bluuebunny/crossref_metadata_2025 huggingface dataset:
Specificity of Lytic Factors for Erythrocytes, Leukocytes and Platelets in a Case of Pancytopenia
Authors: MATOTH Y. , ELIAN E. , NELKEN D. , NEVO A. C.
Published: October 2019

A case of chronic idiopathic pancytopenia in a young girl is presented, in which the pancytopenia was shown to be due to increased destructions of all 3 blood cell types. Antileukocyte and antiplatelet antibodies were demonstrated by transfusion methods as well as by in vitro agglutination, while differential agglutination provided evidence of a plasma factor causing increased red cell destruction.

Cross absorption experiments demonstrated the presence in the patient’s serum of at least 2 separate and distinct antibodies, specific for leukocytes and platelets respectively.

Observations on the phagocytic behavior of leukocytes and on the electrophoretic mobility of leukocytes and platelets exposed to the patient’s serum are reported.
Paroxysmal Nocturnal Hemoglobinuria
Authors: NUSSEY A. M. , DAWSON D. W.
Published: October 2019

1. A fatal case of P.N.H. in a woman of 65 is presented.

2. The subject of P.N.H. is briefly discussed, and

3. The involvement of the bone marrow in the disease process is stressed.
Hemorrhagic Disease with Circulating Inhibitors of Blood Clotting: Anti-AHF and Anti-PTC in Eight Cases
Authors: LEWIS JESSICA H. , FERGUSON JOHN H. , ARENDS TULIO
Published: October 2019

In eight patients suffering from severe hemorrhagic disease it was possible to demonstrate the presence of circulating inhibitors, not heparin-like in nature. Anti-PTC was identified in two PTC deficients, anti-AHF in 5 hemophilics and both, primarily anti-AHF, in one idiopathic case. Possible etiologic factors inducing the appearance of such inhibitors included repeated transfusions of whole blood and plasma in all and a previous pregnancy in the idiopathic case. Two patients showed increases of gamma-globulin and presence of inhibitor in 25-33 per cent ammonium sulfate plasma fractions. Cortisone therapy was ineffective in two cases.
Rituximab Combined with Intravenous or Oral Fludarabine for Previously Untreated Mucosa Associated Lymphoid Tissue (MALT) Lymphoma Patients.
Authors: Salar Antonio , Domingo-Domenech Eva , Estany Cristina , Canales Miguel , Servitge Octavio , Gallardo Fernando , Pedro Carmen , Montalban Carlos
Published: October 2019

Background: Fludarabine as a single agent or in combination with chemotherapy has shown activity in MALT lymphoma. Our ex vivo preliminary results indicate a synergistic antitumor effect on MALT cells with combination of fludarabine and rituximab. The addition of rituximab to other drugs has improved outcomes in several types of NHL without a significant addition of toxicity.

Methods: This study enrolled adult patients with untreated MALT lymphoma who were candidate to receive systemic treatment. Patients received rituximab 375 mg/m2 intravenously (IV) on day 1 and fludarabine 25 mg/m2 (IV) given on days 1–5 (days 1–3 in > 60 years), every 4 weeks; after the first cycle, oral fludarabine was allowed to be given orally at 40 mg/m2 with the same schedule. After three cycles, a work-up was done. Patients in CR received an additional cycle and, if PR, a total of 6 cycles was recommended. Use of G-CSF and prophylactic antibiotics was optional.

