bluuebunny/crossref_metadata_2025_split huggingface dataset:
Superior Acute Myeloid Leukemia-Specific T Cell Responses Using Dendritic Cells Pulsed with Apoptotic Bodies, vs.Tumor Lysates or mRNA.
Authors: Xing Dongxia , Decker William K. , Li Sufang , Champlin Richard E. , MacMannis John D. , Komanduri Krishna V. , de Lima Marcos , Shpall Elizabeth J. 
Published: April 2020

Abstract
Dendritic cells (DCs), which hold promise for cancer immunotherapy have two distinct functions. Immature DCs internalize antigens; while mature DCs trigger native T cell activation. To date, no consensus has been reached concerning the optimal source of tumor antigen nor the mode of antigen uptake for efficient cross-priming of tumor specific response. Reports have demonstrated that human DCs can acquire relevant antigens and stimulate MHC class I-restricted cytotoxic T lymphocytes (CTLs) by phagocytosing apoptotic tumor cells. Apoptosis was established to be the critical trigger of cross-priming in this process. Apoptotic bodies form when cells undergo programmed cell death. In this study, three acute myelogenous leukemia in (AML) antigen preparations were compared in an autologous setting.
Apoptotic bodies, Tumor lysate Tumor mRNA
Methods: DCs were generated by CD14 magnetic selection of peripheral blood progenitor cells (PBPCs) and six days of culture in GM-CSF and IL-4. Tumor lysate was made by repeated freezing and thawing of patient AML cells (leukodepletion product &gt;95% blasts); mRNA was extracted using a kit (Qiagen); apoptotic bodies were generated with UVB irradiation of a fraction of the same AML product. The rate of apoptosis was detected using Annexin V FITC flow cytometry. The DCs were loaded with mRNA by electroporation. The DCs were loaded with lysate and apoptotic bodies by incubation. Autologous T cells stimulated with DCs loaded with the three differing antigen preparations were compared for interferon(INF)-g production in ELISPOT and proliferation assays.
Results: The DCs pulsed with apoptotic bodies; lysate and mRNA expressed similar levels of the costimulatory molecules CD80, CD86, and theDC maturation marker CD83. The internalization rate of apoptotic bodies was about 20% detected by DAPI (label the apoptotic bodies) and DC83 double staining. The lysate pulsed DC and mRNA pulsed DCs were above 90% detected by flow of trace FITC- albumin and tracer GFP mRNA from previous experiments. Yet IFN-g secretion of T lymphocytes was statistically significant higher (P&lt;0.05), using apoptotic body-loaded DC as compared to total lysate or mRNA. The T cell proliferation stimulated by apoptotic body-loaded DC was also better than that of lysate or mRNA loaded DCs (p&lt;0.05).
Conclusions: These preliminary data demonstrate that apoptotic body-loaded DCs induced better Th1 T cell responses than DCs loaded with AML lysate or mRNA. One potential advantage conferred by the use of apoptotic body loaded DCs is that presentation of intracellular proteins in tumor lysates and mRNA that are irrelevant to the induction of anti-tumor responses are eliminated, which may lead to better antigen presentation and consequently an improved Th1 response.
Telomere Length by Tel-PCR in B Cell Malignancies.
Authors: Parker Anton E. , Snell Kimberley , Davis Zadie , Oscier David Graham 
Published: April 2020

Abstract
Previous studies have shown heterogeneity of telomere length among chronic B cell malignancies and within CLL. Longer telomeres are a feature of germinal center-derived tumours and mean telomere length is shorter in unmutated than mutated CLL. The relative contributions of telomere length pre-transformation, germinal centre experience and tumour cell proliferation in determining telomere length remains uncertain. We have used a recently introduced real-time quantitative PCR method to measure the factor by which the ratio of telomere repeat copy number to single gene copy number differs between a sample and that of a reference DNA sample. We compared relative telomere length in 4 types of B cell tumour whose VH genes may be either unmutated or mutated.
