bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced Dose Lenalidomide Is Safe and Effective in Deletion (5q) MDS Patients with Severe Renal Impairment: Report of Two Cases.
Authors: Knop Stefan , Einsele Hermann , Bargou Ralf , Cosgrove Denise , List Alan
Published: October 2019

Introduction: Lenalidomide (Len) is highly active in the treatment of MDS with deletion 5q (del[5q]) (List et al. NEJM 2006). Patients with renal impairment have often been excluded from Len studies because of potential for delayed excretion with increased risk for adverse events. We present 2 MDS patients with 5q− syndrome and renal impairment who were treated with Len at reduced dosage.

Patient 1: A 50-year-old woman presented with shortness of breath on exertion, pallor, and tachycardia. Laboratory tests showed hemoglobin (Hb) 3.9 g/dL with significant macrocytosis; white blood cell count (WBC) of 3.2 × 109/L; and creatinine 2.66 mg/dL (normal value, < 1.1 mg/dL). Bone marrow aspirate was consistent with World Health Organization 5q− syndrome. Abdominal ultrasound revealed unilateral renal agenesis. The patient required 2 packed red blood cell (RBC) units every 2 to 3 weeks. Glomerular filtration rate (GFR) was severely impaired (23 mL/min). Len was initiated at 5 mg/d and increased to 10 mg/d after 4 weeks. The patient showed a fast and sustained hematologic response, Hb of 10–11 g/dL without exogenous erythropoietin, and achieved transfusion independence within 6 weeks. Due to decreased WBC of 1.3 × 109/L, and a platelet count (plt) of 57 × 109/L, indicating thrombocytopenia, Len was adjusted to a daily alternating dose of 5 mg and 10 mg, respectively. Complete cytogenetic remission was achieved by 6 months of treatment.

Patient 2: An 83-year-old female presented with sustained anemia despite treatment with recombinant erythropoietin. Bone marrow findings and peripheral blood features were consistent with the 5q− syndrome. The patient was transfusion dependent and received a single treatment cycle with azacitidine, but developed acute renal insufficiency, accompanied by an elevation in creatinine to 4.8 mg/dL. She continued supportive care measures receiving 2 units of RBC every 4 weeks. Len was initiated at 5 mg daily. GFR at start of therapy was moderately decreased (31 mL/min). After 6 weeks of treatment the patient developed a generalized rash, occupying 30–50% of the body, which subsided with temporary therapy cessation. Laboratory studies at 6 weeks revealed a WBC of 1.58 × 109/L, with an absolute neutrophil count (ANC) of 3.5 × 109/L, Hb of 12.3 g/dL and plt of 42 × 109/L. Because of progressive decline in the patient’s Hb levels Len was resumed 5 months later at a dose of 5 mg 3 times per week. This was well tolerated. Complete cytogenetic remission was achieved after 10 months of follow-up.

Conclusions: These cases show that Len can be administered safely to MDS patients with renal insufficiency and with preserved clinical activity. In addition, the present observations suggest that Len therapy could abrogate the need for use of additional erythropoietin in these patients.
CD200 Expression May Help in Differential Diagnosis between Mantle Cell Lymphoma (MCL) and B-Cell Chronic Lymphocytic Leukemia (B-CLL).
Authors: Palumbo Giuseppe A. , Forgione Giovanna , Parrinello Nunzia , Cardillo Katia , Chiarenza Annalisa , Berretta Salvatore , Stagno Fabio , Villari Loredana , Di Raimondo Francesco
Published: October 2019

Mantle cell lymphoma (MCL) is a B-cell malignancy that shares many morphologic and immunophenotyping features with B-cell chronic lymphocytic leukemia (B-CLL). However, these two entities are characterized by a very different prognosis and response to therapies. Differential diagnosis is assessed by a panel of monoclonal antibodies (moAbs) to screen the expression of CD5, CD23 and Cyclin D1. Nevertheless, some of these antibodies work better on fresh or frozen tissues; for example, on fixed tissues (expecially by B5) CD5 and Cyclin D1 can give equivocal or even negative results. In addition, it is cumbersome to evaluate Cyclin D1 by flow cytometry. CD200 (previously referred to as OX2) is a membrane glycoprotein, belonging to the immunoglobulin superfamily, expressed on T and B lymphocytes (but not on NK cells) and it might play an immunosuppressive role. Its expression has also been reported on human myeloma plasmacells and B-CLL cells. We investigated the expression of CD200 on fresh neoplastic cells of 90 patients (82 B-CLL and 8 MCL in leukemic phase) by flow cytometry, using a mouse IgG1 anti-human moAb (MRC OX-104, BD Pharmingen, U.S.A.). In our series, CD200 was present on neoplastic cells of all 82 B-CLL (expression on 62–100% of CD5+ cells, mean 96%, standard deviation 7%). On the contrary, in MCL patients (Cyclin D1+ and/or t(11;14) FISH+) CD200 was positive in less than 20% of CD5+ cells in 3 subjects (4–18%) and totally absent in the remaining 5 (two-sided Fisher’s exact test p<0.0001). To further extend the study, we examined CD200 by immunohistochemistry on paraffin-embedded formalin-fixed lympoid tissues and Lowy-fixed bone marrow (BM) trephine biopsies from 18 B-CLL (12 lymphnodes and 7 BM samples) and 19 MCL (11 lymphoid tissues and 9 BM) patients, using a goat anti-human IgG affinity-purified polyclonal antibody (R&D Systems, U.S.A.). Again, all B-CLL neoplastic cells were positive for CD200 both in lymphnodes and in BM while all MCL cells were negative, both in lympoid tissues and in BM (two-sided Fisher’s exact test p<0.0001). Notably, in all MCL CD200-negative lympoid tissue biopsies, it was possible to observe CD200+ residual dendritic cells (useful as immunohistochemistry reaction positive control). Moreover, results were consistent in old archival samples and even in non-perfectly prepared tissues referred to our Center. We would propose to add CD200 in flow cytometry and immunohistochemistry routine panels, as it can be of diagnostic utility in distinguishing between MCL and B-CLL, in particular in mantle cell leukemia patients. Finally, these data have to be considered in view of the proposed targeted therapy with anti-CD200, to eventually exclude MCL patients.
Comparison of Gene Expression between T Cells from CLL Patients, Myeloma Patients and Healthy Individuals.
Authors: Choudhury Aniruddha , Osterborg Anders , Mashayakhi Kaveh , Mosfegh Ali , Kiaii Shahryar , Mellstedt Håkan
Published: October 2019

