bluuebunny/crossref_metadata_2025 huggingface dataset:
Radioimmunotherapy (RIT) with 90Y-Ibritumomab Tiuxetan (Zevalin) for the Treatment of Relapsed or Resistant Aggressive Diffuse Large B-Cell Lymphoma (DLBCL) Heavily Pretreated with Rituximab + Chemotherapy: A GIMURELL Experience.
Authors: Botto Barbara , Bellò Marilena , Benevolo Giulia , Cabras Maria Giuseppina , Castellino Claudia , Chiappella Annalisa , Fioritoni Giuseppe , Freilone Roberto , Martelli Maurizio , Orsucci Lorella , Pregno Patrizia , Scapoli Paola , Tonso Anna , Bisi Giovanni , Vitolo Umberto , Gallo Eugenio
Published: November 2019

Introduction: The addition of antibody monoclonal anti CD20 Rituximab to CHOP chemotherapy has resulted in improved CR, progression free survival and overall survival rates for patients affected by diffuse large B cell lymphoma. Recently, RIT with Zevalin has been shown effective in the treatment of relapsed refractory elderly DLBCL. We report the results of a study to evaluate the efficacy and safety of Zevalin in relapsed or chemoresistant aggressive lymphoma previously treated with Rituximab.

Patients and methods: Elegibility criteria were as follows: age over 18 years, refractory or chemoresistant CD20+ aggressive lymphoma (grade III follicular, PML or DLBCL de novo) WHO performance status of 0 to 2, stage II bulky, III or IV, bone marrow involvement < 25%, written informed consent in accordance with institutional guidelines. All patients were previously treated with Rituximab and almost two lines of chemotherapy. Patients with pre-treatment platelet counts of > 150.000/mm3 received Zevalin at 0.4 mCi/kg whereas those with platelets < 150.000/mm3 received 0.3 mCi/kg.

Results: Fourteen patients were treated with RIT: 5 patients were stage II, 3 stage III, 6 stage IV (5 with bone marrow involvement). Six patients had grade III follicular, 4 primary mediastinal and 4 DLBCL de novo. Ten patients received 0.4 mCi/kg and 4 patients 0.3 mCi/kg. Five chemoresistant patients after 2 or more lines chemoimmunotherapy received RIT in combination with BEAM and subsequent autologous stem cell transplantation (ASCT) and 9 patients were treated with Zevalin alone. Two months after RIT we reported CR 4 patients, PR 5 patients and no response/progression 5 patients (ORR 9/14 patients 64%). All 5 patients treated with RIT + BEAM + ASCT achieved a response (CR 2 patients and PR 3 patients ORR 100%). ORR in 9 patients treated with RIT alone was 46% (CR 2 patients and PR 2 patients). No toxic deaths were observed, two patients died of lymphoma (one patients 1 year after Zevalin infusion and the second progressed and died 5 months after RIT). The most common grade 3–4 AE were neutropenia and thrombocytopenia.

Discussion: RIT with 90Y-Ibritumomab Tiuxetan is a safe and effective approach for patients affected by aggressive lymphoma and heavily pretreated with Rituximab + chemotherapy. The effective role of RIT alone or in combination with high dose chemotherapy + ASCT as salvage therapy has to be studied in this subset of patients.
Evaluation of TW-37, a pan Bcl-2 Proteins Small-Molecule Inhibitor, Against Spectrum of Human B-Cell Lines and Patient-Derived Samples.
Authors: Mohammad Ramzi M. , Sun Yuan , Wang Shaomeng , Aboukameel Amro , Al-Katib Ayad M.
Published: November 2019

Non-Hodgkin’s lymphoma (NHL) tumors include a group of heterogeneous diseases with varying natural histories and responsiveness to therapy; nonetheless, overexpression of Bcl-2 protein is seen in more than 80% of NHL. Throughout the years our laboratory succeeded in establishing a panel of B-cell lines representing various maturational stages of NHL. In this study, we have utilized a structure-based strategy to design a new class of potent nonpeptidic small-molecule inhibitor (SMI) of Bcl-2 family. TW-37, a lead compound that was designed to target the BH3 binding groove of antiapopototic Bcl-2 proteins. It binds to Bcl-2, Bcl-XL and Mcl-1 with Ki values of 290 nM, 1110 nM and 260 nM, respectively. TW-37 showed significant antiproliferative effect against Pre-B-Acute Lymphoblastic Leukemia (WSU-pre-B-ALL), Diffuse Large Cell Lymphoma (WSU-DLCL2), Follicular Small Cleaved Cell Lymphoma (WSU-FSCCL), Waldenstrom’s Macroglobulinemia (WSU-WM) and primary cells obtained from lymphoma patients, despite variations in their anti- and pro-apoptotic Bcl-2 proteins (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bim, Bad, BUMA and Bok). The IC50 for TW-37 varied from 165 nM in the WSU-FSCCL to 300 nM in WSU-DLCL2 cells. Apoptosis was independent of proliferative status or pathological classification of B-cell tumor. TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 eterodimerization and induces apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation. Although cell lines and patient samples expressed multiple Bcl-2 family proteins at various levels, TW-37 induced apoptosis was only strongly associated with Bax:Mcl-1 ratio. TW-37 administered to tumor-bearing SCID mice led to significant tumor growth inhibition (T/C), tumor growth delay (T-C) and Log10kill, when used at its maximum tolerated dose (40 mg/kg x 3days) via tail vein. failed to induce changes in the Bcl-2 proteins levels suggests that assessment of baseline Bcl-2 family proteins can be used to prognosticate the response to drug. These findings indicate activity of TW-37 across the spectrum of human B-cell tumors and support the concept of targeting the Bcl-2 system as a therapeutic strategy in the treatment of B-cell lymphoma.
Red Blood Cell Alloimmunization Is Affected by Depletion of Donor White Cell Subsets.
Authors: Campbell-Lee Sally A. , Liu Jinhuan , Ness Paul M. , Baldwin William M.
Published: November 2019