In 24 cases of mutated CLL mean relative length was 4.24 (range 0.16–24.13) compared to 0.81 (range 0.1–3.1) in 21 cases of unmutated CLL, p= 0.004. However, analysis of 8 mutated and 7 unmutated CLL cases using the poor prognosis Vh3-21 gene showed no significant difference between the two subsets. In non-CLL cases, the mean relative telomere length was 0.7 in 11 cases of B-PLL, 1.9 in 10 cases of MCL and 4.06 (range 0.2–22.7) in 29 cases of SMZL. In the latter 3 disorders there was again no significant difference in mean relative telomere length between mutated and unmutated cases. However, when SMZL patients were divided into 2 groups with relative telomere lengths above or below the median, 11/14 patients with stable disease had long telomeres whereas 10/15 patients with progressive disease had short telomeres, p= 0.025. We then compared 26 cases of stage A, mutated CLL, of whom 13 had stable disease for &gt;10 years and 13 had progressive disease. There was no significant difference in telomere length at presentation between the 2 groups nor on follow up testing of samples from 7 patients from each group with a mean of 92 months between test samples. In contrast, 5 cases with stable, stage A, unmutated CLL for &gt;8years had a longer mean relative telomere length of 2.15 (range 1.29–4.12) than the cohort of 21 unmutated cases with progressive disease described above p=0.002.
These data confirm the differences in mean telomere length among different B cell tumours and between mutated and unmutated CLL, but suggest that mutational status per se does not influence telomere length, while in SMZL and unmutated CLL, rate of cell proliferation is reflected in telomere length.
Enhanced Potency of Glycoengineered Anti-CD52 Monoclonal Antibodies (MAbs).
Authors: Dyer Martin J.S. , Moser Samuel , Brünker Peter , Bird Pru , Almond Neil , Püentener Ursula , Wheat Luise M.W. , Bolam Emma , Berrie Eleanor , Grau Roger , Buckby Emma , Kennedy Ben , Stebbings Richard , Hale Geoff , Umana Pablo 
Published: April 2020

Abstract
The CD52 MAb alemtuzumab (Campath) has been licensed for Fludarabine-refractory CLL. Like most commercial therapeutic MAbs, it is produced in mammalian cells (Chinese Hamster Ovary cells) and contains fucosylated carbohydrates attached to the Fc region. In contrast, a fraction of natural human serum IgG lacks fucose on their Fc-oligosaccharides and such antibodies bind with higher affinity to the FcgRIII receptors of immune effector cells. We have produced glycoengineered versions of alemtuzumab with high levels of non-fucosylated Fc oligosaccharides, increased binding affinity to FcgRIII and increased target-cell killing potency via ADCC. Glycoengineering was achieved by stable over-expression of genes encoding carbohydrate-modifying enzymes in an industrial-grade CHO cell line. Two glycovariants were produced: CAMGlyco1, enriched in non-fucosylated, hybrid-type Fc carbohydrates and CAMGlyco2, carrying non-fucosylated oligosaccharides both of hybrid- and complex-type (in approximately equal proportions). Both glycovariants had a proportion of non-fucosylated oligosaccharides leading to markedly enhanced antibody-dependent cellular cytotoxicity (ADCC) in comparison with regular alemtuzumab. CAMGlyco1 mediated lower complement-dependent cytotoxicity (CDC) than CAMGlyco2. The activity of the two glycoforms was assessed in cynomolgus monkeys. The pharmacokinetics of both glycoforms were not significantly different from wild-type alemtuzumab. There was no increased cytokine release with either of the glycoforms. Both CAMGlyco1 and CAMGlyco2 appeared to deplete lymphocytes from both blood and lymph node better than wild-type alemtuzumab. Mean lymphocyte loss from peripheral blood following low dose treatment was 33.7%, 55.9% and 55.0% for alemtuzumab, CAMGlycol1 and CAMGlyco2, respectively (p&lt;0.003). Mean lymphocyte counts per mm3 of lymph node biopsy were 4.6x105, 3.2x105 and 3.6x105 for alemtuzumab, CAMGlycol1 and CAMGlyco2, respectively. A Phase I/II clinical trial comparing CAMGlyco1 directly to alemtuzumab in patients with relapsed CLL has been initiated. Patients receive alternating doses of wild-type alemtuzumab and CAMGlyco1 and rates of decrease in peripheral blood lymphocyte count are monitored. Preliminary results show no increased toxicity. Glycoengineering of CD52 MAbs may allow for enhanced cytolytic efficacy in tumor therapy.