A unique feature of chronic lymphocytic leukemia is the expansion in absolute numbers of circulating T cells, accompanying the increase in the leukemic B cell clone. T cells of CLL patients may have a role in contributing to the microenvironment that favors the survival and progression of the malignant clone. We have looked at the gene profile of highly purified peripheral T cells from indolent CLL patients using Affymetrix global gene arrays and compared them with purified T cells from multiple myeloma patients as well as healthy individuals. T cells from five indolent, treatment-naïve CLL patients were examined for gene expression by Affymetrix-based microarray alongside T cells from 5 stage I, multiple myeloma patients and 5 age-matched healthy volunteers. Differential expression analysis revealed that 356 genes were differentially expressed by T cells from CLL patients. Based on a two-fold or greater difference, 107 genes were specifically upregulated and 247 genes were noted to be specifically downregulated in T cells of CLL patients. The highest level of upregulation was noted for the chemokines XCL1, XCL2, and the cytokine IFN-g. CCL4 and CCL5 were two other important chemokines that also were found to be specifically upregulated in T cells of B-CLL patients as well as KLF6 and TRAF1. The findings of the microarray analysis was further confirmed using quantitative real-time PCR and immunoblotting with specific antibodies. using T cells from an additional 14 CLL, 6 multiple myeloma and 10 healthy individuals. Our results indicate that T cells from B CLL patients may express a variety of genes that contribute to the creation of a microenvironment that sustains the survival of the leukemic clone and may produce soluble factors that inhibit apoptosis and facilitate proliferation of the leukemic B cells.
Quantitative Assessment of DNA Editing Enzymes in B-Cell Lymphomas.
Authors: Wang Y. Lynn , Lu Pin , Chung Sun M. , Cerutti Andrea , Song Zibo , Chadburn Amy , Tam Wayne , Cesarman Ethel , Knowles Daniel M.
Published: October 2019

Somatic hypermutation of immunoglobulin genes (SHM) is a physiological process that helps generate high affinity antibodies in germinal center B cells, and error-prone DNA polymerases are key enzymes involved in this process. Unlike polymerases that are responsible for DNA replication, error-prone DNA polymerases copy DNA with low fidelity leading to variations in immunoglobulin DNA sequences. Chronic lymphocytic leukemia (CLL) has been classified into mutated and unmutated subtypes based on the mutation status of the IGH variable region (IgVH). The latter has been associated with rapid disease progression and unfavorable outcome. Since determination of the IgVH mutations rely on direct sequencing of multiple PCR products and complex data analysis, the assay, although clinically useful, is not provided by many clinical laboratories. To determine whether error-prone DNA polymerases can be used as surrogate markers for IgVH mutation analysis, we examined the levels of several of them to see whether higher levels are associated with higher degree of IgVH mutation. We analyzed 30 CLL samples, of which 18 were unmutated and 12 were mutated. Quantitative PCR was performed to determine the expression levels of error-prone DNA polymerases mu, eta, iota, lambda and zeta. as well as DNA editing enzymes, terminal deoxynucleotidyl transferase (TdT) and activation-induced cytidine deaminase (AID). Statistical analysis revealed no differences in the levels of mRNA expression of any of these seven enzymes between mutated and unmutated cases of CLL. In addition, no differences were found between ZAP-70 positive and negative subgroups. The expression levels of the seven enzymes were then compared among CLL, follicular lymphomas (FL, n=8) and diffuse large B cell lymphomas (DLBCL, n=8). FL and DLBCL, being post-germinal center lymphomas that have undergone SHM, are expected to express higher levels of these enzymes than CLL. However, the statistical analysis revealed that CLL has significantly higher amount of mu, eta, iota, lambda, zeta and TdT expression than DLBCL. CLL also has significantly higher expression of iota, lambda, zeta and TdT than FL. In conclusion, our results suggest that DNA polymerases studied cannot be used as surrogate markers for IgVH analysis. Contrary to the common belief, the mRNA levels of DNA polymerases/DNA editing enzymes do not reflect the SHM activity in different B cell malignancies.than FL. In conclusion, our results suggest that DNA polymerases studied cannot be used as surrogate markers for IgVH analysis. Contrary to the common belief, the mRNA levels of DNA polymerases/DNA editing enzymes do not reflect the SHM activity in different B cell malignancies.
Variable Contribution of Different Monocloncal Antibodies to NK Cell-Mediated ADCC Against Primary CLL Cells.
Authors: Weitzman James , Betancur Monica , Boissel Laurent , Rabinowitz Arthur P. , Klingemann Hans
Published: October 2019

Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of the B-cell antigens CD19, 20 and 22, along with CD5 and CD23. These antigens make the malignant cells an ideal target for monoclonal antibody (mAb) therapy. Although the mechanism of action of mAbs is complex and not fully understood, one well-described action is antibody-dependent cellular cytotoxicity (ADCC). Binding of mAb to its target surface antigen initiates cytotoxicity through the interaction of the Fc portion of the mAb with the Fc receptor (FcR) on natural killer (NK) cells. This triggers release of perforin and granzymes from NK cells, and subsequent killing of the target cells. This study’s objectives were to measure ADCC of 4 different mAbs against primary CLL cells, and determine if cytotoxicity is dependent on antigen density.