Red blood cell (RBC) alloimmunization leads to therapeutic limitations due to difficulties in obtaining compatible RBC components. Better understanding of immune mechanisms responsible for alloantibody formation are necessary in order to develop ways of preventing or treating alloimmunization in high risk patients. A murine model of RBC alloimmunization was developed using B6CBA-F1-Tg Fyb mice that express the human Fyb blood group as donors, and recipient mice of the B6CBA-F1 strain. RBC were obtained from donor mice that were anesthetized and exsanguinated by cardiac puncture. Donor whole blood was collected into sterile syringes containing CPDA-1 (Sigma Aldrich, St. Louis, MO) and was then centrifuged to produce packed RBCs. Recipient mice were transfused 0.3 mL via the tail vein once a week for two weeks with donor RBC that had 108 spleen cells added which were then depleted of either CD4, CD8, CD19 or natural killer (NK) cell subsets (n=6 in each group). Depletions were performed using immunodensity negative selection with murine anti-CD4, CD8, CD19 and DX5 antibodies (StemCell Technologies, Vancouver, BC). Two control groups were transfused with either washed buffy coat depleted RBC (leukodepletion to fewer than 5 × 106 remaining white blood cells (WBC)) or RBC with spleen cells added that was processed through immunodensity negative selection without the addition of antibody (no leukodepletion with 1 × 108 WBC). Serum was collected from recipient mice once a week for four weeks post-transfusion. Circulating IgG anti-Fyb titers were measured by flow cytometry using donor RBC and staining with fluorescein isothiocyanate conjugated rat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA); target cells were gated on by size and side scatter parameters using a FACScan flow cytometer (Becton Dickenson, Franklin Lakes, NJ). Serum samples from week 4 post transfusion demonstrated a mean anti-Fyb titer of 64 in the CD4 depleted group. Mean titers of 256, 512 and 512 were observed in the CD8, CD19 and NK depletion groups, respectively. The leukodepletion control group had a mean anti-Fyb titer of 16, and the non-leukodepletion control group showed a mean titer of 64. These results indicate that donor contaminating WBC affect RBC alloimmunization. CD4 cell removal led to an anti-Fyb titer that was the same as the non-leukodepletion control group. CD8, CD19 and NK cell removal increases anti-Fyb titer and may indicate a variety of inhibitory roles of these cell types.
Compassionate Use Experience with Dasatinib Therapy in Patients after Imatinib and/or Nilotinib Failure.
Authors: Gunawardena Bettina A. , Schlief Sarah , Dempke Wolfram , Wnterhalter Bernd
Published: November 2019

Background: Dasatinib is a potent orally active, multi-targeted tyrosine kinase inhibitor that blocks signal transduction pathways implicated in the proliferation and survival of tumor cells. A European compassionate use trial was conducted to provide dasatinib to patients (pts) who were ineligible to be enrolled into the START-trials.

Methods: From April 2006 to February 2007, 26 pts were included in Germany. Diagnosis at inclusion: 9 Ph+ALL, 6 CML in accelerated phase, 5 CML in myeloid blast crisis, 2 biphenotypic Ph+ALL and 1 chronic phase CML. Additionally, 3 pts with systemic mastocytosis (SM), refractory to imatinib due to D816V mutation were also included. Pts had previously failed several chemotherapy regimens, including imatinib and/or nilotinib. Sixty-one percent were resistant, 35% were refractory and 4% intolerant to imatinib/nilotinib.

Results: Of 26 patients, there were 17 male and 9 female pts. The median age was 56 years (range, 22–80 years). The average daily dose of dasatinib was 137 mg with a median duration of therapy of 69.5d (range 3–252d). Six (23%) pts required transient treatment interruption, 2 (8%) due to non-haematological toxicity (dyspnea and increased hepatic values), 4 (15%) due to haematological toxicities. Twenty-one pts discontinued therapy for the following reasons: study related toxicity (23%), SCT (15%), death (8%), lost to follow-up (11%) and patient’s wish (4%). Two patients still remain on therapy. The response rate was CHR in 42%, complete (CCyR) cytogenetic response in 19%, and PCyR in 4% of the pts. For patients with diagnosed SM one partial HR, one stable disease and one progression have been recorded. Disease progression occurred in 19% of cases. Adverse events were transient and generally mild to moderate in severity. Generally, the observed AE profile was similar to that seen in previous research trials. For all pts, the most common hematological side-effects Grade 3/4 AEs reported were thrombocytopenia (23%), neutropenia (4%), and anemia (4%). Most frequent non-hematological side-effects included dyspnea in 23% of pts (no grade 3–4), edema in 23% (grade 3, 8%), diarrhea in 15% (grade 3, 4%), headache in 15% (no grade 3–4), bone and neck pain in 11% (grade 3, 8%) and pleural effusion in 8% (grade 3, 4%).

Conclusions: Dasatinib therapy appeared to be effective and was well tolerated in this difficult to treat patient population.
Myelodisplastic Syndrome: Is It Associated or Is It a Precursor of Myeloid Malignancies?.
Authors: Corinaldesi Giorgio , Corinaldesi Christian
Published: November 2019

Myelodisplastic syndrome (MDS) is a clonal haematopoietic stem cells disorder considered a pre-leukemic disease (about 30% of MDS turn into acute myeloid leukaemia (AML)) which is frequently associated with cytogenetic abnormalities and chromosomal instability: monosomy 7 or 7q - syndrome, 5q- syndrome, trisomy 8, inv(16), t (8;21), t (15;17), t (12;21), t (9;11), or sub microscopic DNA mutation of gene such as RAS, p53, FLT3, HSPA9, FMS and refractory cytopenias, with dysmyelopoiesis and increased apoptosis. We have identified 15 people aged between 54/67 years with MDS and we have examined their clinical presentation and their associated disorders.

