biochemical analysis, including immunoprecipitation and RT-PCR. MSC from two different sources were analyzed: MSC obtained from bone marrow aspirates from healthy voluntary donors and additionally MSC originating from umbilical cord blood donated for scientific research. We have also shown the presence of vimentin-positive retothelial cells (which are probably the source of MSC) in situ in fresh bone marrow samples.

We demonstrate that MSC were interconnected by occasional gap junctions and frequent adhering junctions. Additionally, we found a unique molecular composition of these adhering junctions, as they comprise the transmembrane glycoproteins cadherin 11, N-cadherin and syndecan-1, together with the cytoplasmic plaque proteins α- and β-catenin and p120ctn. Constitutive complexes of these molecules have been identified.

Our data indicate that MSC communicate with each other through junctions and junctional complexes. We hypothesize that MSC can embark on alternative differentiation pathways with specific junctional and cytoskeletal patterns. Characterization of and understanding the role of such intercellular contacts and their correlation to specific differentiation programs are being conducted.
Blood from Mesenchymal Stem Cell.
Authors: Lange Claudia , Gehling Ursula , Moosmann Sabine , Huss Ralf , Zander Axel R.
Published: April 2020

Mesenchymal stem cells (MSC) have been described to support the maintainance and expansion of clonogenic hematopoietic cells in vitro and as progenitors for nonhematopoietic tissues. They clearly can be distinguished from hematopoietic cells by the surface markers CD45 and CD34. We investigated the potential of human MSC to form blood and endothelial cells. Human MSC in expansion cultures expressed RNA for CD117 and EpoR but not for other hematopoietic antigens. Many transcription factors essential for blood cell differentiation, e.g. c-mpl, Notch-1, Gata-2 and -3, Runx-1 and SCL, were detected in these cells. We used different differentiation strategies: A) Hematopoietic differentiation: 1. Prestimulation in IMDM + fetal calf serum (FCS) + horse serum (HS), mixed with methylcellulose (MC, containing cytokines SCF, IL-3, IL-6, G-CSF, GM-CSF and Epo) and differentiation in MC; 2. Prestimulation in serum free medium supplemented with SCF, TPO, GM-CSF and flt.- 3 ligand and differentiation in serum free MC; B) Endothelial differentiation: Prestimulation in serum free medium supplemented with SCF, TPO, and flt-3L and differentiation in medium + VEGF + bFGF + IGF. Using FACS-analysis, we detected up to 1.8 % CD34-positive cells within 2 weeks. Immunochemically we observed cells positive for CD45, CD34, CD133, CD14, CD16 and CD41. Although hMSC do not form colonies after seeding in MC, after prestimulation we observed colonies with the typical appearance of BFU-E which were GlycophorinA positive. Under endothelial conditions we detected cells positive for CD34 and KDR or CD31 and vWF. However, cell numbers of positive cells were low and therefore RT-PCR results were not very reliable. To confirm the differentiation capacity of MSC we generated CD45.2 mouse MSC (P9 what took about 6 month) and transplanted into lethally irradiated CD45.1 recipient mice. Four weeks after transplantation mice showed normal white blood cell counts but still decreased platelet counts. Analysis of donor chimerism 29 weeks after transplantation revealed up to 36 % (14.3±7.9%) donor cells in peripheral blood, mainly in the CD11b positive myeloid population but not in lymphoid cells. In thymus and bone marrow, marginal donor chimerism (3±0.8%) was detected, but the number in c-kit+ cells reached up to 28.2% (7.9±6.8%). In B-cells and erythroid progenitors chimerism was low but stable, but not in CD3+ cells. Taken together, we have shown that under the right conditions MSC are able to form blood and endothelial cells and reconstitute animals. We suggest that MSC could be an yet unrecognised precursor for both cell types and may contain the long searched hemangioblast in adult organisms.
TGF-β Receptor I Kinase Inhibitor Downregulates Cytokine Secretion and Multiple Myeloma Cell Growth in the Bone Marrow Microenvironment.
Authors: Hayashi Toshiaki , Hideshima Teru , Podar Klaus , Richardson Paul , Munoz Olivier , Hamasaki Makoto , Ishitsuka Kenji , Yasui Hiroshi , Murphy Alison , McEnroe Glenn , Chakravarty Sarvajit , Dugar Sundeep , Higgins Linda S. , Chauhan Dharminder , Anderson Kenneth C.
Published: April 2020

