bluuebunny/crossref_metadata_2025 huggingface dataset:
Durable Responses with Bortezomib in Patients with Relapsed or Refractory Mantle Cell Lymphoma (MCL): Updated Time-to-Event Analyses of the Multicenter PINNACLE Study.
Authors: Goy Andre , Bernstein Steven , Kahl Brad , Djulbegovic Benjamin , Robertson Michael , de Vos Sven , Epner Elliot , Krishnan Amrita , Leonard John , Lonial Sagar , Stadtmauer Edward , O’Connor Owen , Shi Hongliang , Boral Anthony , Fisher Richard
Published: October 2019

Background: We previously reported substantial activity with single-agent bortezomib (VELCADE®; Vc) in patients (pts) with relapsed or refractory MCL in the PINNACLE study (JCO2006;24:4867–74), which resulted in approval of Vc for MCL pts following ≥1 prior therapy. All pts have now completed treatment. Here we report updated time-to-event data in all pts, and by response category, with extended follow-up.

Methods: 155 pts (median age 65 yrs; 55%/41%/4% with 1/2/≥3 prior therapies; 77% Stage IV MCL; 55% positive bone marrow) received Vc 1.3 mg/m2 on days 1, 4, 8, and 11 of 21-day cycles; of these, 141 were response-evaluable. Response and progression were determined by modified International Workshop Response Criteria using independent radiology review.

Results: After a median follow-up of 26.4 mo, 55 pts (35%) remained in follow-up; 93 (60%) had died, 2 (1%) had withdrawn consent, and 5 (3%) were lost to follow-up. Pts received a median of 4 treatment cycles (range 1–21; 8 in responding pts). Median time to first response was 1.3 months. Median duration of response (DOR) was 9.2 mo in all responders and has not been reached in pts achieving CR/CRu. Median time to progression (TTP), time to next therapy (TTNT; first Vc dose to start of next therapy), and overall survival (OS) are shown in the table for all pts and by response.

Survival rate at 12-mo was 69% overall and 91% in responding pts. In pts refractory to their last therapy (no response or response with TTP <6 mo; n=58), median DOR was 5.9 mo, median TTP was 3.9 mo, median TTNT was 4.6 mo, and median survival was 17.3 mo. Safety profile was similar to previously reported; most common grade ≥3 AEs were peripheral neuropathy (13%), fatigue (12%), and thrombocytopenia (11%). The most common AE resulting in Vc discontinuation was peripheral neuropathy (10%). Twelve (8%) pts died on-study, including 5 (3%) considered related to Vc.

Conclusions: Vc provides durable responses plus prolonged time off-therapy and survival in responding pts, suggesting substantial clinical benefit in relapsed/refractory MCL.

Median TTP, TTNT, and OS (months) in all pts and by response All pts (N=155) Responders (N=45) CR/CRu (N=11) PR (N=34) SD (N=52) PD (N=34) NE, not estimable TTP 6.7 12.4 NE 9.1 6.9 1.2 TTNT 7.4 14.3 23.9 13.3 7.0 2.3 OS 23.5 35.4 36.0 35.1 27.8 13.7
Differential Role of the Transcription Factor ZBP-89 in Hemangioblast Fate Determination: ZBP-89 Is a Direct Regulator of SCL.
Authors: Li Xiangen , Shelley Carl Simon , Arnaout M. Amin
Published: October 2019

Several molecular pathways have been identified that regulate distinct stages in the developmental progression from mesoderm to the formation of the hematopoietic and vascular lineages. Our previous work indicated that ectopic expression of the zinc finger transcription factor ZBP-89 promotes hematopoietic lineage development and represses endothelial cell lineage differentiation from hemangioblasts in murine embryonic stem cells. Here we evaluated the functional consequences of stable knockdown of ZBP-89 in embryonic stem cells (ESC) on hematopoietic and vascular development. Stable knock down of ZBP-89 in ESC significantly decreased the number of Blast Colony Forming Cells (BL-CFC) hemangioblasts, as well as primitive and definitive hematopoietic progenitor colonies BFU-E, GM-CFU, G-CFU, M-CFU and GEMM-CFU in vitro. In contrast, sprouting angiogenesis was markedly increased in EB cultures. Flow cytometric analysis of the lineages derived from ZBP-89 deficient EB cultures showed that the early (C-kit+Sca-1+) and definitive (CD45+) hematopoietic stem cells populations were reduced, but the endothelial cell population (CD31+ VE-Cadherin+) was increased. RT-PCR analysis of EB cultures revealed a direct correlation between the expression levels of ZBP-89 and hematopoietic markers (including SCL and Runx1) but an inverse correlation with the vascular marker CD31, with no change in Oct4 expression level. To investigate the mechanism underlying the role of ZBP-89 in hematopoiesis, the effect of ZBP-89 on expression of SCL, a master regulator of hematopoiesis, was examined. The murine SCL promoter transduced into the ZBP-89-expressing MEL cell line drove luciferase gene expression. ZBP-89 knockdown in MEL cells markedly reduced SCL expression. ChIP analysis showed that endogenous ZBP-89 protein bound directly to the murine SCL promoter in MEL cells. Thus ZBP-89 plays a central role in fate determination of hemangioblasts; its induction suppresses angiogenesis but enhances differentiation of hemangioblasts along the hematopoietic pathway, an effect mediated through the regulated expression of SCL.
Generation of Megakaryocytes from Human Embryonic Stem Cells.
Authors: French Deborah L. , Jirouskova Marketa , Kennedy Marion , Coller Barry S. , Keller Gordon
Published: October 2019