RA: refractory anaemia (7 patients) RARS: refractory anaemia with ringed sideroblasts (1 patient) 5q-syndrome (1 patient) RAEB refractory anaemia with excess blast: type 1 (5–10%) (3 patients); type 2 (11–20%) (1 patient) E) RAEB-T refractory anaemia in transformation (1 patient) F) CMML chronic myelomonocytic leukemia (1 patient)

The disease is characterized by chronic tiredness, headache, shortness of breath, increased susceptibility to infection especially in the lung with a prolonged fever, easy bruising, nosebleeds, pinpoint red spots, bleeding, splenomegaly, hepatomegaly, abdominal distress and pain are also common. VEGFR-1 and VEGFR-2 are strongly expressed in megakaryocytes, HLA-DR2 and HLA-DR15 are higher, immune dysfunction in MDS increases activated cytotoxic T-cells with higher percentage of CD8+CD28-, CD8+CD28- CD57+ with an increased level of CD4, and Fas ligand/Apo-1 (CD95). The RA, RARS, 5q-syndrome are more indolent disease with a lower rate of progression to AML and a prolonged clinical course; the RAEB, RAEB-T are slightly more advanced with a short course; in the CMML the progression is faster and it is frequently associated with the secondary chromosomal perturbation t(5;12), (q33;p13), or with the loss of specific portion 3p-, 7q- (7q22-7q33-). The goals of the therapy are to control symptoms and to decrease the progression to myelogenous leukaemia; our results show that a low intensity treatment in the low and intermediate groups with 5-azacitidine 75 mg/m2/day produces a high rate of response (72%, of which: complete remission: 18%, partial remission: 34%, improved: 20%), relapse 24%, disease-free survival at 3 years of 64%; with a low incidence of side effects (nausea 68%, vomiting 60%, chest pain 28%); on the other hand in the high risk group the 5-azacitidine with gentuzumab reverted to normal about 90% of the patients (complete remission: 6%, partial remission: 28%, improved: 56%), disease free survival at 3 years 52%, relapse 48%; however, the only curative treatment is stem cells transplantation from a tissue matching donor. Finally, it is very important to provide a supportive care with erythropoietin, G-CSF or GM-CSF, red cells transfusions, administration of broad-spectrum antibodies mostly for controlling symptoms, but also for preventing or treating complications.
Clinical and Prognostic Relevance of Magnetic Resonance Imaging of the Spine in Newly Diagnosed Multiple Myeloma Patients Receiving Autologous Stem Cell Transplantation.
Authors: Tosi Patrizia , Zamagni Elena , Tacchetti Paola , Salizzoni Eugenio , Perrone Giulia , Ceccolini Michela , Brioli Annamaria , Pallotti Maria C. , Pantani Lucia , de Vivo Antonio , Baccarani Michele , Cavo Michele
Published: November 2019

Magnetic resonance imaging (MRI) of the spine has demonstrated to be a useful tool for correct staging of multiple myeloma (MM), as it is more sensitive than plain x-rays in detecting vertebral lesions. Furthermore, different patterns of bone marrow involvement (focal, diffuse or normal) can be depicted, and this could contribute to better define the prognosis in newly diagnosed patients. In the present study we prospectively evaluated the clinical and prognostic role of spinal MRI in 120 newly diagnosed MM patients (68M, 52F, median age = 56yrs) that subsequently received high-dose chemotherapy and autologous stem cell transplantion, either single (n=28) or double (n=92). Pattern of marrow involvement was focal in 72 cases (60%), diffuse in 29 (24%) and negative in 19 (16%). Patients with a diffuse pattern showed a significantly higher bone marrow plasma cell infiltration (p=0.05) and beta2 microglobulin values (p= 0.04) as compared to patients with a focal pattern; stage III disease according to ISS was observed in 29%, 8% and 1% of patients with a diffuse, focal or negative pattern, respectively. Response rate to treatment program was similar in the three groups of patients, with a stringently defined CR obtained in 34% of patients with a focal pattern, 29% in those with a diffuse pattern and 35% in patients with a negative MRI. Median progression-free survival showed a trend in favor of patients with a negative MRI (54 months) as compared to those with a focal or diffuse pattern (42 and 38 months, respectively). A focal pattern of bone marrow involvement was associated with a significantly higher probability of experiencing an overt vertebral lesion (73% vs 48% in patients with diffuse pattern, p=0.03), including either a compression fracture or a vertebral mass. Consistently with this finding, also extra-spinal bone lesions were more common in patients with a focal pattern (66%) as compared to patients with a diffuse or negative pattern (42% and 6% respectively) (p=0.05). Serum crosslaps were significantly increased in patients with a focal pattern (7032 pmol/L±635SE vs 5431pmol/L±812SE in those with a diffuse pattern, p=0.04). According to our data, a diffuse pattern of bone marrow involvement could be predictive of a more aggressive disease, even though these data needs to be confirmed in a larger series of patients. A focal pattern of bone marrow involvement at diagnosis could help to identify MM patients more prone to develop skeletal complications, so that a careful monitoring and bisphosphonate therapy should be recommended.
Stroke in a Multiple Myeloma Cohort.
Authors: Keyrouz Salah G. , Saba Sadia , Zangari Maurizio , Schmidley James W.
Published: November 2019

Background: Neurologic complications of multiple myeloma (MM) are numerous, however little is known about strokes occurring in the course of MM and its treatment.

Methods: 2877 MM patients were seen for an initial evaluation at our institution over a 5-year period (1998–2002). Patients were identified using discharge summaries by combining ICD-9 codes for MM and stroke. They were included if they had clinical and radiological features of acute stroke. Ischemic stroke was defined as a new acute neurologic deficit lasting > 24 hours, irrespective of diffusion-weighted MRI results. Diagnosis of hemorrhagic stroke was made in patients with new acute neurologic deficit plus evidence of hemorrhage on CT scan. We retrospectively reviewed medical records for demographics, type of MM and treatment, stroke type, clinical features, relevant imaging and laboratory data, and outcomes.