Methods: Mononuclear cells from 16 patients with untreated CLL were separated by density gradient separation and served as targets. The antigen density for CD20, 22 and 23 of each patient sample was quantified by flow cytometry. Effector cells for ADCC were NK-92 cells that do not express FcR, and a high affinity Fc receptor-expressing NK-92 variant (NK92.26.5) that also expresses inhibitory receptors (i.e. KIR). A fluourochrome-based flow cytometric assay determined ADCC by subtracting NK-92 induced cytotoxicity from NK-92.26.5 induced killing. Monoclonal antibodies tested were Rituximab (anti-CD20, Biogen/IDEC), Veltuzumab (anti-CD20, Immunomedics), Epratuzumab (anti-CD22, Immunomedics), and Lumiliximab (anti-CD23, Biogen/IDEC).

Results: Mean ADCC of the four antibodies tested against 16 primary CLL cells were: 46.5% for Rituximab; 43.4% for Veltuzumab; 5.8% for Epratuzumab; 8.8% for Lumiliximab. Cytotoxicity of NK-92 and NK-92.26.5 against CLL cells without antibody ranged from 2–6%. Mean antigen density on the 16 CLL patient specimens were: CD20: 27,900 (range: 10,000–56,100); CD22: 820 (600–1300); CD23: 9870 (2340–14,800).

Conclusions: We have developed a reliable in vitro assay to measure ADCC of mAbs against CLL cells. Our results indicate that ADCC contributes to cytotoxcity of CLL in vitro, and suggest that the magnitude of ADCC depends upon the surface antigen targeted. Anti-CD20 antibodies had significantly greater ADCC than the anti-CD22 and CD23 antibodies. This pattern was consistent in all patient cells tested. A similar pattern was observed with the surface antigen density on CLL cells tested, suggesting a correlation between cell surface antigen density and ADCC. Our results also suggest that resistance of primary CLL cells toward NK-mediated killing can be overcome by monoclonal antibodies even in the presence of inhibitory KIR expression on NK cells.
B-Type Natriuretic Peptide (BNP) and Mitral Velocity E/A Ratio (MV E/A) Are Two Important Noninvasive Functional Indices of Cardiac AL Amyloidosis.
Authors: Kabos Peter , Bashir Qaiser , Chen Robert , Lindenfeld JoAnn , Myint Han , Lee Choon Kee
Published: October 2019

Cardiac AL amyloidosis is characterized as global myocardial dysfunction, oftentimes complicated by pulmonary events during stem cell mobilization and autotransplant. Although BNP or N terminal proBNP has been shown to be prognostically significant for survival following autotransplant, pathophysiologic processes of the heart for elevated levels of these molecules are unclear. In the current study, we sought to evaluate functional cardiac indices assessed by two-dimensional and Doppler echocardiography and to correlate to BNP and clinical events. Evaluations were done on 39 serial echocardiogrmas obtained between July 2006 and July 2007 from 5 patients with established cardiac AL amyloidosis as a part of systemic amyloidosis (3 primary; 2 secondary to free kappa light chain multiple myeloma). Their median age was 78 years (range 48 to 80). One patient presented with pulmonary edema initially and three others developed pulmonary edema (n = 2) or congestion with pleural effusion (n = 1) during the course of therapy. Two patients underwent melphalan-based autotransplant, and three received melphalan 50 mg/m2 up to 2 cycles every 3 months without stem cell infusion. The median BNP at diagnosis was 1110 pg/mL (range, 115 to 2540); right ventricular diastolic diameter (RVDd) 3.8 cm (2.9 to 4.0); left ventricular end diastolic volume (LVEDV) 88 mL (50 to 125); ejection fraction 60% (53 to 77); MV E/A 2 (1.3 to 2.9); right ventricular systolic pressure (RVSP) 48 mmHg (40 to 68). In multiple regression analyses, BNP levels were most significantly correlated to the RVDd, β = 0.58, p < 0.05. The RVSP as a surrogate index of pulmonary arterial pressure was shown most dependent on the left ventricular diastolic filling assessed by the MV E/A (β = 0.93, p < 0.05) that in turn was dependent on the LVEDV (β = 2.68, p < 0.05). None of the myocardial wall measurements and left ventricular indices except these were found to be significant. Although the RVDd was dependent on the RVSP (β = −1.17, p < 0.05), both BNP and RVSP were independent of each other (β = −0.25, p = 0.6). RVSP levels were significantly elevated in 4 patients who had pulmonary complications: 50, 55, 57, 68 mmHg, respectively. The MV E/A was elevated at or above 2.5 at the time of these episodes: 2.5, 2.9, 2.9, 3.7, respectively. Decrease in both BNP and RVSP appeared to be minimally related to improving serum free light chain levels following therapy: β = 0.29, p = 0.08 and β = 0.34, p = 0.05, respectively. There was no correlation between the MV E/A and serum free light chain level either, β = 0.19, p = 0.3. The data suggests that the MV E/A and RVSP may be useful indices for monitoring patients for potential pulmonary complications, and the BNP appears to reflect more of the right-sided heart events. Further studies are needed to confirm the current findings.
An Observational Post Authorization Study on the Use of Bortezomib in Multiple Myeloma Patients in The Netherlands: Results of an Interim Analysis.
Authors: Biesma Douwe H. , Lokhorst Henk M. , van der Hem Klaas , van der Heul Cees
Published: October 2019

Introduction: Bortezomib (Velcade®) is a relatively new drug in the treatment of patients with Multiple Myeloma (MM). Most data on efficacy and safety are from randomised controlled studies in selected groups of patients with MM.

Aim of the study: To evaluate safety and efficacy of bortezomib in daily practice after its registration for second and later line therapy in patients with MM.

Materials and methods: In an observational, multicentre study, the efficacy and safety of treatment with bortezomib was assessed in MM patients. An interim analysis was performed on data of the first 50 patients enrolled from November 04 until March 06.