The ability to generate multiple cell types from human embryonic stem cells (hESC) in culture offers an unprecedented opportunity to produce an unlimited supply of cells for research and clinical purposes. Megakaryocytes comprise only 0.02–0.05% of the nucleated cell population in the bone marrow, thus making them a difficult cell to isolate and study. The ES cell technology provides a resource for generating megakaryocyte progenitors for in vitro and in vivo analyses. For this approach to be successful, reproducible differentiation schemes that generate sufficient numbers of cells are necessary. With an embryoid body (EB)-based protocol, in serum-free media we are able to generate hematopoietic populations with megakaryocyte potential from two different hESC lines; H1 and HES2. Using CD41 as an early marker of definitive hematopoiesis, we found that 4 to 12% of the day 11–12 EB population expressed high levels of the marker (CD41Hi) whereas 10 to 20% expressed low levels of the marker (CD41Lo). Cells from the CD41Hi but not the CD41Lo population expressed the megakaryocyte marker GPIb. To further analyze these populations, both were isolated from day 12 EBs by cell sorting and cultured in media containing TPO and SCF. Following several days in culture, the CD41Hi population expressed increasing levels of GPIb, as determined by flow cytometry and real time PCR. PF4 was detected in the sorted CD41Hi population and levels increased 10-fold following culture. These cells also adhered to fibrinogen and collagen and stained positive for von Willebrand factor (vWF) and P-selectin. Following activation by PAR1, the cells bound soluble fibrinogen and expressed P-selectin on their surface. Few to no progenitors were detected in the CD41Hi fraction by colony assays suggesting that this population contained cells that were already committed to the megakaryocyte lineage. Incubation of the CD41Lo population of cells in the presence of TPO resulted in 70–80% of the cells expressing increased levels of CD41 and GPIb. Immunostaining revealed vWF and P-selectin expression following adhesion of the cells to fibrinogen and collagen. Both fibrinogen binding and P-selectin surface expression were detected following activation with PAR1. Quantitative PCR analysis demonstrated low levels of PF4 message in the sorted population with an increase of 3-fold following incubation in the presence of TPO. Progenitor cells were detected in colony assays suggesting that this population of cells contained megakaryocyte-like progenitors. Taken together, these finding demonstrate the efficient and reproducible generation of megakaryocytes from hESC using a serum-free EB protocol.
HoxA2 Regulates Proliferation of an Embryonic Megakaryocyte Progenitor, Which Can Effectively Produce Platelets In Vitro.
Authors: Iacovino Michelina , Osawa Mitsujiro , Xu Zhaohui , Prather Melissa G. , Kyba Michael
Published: October 2019

Megakaryocytes are rare cells in the bone marrow, and are solely responsible for thrombopoiesis throughout the life of the organism. Various transcription factors regulating megakaryopoiesis have been identified, including GATA1, Fli1 and NF-E2, which are important for the maturation and differentiation of megakaryocytes and SCL and FOG1, which play important roles in the specification and proliferation of the early megakaryocyte progenitor but no single factor capable of sustaining long-term proliferation of the megakaryocyte progenitor has been identified. The rarity of this progenitor, and the difficulty of proliferating megakaryocytes in vitro have seriously hampered the effort to produce large quantities platelets in vitro, and to date the only source of platelets for transplantation remains human donors. In this study, through a gain of function screen of Hox genes in which each paralog group was tested, we identified HoxA2 as an important transcription factor for the proliferation of early embryonic megakaryocytic progenitors. HoxA2 promoted the outgrowth of undifferentiated megakaryocyte progenitors when transduced into precirculation yolk sac stem and progenitor cells, however it had no effect on megakaryocytic progenitors derived from definitive HSCs of adult bone marrow. To study this unique embryonic regulatory circuit further, we generated an inducible mouse embryonic stem cell line, in which the expression of murine HoxA2 is regulated by doxycycline. Embryoid body differentiation of these cells, followed by sorting and reculture of the Kit+/CD41+ hematopoietic progenitor fraction at day 6 in the presence of continual HoxA2 expression allowed undifferentiated c-Kit+/CD41+/CD45+ megakaryocyte progenitors to proliferate for upwards of 2 months. When doxycycline is removed, numerous gene expression changes take plase, and these progenitors differentiate into mature c-Kitneg/CD41high/CD45neg megakaryocytes, which undergo proplatelet formation, releasing large quantities of platelets (∼107/ml) into the medium. As this rate of production can be sustained for weeks, a highly efficient platelet bioreactor is enabled. The platelets express the integrin αIIb-β3 (GPIIb-IIIa complex), and upon stimulation they bind fibrinogen, demonstrating functionality. We are currently investigating the transplantation potential of these cells.Our data suggest that adult and embryonic megakaryopoiesis have distinct regulatory circuits, and demonstrate a novel and uniquely embryonic role for HoxA2, which opens up the possibility of producing industrial quantities of platelets in vitro for therapeutic purposes.
Coagulation Factors, Postmenopausal Hormone Replacement Therapy and the Risk of Venous Thrombosis: The WHI Clinical Trials of Postmenopausal Hormone Therapy.
Authors: Cushman Mary , Larson Joseph , Rosendaal Frits R. , Phillips Lawrence S. , Howard Barbara V. , Curb J. David , Hays-Grudo Jennifer , Baird Alison , Eaton Charles B. , Heckbert Susan R. , Stafford Randall S.
Published: October 2019

Background. Postmenopausal estrogen (E) therapy, especially in combination with progestin (P) doubles the relative risk of venous thrombosis (VTE). Risk with hormones is higher with increasing age, obesity and with factor V Leiden. We studied coagulation markers as susceptibility factors for postmenopausal hormone-related VTE.

Methods. The Women’s Health Initiative program included two placebo-controlled double-blind randomized trials of two E regimens, E (conjugated equine estrogens) or E+P (E + medroxyprogesterone acetate), in 16,608 postmenopausal women aged 50–79. We performed a nested case control study that measured baseline levels of coagulation markers in 215 women who developed VTE during follow up and 867 age-matched controls. The joint effects of treatment assignment to either E regimen vs placebo and prespecified abnormal levels of each coagulation factor on relative risk of VTE were estimated by logistic regression adjusting for age, race, body-mass index and type of E regimen.