Results: The 11 patients that we identified included eight with ischemic strokes and three with subarachnoid hemorrhages (SAH). There were no patients with intraparenchymal hemorrhage. The overall incidence of stroke in this cohort was 76/100,000 per year, whereas the incidence of ischemic stroke was 56/100,000 per year. The mean age of patients with ischemic stroke was 59 years. Seven had one or more stroke risk factors and an equal number had received thalidomide. The latter were given small doses of coumadin as part of the treatment protocol; at the time of the stroke, INR range was 0.9–1.4. Based on clinical, radiological and other laboratory features, of the eight patients with ischemic strokes, two were presumed cardio-embolic (one had infective endocarditis), five were thrombotic, and one was caused by hypoperfusion. Two patients had findings of severe stenosis/occlusion in vessels corresponding to the infracted brain territory (internal carotid and basilar arteries). Two patients had documented normal plasma viscosity and three had evidence of extra-cranial thrombosis at the time of the stroke. In the three patients with SAH, hemorrhage occurred in the setting of trauma and thrombocytopenia, although one patient had an incidental anterior communicating artery aneurysm. Seven patients were left with minor or no deficits and four died, two in each of the groups.

Conclusion: Overall, strokes did not appear to be more common in MM patients than in the general population, and the pathophysiology was likewise, not different than in patients without MM, with the exception of one patient with infective endocarditis, which is probably directly related to MM or its treatment. The highly selected referral population at our myeloma institute, and the retrospective nature of the study probably underestimate the incidence of stroke. The role of thalidomide in stroke remains unknown. Thrombocytopenia and trauma are probable risk factors for subarachnoid hemorrhage in MM patients. Fatal outcomes were frequent, but when death did not occur, the deficits were minor.
Update of Experience with Fludarabine, Cyclophosphamide, Mitoxantrone Plus Rituximab (FCM-R) in Frontline Therapy for Chronic Lymphocytic Leukemia (CLL).
Authors: Faderl Stefan , Wierda William , Ferrajoli Alessandra , O’Brien Susan , Ravandi Farhad , Kantarjian Hagop M. , Showalter Brandi L. , Lerner Susan , Keating Michael J.
Published: November 2019

Combination therapy has developed into a gold standard of CLL therapy. Chemoimmunotherapy combinations currently achieve the highest rates of complete remission (CR) and molecular responses. Various combinations have been tried to maximize outcome. Based on our previous results with fludarabine, cyclophosphamide, rituximab (FCR) and more recent reports of favorable results with FC + mitoxantrone (M), we have designed an FCM-R (plus pegfilgrastim) combination for pts with previously untreated and symptomatic CLL. All patients were younger than 70 years with b2M levels < 4 mg/L. Pts received F 25 mg/m2 i.v. d2–4, C 250 mg/m2 i.v. d2–4, M 6 mg/m2 i.v. d2, R 375 mg/m2 i.v. d1, and pegfilgrastim 6 mg s.c. d4. For courses 2–6, FCM started day 1 together with R 500 mg/m2, and pegfilgrastim on d3. Courses were repeated q4–6 wks. The primary objective was assessment of clinical responses (NCI-WG criteria) and flow cytometry (CD5/CD19) response rate at 3 and 6 months as well as toxicity. Thirty-one pts were enrolled of whom 30 were treated. The median age was 57 yrs (range 38–69). Fourteen pts (48%) were male. Four pts (14%) had Rai stage ≥ 3. Median b2M was 2.6 mg/L (1.4–4) and the median WBC 59.9 × 109/L (5.6–355). Two pts had 11q23 and 17p- abnormalities by cytogenetics/FISH. Unmutated IgVH occurred in 12/17 (71%) pts; ZAP-70 immunohistochemistry was positive in 11/19 (58%) pts. Twenty-nine pts were evaluable for response at 3 months and 30 at completion of therapy. Twenty-eight pts (97%) responded at 3 mos (41% CR, 17% nPR, 39% PR); 10 pts (34%) had <1% CD5/CD19+ cells in the marrow. Response rates at completion of therapy: 77% CR, 10% nPR, 10% PR for an OR rate of 97%. Seventeen pts (57%) had < 1% CD5/CD19+ cells in the marrow at the end of therapy. Grade >/= 3 neutropenia occurred in 19 (63)% of the pts, thrombocytopenia in 2 (7%). Infectious episodes were seen in 13 pts (45%). Seven (23%) did not complete 6 courses because of ongoing cytopenias. In conclusion, FCM-R has a high CR rate in symptomatic frontline patients with CLL. Time-to-event parameters and a comparison with FCR will be presented.
A Long-Term (5-Year) Sustained Efficacy of Tocilizumab for Multicentric Castleman’s Disease and the Effect on Pulmonary Complications.
Authors: Nishimoto Norihiro , Honda Osamu , Sumikawa Hiromitsu , Johkoh Takeshi , Aozasa Katsuyuki , Kanakura Yuzuru ,   Japanese MRA Study Group on MCD
Published: November 2019