Increasing the Dose Intensity of Alemtuzumab (CAMPATH-1H) in Chronic Lymphocytic Leukemia (CLL): The CAM1515 Study.
Authors: Dyer Martin J.S. , Buckby Emma , Fermahan Pam , Walewska Renata , Kennedy Ben 
Published: April 2020

Abstract
Background: The doses and timing necessary for effective cytoreduction with monoclonal antibodies remain largely unknown. The human IgG1 CD52 MAb alemtuzumab is normally given intravenously (IV) three times per week at a standard dose of 30mg, following dose escalation. However, comparable results may be observed with fewer side effects using subcutaneously (SC) administered MAb, although the pharmacokinetics of SC administered MAb are slower than IV and therapy may have to be administered on a thrice weekly schedule for up to 18 weeks to achieve maximal efficacy. Efficacy with alemtuzumab has been shown to relate directly to serum levels of the MAb. We wished to assess whether more rapid cytoreduction with acceptable toxicities might be attained with daily alemtuzumab dosing without dose escalation.
Patients and methods: Patients with CLL (either as first-line therapy or at first or subsequent relapse) received SC alemtuzumab for 15 consecutive days at a dose of 30mg daily and thereafter three times per week for up to 8 weeks. To minimize adverse skin and possible systemic reactions, dexamethasone (12mg) was given along with acetaminophen (1g) for the first two injections only.
Results: 7 patients (aged 55– 73, 6 male 1 female) have been treated comprising 3 patients with Binet stage A progressive and 4 stage C. Use of steroid premedication for the first two doses virtually abolished all acute toxicities with only one patient showing any significant cutaneous reaction. CMV PCR was performed weekly. 4 patients showed low-level CMV (up to 7000 viral copies/ml) reactivation in the absence of any symptoms. All were treated successfully with oral valganciclovir 900mg bd whilst continuing with alemtuzumab without interruption. All 7 patients showed rapid reductions of peripheral blood lymphocytes as shown in the figure. Rates of drop in lymphocye count were much more rapid with daily than with conventional thrice weekly dosing schedules. Despite the more rapid reductions in lymphocytes, no patient developed tumor lysis. All patients showed significant reduction in bone marrow infiltration after the 15 days of daily therapy although a further 8 weeks of therapy was necessary to induce CR.
Conclusions; these data indicate that daily SC dosing with alemtuzumab results in rapid cytoreduction without increased adverse events. Prolonged daily dosing may result in enhanced efficacy of alemtuzumab in CLL.
Figure Figure
Pharmacokinetic Analysis of Subcutaneous Alemtuzumab as Consolidation Therapy To Achieve MRD-Negative Responses.