Results. Low levels of protein C and free protein S (<5th percentile), high D-dimer (top quartile), and high plasmin antiplasmin complex (PAP) and prothrombin fragment 1–2 (top decile) were all associated with risk of VTE with adjusted odds ratios (95% CI) of 2.0 (1.0–4.1), 2.9 (1.5–5.6), 2.8 (2.0–4.0), 2.5 (1.6–4.0) and 1.9 (1.2–3.1), respectively. Elevated factors II, VIII, IX and fibrinogen were not VTE risk factors. Compared to women with normal coagulation marker levels assigned to placebo, the joint odds of VTE with either E regimen plus an abnormal coagulation marker were more than additive compared to the separate effects of hormones and coagulation abnormalities for low protein C, low free protein S, and elevated D-dimer, PAP and F1–2. The odds ratios of VTE with the combination of an abnormal coagulation factor and assignment to hormones were (in order listed in prior sentence), 4.5 (95% CI 2.0–10.2), 6.7 (3.0–14.5), 6.1 (3.7–10), 5.8 (3.2–10.5) and 4.4 (2.4–7.7).

Conclusions. We report new findings of elevated F1-2 and PAP as VTE risk factors in women in this prospective study nested in trials of E or E+P versus placebo. Protein C or S values below the 5th percentile were also clinically relevant even though they do not represent inherited deficiency. Lower protein C and free protein S, and higher D-dimer, F1-2 and PAP all identified women at increased risk of VTE with hormones. If our findings are confirmed in management studies, measurement of these factors might assist women with decision-making on safety of E or E+P.
Update of a GITIL Cohort Study: Frontline High Dose Sequential Chemotherapy with Rituximab and Autologous Stem Cell Transplantation Induces a High Rate of Long-Term Remissions in Patients with Mantle Cell Lymphoma
Authors: Cortelazzo Sergio , Magni Michele , Tarella Corrado , Mian Michael , Billio Atto , Rossi Andrea , Marchesi Maddalena , Gallamini Andrea , Corradini Paolo , Ciceri Fabio , Parvis Guido , Majolino Ignazio , Patti Caterina , Mirto Salvo , Benedetti Fabio , Pizzolo Giovanni , Gianni Alessandro M. , Rambaldi Alessandro
Published: October 2019

We updated a cohort study with the aim of evaluating if an up-front Rituximab supplemented high-dose sequential chemotherapy (R-HDS), supported by autologous bone marrow transplantation (ASCT) can induce log-term remissions in Mantle Cell Lymphoma (MCL), with an acceptable toxic profile. From 1997 to 2005, 77 consecutive MCL patients, <61 year old, considered suitable for ASCT in 11 Italian cancer centres were enrolled in this study (Group 1). Their clinical outcome was compared with that of 79 age-matched historic controls treated between 1978 and 1999 with standard-dose anthracyclin or fludarabine containing regimens (group 2). The majority of both groups had an advanced stage, bone marrow infiltration and >1 IPI risk factors, while >1 extranodal sites prevailed in Group 1 and a poor ECOG-PS in Group 2. After 2–3 cycles of either doxorubicin- or cisplatin-containing chemotherapy, Group 1 received R-HDS including: HD-cyclophosphamide (CTX) 7 gr/sqm and HD-Ara-C (2 g/sqm every 12 hours for 6 days), followed by HDS HD-melphalan (180 mg/sqm) and/or HD-mitoxantrone plus melphalan (60 and 180 mg/sqm) and ASCT. Rituximab (375 mg /sqm) was given for a total of 6 doses, twice after HD-CTX and HD-Ara-C, as in vivo purging before CD34+ cells harvest and twice after ASCT. Sixty-nine patients (90%) in Group 1 completed the planned program and a median number of 6.3 x 10^6 cells CD34+/kg were transplanted. In Group 2, 85% of patients received the planned chemotherapy. The CR rate was 86% in Group 1 and 35% in Group 2 and the treatment-related mortality was 1.3% and 0.8%, respectively. Moreover, in Group 1, 4 patient developed sMDS, and 4 died of solid tumors (n=3), or septic shock (n=1). After a median follow-up of 50 months (range 3–111) in Group 1 and 37 months (range 1–141) in Group 2, the rate of 1st CCR was 65% and 14%, respectively. The 5-year projected OS, EFS and DFS were 74%, 61% and 70%, in Group 1 and 31%, 14% and 25%, in Group 2, respectively. Cox multivariate analysis failed to identify within potential prognostic markers factors predictive for OS and EFS. In conclusion, R-HDS induces long-term remissions with a manageable toxicity in patients with newly diagnosed MCL. Studies comparing this program with other intensive regimens are needed for ascertaining the role of R-HDS in the treatment of this otherwise incurable disease.
Early Results of a Phase II Trial Testing Low Dose Total Body Irradiation (LTBI) after Chemoimmunotherapy in Elderly High Risk Patients with Diffuse Large B-Cell Lymphoma (DLBCL)
Authors: Safwat Akmal , Specht Lena , Hansen Flemming , Hansen Mads , Jurlander Jesper , Boesen Anne Marie , d’Amore Francesco
Published: October 2019

Background: The addition of rituximab (R) to bi-weekly CHOP (CHOP-14) in the RICOVER-60 trial resulted in improved time to treatment failure in 828 elderly (61–80 years) DLBCL patients (Blood106:9a, 2005). We investigated the addition of LTBI in a dose of 1,6 Gy given as adjuvant therapy in high-risk elderly patients with DLBCL treated according to the best performing arm of the RICOVER-60 trial (R-CHOP-14 x 6 + 2xR).