Multicentric Castleman’s disease (MCD) is an atypical lymphoproliferative disease, which is closely associated with dysregulated overproduction of interleukin-6 (IL-6). These patients have numerous complications including secondary amyloidosis, paraneoplastic mucocutaneous disease, renal failure, and pulmonary manifestations such as diffuse lymphoid hyperplasia. We previously reported on a 60 weeks study of the humanized anti-human IL-6 receptor monoclonal antibody tocilizumab (TCZ) in patients with MCD and showed that TCZ provides rapid clinical improvement in constitutional symptoms as well as biochemical markers such as c-reactive protein, erythrocyte sedimentation rate, serum amyloid A, hemoglobin, and albumin levels. In this long-term follow-up over 5 years, we investigated the efficacy and safety of TCZ. Patients received TCZ monotherapy at 8 mg/kg intravenously every 2 weeks for 5 years. The dose and dosing interval were allowed to be adjusted according to disease activity. Pulmonary diffuse lymphoid hyperplasia was examined by high resolution computed tomography (HRCT) and evaluated and scored by 2 independent radiologists who were blinded to the time sequence of the images and clinical response of each patient. This study complied with all provisions of the Declaration of Helsinki and was conducted in accordance with Good Clinical Practice guidelines. Thirty-five patients were enrolled in this clinical trial and 30 patients (86%) continued TCZ treatment over the period of 5 years. The reduction in size of enlarged lymph nodes as reported previously was sustained. Clinical improvement in constitutional symptoms as well as the improvement of laboratory markers was maintained. Twenty-two patients had received corticosteroids at baseline (mean corticosteroid dose was 16.5 mg/day prednisolone equivalent), and 11 patients (50%) were able to discontinue corticosteroid therapy without flare. Pulmonary diffuse lymphoid hyperplasia was identified in 31 patients at baseline. Significant improvement was observed in the scores of centrilobular nodules, interlobular septal thickening, bronchovascular bundle thickening and ground glass attenuation with 50 to 60% reductions evaluated by HRCT over the 5 year period. No statistically significant change was observed in scores of cyst and airspace consolidation. TCZ was generally well tolerated during this 5 years treatment. The majority of adverse events (AEs) were mild to moderate in severity. Most frequently reported AEs were mild to moderate infections such as common cold and urinary tract infections (2.3 AEs / patient year). In conclusion, in this 5 years extension study tocilizumab demonstrated an excellent long term efficacy on inflammatory manifestations and pulmonary diffuse lymphoid hyperplasia. Tocilizumab treatment also showed a very good safety profile.
Surviving Host CD4+CD25+Foxp3+ Cells Following Ablative Conditioning Expand and Comprise the Major Component of the Treg Compartment during the Lymphoid Reconstitution Period Following HCT.
Authors: Bayer Allison L. , Jones Monica , Urbieta Maite , Chirinos Jackeline , Schreiber Taylor , De Armas Leslie , Malek Thomas R. , Levy Robert B.
Published: November 2019

The capacity of CD4+CD25+Foxp3+ (Treg) cells to regulate adaptive and innate immune responses has led to studies investigating their application to regulate allogeneic T cell responses arising during hematopoietic stem cell transplants (HCT). With respect to HCT, a fundamental clinical concern is the reconstitution of the lymphoid compartment in recipients, particularly T cells since this process can be exceedingly delayed. We have previously found that host Treg cells can regulate resistance to engraftment following HCT, demonstrating that such cells survive and function at least transiently in recipients. The present studies investigated the residual host Treg compartment following HCT. Utilizing varying levels of total body irradiation (5.0 – 14Gy), we observed:

recipient CD4+CD25+Foxp3+ cells can survive ablative as well as reduced intensity conditioning, the surviving, i.e. residual Treg cells undergo expansion as assessed by BrdU uptake and cell numbers, and these cells comprise the majority of the Treg compartment in recipients for several months post-HCT during which time donor derived Treg cells gradually arise and cede this compartment.

Residual Tregs also dominated the compartment following allogeneic HCT of MHC-matched bone marrow depleted of T cells. To assess the functional capacity of the residual Treg cell compartment, the development of autoimmune disease following transplant of IL-2Rβ −/− (CD122−/−) bone marrow into syngeneic recipients with and without residual Tregs was examined. Autoimmune disease symptoms and T cell alterations were prevented in B6-wt but not T cell deficient recipients. Interestingly, the failure to transfer autoimmune disease following IL-2Rβ −/− HCT into lethally conditioned B6-CD4−/− recipients was associated with the presence of a peripheral CD8+FoxP3+ population not detected in B6-wt mice or the B6-wt mice transplanted with IL-2Rβ −/− BM. This finding indicates that in the genetic absence of CD4+ T cells, a CD8 regulatory population appears to emerge. In total, our observations support the notion that functioning host Tregs initially occupy a niche in the transplant recipient permitting lymphopenic expansion and an extended period of contribution to this compartment. Notably, this contribution reflected much greater levels than conventional T cell populations - even in aggressively conditioned recipients. Finally, these findings imply that the presence of host regulatory cells may be important to consider with respect to eliciting anti-tumor responses and vaccination in recipients during the early period post -hematopoietic cell transplantation.
MEK Inhibitor AZD6244 Induces Cell Growth Arrest and Synergizes Nutlin-3a-Mediated Cell Death by Upregulating p53 and PUMA Levels in Acute Myelogenous Leukemia.
Authors: Zhang Weiguo , Konopleva Marina , Schober Wendy D. , McQueen Teresa , Andreeff Michael
Published: November 2019