Transforming growth factors (TGFs) have pleiotropic biologic effects on tumor cells and their environment. In multiple myeloma (MM), we have reported that bone marrow stromal cells (BMSCs) from MM patients produce more TGF-β1 than BMSCs from healthy donors, which in turn induces interleukin-6 (IL-6) secretion. In this study, we delineate the functional squelae of TGF-β1 in MM, and importantly, show that the TGF-β receptor I kinase inhibitor SD-208 significantly decreases secretion of both IL-6 and vascular endothelial growth factor (VEGF) from BMSCs, as well as tumor cell growth triggered by MM cell adhesion to BMSCs. Cytokine production and MM cell proliferation triggered by TGF-β1 or adhesion to BMSCs were examined in the presence or absence of SD-208 using ELISA and 3H thymidine incorporation assay, respectively. Effects of SD-208 on TGF-β1-induced signaling pathways triggering IL-6 and VEGF transcription in BMSCs were delineated using immunofluorescence staining, immunoblotting and DNA-binding assay. We here show that adhesion of MM cells to BMSCs triggers secretion of TGF-β1, which further upregulates IL-6 and VEGF secretion in BMSCs. These cytokines in turn mediate MM cell growth, survival, drug resistance, and migration. Importantly, SD-208 significantly inhibits not only transcription but also secretion of both IL-6 and VEGF from BMSCs triggered by either TGF-β1 or adhesion of MM cells to BMSCs. Moreover, SD-208 decreased tumor cell growth triggered by MM cell adhesion to BMSCs. SD-208 works, at least in part, by blocking TGF-β1-triggered nuclear accumulation of Smad2/3 and hypoxia-inducible factor 1α, as well as related production of IL-6 and VEGF, respectively. These studies indicate that SD-208 inhibits production of cytokines mediating MM cell growth, survival, drug resistance, and migration in the BM milieu, thereby providing the preclinical rationale for clinical evaluation of SD-208 to improve patient outcome in MM.
Serum Protein Profiling with Mass Spectrometry for the Diagnosis of Myelodysplastic Syndromes.
Authors: Aivado Manuel , Spentzos Dimitrios , Germing Ulrich , Alterovitz Gil , Meng Xiao-Ying , Grall Franck , Giagounidis Aristoteles A.N. , Klement Giannoula , Steidl Ulrich , Otu Hasan H. , Iking-Konert Christof , Czibere Akos , Prall Wolf C. , Shayne Michelle , Ramoni Marco F. , Gattermann Norbert , Mitsiades Constantine S. , Haas Rainer , Fung Eric T. , Libermann Towia A.
Published: April 2020

Surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) has facilitated disease-specific serum protein profiles, which may become instrumental for diseases that are difficult to diagnose. The diagnosis of MDS can be very difficult since bone marrow dysplasia and peripheral cytopenia, the hallmarks of MDS, can be observed due to many reasons other than MDS. Moreover, cytomorphological evaluation of dysplasia requires extensive experience and is thus dependent on the examiner. Between 2–16% of the patients have a fibrotic bone marrow, which can impede appropriate bone marrow aspiration and smear. Finally, while cytogenetic examination can strongly support the diagnosis of MDS, only 40–50% of the patients have an abnormal karyotype. In the present study, we employed a SELDI-TOF MS-based procedure termed Pattern Track™ in order to analyze a total of 218 serum samples. We generated serum protein profiles from a first sample set comprising 74 patients with MDS, 39 control patients with cytopenia for reasons other than MDS, and 24 healthy persons. We fractionated their serum by means of anion exchange chromatography and applied the resulting serum fractions to weak cationic exchange as well as to reversed phase chromatography ProteinChip™ arrays. We randomly split this dataset into a learning (n=72) and a first independent validation set (n=41). Then, we used a k-nearest-neighbor algorithm to build a class predicting profile that consisted of 81 protein peaks. That profile was tested by leave-one-out cross validation and predicted the diagnosis MDS with an accuracy of 81.9% in the learning set (Fisher’s test, P=0.0000003). Then, we tested the profile on our first independent validation set and obtained a similar accuracy of 80.5% (P=0.0002). Its diagnostic performance and long-term reproducibility were confirmed by successfully applying it to a prospectively collected second independent validation set consisting of 81 new samples (P=0.0000006). Eventually, following serial chromatography, 1D gel electrophoresis, and tryptic peptide fingerprinting, we discovered the identity of 2 members of the profile. We conclude that our predicting serum protein profile represents a novel, non-invasive aid in distinguishing patients with MDS from patients with cytopenia for reasons other than MDS.
Sequential Cytogenetic Analysis of 322 Patients with Myelodysplastic Syndromes. Delineation of Genetic Evolution and Clinical Implications.
Authors: Steidl Christian , Schabla Rainer , Germing Ulrich , Hildebrandt Barbara , Noesslinger Thomas , Pfeilstocker Michael , Haas Peter , Lübbert Michael , Truemper Lorenz H. , Haase Detlef
Published: April 2020

INTRODUCTION: Myelodysplastic syndromes are dynamic diseases affecting the stem cell compartment in bone marrow presenting with different clinical courses ranging from stable, indolent disease to rapid progression to acute myeloid leukemias. So far, only 3 studies on karyotype analysis in MDS with a minimum of 30 patients have been published. Most knowledge about genetic evolution in MDS is based on the description of parallely existing subclones within one single examination. Thus, little is known about the real frequency, the time spans and the clinical impact of karyotype evolution.

MATERIALS AND METHODS: So far, data from 322 patients with MDS or secondary AML and at least two successfully performed classical cytogenetic analyses are available from four centres of the Competence Network Acute and Chronic Leukemias. As yet, we retrospectively examined 268 patients out of this data set. Karyotype evolution (KE) was defined as acquisition of additional aberrations, expansion of an aberrant clone (>20%) or development of a completely aberrant karyotype after an initial mosaic karyotype.

RESULTS: In 44 cases (16%) KE was observed. In the mean 2.8 (range 2–9) cytogenetic examinations have been performed. In 27 cases additional aberrations occurred and in 17 cases the abnormal clone expanded in a subsequent analysis. Compared to stable courses, patients with KE had a tendency towards a shorter survival (p=0.15).

In the group of patients with expansion of the aberrant clone the most frequent karyotypes were −7/7q- (4x), complex (3x), 5q- (3x) and +8 (3x). The most frequent karyotypes in which during the course of the disease additional aberrations occurred were complex (4x) and karyotypes with two miscellaneous aberrations (4x). The most frequent additional aberrations were 5q- (3x) and −17/17p- (3x).

CONCLUSIONS: In sequential cytogenetic examinations KE is a frequent event. Patients with KE tend to have a shortened survival. In our collective no long-term survivor could be observed in the group displaying KE regardless of the therapy strategies (excluding allogeneic transplantation). In this multicentric study which encompasses the largest data base on sequential analyses in MDS to date, frequency, evolution patterns and prognostic relevance of karyotype changes have been studied allowing a better insight into the genetic dynamics of MDS.
Myelodysplastic Syndrome with Bone Marrow Hypoplasia Is Associated with Much Higher Frequency of Apoptosis in CD 34+ Cells.
Authors: Oyake Tatsuo , Ito Shigeki , Kowata Shugo , Murai Kazunori , Ishida Yoji
Published: April 2020