Methods: A phase II trial including pts >60 yrs with stage II-IV, CD20-positive DLBCL was performed between 2003 and 2007. Pts received 6x R-CHOP-14 and 2 x R alone followed by LTBI given as 2 courses of 4 daily fractions of 0,2 Gy separated by 2 weeks of rest.

Results: We report the results of the first 36 patients. The median age was 69 years; 56% had ≥ 1 extranodal lesion; 67% had stage III or IV; 48% had B symptoms and 37% had Bulky (> 7.5 cm) disease; 54% had ECOG score ≥ 1; 75% had elevated LDH and 63% had IPI of >2, while 73% stained positive for BCL2 in pre-therapeutic histological samples. One patient got off study because of the discovery of breast cancer and one patient refused to get LTBI. Twenty one pts achieved a CR or CRu at the end of chemotherapy (58%), 10 (27%) were in PR while only one patient progressed under chemotherapy. All 10 PR pts achieved CR in the first follow up after LTBI. Four pts relapsed within 12 months after achieving CR. All treatment failures occurred in patients with IPI 3&4. Of the administered R-CHOP-14, Rituximab and LTBI cycles, 95%, 100% and 96%, respectively, were given in full dose and on time. CTC Gr 3–4 neutropenia occurred following 50 of 250 R-CHOP-14 cycles (20%). There were 3 toxic deaths (7.8%) due to sepsis occurring during chemotherapy. CTC Gr. 3–4 thrombocytopenia was seen in 8 pts following the last LTBI cycle (22%) and could be prolonged. The 2-Y Overall survival (OS) was found to be 79% (SE ± 8%) while the 2-Y disease free survival (DFS) was 74% (SE ± 10%).

Conclusion: In a selected DLBCL group of elderly patients with high-risk prognostic profile, R-CHOP-14 followed by LTBI, although sometimes associated with severe infections and thrombocytopenia, seems to achieve high response rates and provide correspondingly high OS and DFS values.
Evidence for QTLs Influencing the Endogenous Thrombin Potential (ETP) and Protein C Concentrations in a Dutch Family with Unexplained Thrombophilia.
Authors: Tanck Michael W.T. , Wichers Iris M. , Meijers Joost C.M. , Büller Harry R. , Reitsma Pieter H. , Middeldorp Saskia
Published: October 2019

Introduction: In Dutch families with unexplained thrombophilia (GENES study), plasma levels of the endogenous thrombin potential (ETP) discriminated affected from non-affected family members. A high variance, i.e. >60%, attributable to genetic factors was found for the ETP in one extended family. Increases in ETP levels could not be readily explained by known determinants of ETP from the coagulation cascade or anticoagulant pathways. In the present study, a genome-wide scan was performed in this family to identify quantitative trait loci (QTL) influencing ETP and several other coagulation factors.

Methods: Phenotypic data, plasma, and DNA were available for 135 of the 185 individuals in the pedigree. Patients using anticoagulation medication were excluded from the analyses. If necessary, data were transformed prior to the analyses. Multipoint Identity by Descent (IBD)-estimation and variance component multipoint (bivariate) linkage analysis including covariates were performed using SOLAR. LOD scores were empirically adjusted.

Results: For ETP levels, suggestive evidence for linkage was found at 36 cM (one-lod support interval: 1 – 61 cM) on chromosome 20 (LOD = 2.57). Interestingly, significant evidence for linkage with protein C levels (LOD=6.6) was found in the same region at 48 cM (41 – 59 cM). A bivariate LOD score of 5.8 (p<2x10−6) was obtained for this region of chromosome 20, but the correlation between the two traits due to QTL effects was low (0.10 ± 0.28, p=0.734). This finding, together with the fact that the test for complete pleiotropy had to be rejected (p<0.0001), indicates that ETP and protein C levels are most likely influenced by two different QTLs (i.e. coincident linkage) on chromosome 20.

Conclusion: In this Dutch family, ETP and protein C levels were influenced by two putative major genes, both localized in the same region of chromosome 20. This region is known to harbor several liver-enriched transcription factors, e.g. hepatocyte nuclear factor (HNF) 3b (20p11) and HNF4a (20q12-q13.1). Since most of the plasma proteins that determine the level of ETP are synthesized in the liver, these candidate genes are now further explored within this family with unexplained thrombophilia and inherited high ETP levels.
In Vivo Permanent Marking of Cells with RAG1 Activation History Demonstrates That Lymphoid and Myeloid Derived Dendritic Cells Operate an Identical Developmental Program after Their Lineage Commitment.
Authors: Chong Yong , Iino Tadafumi , Mizuno Shin-ichi , Shigematsu Hirokazu , Arinobu Yojiro , Iwasaki Hiromi , Akashi Koichi
Published: October 2019