Raf/MEK/Erk signaling is constitutively activated in the majority of acute myeloid leukemias (AML), providing rationale for targeting this pathway with therapeutic intent. We investigated growth-inhibitory and pro-apoptotic effects of AZD6244, a small molecule MEK1/2 inhibitor, as well as potential synergistic effects with the MDM2 antagonist Nutlin-3a in AML. AZD6244 inhibited proliferation of AML cells by arresting cells in G1 phase, which resulted from inhibition of cell cycle checkpoint proteins cyclin E and cdk2, and was associated with the modulation of cyclin D1, cyclin A, cyclin B1, p27 and Rb phosphorylation levels. AZD6244 also induced apoptotic cell death in AML cell lines with constitutive activation of MEK/ERK signaling (OCI/AML3, HL60 and MOLM13) via decrease of anti-apoptotic Mcl-1 protein expression and upregulation of pro-apoptotic Bax, Bak, Bad and Bim proteins. Next we tested the hypothesis that activation of p53 signaling with a small molecule MDM2 inhibitor Nutlin-3a would enhance the pro-apoptotic effects of MEK inhibition in AML. Combinations of AZD6244 and Nutlin-3a triggered profound apoptotic responses in AML cells with wild-type p53 (OCI/AML3, MOLM13 and Baf3-FLT3-ITD), with combination indices (averaged ED50=0.37, ED75=0.38, ED90=0.43) suggesting strong synergism. In contrast, no synergism was seen in p53 null cell HL60 (ED50=1.27, ED75=1.38, ED90=1.66) or in OCI-AML3 cells stably transduced with p53 shRNA. Mechanistically, combined blockade of MEK pathway and activation of p53 signaling resulted in marked upregulation of p53 expression and of the p53 target protein PUMA, while only partially suppressing Nutlin-induced p21 upregulation. This resulted in loss of the mitochondrial membrane potential, cytochrome c release and cleavage of caspase-9 and -3. Importantly, combined AZD6244 and Nutlin-3a treatment potently induced apoptosis in blasts from primary AML patients. In summary, AZD6244 induced growth-inhibitory and pro-apoptotic effects in AML cells with constitutive activation of MEK/ERK signaling. These effects were synergistically enhanced by simultaneous activation of p53 signaling by Nutlin-3a, resulting in the activation of the intrinsic apoptotic pathway. Altogether, these results strongly suggest potential activity of AZD6244 alone or in combination with MDM2 antagonists as novel mechanism-based therapeutic agents in AML therapy.
The Decrease of Regulatory T Cells through the Low Levels of FOXP3 Expression Mediated by NFAT1 in Infancy with Autoimmune Neutropenia.
Authors: Nakamura Kazuhiro , Miki Mizuka , Karakawa Syuhei , Onodera Rie , Kurita Emi , Hiraoka Asako , Sato Takashi , Kobayashi Masao
Published: November 2019

CD4+ /CD25+ regulatory T cells have been recognized to regulate the maintenance of self-tolerance, and several human autoimmune diseases in adulthood. To date, little is known about the standard value of regulatory T cells in infancy and childhood and about its involvement in pediatric autoimmune disease. Autoimmune neutropenia (AIN) in infancy is a common disorder characterized by chronic neutropenia, the detection of antineutrophil antibodies in sera, and spontaneous resolution of neutropenia within several months to a few years. In this study we examined the frequencies of regulatory T cells in various normal age groups and AIN patients to elucidate the involvement of regulatory T cells in AIN known as a representative autoimmune disease in infancy and early childhood. Blood samples from healthy neonates (n = 22), infants (1 month through 3 years in age, n = 21), adults (n = 12) and AIN patients (1 month through 3 years in age, n = 22) were obtained after informed consent. The frequencies of regulatory T cells were assessed by the expression of CD4, CD25, and FOXP3 using flow cytometry. The mRNA expressions of FOXP3 and its transcription factor, NFAT in CD4+ /CD25+ regulatory T cells were determined by quantitative real-time PCR. The frequency of CD4+ /CD25high regulatory T cells known as pure regulatory T cells fraction was highest in neonates and decreased by aging. Notably, the number of CD4+ /CD25high regulatory T cells in AIN patients significantly decreased, compared with that of age-matched healthy infants (P < 0.01) and the ratio of intracytosolic FOXP3 expression of each regulatory T cell fraction in AIN patients was lower than that of age-matched healthy infants (P < 0.05). Next, we purified CD4+ /CD25+ T cells by using FACS-Aria (more than 95% of purity) and the mRNA expressions in CD4+ /CD25+ T cells were quantified. The expressions of FOXP3 and NFAT1 mRNA in AIN patients were significantly lower than those of healthy infants (P < 0.05). The low level of FOXP3 mRNA in patients well correlated with the expression level of NFAT1 mRNA, implying that the expression of FOXP3 was mediated by NFAT1. Furthermore, the decreased frequency of CD4+ /CD25high regulatory T cells and FOXP3-positive cells in AIN patients were restored to normal levels when the neutrophil count was spontaneously recovered. These results strongly suggest that regulatory T cells play an important role in the immunopathophysiology of AIN in childhood.
Lenalidomide Is Active in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL) Carrying Unfavorable Chromosomal Abnormalities.
Authors: Ferrajoli Alessandra , Keating Michael J. , Wierda William G. , O’Brien Susan M. , Padmanabhan Swaminathan , Czuczman Myron S. , Chanan-Khan Asher A.
Published: November 2019

Genomic abnormalities, according to the Dohner hierarchical classification, are an important prognostic factor in chronic lymphocytic lymphoma (CLL). Specific chromosomal aberrations, such as a deletion 17p (P53 abnormalities) and deletion 11q (ATM defects), are associated with aggressive disease and inferior response to purine analogue-based chemotherapy. Lenalidomide is an immunomodulatory agent that has demonstrated clinical efficacy in patients with relapsed or refractory CLL in 2 separate phase II clinical trials (Chanan-Khan et al. JCO 2006; Ferrajoli et al. Blood 2006 abst). To investigate the clinical activity of lenalidomide in patients with high-risk cytogenetics (17p or 11q abnormalities), we reviewed data from 2 clinical studies. On these studies, lenalidomide was given orally either at 10 mg daily for 28 days followed by 5 mg increments every 28 days to a maximum dose of 25 mg (study A) or given at 25 mg on day 1–21 of each 28 day cycle (study B). The presence of deletion 17p or deletion 11q was demonstrated by interphase fluorescence in situ hybridization (FISH) analysis. Clinical responses were assessed using the NCI-WG 1996 criteria. Among the 80 patients treated, 40 patients with deletion 17p or deletion 11q were identified. Their characteristics are described in Table 1.

The overall response rate in patients carrying deletion 17p or 11q was 35% (14/40) and responses are depicted in Table 2. Median response duration was 12 months in both studies.

Based on our experience, single agent lenalidomide induces complete and partial responses in patients with unfavorable genomic aberrations, a group characterized by a poor outcome in several studies. The activity of this agent warrants further evaluation in this patient population.