Results: All results are given as median ± SD. The patients’ age was 65 yrs, Karnofsky Performance Score was 80%, time from initial diagnosis was 3.4 years, 62% of the patients had more than 2 previous treatment lines, 88% of the patients had disease stage III. Patients were treated with 4 cycles (95% CI: 4.0 – 5.4) of bortezomib. The patients had Response Rate (RR; based on serum M-protein values) of 64% (2.0% nCR, 2.0% VGPR, 22% PR, 38% MR). Best RR remained 64% with an increasing depth of response (2% CR, 8% nCR 6.0% VGPR, 32% PR, 16% MR); 6 patients were not evaluable for response. Time to initial response was 45 days; time to best response was 63 days. The duration of response (DOR) was 6.4 months. Also, a relation between treatment duration and DOR was found (correlation coefficient 0.4118, p-value 0.0192). Overall survival was 1.3 yrs and PFS 6.4 months. The treatment was generally well tolerated. Most common adverse events (AE’s) were in order of decreasing frequency thrombocytopenia, nausea, constipation, diarrhoea, fatigue, malaise, polyneuropathy, pyrexia, anaemia, dizziness and leukopenia. Only 6.1% of all AE’s resulted in a permanent discontinuation of bortezomib; 13% of all AE’s were reported as serious of which 54.7% were considered as grade 3 or higher (CTCAE) and at least possibly related to bortezomib in 45.3%. In one patient (2%), Herpes zoster was reported.

Conclusion: Response Rates in this study were comparable to those in other community studies and higher than reported in previous clinical studies. The somewhat shorter DOR observed versus previously reported studies might have been caused by a shorter duration of bortezomib treatment, although more data is needed to derive a final conclusion. Overall the treatment with bortezomib was well tolerated and overall survival was as expected in this patient population. The clinical significance of the observed relation between treatment duration and response duration requires further exploration.
Radioimmunotherapy for Myeloablation Before SCT in Paediatric Patients with Malignant and Non-Malignant Diseases.
Authors: Schulz Ansgar S. , Gatz Susanne , Schuetz Catharina , Blumstein Norbert , Glatting Gerhard , Kull Thomas , Reske Sven , Debatin Klaus-Michael , Friedrich Wilhelm
Published: October 2019

Radioimmunotherapy (RIT) using radio-labelled monoclonal antibodies can be used for targeted radiation of the bone marrow in the context of SCT. We explored this treatment in 32 pediatric patients with malignant and non-malignant bone marrow disorders with very high risk patterns from disease or comorbidity. For RIT the Yttrium-90 labelled anti-CD66 (n=30) or anti-CD45 (n=2) monoclonal antibodies were used. Dosimetry was performed about 3 weeks before SCT and showed favourable organ distribution. RIT was administered on an outpatient basis 12 to 15 days before SCT. The mean BM dose was 27 Gy (range 11–46) in malignant diseases and 17 Gy (range 16–20) in non-malignant diseases. Patient characteristics were as follows:

malignant diseases (n=16): 7 ALL, 8 AML, 1 Neuroblastoma °IV; disease status pre-BMT: 7 CR2, 2 CR>2, 7 PR/NR; non-malignant diseases (n=16): 12 immunologic disorders (SCID, CID, Hyper-IgM-Syndrome, Hyper-IgE-Syndrome, CGD-Syndrome, Griscelli-Syndrome, IFN-γ-Receptor-Deficiency.), 4 hematologic disorders (Thalassemia major, congenital haemolytic anaemia).

Additional risk factors were active aspergillosis (n=4), acute pneumonia (n=5), liver fibrosis (n=4) and previous transplant (n=7). RIT was followed by high dose conditioning (based on TBI or high dose Busulfan) in 9 malignant cases or by reduced intensity conditioning (based on Fludarabin ± Melphalan) in 7 malignant and all non-malignant cases. Donors were HLA-genoidentical (n=10), matched unrelated (n=15), mismatched related (n=1), mismatched unrelated (n=2) or HLA-haploidentical relatives (n=4). RIT was well tolerated in all patients and no sign of significant additional or unexpected toxicity was observed. Myeloablation was achieved in all patients. All but 2 patients, who rejected their HLA-haploidentical grafts, showed a primary engraftment within the normal time frame after SCT. Six patients died, 3 from TRM and 3 from relapse. Eleven out of 16 patients with malignant diseases survive 3–53 months (median 26 months), 15 out of 16 patients with non-malignant diseases survive 3–30 months (median 11 months). Interestingly, all but one of the 23 patients treated by RIT and reduced intensity chemo-conditioning survive (1 y estimated survival 0.9, 95% CI 0.51–0.99). Taken together, targeted immunotherapy is a promising approach for myeloablation in pediatric patients with high comorbidity and/or high relapse risk.
Syk Tyrosine Kinase Is Overexpressed in the Majority of Peripheral T- and NK-Cell Lymphomas, and Represents a Potential Therapeutic Target.
Authors: Feldman Andrew L. , Sun David X. , Law Mark E. , Novak Anne J. , Attygalle Ayoma D. , Thorland Erik C. , Fink Stephanie R. , Vrana Julie A. , Caron B. Lynn , Morice William G. , Remstein Ellen D. , Grogg Karen L. , Kurtin Paul J. , Macon William R. , Dogan Ahmet
Published: October 2019