Dendritic cells (DCs) have been shown to arise from both myeloid and lymphoid pathways, by evaluating the in vivo reconstituting potential of myeloid- and lymphoid-committed progenitor populations. However, evaluation of DC development after conventional adoptive transfer experiments may not correctly represent normal DC development including lineage contribution toward the formation of the DC pool. By crossing RAG1-Cre knockin with yellow fluorescence protein (YFP)-floxed reporter lines, we developed a mouse line in which cells with a history of RAG activation should be permanently marked with YFP as a result of lymphoid commitment. Lymphoid-derived DCs were successfully marked as YFP+ in vivo. We found that only ∼10% of conventional DCs (cDCs) and ∼20% of plasmacytoid DCs (pDCs) were YFP+, even in the thymus. This is a formal evidence that the majority of DCs in steady-state hematopoiesis originate from stages that have committed to the myeloid lineage. The lineage origin of DCs did not affect their functional abilities, including antigen-presentation and cytokine production. We then profiled the gene expression of the YFP+ and YFP- DCs at a genome wide level by DNA microarray analysis. Unexpectedly, the gene expression profiles of lymphoid and myeloid-derived DCs were virtually identical, irrespective of their organs. In contrast, the genetic programs were apparently different between splenic and thymic DCs, regardless of their lineage origin. Moreover, thymic DCs contained a number of more activated genes, such as MHC class II-related ones, as compared to splenic DCs. These results suggest that lymphoid and myeloid DCs might use a common developmental program that can be activated even after lymphoid or myeloid commitment. Thus, DCs are a unique cell population independent of other conventional lineage cells. Lineage-restricted DCs might be merged into an identical pool, distributed to various tissues, and finally fated by the microenvironment of their sites.
Marrow-Derived Mesenchymal Stromal Cells Can Block an Antigen-Driven Humoral Response by Suppressing Plasma Cell Activity Via Production of CCL2.
Authors: Rafei Moutih , Hsieh Jeremy , Li MengYang , Galipeau Jacques
Published: October 2019

Marrow-derived Mesenchymal Stromal Cells (MSCs) have been demonstrated to possess powerful immunomodulatory suppressive properties. In vitro studies by many groups have shown that MSCs can suppress th1 immune responses as exemplified by in vitro blockade of a 2-way mixed lymphocyte reaction (MLR) by an assortment of mechanisms including production of soluble factors such as: nitric oxide, transforming growth factor-β, IGF-1 and hepatocyte growth factor. Lately, interesting studies have also demonstrated the potency of MSCs in modulating humoral immunity by inhibiting B-cell migration, proliferation as well as immunoglobulin secretion in vitro. We here sought to further define the ability of autologous MSCs in modulating an ovalbumin (OVA) antigen-specific humoral response in normal immune competent C57BL/6 mice. Immunologically naïve mice were vaccinated with 50 ug of recombinant chicken ovalbumin protein followed by a boost dose 2 weeks later. All mice developed a robust IgM and IgG anti-OVA antibody response as measured in serial plasma samples over time. Following establishment of anti-OVA humoral immunity, test mice (n=10) were administered 1.5 million autologous MSCs in the peritoneal cavity twice at one month interval. Compared with same-treated controls (n=10), we found that MSC treated mice significantly suppressed anti-OVA IgG titer. This inhibitory effect requires metabolically active MSCs since live MSCs inhibited anti-OVA IgG secretion in ELISPOT assays, whereas paraformaldehyde fixed MSCs had no effect. Interestingly, the addition of MSC conditioned media directly on spleen-derived plasma cells derived form OVA immunized mice inhibited OVA-specific IgG secretion in vitro. A cytokine array screen on MSC secretome identified CCL2 as a possible effector molecule in suppressing plasma cell activity, and we found that anti-CCL2 neutralizing antibodies abolished the suppressive effect of MSCs on plasma cells. In conclusion, we have found that MSCs can suppress a pre-established humoral response to a defined antigen in vivo. This effect is contact independent, and requires metabolically active MSCs. MSC-derived CCL2 appears to be a key suppressor of antigen-specific immunoglobulin in this system.
Treatment of Indolent Non-Hodgkin’s Lymphoma in Germany - Results of a Representative Population-Based Survey.
Authors: Dreyling Martin , Fetscher Sebastian , Kornek P. , Nusch Arnd , Kornacker Martin , Angermund Ralf , Pliskat Harold , Kellermann Lenka , Kegel Thomas
Published: October 2019

In advanced stage indolent lymphoma, therapeutic approaches may vary from watch and wait, antibody monotherapy, conventional chemotherapy or dose-intensified consolidation up to allogeneic strategies. In this nation-wide survey, representative hematological/oncological centers monitored current treatment strategies under routine conditions. 495 centers involved in the treatment of indolent lymphoma including university hospitals (UH), community hospitals (CH), and office-based hematologists (OBH) were contacted. 13% of identified centers provided information on 741 patients corresponding to 10% of the expected national prevalence. Detailed data on 576 unselected patients (median age 67 years, range 17 to 95) with treatment decision in the second and third quarter of 2006 (start, change or end of therapy) of 46 representative centers (2 UH, 25 CH, and 19 OBH) were included in this analysis. Data were verified by monitoring anonymized patients source data. Median age was 67 with hypertension (28%), coronary heart disease (14%), diabetes (11%), heart failure (8%), cardiac arrhythmia (7%) and renal impairment (7%) being the most frequent concomitant diseases at time of diagnosis. Histology included 39% follicular lymphoma, 26% chronic lymphocytic leukemia (CLL), 10% marginal zone, 9% mantle cell lymphoma, and 16% other histologies. Aim of initial therapy was curative in 35%, aiming at improved survival in 62% and palliation in 54% of patients. Radiation (10%), antibody monotherapy (4%), chemotherapy (33%) and combined immuno-chemotherapy (31%) were the most frequent approaches. Applied chemotherapies included CHOP (46%), fludarabine combinations (F/FC/FCM: 15%), chlorambucil (14%), CVP/COP (9%), Bendamustin (4%), with maintenance (12%) and autologous/allogeneic stem cell consolidation both in 3% of patients. In first relapse, complex regimen including immuno-chemotherapy (49%), maintenance therapy (16%), and autologous/allogeneic transplantation (14%/4%) were more frequently planned. As expected, significant differences were observed between follicular, mantle cell lymphoma and CLL. Interestingly, supportive measures including antibiotics (34%), erythrocyte transfusions (32%), G-CSF (22%), immunoglobulins (19%), antifungal drugs (13%), and erythropoietin (10%) were frequently applied already in first line therapy. Overall response was 83% (FL: 97%, MCL: 95%, CLL: 74%) with a 39% CR rate. Only 10% of first line patients were treated within studies (UH: 19%, CH: 5%, OBH: 13%). In this population-based survey, patient characteristics differed significantly from published study cohorts as did clinical strategies and therapeutic approaches. Thus, clinical studies more relevant to the treatment of medically compromised patients are urgently warranted.
Primary Central Nervous System Lymphoma (PCNSL): Intent-to-Treat Analysis of Monocenter Long-Term Experience.
Authors: Korfel Agnieszka , Kiewe Philipp , Fischer Lars , Mertus Peter , Thiel Eckhard
Published: October 2019

Background This is a retrospective, long-term single-center analysis of immunocompetent patients with PCNSL treated at our institution.