Table 1. Characteristics of 40 patients with unfavorable cytogenetics Study A Study B N=24 N=16 Median age, years (range) 63 (49–86) 62 (56–75) Rai stage III/IV 13 (54%) 4 (25%) Median no. of prior therapies (range) 3 (1–15) 3 (1–10) Median beta 2 microglobulin (range) 4.5 (1.9–10.1) 4 (2–10) Table 2. Responses according to NCI-WG criteria in 40 patients with unfavorable cytogenetics. Response Study A Study B 17p- (N=8) 11q- (N=16) 17p-/11q- (n=24) 17p- (N=6) 11q- (n=10) 17p-/11q- (N=16) CR, n 0 2 2 0 1 1 PR, n 1 4 5 3 3 6 OR, n (%) 1 (13) 6 (38) 7 (29) 3 (50) 4 (40) 7 (44)
Alemtuzumab Increases Serious Infections in Patients with Previously Untreated Chronic Lymphocytic Leukemia (CLL) Receiving Fludarabine-Based Therapy: A Comparative Analysis of 3 Cancer and Leukemia Group B Studies (CALGB 9011, 9712, 19901).
Authors: Morrison Vicki A. , Peterson Bercedis L. , Rai Kanti R. , Byrd John C. , Larson Richard A.
Published: November 2019

Infections remain a critical issue in the care of CLL patients (pts). The specific agents used and their resultant immunosuppression impact the spectrum of infection. We compared the infectious complications reported in previously untreated CLL pts enrolled on 3 sequential CALGB treatment trials. On CALGB 9011, 188 pts received single agent fludarabine (F) therapy. On CALGB 9712, 104 pts received concurrent or sequential fludarabine plus rituximab (FR). On CALGB 19901, pts were to receive 4 cycles of F, followed by 6 weeks of alemtuzumab consolidation (FA); of the 85 enrolled patients, 59 proceeded to alemtuzumab consolidation. Infections requiring hospitalization and/or parenteral antibiotics were tabulated. The 85 pts on study 19901 (FA) had significantly more of these infections during protocol therapy (32/85, 38%) compared to the 188 pts receiving F on study 9011 (43/188, 23%, p=0.01). Similarly, the 19901 study pts also had more infections than the 104 FR-treated pts on study 9712 (21/104, 20%, p=0.0007). Among the 85 pts on study 19901, 14 (16%) had infections only during the F phase of therapy, 14 (16%) had infections only during alemtuzumab consolidation, and 4 (5%) had infections during both phases of therapy. The occurrence of cytomegalovirus (CMV) and Pneumocystis (PCP) infections was also examined on these trials. No CMV and only 3 PCP infections were diagnosed among the 188 pts receiving F on study 9011. For study 9712, 3/104 pts had PCP (one on concurrent FR, two on sequential FR), and 1 pt had CMV pneumonia on sequential FR. Among pts on study 19901, one case of PCP and no cases of CMV occurred during the F phase of therapy. However, 12/59 pts developed CMV infection during alemtuzumab consolidation. Four had resolution of infection and completed alemtuzumab therapy, while 8 were hospitalized and did not complete this therapy. We conclude that the addition of rituximab to F therapy does not increase the risk or severity of infections in previously untreated CLL pts, but the use of alemtuzumab consolidation following initial F therapy does increase the overall risk of serious infectious complications, and especially CMV infections. These issues are important not only for prophylactic antimicrobial therapy and monitoring during and after therapy, but also for the potential use of alemtuzumab as a consolidation therapy to eradicate residual CLL after F treatment.
Factor VIII, a Cofactor for Accelerating Proteolytic Cleavage of von Willebrand Factor by ADAMTS13 Metalloprotease.
Authors: Cao Wenjing , Krishnaswamy Sriram , Lenting Peter , Zheng X. Long
Published: November 2019

von Willebrand factor (VWF)-cleaving protease (ADAMTS13), a 195-kDa plasma glycoprotein, preferentially cleaves “unusually large” VWF multimers and generates a range of VWF multimers with sizes from approximately 500,000 Daltons to 20 million Daltons. However, the molecular basis how ADAMTS13 prefers the larger VWF multimers to the smaller ones under physiological condition remains unknown. We hypothesize that coagulation factor VIII, which binds VWF in high affinity, may alter the susceptibility of VWF to ADAMTS13 protease. In the present study, a plasma-derived or recombinant VWF at final concentration of 150 nM was incubated for 3 min with 50 nM of recombinant ADAMTS13 in the absence and the presence of various concentrations of recombinant factor VIII (0–40 nM) in 50 mM HEPES, pH 7.5 containing 150 mM NaCl and 0.5 mg/ml bovine serum albumin under constant vortex-induced mechanic shear stress at rotation rate of 2,500 rpm. The specific cleavage products (the dimer of 176 kDa) were determined by Western blotting with rabbit anti-VWF IgG, followed by a fluorescence (IRDye800)-labeled anti-rabbit IgG and an infrared fluorescent image system. We showed that the rate of proteolytic cleavage of VWF by ADAMTS13 was markedly accelerated by an addition of recombinant factor VIII into the reaction. This augmenting effect was factor VIII concentration-dependent. The maximal augmentation in proteolytic cleavage of VWF by ADAMTS13 in the presence of 20 nM of recombinant factor VIII was approximately 10 fold over the baseline in the absence of factor VIII. The concentration of factor VIII that achieved half of the maximal effect on proteolytic cleavage of both plasma and recombinant VWF was nearly identical (approximately 3.0 nM). The B domain-deleted factor VIII appeared as efficacious as wild type factor VIII in accelerating the proteolytic cleavage of plasma-derived VWF by ADAMTS13, suggesting that the large, central B-domain of factor VIII is not required for its cofactor activity. Moreover, after being activated by thrombin, the activated factor VIII retained its cofactor activity initially, but rapidly lost its ability to enhance the proteolysis of VWF by ADAMTS13 within minutes, which was correlated with the dissociation (or inactivation) kinetics of the activated factor VIII heterotrimer. These data demonstrate that both factor VIII and activated factor VIII may be the cofactors that regulate ADAMTS13 protease function under physiological condition. The findings may provide a novel insight into how the larger VWF multimers are more susceptible to ADAMTS13 protease, likely because the larger VWF multimers are able to bind more factor VIII. The findings may also help design an optimal therapeutic regimen for treatment of thrombotic thrombocytopenic purpura.
Expression of Coagulation FVIII and FIX in Human Adipose Stromal Cells (hASC) and hASC-Derived Cells - Potential Autologous Cell Therapy for Hemophilia.
Authors: Rosen Elliot D. , Liang Zhong , Song Hui , Bischoff Daniella , Kamocka Malgorzata , Zollman Amy
Published: November 2019