Authors: Regazzi Mario , Montagna Michela , Avanzini Antonia , Scarpati Barbara , Colosimo Anna , Veronese Silvio , Tedeschi Alessandra , Miqueleiz Sara , Ricci Francesca , Intropido Liliana , Morra Enrica , Montillo Marco 
Published: April 2020

Abstract
Persistence of minimal residual disease (MRD) after therapy has been shown to be a powerful prognostic factor for relapse among patients with chronic lymphocytic leukemia (CLL). Treatment with the anti-CD52 monoclonal antibody alemtuzumab (Campath®), either as a single agent or in combination with fludarabine, has been shown to induce MRD-negative responses, even among patients who are not responsive to traditional therapeutic interventions. Dramatically improved responses and MRD negativity have also been achieved using alemtuzumab as consolidation therapy, to purge MRD after initial therapy with a fludarabine-based regimen. The pharmacokinetics (PK) of alemtuzumab administered subcutaneously to achieve MRD negativity after induction therapy with fludarabine are reported here. Of 41 patients (median age, 54 years) who received consolidation therapy with alemtuzumab, all 16 patients participating in the PK analysis were MRD-positive after receiving induction therapy as measured by PCR consensus primer. At least 8 weeks following the completion of induction therapy, dose escalated alemtuzumab 10 mg was administered for 6 weeks, 3 times a week. Plasma samples were collected from 16 patients on Days 1, 3, 5, 15, 17, 22 and 31, and ELISA was used to assess the PK of alemtuzumab. On Day 15, blood samples were drawn at 1, 2, 3, 4, 6, 8 and 12 hours after alemtuzumab administration. Alemtuzumab plasma concentrations gradually increased during the first 2 weeks, and reached concentrations 25.5-fold higher on Week 3. The median pre-dose concentrations (Cpre-dose) were: 0.01 μg/mL (range, 0–0.07 μg/mL) on Day 3, 0.58 μg/mL (range, 0–1.2 μg/mL) on Day 15, 0.51 μg/mL (range, 0–2.61 μg/mL) on Day 17, and 1.09 μg/mL (range, 0.19–3.08 μg/mL) on Day 31. The median Cmax plasma concentration was 1.05 μg/mL (range, 0.097–1.75 μg/mL) and the median Cpre-dose was 0.58 μg/mL (range, 0–1.2 μg/mL). An analysis was performed to correlate quantitative response as represented by polyclonal complete remission and AUC0–12 values. Preliminary data indicate that the systemic exposure associated with effective therapy corresponds with an AUC0–12 value higher than 5 μg•h/mL on Day 15 of the administration schedule. Therefore, complete response (CR) rates, expressed as percentage of patients with AUC0–12 values correlating with effective treatment, increased with higher levels of systemic exposure to alemtuzumab.
Rituximab® and High Dose Methylprednisolone (HDMP) as a First Line Treatment for Patients with Chronic Lymphocytic Leukemia.
Authors: Castro Januario E. , Bole Jan E. , Prada Carlos E. , Loria Olivier , Kipps Thomas J. 
Published: April 2020

Abstract
High-dose methylprednisolone (HDMP) and the anti-CD20 mAb Rituximab® each can effect partial responses in patients with chronic lymphocytic leukemia (CLL). Previously, we noted that these two agents used in combination were highly effective in treating CLL patients with relapse/refractory disease. We examined whether patients with CLL could respond to these agents when used in combination as a first line therapy. To mitigate the toxicity associated with HDMP, the dose of methylprednisolone was reduced to 60% of that used in salvage regimens. Sixteen patients with CLL were treated with three four-week cycles of methylprednisolone at 1 gm/m2 daily for 3 days and weekly Rituximab® at 375 mg/m2 for four weeks. All patients satisfied NCI-working group criteria for requiring treatment. The median age of the patients was 60 years, the male to female ratio was 4:1, the ECOG performance status was ? 2. Eighty-six percent of the patients had high-risk disease prior to therapy, as per the modified Rai classification. Fifty-six percent of the patients had CLL cells that expressed ZAP-70 and/or unmutated immunoglobulin variable region genes. Response assessment was performed at the end of each cycle, two months after completion of treatment, and each 3–6 months thereafter until the patient experienced disease progression and/or required further treatment. Objective responses were observed in 14 out of 16 patients (Overall response rate 93%), with 1 patient achieving a complete response (CR) without disease detectable in the marrow, 1 patient achieving a nodular PR, 12 patients obtaining an excellent PR only with minimal residual disease in the bone marrow, and 1 patient having stable disease, as per the NCI-working group criteria. We observed significant reductions in the white blood cell (WBC) counts, increases in hemoglobin, elevations in platelet counts, and dramatic reduction and resolution in lymphadenopathy and splenomegaly. The median time to progression (TTP) after 12 months of follow up has not been reached. Overall, the treatment was well tolerated and all but one patient completed 3 cycles of therapy. Most adverse events were Grade I-II (fluid retention, cough, transient hyperglycemia, fatigue). We also observed 2 transient episodes of grade III–IV toxicity secondary to infection, resulting in pneumonia or sinusitis that resolved completely after antibiotic treatment. Pharmacodynamic studies showed that leukemia cells from patients responding to this combination regimen were induced to express pro-apoptotic molecules, such as Bid, Apaf-1, Smac-Diablo, and to downregulate expression of anti-apoptotic molecules, such as XIAP and Mcl-1. These data indicate that modified-dose HDMP/Rituximab® may be an effective and well-tolerated first line treatment for patients with CLL.