Myelodysplastic Syndrome (MDS) is a clonal disorder characterized by dysplastic changes and ineffective hematopoiesis. The ineffective hematopoiesis is considered as the results of excessive apoptosis of bone marrow (BM) hematopoietic cells. Recently, immunosuppressive therapy is effective in some hypoplastic MDS patients, resulting in the dramatic improvement of complete blood counts. To elucidate the difference between MDS patients with hypoplasia and normo/hyperplasia in BM, we measured the frequency of apoptosis in each lineage CD34+ BM cells in 35 MDS (12 RA patients with hypoplastic BM, 9 RA, 7 RAEB, and 7 RAEB-t patients with normo/hyperplastic BM) at diagnosis by three color flow cytometric analysis. Apototic cells were analyzed by PE labeled AnnexinV. The lineage cell population was identified as CD34+/GlycophorineA+, CD34+/CD33+, CD34+/CD41+, CD34−/GlycophorineA+, CD34−/CD33+ and CD34−/CD41+. In this study, the higher frequency of apoptosis was observed in each lineage CD34+ cells in all MDS patients (n=35, median: 32.2% (range: 6.3–80.5%) in erythroid, 38.0% (8.8–93.3%) in myeloid, 41.4% (10.2–78.4%) in megakaryocytic lineage, p<0.05, respectively), compared to that in normal controls (n=10, 8.5% (1.5–9.9%) in erythroid, 8.5% (2.2–8.8%) in myeloid, 7.7% (4.4–9.3%) in megakaryocytic lineage, respectively). While much higher frequency of apoptosis was observed in each lineage CD34+ cells in hypoplastic MDS patients (n=12, 49.1% (31.2–80.5%) in erythroid, 66.0% (37.8–93.3%) in myeloid, 68.5% (43.4–78.4%) in megakaryocytic lineage, p<0.05, respectively), compared to that in normo/hyperplastic MDS patients (n=23, 28.7% (6.3–69.4%) in erythroid, 30.0% (8.8–61.7%) in myeloid, 29.8% (10.2–58.1%) in megakaryocytic lineage, respectively). The increased frequency of apoptosis in each lineage CD34+ cells decreased after immunosuppressive therapy (n=5, 25.3% (19.8–36.4%) in erythroid, 35.2% (20.1–42.0%) in myeloid, 38.5% (30.6–47.9%) in megakaryocytic lineage, respectively). Our findings suggested that the excessive apoptosis occurred mainly in CD34+ cells in hypoplastic MDS as well as in non-hypoplastic MDS. Much more increased frequency of excessive apoptosis in CD34+ cells resulted in BM hypoplasia in hypoplastic MDS patients. This method is useful to evaluate quantitatively the ineffective hematopoiesis in BM hematopoietic cells.
Generation of Regulatory T Cells through Intravenous Delivery of Autologous Apoptotic Cells.
Authors: Peritt David , Campbell Kim , Krutsick Amy , Huber Janine , Thienel Ulrich , Schwarz Agatha , Maeda Akira , Schwarz Thomas
Published: April 2020