Results: Characteristics of all 18 pts: median age: 59 years (range: 32–83); 7 male, 11 female; PS 0 (94%); site of lymphoma origin: stomach (61%), skin (16%), lung (11%), parotid gland (11%); stage: I (66%), II (16%) and IV (16%). A total of 82 cycles of R-F were administered; 2 pts received 2 cycles, 9 pts 4 cycles, 7 pts 6 cycles. 17 pts are evaluable for response. Overall response rate was 100% with 94% achieving CR. In the pt in PR, symptoms resolved and received no further treatment. One pt with parotid gland involvement at dx relapsed at 6 m from the end of treatment in two unaffected areas at dx (breast and bone marrow). Median follow-up from starting treatment is 15 m (range: 17–27). PFS rate is 93% (CI95%: 79–100%) at 12 m and OS rate is 100% at 12 m. Tolerance to oral fludarabine was excellent with many patients preferring this formulation. Mild neutropenia was the most common toxicity, usually presenting after the third cycle. 1 pt developed a prolonged grade 4 neutropenia after the 6th cycle. No blood transfusions were required. 3 pts developed grade 2 respiratory infection, but none pt had to be admitted.

Conclusions: These preliminary data indicate that the RF regimen, either with intravenous or oral fludarabine, was well tolerated even in elderly patients. This combination is very active for the treatment of untreated MALT lymphoma, even with fewer cycles than initially planned. A phase II national multicenter study is being planned.
Single Institution Outcomes for Early Stage Follicular Lymphomas Treated with Radiation Therapy Alone as Initial Therapy.
Authors: Owen Tymothy E. , Williams Gaynor , Nugent Zoann , Rubinger Morel , Ahmed Naseer
Published: October 2019

Objectives: Less than 10% patients with follicular lymphoma (FL) have stage I or II disease and radiation therapy is frequently used as the initial treatment modality. There is limited data on the outcomes of such treated patients. This study was undertaken to evaluate the outcome of patients with limited follicular non-Hodgkin’s lymphomas (NHL) at our institution treated with radiation therapy (RT) alone as initial management.

Methods: Demographic, histological subtype and treatment factors were retrospectively reviewed from charts of patients treated at CancerCare Manitoba between 1982 and 2003. Thirty-four patients with stage I–II, grade I–II FL treated initially with RT at our institution within the specified time period were analysed.

Results: The median follow up time was 7.4 years. RT was delivered using involved or extended field techniques. Doses ranged from 15 Gy to 48 Gy, with a median dose of 35 Gy. The 5 and 10 year overall survival (OS) for all patients is 80% and 64%, respectively. Twelve patients had recurrences, with a median time from radiation to recurrence of 1.43 years. The majority of recurrences were outside the initial treatment field (93%). Patients with stage I versus II disease have had a 5 year freedom from recurrence of 79% and 45%, respectively (P=0.03). Those patients < 60 years versus ≥ 60 years have an OS of 83% and 78%, respectively (P=0.03). Hemoglobin levels and LDH did not predict survival within this data set.

Conclusions: After RT alone the 10 year freedom from recurrence may be as high as 52% in patients with early stage FL. There was a significant improvement in OS with age <60. There is also improvement in time to recurrence with Stage I versus Stage II disease. This data is consistent with previously published data on RT for early stage FL.
Safety of Intrathecal Liposomal Cytarabine (Depocyte®) Injection Given Prophylactically in Patients with High-Risk Diffuse Large B-Cell Lymphoma: A Report of 22 Patients in Spain.
Authors: Canales Miguel A. , Salar A. , Diaz J.A. , Ferreiro J.J. , Ferrer S. , Llorente A. , Caballero D. , Garcia-Marco J.A. , Palomera L. , Sanchez-Blanco J.J. , Gonzalez-Barca E.
Published: October 2019