Methods All 72 consecutive patients diagnosed with PCNSL between January 1994 and February 2005 were scheduled to receive high-dose methotrexate (HDMTX; 1.5–4 g/m2) based chemotherapy.

Results The median age of the patients was 62 years, the median Karnofsky performance status (KPS) was 70%. Twelve patients did not receive HDMTX based chemotherapy due to poor physical condition or renal insufficiency. Of 60 patients treated with HDMTX based chemotherapy, 9 were followed by whole brain irradiation (WBI). Of 54 patients evaluable for response 35 (65%) responded (52% CR and 13% PR), and 19 (35%) did not. At a median follow-up of 58.7 months, median progression-free survival (PFS) was 9 months, and median overall survival (OAS) was 41.4 months. Eight patients have survived 60 months or longer (median age 48, range 19–76; median KPS 80, range 40–100), 5 of whom relapsed after primary therapy at a median of 5 months (range, 3–39.7) after initial diagnosis. According to the Memorial Sloan-Kettering Cancer Center (MSKCC) prognosis score, patients could be divided into three groups with significantly different OAS: 52.9 months for patients younger than 50 years, 42.4 months for patients ≥50 years and with KPS ≥70, and 5.2 months for patients ≥50 years and KPS <70 (p=0.009; log-rank test). Eleven of 17 patients alive without cerebral lymphoma after 27–84 months tested exhibited clinical deficits attributable to late neurotoxicity.

Conclusions This study providing long-term data shows that a relatively moderate HDMTX-based chemotherapy can result in prolonged survival and probably cure even in older and early relapsing patients. However, the probability of late neurotoxicity with prolonged survival is considerable. The comparison of our results to other retrospective studies underscore the importance of treating PCNSL patients preferably at experienced institutions. The MSKCC score proved useful to predict survival.
Preclinical Results of the Camptothecin-Polymer Conjugate IT-101 in Multiple Human Lymphoma Xenografts.
Authors: Numbenjapon Tontanai , Wang Jianyi , Colcher David , Schluep Thomas , Davis Mark E. , Duringer Julienne , Forman Stephen J. , Raubitschek Andrew
Published: October 2019

Camptothecin (CPT), a natural alkaloid isolated from Camptotheca acuminata, has potent broad spectrum antitumor activity by inhibiting type I DNA topoisomerase. It has not been used clinically because it is water-insoluble and highly toxic. As a result, irinotecan (CPT-11), a water-soluble analogue of CPT, has been developed and used as salvage chemotherapy in patients with refractory/relapsed lymphoma, but with only modest activity. For intravenous administration, we have developed IT-101, a ca. 40 nm diameter nanoparticle that is an assembly of cyclodextrin-based polymer conjugates of 20-(S)-CPT. The purpose of this study is to compare the preclinical efficacy of IT-101 with CPT-11 both in vitro and in vivo. Fluorescence microscopy studies demonstrated that incubation of a human lymphoma cell line with IT-101 resulted in the uptake and intracellular accumulation of CPT. Based on the demonstration of in vitro cytotoxity of IT-101 against multiple human lymphoma cell lines, we initiated experiments in xenograft models of lymphoma. In subcutaneous human xenograft models, a single cycle of three weekly doses of intravenous IT-101 at 10 mg/kg (CPT equivalents) showed significantly potent antitumor activity against Daudi, Karpas 299, and L540 cell lines compared to three weekly doses of intraperitoneal CPT-11 at its maximum tolerated dose in mouse of 100 mg/kg (P < 0.0001, P = 0.0072, and P < 0.0001, respectively). In the same animal models, IT-101 led to pathologically complete remissions in 78%, 44%, and 78% of animals inoculated with Daudi, Karpas 299, and L540 cell lines, respectively. In disseminated human xenograft models, IT-101, administered using the same dosing schedule, significantly prolonged the survival of animals intravenously injected with either Daudi or Karpas 299 cell lines when compared to CPT-11 (P < 0.0001 and P = 0.0049, respectively). The promising present results in a variety of lymphomas provide the basis for a phase I/II clinical trial in patients with refractory/relapsed lymphoma.
Targeted Therapy with Arsenic Trioxide and Interferon alpha Eradicates Leukemic Cells in SCID Mice Model of Adult T-Cell Leukemia/Lymphoma.
Authors: Hajj Hiba El , Hasegawa Hideki , El-Sabban Marwan , Zaatari Ghazi , Saab Shehrazad , Mahfouz Rami , Nasr Rihab , Kfoury Youmna , Hermine Olivier , de Thé Hugues , Hall William , Bazarbachi Ali
Published: October 2019