Peripheral T- and NK-cell lymphomas (PTCLs) are fatal in the majority of patients, and conventional chemotherapy is minimally effective. Targeted inhibition of tyrosine kinases (TKs) might be a useful treatment strategy, but TK overexpression by PTCLs has not been well characterized (except ALK). Recently a novel translocation t(5;9) fusing the ITK and SYK genes was reported in a subset of unspecified-type PTCLs, associated with high SYK gene expression and Syk protein positivity. Syk is a non-receptor TK important in immunoreceptor signal transduction, providing proliferation and survival signals in a variety of hematopoietic cells. Syk is absent or expressed at very low levels in most normal post-thymic T cells, where this signal transduction role is fulfilled by ZAP70. Since Syk TK inhibitors are now in clinical trials for other diseases, we sought to determine the incidence of t(5;9) and Syk overexpression in PTCL. Tissue microarrays were constructed using triplicate cores from paraffin-embedded tissue blocks. Cases were studied using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Additional studies were performed using flow cytometry and Western blot. There were 141 PTCLs, including 35 angioimmunoblastic, 66 unspecified, 21 anaplastic large cell, 2 enteropathy-associated, 5 hepatosplenic, 5 extranodal NK/T, and 7 other. No evidence of t(5;9) was identified by FISH in 86 informative cases. Normal T-cell subsets from peripheral blood, spleen, and lymph node were negative for Syk by IHC, flow cytometry, and Western blot. Syk was positive by immunohistochemistry in 133 of 141 (94%) PTCLs. All 8 Syk negative cases were extranodal, cytotoxic PTCLs. Western blot on frozen tissue correlated with IHC results in paraffin. All 4 Syk-positive cases tested showed phosphorylation of tyrosine residues 525/526, known to be required for Syk activation. We conclude that Syk is overexpressed in most PTCLs, including the vast majority of nodal cases. Syk may substitute for ZAP70 in these tumors and provide ongoing proliferation and survival signaling. The overexpression of Syk and phosphorylation of its Y525/526 residues suggest Syk may be a suitable target for pharmacologic TK inhibition as a treatment option for patients with PTCL.
Gain-of-Function Mutations and Copy Number Increases of Notch2 in Diffuse Large B-Cell Lymphoma.
Authors: Lee Suk Young , Kumano Keiki , Nakazaki Kumi , Sanada Masashi , Yamamoto Go , Nannya Yasuhito , Suzuki Ritsuro , Ota Satoshi , Ota Yasunori , Izutsu Koji , Sakata-Yanagimoto Mamiko , Hangaishi Akira , Yagita Hideo , Fukayama Masashi , Seto Masao , Kurokawa Mineo , Ogawa Seishi , Chiba Shigeru
Published: October 2019

[Background] Signaling through the Notch receptors, triggered by the binding of ligands expressed on neighboring cells, has a key role in determining cell fate in a variety of cell lineages, including lymphocytes. The physiologic roles of two of the four mammalian Notch genes, Notch1 and Notch2, have been clarified in mouse models. Notch1 is preferentially expressed in immature T cells and is essential for specification of early hematopoietic progenitors toward the T-cell fate and for early T-cell development in the thymus. In contrast, Notch2 is preferentially expressed in mature B cells and is required for the generation of a mature B-cell subset, known as splenic marginal zone B (MZB) cells. Deregulation of Notch1 function results in the development of T-cell acute lymphoblastic leukemia (T-ALL) in a number of mouse experimental models, and more importantly, more than 50% of childhood and 30% to 40% of adult human T-ALL cases carry Notch1 mutations that lead to deregulated activation of Notch signaling, indicating that accelerated Notch signaling contributes to the development of human neoplasms.

[Results and Discussion] We screened Notch2 gene mutations at the heterodimerization and PEST domains in 109 B-cell lymphoma samples, and found mutations in 5 samples, all of which were diffuse large B-cell lymphomas (DLBCL; in DLBCL, 5 of 63 samples, ∼8%). These mutations lead to partial or complete deletion of the PEST domain, or a single amino acid substitution at the C-terminus of Notch2 protein. All five DLBCL cases with Notch2 mutations were uniformly immunohistochemically negative for CD10 and positive for BCL6 and MUM-1, whereas only 10 out of 24 Notch2 mutation-negative subjects showed this staining pattern. This observation might imply a link between Notch2 mutations and a fraction of non-germinal center B-cell-like DLBCL (non-GCB-DLBCL), according to the immunohistchemistry-based DLBCL subclassification. Interestingly, high-density oligonucleotide microarray analysis revealed that two of the five Notch2 mutation-carrying samples had an increased copy number of the mutated Notch2 allele, and in another sample of the five the total copy number of the Notch2 allele was increased. Furthermore, in the Notch activation-sensitive luciferase reporter assay in vitro, mutant Notch2 receptors showed increased activity compared with wild-type Notch2. These findings implicate Notch2 gain-of-function mutations in the pathogenesis of a subset of B-cell lymphomas, and suggest broader roles for Notch gene mutations in human cancers.
A Multi-Site, Prospective Cross-Over Study of Intranasal Desmopressin and Oral Tranexamic Acid in Women with Menorrhagia and Abnormal Laboratory Hemostasis.
Authors: Kouides Peter A. , Heit John A. , Philipp Claire S. , Stein Sid F. , Lukes Andrea S. , Byams Vanessa R. , Dowling Nicole F. , Evatt Bruce L. , Miller Connie R. , Owens Sally , Skerette Nyasha , Kulkarni Roshni
Published: October 2019

Introduction: Menorrhagia is a common health problem affecting ∼5-10% of women. The optimal management of menorrhagia among women with abnormal laboratory hemostasis is uncertain. The goal of this study was to determine the effect of intranasal desmopressin (IN-DDAVP) vs. oral tranexamic acid (TA) on menstrual blood loss (MBL) and quality of life (QOL).

Methods: In a cross-over study design, 117 consenting women 18–50 years of age with menorrhagia (defined as subjective report of heavy bleeding and a pictorial blood assessment chart [PBAC] score > 100) with a negative gynecological evaluation and abnormal laboratory hemostasis (62% platelet dysfunction, 15% von Willebrand disease, 23% other coagulation defect or combined) were randomly assigned to initial IN-DDAVP or TA therapy each for two menstrual cycles, followed by cross-over to the second study drug for an additional two cycles. MBL by PBAC was measured at baseline and after each menstrual cycle while QOL was assessed by four validated instruments: Health Related Quality of Life (HRQOL), SF-36®, Center for Epidemiologic Studies Depression Scale (CES-D) and the Ruta menorrhagia questionnaire.