Pleuripotent Adipose Stromal Cells (ASCs) have the capacity to differentiate into mesodermal lineage cells such as adipocytes, myocytes, osteocytes and chondrocytes. In addition, the ASCs can trans-differentiate in vitro into hepatogenic, neurogenic, endothelial and endocrine cells. Since hASCs are relatively easy to isolate in large quantity and have high proliferative potential, they provide a potential source of stem/progenitor cells for autologous cell therapy for the regeneration of injured tissue. The goal of this study is to isolate and engraft adipose stromal cells (ASCs) to generate new tissue capable of secreting therapeutic plasma proteins. In particular, can one engraft genetically modified ASCs in a recipient that secrete coagulation factors for the treatment of hemophilia? Hemophilia A and B result from too low levels of plasma Factor VIII or FIX activity, respectively. The aim of the project is to generate hASC derived tissue capable of secreting FVIII or FIX to support hemostasis in hemophilic mice. Unlike murine ASCs, undifferentiated human ASCs express FVIII at low but significant levels (0.03 ug/ml per 105 cells in 48 hours; ∼30% of normal human plasma FVIII level). ASCs were transfected with a plasmid expressing a modified B-domainless FVIII cDNA. Following differentiation into preadipocytes, the level of FVIII secretion increased 3-fold. In preliminary studies, expression of FIX is detectable in hASCs and the level increases following differentiation into preadipocytes. These results indicate that ASCs and preadipocytes are capable of gamma carboxylation. FIX expression was further increased following infection with a lentivirus carrying a FIX expression cassette. Since HGF has been shown to elicit a potent proangiogenic response (Cai et al., in press), hASCs predifferentated into preadipocytes were mixed in hydrogel containing HGF and injected subcutaneously into SCID mice. Oil red O staining cells were recovered in a cellular nodule at the injection site 3 weeks post-transplantation. Preliminary studies indicate that co-injection of endothelial progenitor cells (EPCs) with ASCs promotes the development of more highly vascularized nodules. In summary, hASCs and hASC-derived preadipocytes cultured ex vivo express low levels of coagulation Factors VIII and IX. These levels can be increased following transfection with FVIII or FIX expression vectors. Furthermore, transplantation of hASCs cultured in adipogenic media results in the formation of preadipocyte/adipocyte containing nodules. These results suggest it may be possible to generate tissue derived from genetically modified hASCs capable of secreting coagulation factors for the treatment of hemophilia.
The Studies in Various Murine Strains with Defects in Activation of Complement Cascade (CC) Reveal Both Pivotal and Pleiotropic Role of CC in Mobilization of Hematopoietic Stem/Progenitor Cells.
Authors: Wysoczynski Marcin , Reca Ryan , Wan Wu , Kucia Magda , Botto Marina , Ratajczak Janina , Ratajczak Mariusz Z.
Published: November 2019

We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPC) by i) immunoglobulin (Ig)-dependent pathway and/or by ii) alternative Ig-independent pathway and, as result of this, iii) several potent bioactive CC anaphylatoxins (C3a, desArgC3a, C5a and desArgC5a) are released (Blood2003;101,3784; Blood2004;103,2071; Blood2005;105,40). To learn more on the role of CC and innate immunity in this process, we compared mobilization in mice that possess defects in CC activation by i) classical pathway (C1q−/−, Ig-deficient), ii) both classical and alternative pathway (C2fB−/−) and in animals iii) that do not generate CC-derived anaphylatoxins (C3−/−, C5−/−). For mobilization, we employed G-CSF and zymosan that activate classical and alternative pathways of CC, respectively. First, we found by ELISA that CC activation in fact correlates with the level of HSPC mobilization. Next, studies in mice deficient in CC activation revealed that CC plays both pivotal and pleiotropic roles in this process. Accordingly, while C1q−/− and C3−/− mice turned out to be easy mobilizers, mobilization was very poor in Ig-deficient, C2fB−/− and C5−/− mice that demonstrate that C3 and C5 cleavage fragments differently control the mobilization of HSPC. To explain this at molecular level, we found that C3 cleavage fragments (C3a, desArgC3a) directly interact with HSPC and increase their responsiveness to SDF-1 gradient and thus prevent uncontrolled egress of HSPC from BM. It explains why C1q−/− and C3−/− mice that do not generate C3 cleavage fragments in BM release easily HSPC into circulation. In contrast, C5 cleavage fragments (C5a, desArgC5a) increase permeability of BM-endothelium and thus are crucial for the egress of HSPC from BM to occur. This explains why mice that do not activate efficient CC such as Ig-deficient, C2fB−/− and C5−/− animals are poor mobilizers. We conclude that the mobilization of HSPC is i) dependent on C activation by the classical or alternative pathway and balanced differently by C3 and C5 cleavage fragments that enhance retention or promote egress of HSPC respectively. Thus, modulation of C activation in BM may help to develop new more efficient strategies for both HSPC mobilization and their homing/engraftment.