HTLV-I associated adult T-cell leukemia (ATL) is a severe, chemotherapy-resistant malignancy associated with poor prognosis. Recently, HTLV-I Tax transgenic mice, accurately reproducing human ATL disease were generated. To develop a more consistent and rapid model of disease development, direct transfer of Tax transgenic cells into SCID mice was assessed and showed that they die within 28 days, having developed both an extremely aggressive leukemia with characteristic flower cells, and extensive lymphomatous infiltration of the spleen, lymph nodes, bone marrow, liver, kidney and lung by malignant T lymphocytes highly expressing CD25. Furthermore, and as in ATL patients, we observed marked hypercalcemia and high level of LDH. We have previously identified in vitro several potential targeted therapies for ATL. To define the optimal schedule and drug combination to be evaluated in clinical trials, we tested the following drugs using the in vivo ATL SCID model: zidovudine, interferon alpha (IFN), arsenic trioxide, the proteasome inhibitor bortezomib, and the combinations of zidovudine and IFN, or of arsenic and IFN. The combination of zidovudine and IFN had no effect on the survival of ATL SCID mice confirming the hypothesis that this combination targets the viral replication since these animals have Tax-transgenic leukemic cells but no entire HTLV-I retrovirus, contrary to ATL patients. Inhibition of NF-kappaB using bortezomib or arsenic alone almost doubled the mice survival but was not sufficient to eradicate ATL malignant cells. Strikingly, the most relevant effect on the mice survival was obtained when we used the combination of arsenic trioxide and interferon alpha, which targets both the HTLV-I Tax oncoprotein and the constitutive activation of the NF-kappaB pathway. Indeed, after 6 months of follow up, this combination totally cured 40% of the treated mice, and tripled the survival of the remaining 60%. These impressive results suggest that double targeting of Tax and NF-kappaB by the combination of arsenic trioxide and interferon alpha may suffice to cure ATL. This combination is now tested in newly diagnosed ATL patients.
The MDM2 Antagonist Nutlin-3 Is Lethal to Mantle Cell Lymphoma with Wild Type p53.
Authors: Tabe Yoko , Sebasigari Denise , Rudelius Martina , Pittaluga Stefania , Raffel Mark
Published: October 2019

Mantle cell lymphoma (MCL) has one of the poorest overall prognoses of the non-Hodgkin lymphomas, and is resistant to standard chemotherapy. P53 mutations are rare in typical mantle cell lymphoma and occur in only about 1/3 of the less common blastoid variant. The relatively low overall rate of p53 mutation in MCL suggests that this lymphoma may be a good candidate for biologic therapies that upregulate p53 and lead to cell death. Nutlin-3 is a novel small-molecule antagonist of MDM2 that efficiently activates p53 by blocking the MDM2-p53 interaction. In this study, we investigated the effects of nutlin-3 in 5 MCL cell lines with known p53 mutation status (wt-p53: REC-1, Z138, Granta-519, JVM-2; mt-p53: Jeko-1). IC50s for all cell lines were determined, and the activation of p53 and multiple p53 target genes were studied by western blot analysis. Cell proliferation and apoptosis were assessed using the MTT test and flow cytometry. Treatment with Nutlin-3 resulted in a marked reduction in cell proliferation/viability, and an increase in the apoptotic fraction, in a time and concentration-dependent manner in wt-p53 cells (IC50 at 48 hrs; 7.5 uM for Granta-519, 9.2 uM for REC-1, 0.5 uM for Z138, and 5.7 uM for JVM-2), while mt-p53 Jeko was resistant to nutlin-3 treatment (IC50>60 uM), and showed no increase in the apoptotic cell fraction. In the wt-p53 MCL cell lines, nutlin-3 treatment increased the cellular levels of p53, and several p53 dependent proteins including p21, MDM2 itself and the proapoptotic BH3-only protein Puma, while there was no change in the levels of these proteins in Jeko-1. Recently, attention has been focused on novel p53 target genes that function to inhibit cell growth and proliferation, rather than by inducing apoptosis. These include the AKT-mTOR regulators PTEN, TSC2 and AMPKβ1. We saw no increase in these novel p53 targets in any of the cell lines analyzed, suggesting that nutlin-3-activated p53 activates only a subset of its possible targets. These findings demonstrate that nutlin-3 successfully activates wt-p53 in mantle cell lymphoma leading to the upregulation of traditional targets such as p21 and proapoptotic proteins including Puma, and result in apoptotic cell death. The data suggest that p53 activators such as nutlin-3 may be effective agents in the treatment of mantle cell lymphoma.
Localization and Functional Characterization of the Deoxyhemoglobin Binding Site on Human Erythrocyte Band 3.
Authors: Chu Haiyan , Breite Andrew , Ciraolo Peter , Franco Robert S. , Low Philip S.
Published: October 2019

Band 3, the major protein of the human erythrocyte membrane, associates with multiple metabolic, ion transport, and structural proteins of the cell. Functional studies demonstrate that the oxygenation state of the erythrocyte regulates metabolic, ion transport and mechanical properties of the cell. Because deoxyhemoglobin, but not oxyhemoglobin, binds band 3 with high affinity, these observations raise the hypothesis that hemoglobin (Hb) might regulate erythrocyte properties through its oxygenation-dependent association with band 3. To explore how reversible association of deoxyHb with band 3 might modulate erythrocyte properties, we have characterized the binding site of deoxyHb on human erythrocyte band 3. We report that:

the deoxyHb binding site on the erythrocyte membrane comprises residues 12–23 of band 3; mutation of residues on either side of this sequence greatly enhances affinity of deoxyHb for band 3, suggesting that evolution of a higher affinity interaction was possible had it been beneficial; Hb does not bind to two similar sequences in band 3 despite their high sequence homology to residues 12–23, and the Hb binding site on band 3 lies proximal to binding sites for glycolytic enzymes, band 4.1 and ankyrin, suggesting possible mechanisms through which multifarious erythrocyte properties might be regulated by Hb oxygenation.