Results: There was a statistically significant decrease in the PBAC for both treatments. On average, the estimated decrease in the PBAC from baseline for IN-DDAVP was −66.0 (CI = (−89.5, −42.6)) and for TA, −107.8 (CI = (−131.5, −84.1)). The decrease in the PBAC score was larger for TA with a difference of 41.8 (p-value = 0.0002, CI = (20.4, 63.2)) between the two treatments. In the multivariable analysis, the test for treatment-cycle number interaction was not significant, suggesting no carry-over effect. The test for a treatment type effect was significant (p<0.0001) suggesting a greater reduction in PBAC score with TA compared to IN-DDAVP. Both IN-DDAVP and TA generally improved QOL as assessed by each of the four instruments. Over-all, the Ruta Menorrhagia Questionnaire and the Center for Epidemiologic Studies Depression Scale measurements showed the most statistically significant improvements.

Conclusion: Both TA and IN-DDDAVP reduce MBL and improve QOL in females with menorrhagia and abnormal laboratory hemostasis.The significance of these findings are underscored by the fact that prior studies of menorrhagia treatment in these patients have not included validated QOL instruments. The marked improvement in MBL and QOL noted with TA and IN-DDAVP appears to justify hemostasis screening in women presenting with menorrhagia for whom no gynecological cause can be found.
Children with t(12;21)/TEL-AML1-Positive Acute Lymphoblastic Leukemia Exhibit a Distinct Germline Genomic Signature.
Authors: Trevino Lisa R. , Yang Wenjian , Hunger Stephen , Carroll William L. , Devidas Meenakshi , Willman Cheryl L. , Downing James , Evans William E. , Pui Ching-Hon , Relling Mary V.
Published: October 2019

High-throughput genome-wide studies have identified significant associations between inherited genetic variations and complex diseases. To delineate the heterogeneous nature of pediatric acute lymphoblastic leukemia (ALL), we performed a genome-wide interrogation of approximately 600K single nucleotide polymorphisms (SNPs) in 470 Caucasian children (using > 99% European ancestry genetic markers to define race) with newly diagnosed ALL to identify the contribution of germline SNP genotypes to ALL subtype. Our patient population included children enrolled on St Jude Children’s Research Hospital Total XIIIB and Total XV ALL protocols and the COG9906 ALL protocol. The study cohort included 47 cases with the t(12;21)/TEL-AML1 (aka ETV6/RUNX1) translocation, 116 with hyperdiploidy > 50 chromosomes, 46 with T-lineage ALL, and 139 with non-hyperdiploid B-lineage ALL (without known translocations). Using genotype data from the Affymetrix 100K and 500K Mapping Array sets and logistic regression analyses, we found that only patients with TEL-AML1-positive ALL demonstrated a distinct germline SNP signature (p = 0.043, 1000 permutations) compared to patients with other ALL subtypes. We identified 1,046 germline SNPs whose allele frequencies discriminated TEL-AML1 ALL from non-TEL-AML1 ALL (p < 0.005). For example, the odds ratio associated with the risk of TEL-AML1 vs. other ALL subtypes for the T vs. C allele at the ITPR2 SNP (rs12814812) was 3.5 (p=0.000019), and that for the G vs. A allele at the IL26 SNP (rs11570906) was 3.1 (p = 0.000111). These 1,046 SNPs represent 224 unique genes. Among these 1,046 SNPs, 28 cis SNPs were significantly associated with the mRNA expression of 19 genes in diagnostic leukemic lymphoblasts. Of these 19 genes, the gene expression of the growth factor, ANGPT2;, coagulation factor, F13A1; and the enzyme, AGA, differed significantly between TEL-AML1-positive and negative cases. We also identified one intronic SNP in each of the translocation target genes (RUNX1 and ETV6) whose genotype frequencies differed between TEL-AML1-positive and negative cases (p = 0.01 and 0.03 respectively). Conversely, SNPs in high linkage disequilibrium with previously hypothesized risk alleles in MTHFR and NQO1 did not differ in allele frequencies between the two subgroups (p > 0.05). It is well recognized that TEL-AML1-positive ALL represents a fourth of childhood cases but is exceedingly rare in adults, and hence, it is not surprising that the TEL-AML1-positive ALL subtype may be more likely to be affected by inherited genomic variability compared to other subtypes. We conclude that inherited genetic variation may contribute to risk of or pathogenesis of the TEL-AML1 subtype of childhood ALL.
Missplicing of Glycogen Synthase Kinase 3β: A Potential Mechanism of Blast Crisis Chronic Myeloid Leukemia Stem Cell Generation.
Authors: Abrahamsson Annelie , Geron Ifat , Gotlib Jason , Dao Kim-Hien , Giles Francis , Newton Isabel , Kavalerchik Edward , Durocher Jeffrey , Creusot Remi , Karimi Mobin , Jones Carol , Zehnder James , Keating Armand , Negrin Robert , Weissman Irving L. , Jamieson Catriona H.M.
Published: October 2019

Recent evidence suggests that a rare population of self-renewing cancer stem cells (CSC) is responsible for cancer progression and therapeutic resistance. Chronic myeloid leukemia (CML) represents an important paradigm for understanding the genetic and epigenetic events involved in CSC production. CML progresses from a chronic phase in hematopoietic stem cells (HSC) that harbor the BCR-ABL translocation, to blast crisis, characterized by aberrant activation of β-catenin within granulocyte-macrophage progenitors (GMP). Here we show that blast crisis CML myeloid progenitors can serially transplant leukemia in immunocompromised mice, and thus are enriched for leukemia stem cells (LSC). Genomic DNA and cDNA sequencing analysis of Wnt/β-catenin pathway regulatory genes including APC, GSK3β, axin 1, β-catenin, LEF-1, cyclin D1 and c-myc in 49 normal and 51 CML HSC, GMP and lineage-positive populations revealed that in 57% of blast crisis samples a novel in-frame splice deletion of the GSK3β kinase domain occurred in blast crisis CML GMP, but not blasts found in blast crisis or normal progenitors. CML progenitors with misspliced GSK3β have enhanced β-catenin expression as well as serial engraftment potential. We propose that chronic phase CML is initiated by BCR-ABL expression in an HSC clone but that progression to blast crisis may include missplicing of GSK3β in GMP LSC, enabling unphosphorylated β-catenin to participate in LSC self-renewal. Missplicing of GSK3β represents an unique mechanism for the emergence of blast crisis CML LSC, is a potential diagnostic indicator of disease progression and represents a novel therapeutic target in blast crisis CML. As the first description of GSK3β dysregulation in cancer, this research provides a focal point for investigating whether GSK3β missplicing also triggers β-catenin activation in other CSC.
Reduced Oxidative Stress and Enhanced Nitric Oxide (NO) Bioavailability in Transgenic-Knockout Sickle Mice Expressing Fetal Hemoglobin (HbF) Is Mediated by Decreased Sickling and Hemolysis.
Authors: Dasgupta Trisha , Fabry Mary E. , Kaul Dhananjay K.
Published: October 2019