Alemtuzumab Is Effective in Patients with Advanced, Pretreated B-Cell Chronic Lymphocytic Leukemia: A Nationwide Study of Routine Use in Austria.
Authors: Fiegl Michael , Falkner Andreas , Hopfinger Georg , Brugger Stefan , Zabernigg August , Haslbauer Ferdinand , Andel Johannes , Thaler Josef , Fridrik Michael , Bauer Franz , Mian Michael , Tatzreiter Georg , Zojer Niklas , Demirtas Dita , Postner Gerhard , Auberger Jutta , Gastl Günther , Greil Richard 
Published: April 2020

Abstract
It is often questionable as to whether or not therapeutic benefit documented in prospective studies reflects that which is observed in routine use. Four prospective studies have explored the efficacy of alemtuzumab in heavily pretreated B-CLL patients, reporting response rates (RR) between 33% to 55%, and median survival between 13–28 months. Here, we report clinical outcomes after therapy with alemtuzumab of 106 consecutive, unselected B-CLL patients treated in 25 centers in Austria. Seventy-two patients were male and 34 female, the median age was 66 years (range, 46–88 years). The majority of patients had advanced stage disease, with 58% having Rai stage IV disease. The patients had received a median of 3 prior therapies (range, 1–11). Alemtuzumab was administered according to guidelines, but in the majority of cases, dosing was individualized, according to tolerability, side effects, and economic considerations. Thus, the median duration of therapy was 7 weeks (range, 2–24 weeks), and median dose was only 390 mg (range, 11–1333 mg). Therapeutic response was determined, strictly adhering to the NCI criteria (duration of complete response [CR]/partial response [PR]/stable disease [SD] ≥ 2 months; adequate radiologic studies of all non-palpable manifestation; marrow for CR). CR, PR, and SD were achieved in 5%, 17%, and 34% of patients (RR 22%). Progressive disease occurred in 34%, and responses were not evaluable in 9%, due to early death. In the 59 patients with overall CR/PR/SD, peripheral blood responses were as follows: 63% CR, 25% PR, and 12% SD. Survival times were encouraging and comparable with other studies. The median survival for all patients was 19 months; 15 months for fludarabine-refractory patients (n = 67) and 31 months for fludarabine-sensitive patients (n = 24; P = 0.04). Survival was dependent on response (P = 0.0001), the number of previous therapy lines (0–3 vs &gt;3; P = 0.0001), and the presence of bulky disease (P = 0.005). Route of administration (IV: 49%; IV to SC 38%; upfront SC 13%) did not influence outcome. Toxicities were within the expected range, with grade 3/4 infections in 37% of patients; grade 4 neutropenia and thrombocytopenia in 25% and 23% of patients, CMV reactivation in 14% (CMV disease in 3%) of patients, and early death (&lt;2 months after last dose) in 12% of patients. We conclude that in the routine clinical setting, alemtuzumab is a safe and effective treatment with profound prolongation of survival in pretreated B-CLL patients. This retrospective study is an important contribution to continuous, nationwide quality control.