We conclude that nature has evolved a deoxyHb binding site on band 3 whose affinity is regulated by O2, enabling the reversible association of deoxyHb with band 3 to serve as a regulator of the oxygenation-dependent properties of the cell.
CD133+ Purified Hematopoietic Stem Cells in Co-Culture with Mesenchymal Stromal Cells - The Cell to Cell Contact Matters.
Authors: Alakel Nael , Jing Douhui , Bornhaeuser Martin , Ehninger Gerhard , Ordemann Rainer
Published: October 2019

Hematopoietic stem cells (HSC) are defined by their capacity of self-renewal and differentiation. In recent years it became aware that cell to cell contact mediated communication between mesenchymal stromal cells (MSC) and HSC is important for homeostasis of hematopoiesis. MSC play a crucial role in the so called bone marrow niche giving rise to the majority of marrow stromal cell lineages. In vitro we investigated the impact of MSC on CD133 purified HSC expansion and differentiation in terms of phenotype, (CXCR4) chemokine receptor expression, migration capacity and clonogenicity. After one week of co-culture with MSC adherent and non-adherent HSC were isolated and analyzed separately. As expected in co-culture experiments with MSC a significant higher expansion of TNC was observed in comparison to the expansion culture without MSC (cytokine driven), 37±6 fold vs. 19±3 fold, p<0.001. However the co-culture expansion was accompanied by a significant increase of differentiation and maturation of the progenitor cells. Therefore we were focusing on the adherent cell fraction of the co-culture system. According to FACS analysis results, adherent HSC in the co-culture display a more immature phenotype than non adherent cells (CD34+CD38−). Next colony assays and LTC-IC were performed showing that the adherent cell fraction contained a significant higher proportion of primary and secondary CFU-C, indicating that this cell fraction contains the majority of clonogenic HSC. The CXCR4 - SDF-1 axis plays an important rule in homing, mobilization and engraftment of the hematopoietic stem cells. Therefore we looked for CXCR4 expression in the two cell fractions using FACS analysis. The adherent cells expressed consistently and significantly more CXCR4 than the non adherent cells; 43±4% vs 15±2%, p<0.001. Data could be confirmed by performing western blot. Due to this data we evaluated the capacity of HSC migration toward SDF-1 gradient using transwell migration assay. The migration capacity of the adherent cells was significantly higher than that of the non adherent cell fraction correlating with the CXCR4 expression; 13±1% vs. 6±1%, p<0.001. We checked the expression of different cell adhesion molecules by FACS analysis. CD44 and CD49e (integrin α5) were significantly higher expressed in adherent cells comparing with non-adherent cells. Our data indicate that the cell-to-cell interaction mediated impact on CD133+ selected HSC leads to an enhanced clonogenic capacity and migratory potential. Further experiments are needed to investigate engraftment and NOD/SCID repopulation potential of the adherent and non-adherent cells after ex-vivo expansion.
Pharmacodynamic Analysis of the Anticoagulant Effects of Idraparinuxparinux after Termination of 6 and 12 Months Therapy for Prevention of Recurrent Venous Thromboembolism: Observations after the vanGogh Trials.
Authors: Harenberg Job , Joerg Ingrid , Hagedorn Antje , Giese Christina , Weiss Christel , Mikus Gerd
Published: October 2019

Idraparinux is a synthetic, polymethylated O-methyl, O-sulphate pentasaccharide with a higher affinity to antithrombin and has been investigated in patients with acute venous thromboembolism (VTE) for prophylaxis of recurrent events (vanGogh studies). 2.5 mg Idraparinux was given once weekly subcutaneously in patients with a creatinine clearance > 30 ml/min and continoued with 1.5 mg Idraparinux from the second injection in patients with lower creatinine clearance. This treatemnt was randomly compared to bodyweight adjusted enoxaparin followed by INR-adjusted warfarin for 3 to 6 months to prevent symptomatic recurrent VTE. The vanGogh extension trial randomised patients of the vanGogh trials or other patients with a 3 to 6 months course of warfarin after an initial first event of VTE for an additional double blind 6 months therapy of Idraparinux compared to placebo. The pharmacodynamic parameters of factor Xa (aXa) inhibition using the S2222 chromogenic substrate and Heptest assay were determined from plasma samples of 23 patients obtained after termination of a 6 or 12 months (Idraparinux for 6 months each in the DVT/PE and extension studies) therapy up to 15 months. The elimination half life of Idraparinux was 60±14 days (d, mean+SD) after termination of therapy (S2222) and 107±34 d (Heptest, p<0.0001) and did not differ between 6 and 12 months of treatment. The mean residence time differed between S2222 (75±31 d) and Heptest (121±49 d, p<0.0001) and not between 6 and 12 months. The Cmax was lower after 6 months of therapy compared to 12 months (S2222: 0.26±0.10 vs 0.44±0.08 μg/ml, p=0.0013, Heptest: 0.35±0.13 vs 0.54±0.11 μg/ml, p=0.0085). The volume of distribution was higher after 6 months of therapy compared to12 months (S2222: 8.5±3.5 vs 4.1±0.6 liter, p=0.0052, Heptest: 9.8±4.5 vs 4.7±1.3 liter, p=0.0145) without differences between the assays. The clearance differed between assays (S2222: 0.09±0.06. Heptest: 0.06±0.03 ml/min, p<0.0001) and was higher after 6 than after 12 months of treatment (S2222; 0.11±0.06 vs 0.05±0.02 ml/min, p=0.0088, Heptest: 0.07±0.03 vs 0.03±0.01 ml/min, p=0,0145). The data show a very long pharmacodynamic elimination half-life of 60 days or longer after termination of therapy after once weekly injection of Idraparinux. The half live is longer when determined by Heptest indicating also non-anti-factor Xa activities of Idraparinux. The steady state of Idraparinuxparinux is reached between 6 and 12 months of therapy or later. The low VTE-recurrence rate of 0.9% of patients randomized to placebo in the vanGogh Extension trial and after an initial course of 6 months of Idraparinux in the DVT/PE trial can be explained by the long half life of Idraparinux.