The primary event in the vaso-occlusive pathophysiology of sickle cell disease (SCD) is polymerization of hemoglobin S under deoxygenated conditions. In SCD, sub-clinical transient vaso-occlusive events caused by red cell sickling are likely to be more frequent resulting in “reperfusion injury” that generates reactive oxygen species and results in chronic oxidative stress that will contribute to multiple organ damage. In fact, previous studies have suggested that sickling is etiologic to repefusion injury and oxidative stress (Kaul and Hebbel, JCI, 2000), although the effect of antisickling therapy on oxidative stress has not been evaluated. Increasing the levels of antisickling fetal hemoglobin (HbF) by hydroxyurea therapy markedly reduces polymer formation. HbF exerts an ameliorating effect in sickle cell disease patients both on red cells and in the prevention of multiple organ damage. Here, we hypothesize that induction of HbF by genetic manipulation (in the absence of pharmacological manipulation) will reduce organ oxidative stress by reducing sickling and hemolysis, and thereby increase NO bioavailability. To test our hypothesis, we measured activity of selected antioxidants and lipid peroxidation (LPO) in BERK mice expressing exclusively human α- and βS-globins and varying levels of HbF, i.e., BERK (<1% HbF), BERKγM (20% HbF) and BERKγH (40% HbF). Percent sickled cells in venous samples (drawn in 2.5% glutaraldehyde solution in 0.1M cacodylate buffer) showed a distinct decrease with increased %HbF (P<0.05, multiple comparisons). Consistent with maximal sickling, BERK mice showed 5.4–6.9-fold increase in LPO in various tissues (muscle, kidney and liver) compared with C57BL controls (P<0.001). In contrast, BERKγM and BERKγH mice showed a marked decrease (73% and 80%, respectively) in LPO compared with BERK mice (P<0.001). Also, activity/levels of antioxidants (superoxide dismutase [SOD], catalase, glutathione peroxidase [GPx] and reduced glutathione [GSH]) showed significant decreases in BERK mice (P<0.001–0.00001). On the other hand, BERKγM and BERKγH mice showed significant increases in antioxidant activity (P<0.05–0.0001). Induction of HbF was associated with increased levels of NO metabolites (NOx) and reduced hemolysis; the latter is in agreement with our previous observations in BERKγM mice (Kaul et al. JCI, 2004). These results strongly suggest that reduced sickling and hemolysis in the presence of HbF cause increased NO bioavailability. NO is well known to exert antioxidative effects. Thus, we show for the first time that the induction of antisickling HbF leads to an increase in NO bioavailability and a decrease in oxidative stress, and that these protective effects are mediated primarily by reduced intravascular sickling.
Sulindac Sulfide Reverts the Activation of the WNT-Signaling and Aberrant Self Renewal Induced by the AML-Associated Fusion Proteins PML/RARα and PLZF/RARα.
Authors: Steinert Gunnar , Seshire Anita , Hoelzer Dieter , Ruthardt Martin , Puccetti Elena
Published: October 2019

Leukemia-specific translocations - t(15;17), t(11;17), or t(8;21) - lead to the expression of leukemia associated fusion proteins (LAFP) such as PML/RAR, PLZF/RAR and AML-1/ETO. LAFP induce and maintain a leukemic phenotype by blocking terminal differentiation of hematopoietic progenitors and by increasing the self renewal potential of the leukemic stem cells (LSC). The key mechanims by which LAFP increase LSC self renewal is the activation of the Wnt-signaling pathway by up-regulating of γ-catenin and β-catenin at a transcriptional level. The aberrant activation of Wnt-signaling by the LAFP decisively contributes to the pathogenesis of AML. To disclose whether a “leukemic stem cell therapy” is effective we targeted the Wnt-signaling by Sulindac Sulfid (SuSu) in PML/RAR- or PLZF/RAR- (X-RAR) positive stem cell models. SuSu represents the active metabolite of Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), known to inactivate the Wnt-signaling. SuSu was used at a clinically achievable concentration of 50–100 μM. As leukemia models we used U937 cells stably expressing the X-RAR and NB4 cells. As stem cell models we used i.) the aldehyde dehydrogenase positive/CD34+/CD38- fraction of the KG-1 cells stably expressing the X-RAR; ii.) Sca-1+/lin- murine HSC retrovirally transduced with the X-RAR in semi solid medium. The amount of total γ-catenin and β-catenin and activated β-catenin was determined by immunoblotting. We report that SuSu i.) down-regulated not only β-catenin but also γ-catenin in X-RAR expressing U937 and KG-1 cells; ii.) reduced the active form of β-catenin in the presence of X-RAR; iii.) induced a high apoptosis rate in PML/RAR-positive NB4 cells; iv.) reduced the “stem cell fraction” of KG-1 cells expressing X-RAR but not of mock transfected controls; iv.) reduced the self renewal potential of X-RAR-positive HSC as revealed by a significantly reduced replating efficiency. Here we provide evidence that the exposure to therapeutically achievable dosages of a NSAID revert the aberrant activation of the Wnt-signaling by LAFP. The significant reduction of the aberrant self renewal potential of HSC in the presence of X-RAR further support that the inhibition of the aberrantly activated Wnt signaling in AML might be a valid molecular therapy approach which has to further validated in in vivo leukemia models and in a more clinical setting.
Role of CD137/4-1BB and Its Ligand in the NK Cell Mediated Immune Surveillance of Acute Myeloid Leukemia.
Authors: Baessler Tina , Krusch Matthias , Baltz Katrin M. , Schmiedel Benjamin J. , Schmetzer Helga M. , Kanz Lothar , Salih Helmut R.
Published: October 2019