The Novel Cancer Drug Seliciclib Engages the Mitochondrial Apoptosis Pathway Via the Mcl-1/Noxa Axis in CLL.
Authors: Hallaert Delfine , Spijker Rene , Derks Ingrid A.M. , Jak Margot , de Goeij Bart , de Boer Jan Paul , de Jong Daphne , Green Simon R. , van Lier Rene , Eldering Eric , Van Oers Marinus H.J. 
Published: April 2020

Abstract
Seliciclib (cyc202, R-roscovitine) is a novel cyclin dependent kinase inhibitor currently in pre-clinical testing. It indirectly inhibits RNA polymerase II and has been shown to be effective against B-cell malignancies by inducing apoptosis and Mcl-1 degradation. The aim of this study was to dissect the underlying apoptotic pathway(s) of seliciclib in chronic lymphocytic leukemia (B-CLL). Treatment of CLL cells with &gt;10 μM seliciclib in vitro induced apoptosis within 24 hours, irrespective of IgVH mutation status (n=20). Gene profiling by means of RT-MLPA, did not reveal involvement of p53 as indicated by the absence of Puma upregulation. None of the &gt;30 other apoptosis genes tested were induced; instead signals for certain labile mRNAs (Mcl-1, A1/Bfl-1, PI-9) were clearly decreased upon seliciclib. Detailed investigation of B cell lines overexpressing various anti-apoptotic proteins (Bcl-2, caspase-9-DN, Flip, FADD-DN), indicated that seliciclib activated the mitochondrial but not the death receptor pathway. Neither mitochondrial activation nor the rapid degradation of Mcl-1 protein could be prevented by caspase inhibition (zVAD) or overexpression of inactive caspase-9 (DN). This indicated that Mcl-1 decline is an upstream, caspase-independent event. Immuno-precipitation demonstrated that pro-survival Mcl-1 is engaged by pro-apoptotic Noxa and Bim. The specific contribution of Noxa was confirmed by RNAi, resulting in inhibition of apoptosis after seliciclib, but not after staurosporin, CD95- or BCR triggering.
These findings demonstrate that seliciclib induces rapid, p53-independent apoptosis via Mcl-1 degradation and mitochondrial activation. The involvement of Noxa, which is highly expressed in CLL, suggests this BH3-only protein may be a novel therapeutic target.
Comparison of Viral Vectors for Gene Transfer into CLL Cells: Efficient Transduction with Adeno-Associated Virus-8 (AAV-8).
Authors: Jewell Andrew P. , Cochrane Melanie , McIntosh Jenny , Benjamin Reuben , Nathwani Amit 
Published: April 2020

Abstract
Chronic Lymphocytic Leukaemia (CLL) remains largely incurable despite recent advances in therapy, and therefore alternative strategies are of interest in treating this disease. One such alternative is the use of gene therapy, but this relies on developing efficient gene transfer technologies. We have compared several viral vectors coding for green fluorescent protein (GFP) for their ability to transduce CLL cells. Three serotypes of adeno-associated virus (AAV) were used, AAV-2, AAV-5 and a relatively new isolate AAV-8, an EI-EIII deleted adenoviral 5 based vector, AV-5, all with GFP regulated by the CMV promoter, and a VSVG pseudotyped lentiviral vector in which GFP expression is controlled by EF1a promotor/enhancer complex. AV-5 resulted in variable GFP expression, 24.1±3.4%, n=10 but caused cell death at high multiplicities of infection (MOI). The lentiviral vector resulted in GFP expression of 23.5±2.6%, n=12, at the highest titre used, and expression declined in a distinct dose-dependent manner as titres were reduced. Of the AAV vectors, AAV-8 was the most efficient with GFP expression at 41.3±1.0% n=14. We conclude that AAV-8 is a promising viral vector for efficient transduction of CLL cells.
Figure 1. Percentage GFP expression for three viral vectors. Three different MOI’s were used at log dilutions. Figure 1. Percentage GFP expression for three viral vectors. Three different MOI’s were used at log dilutions.