Extracorporeal photopheresis (ECP) is approved for the palliative treatment of skin manifestations associated with cutaneous T cell lymphoma. As reported in the literature, ECP has shown promise as a treatment for such immune-mediated inflammatory disorders as graft versus host disease, transplantation rejection, and autoimmune diseases. ECP involves the reinfusion of autologous, apoptotic peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen (8-MOP) and UVA light. The biological mechanism of action of ECP, however, remains unresolved. We have evidence to suggest that delivery of ECP-treated apoptotic cells modulates immune responses, possibly through generation of regulatory T cells. When co-incubated with ECP-treated cells, activated dendritic cells produce reduced levels of proinflammatory cytokines, such as IL-12, while TGFβ levels were modestly increased. Activation of CD4+ T cells in the presence of allogeneic dendritic cells and ECP-treated cells promotes generation of a population of T cells that can suppress proliferation of, and IFNγ production by, naïve syngeneic T cells. To confirm these findings in vivo, we employed a murine contact hypersensitivity model. ECP-treated or control spleen and lymph node cells from mice sensitized with the hapten dinitrofluorobenzene (DNFB) were injected intravenously into naïve recipients. Compared to controls, mice that received ECP-treated cells demonstrated significantly less ear swelling following sensitization and challenge with DNFB. Suppression of ear swelling was specific for DNFB and cell-mediated, as demonstrated by the ability to transfer DNFB tolerance to naïve mice, which could appropriately respond to the unrelated hapten oxazalone. Transfer of this tolerance was abrogated by depletion of either CD4+ or CD25+ T cell populations. Collectively, these results suggest that delivery of ECP-treated cells promotes the generation of regulatory T cells that are capable of modulating immune responses. Therakos sponsored Phase II trials for the prevention and treatment of GvHD are concluding and an international blinded pivotal phase III study is planned for 2005.
Oncolytic Measles Virus Selectively Targets CD46 Overexpression on Myeloma Cells.
Authors: Peng Kah-Whye , Timm Michael M. , Dingli David , Dispenzieri Angela , Gertz Morie , Witzig Thomas E. , Greipp Philip R. , Russell Stephen J.
Published: April 2020

Multiple myeloma remains incurable and novel therapeutics are urgently needed. We have shown that live attenuated Edmonston strain measles virus (MV-Edm) is potently oncolytic in multiple myeloma xenograft models and can efficiently infect CD138 sorted primary myeloma cells inducing extensive intercellular fusion which leads to cell death (Blood, 2001, v98, p2002). In contrast, the virus causes minimal damage to normal PHA-stimulated peripheral blood lymphocytes, fibroblasts and smooth muscle cells. To facilitate non-invasive imaging of sites of viral gene expression, we introduced the human thyroidal sodium iodide symporter (NIS) gene into the viral genome (MV-NIS). MV-NIS infected myeloma cells efficiently concentrate radioiodine which provides a basis for noninvasive imaging and destruction of infected tumors using beta emitters such as 131I (Blood, 2004, v103, p1641). Clinical translation of MV-NIS in patients with refractory myeloma is planned. However, the mechanism underlying the tumor selectivity of MV-Edm has not previously been defined. MV-Edm uses either of two cellular receptors for cell entry and cell fusion; CD46, a regulator of complement activation, is expressed on all nucleated cells, wheras SLAM (signaling lymphocyte activation molecule) is expressed only on activated B lymphocytes, T lymphocytes, and monocytes, and is not present on myeloma plasma cells. Using a panel of CHO cell transfectants, we demonstrated that MV-Edm -mediated cell killing increases exponentially above a threshold CD46 receptor density (Can Res, 2004, v64, p4919). We subsequently obtained bone marrow aspirates from 18 myeloma patients and performed multi-parameter flow cytometry to determine the relative expression levels of CD46 on the myeloma plasma cells versus the rest of the hematopoietic cells in the bone marrow. The myeloma cells expressed significantly higher levels (5-fold) of CD46 receptors (MFI=294.9 + 204.4, means ± SD) on their cell surfaces compared to hematopoietic cells of the erythroid, myeloid and lymphoid lineages (MFI=58.6 ± 9.0). Analysis of the myeloma array data generated by the Shaughnessy lab indicated that, in comparison to plasma cells from healthy volunteers (2997 ± 1053, n=30, means ± SD), primary myeloma cells express 2-fold elevated levels of CD46 mRNA (6592 ± 4029, n=72, p=0.001, Wilcoxon signed rank test). Taken together, these findings provide strong support for the hypothesis that selective destruction of myeloma cells by oncolytic measles viruses is a direct consequence of their overexpression of the cellular receptor CD46.