NK cells play an important role in the reciprocal interaction of tumor cells with the immune system and participate in the surveillance and eradication of hematological malignancies including acute myeloid leukemia (AML). NK cell reactivity is governed by a balance of activating and inhibitory receptors including various members of the TNF receptor (TNFR) superfamily. The TNFR superfamily member CD137/4-1BB has been shown to stimulate proliferation and IFN-γ production, but not cytotoxicity of NK cells in mice. Surprisingly, yet nothing is known regarding the consequences of CD137-CD137 ligand (CD137L) interaction for NK cell reactivity in humans. In this study we demonstrate that CD56dimCD16+ but not CD56brightCD16− NK cells express CD137 upon stimulation with the activating cytokines IL-2 and IL-15 with peak expression between 48 and 60h. Furthermore, we found that 5 of 7 investigated AML cell lines and 16 of 51 (33%) primary AML cells of patients expressed substantial CD137L levels, while no CD137L expression was detected on CD34+ cells of healthy donors (n=5). CD137L expression was not restricted to a specific French-American-British (FAB) subtype, but was significantly (p<0.05, one-way ANOVA) associated with monocytic (FAB M4, M5) differentiation. In addition, no association with a particular cytogenetic abnormality or with expression of MHC class I was observed. Reverse signaling via CD137L into AML cells (n=10) significantly induced the release of the immunoregulatory cytokines IL-10 and TNF (both p<0.05, Mann-Whitney U-test). Surprisingly and in contrast to available data regarding the function of murine CD137, we found that in humans blocking CD137-CD137L interaction caused a significant increase in NK cell cytotoxicity and IFN-γ production about 50% (both p<0.05, Mann-Whitney U-test) in coculture assays with CD137L-expressing patient AML cells and AML cell lines. The inhibitory effect of CD137 on NK cell reactivity was further confirmed in cocultures of NK cells with CD137L-transfectants and by triggering CD137 with an agonistic monoclonal antibody. This indicates that CD137 mediates opposite effects in murine compared to human NK cells. Furthermore we conclude that CD137L expression substantially influences tumor immunoediting by AML cells and diminishes NK cell reactivity against AML.
A Novel Therapy of Clofarabine in Combination with Etoposide and Cyclophosphamide Shows Activity in Pediatric Patients with Refractory or Relapsed Acute Leukemia.
Authors: Hijiya Nobuko , Gaynon Paul , Fernandez Manuel , Rytting Michael , Chu Roland , Barry Elly , Thomson Blythe , Cooper Todd , Kadota Richard , Chaleff Stanley , Steinherz Peter , Abichandani Rekha , Carroll William
Published: October 2019

Clofarabine as a single agent has demonstrated activity in childhood acute leukemia. In the current phase I/II study, clofarabine was added to the widely used combination of etoposide and cyclophosphamide. The phase I component of the study is completed. Patients between 1 and 21 years old with refractory or relapsed acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML) were enrolled in phase I. A standard 3+3 design was followed to determine the safest dose when used in combination. All 3 drugs were administered via IV infusion daily for 5 consecutive days in induction and 4 consecutive days in consolidation. Patients received up to 2 induction cycles (depending on the response following cycle 1), followed by consolidation (up to a maximum of 8 cycles, including induction). The initial doses (cohort 1) were: clofarabine 20 mg/m2/day, etoposide 75 mg/m2/day and cyclophosphamide 340 mg/m2/day. Clofarabine was increased to 30 mg/m2/day in cohort 4 and 40 mg/m2/day in cohort 5 after cyclophosphamide and etoposide were escalated to their respective target dose levels (440 mg/m2/day and 100 mg/m2/day) in cohorts 2 and 3. Twenty-five patients (ALL: 20 patients; AML: 5 patients) were enrolled in the 5 cohorts, and response data based on investigator’s assessment are available on the first 22 patients (ALL: 18 patients; AML: 4 patients). The median number of prior induction regimens was 2, and 3 patients had a prior hematopoietic stem cell transplant (HSCT). Data show complete remission (CR) in 7 patients (ALL: 7 patients; AML: 0 patients) and complete remission without platelet recovery (CRp) in 6 patients (ALL: 2 patients; AML: 4 patients) for an overall response rate of 59% (ALL: 50%; AML: 100%). Four patients proceeded to HSCT after treatment. One patient in cohort 4 experienced a dose-limiting toxicity (DLT) that resolved (grade 3 elevation of lipase and abdominal pain). In addition, a second patient in cohort 4 experienced a lipase elevation that did not meet DLT criteria, leading to a cohort expansion to 10 total patients. One patient in cohort 5 (n=6) experienced a DLT of prolonged bone marrow aplasia leading to cohort expansion. Common toxicities observed in ≥20% of patients included febrile neutropenia, abdominal pain, diarrhea, nausea, vomiting, pyrexia, anorexia, hypokalemia, headache, anxiety and rash. As determined by the independent data monitoring committee, the recommended phase II doses of clofarabine, cyclophosphamide, etoposide 40mg/m2/day, 440 mg/m2/day, and 100 mg/m2/day, respectively. Encouraging preliminary efficacy results and an acceptable safety profile of the combination regimen warrants continuation of the phase II portion of the study, which is actively enrolling patients.