Patients with diffuse large B-cell lymphoma (DLBCL) who develop lymphomatous meningitis (LM) during or after first-line treatment have a poor prognosis, with CNS relapse occurring within 1 year in approximately 80%. Risk factors have been defined and prophylaxis is recommended in patients with high-risk DLBCL. Liposomal cytarabine (DepoCyte®), a sustained-release preparation of cytarabine for intrathecal (IT) injection, has been shown to be well tolerated and effective in the treatment of LM. Its long CSF half-life allows liposomal cytarabine to be given less frequently than conventional therapy, reducing discomfort for patients and the risks associated with repeated lumbar punctures. The potential of liposomal cytarabine to improve the outcome of prophylaxis against LM is being investigated into a multicenter and prospective trial in patients with high-risk DLBCL in Spain. Preliminary safety results are reported in 22 patients (median age 67 years; range 18–79; 14 male) with newly diagnosed DLBCL at high risk of developing LM (defined as the presence of at least one of the following criteria: retroperitoneal mass >10 cm, Waldeyer’s ring or paranasal involvement, involvement of >30% bone marrow, testicular involvement) who received prophylactic IT liposomal cytarabine during treatment with R-CHOP14 regimen between June 2006 and July 2007 at 10 centers in Spain. Liposomal cytarabine 50 mg was administered during the first day of treatment at first, second and sixth cycles of R-CHOP14 scheme (study days 1, 15 and 71). The median number of doses administered was 3 (range 1–3). Seventeen patients received corticosteroid as prophylaxis for chemical arachnoiditis: 16 dexamethasone (4 mg IT [n = 9] or PO at varying dosages [n = 7]); and 1 patient received IT hydrocortisone (20 mg). The remaining patients did not receive specific corticosteroid prophylaxis for chemical arachnoiditis. Overall, liposomal cytarabine was well tolerated. Six patients experienced minor side effects including headache (Grade 1/2, n = 4; grade 3/4, n = 2) and nausea/vomiting (Grade 3/4, n = 1). No signs of neurological progression or relapsed were observed. These preliminary observations indicate that IT injection of liposomal cytarabine (DepoCyte®) is well tolerated and can be administered safely in combination with dose-dense regimens. Longer-term follow-up will be needed to confirm these encouraging observations.
Radioimmunotherapy (RIT) of Refractory or Relapsed Hodgkin’s Lymphoma (HL) with 90Yttrium-Labelled Antiferritin Antibody.
Authors: Decaudin Didier , Lévy Rafaël , Lokiec François , Madar Olivier , Brossel Rémi , Morschhauser Franck , Songeur Valentine , Djeridane Malika , Kadouche Jean , Pecking Alain
Published: October 2019

The aim of this study was to evaluate the safety and efficacy of radiolabelled DTPA-chelated rabbit polyclonal antiferritin antibody (Ab) in relapsed or refractory HL. The protocol included a first intravenous injection of 111Indium-labelled antiferritin Ab followed by immunoscintigraphy at 4, 48, and 72 hours and intravenous injection of 90Yttrium-labelled antiferritin Ab in the case of tumour targeting. Ten patients were included in the study: median number of chemotherapy regimens: 3; number of autografted pts: 8; number of previously irradiated pts: 9; response to last chemotherapy: 6 PR and 4 progressions. All immunoscintigraphies showed tumour targeting. Nine patients were treated, as the last patient died from progressive HL before therapeutic injection. Median injected activity was 12 MBq/kg (0.32 mCi/kg). Among the ten patients who were included in the study, 1 CR and 6 PR were observed (ORR 70%) with a median duration of response of 8 months (range: 7–12 months). Toxicity was mainly haematological, with grade 1 or 2 neutropenia and anaemia, and grade 2 and 3 thrombocytopenia. The pharmacokinetic study showed that the half-lives of 111Indium and 90Yttrium were almost identical. These results confirm those previously reported in the literature and show the therapeutic potential of rabbit polyclonal antiferritin Ab in relapsed or refractory HL. On the basis of all these results, MATBioPharma proposed to test a radioimmunotherapy with polyclonal antiferritin antibodies (Abs) in patients with refractory or relapsed HL. The treatment is constituted with chelated rabbit polyclonal antiferritin Abs to be loaded with 111Indium for the diagnosis of the tumour(s) by immunoscintigraphy and with 90Yttrium for the treatment of the tumour(s). A phase I study is still ongoing at the Institut Curie / Centre René Huguenin (France) to evaluate the safety and tolerability of ascending doses of 90Yttrium antiferritin until the maximum tolerated dose (MTD) is reached and to select a dose for further investigation (one dose step below MTD). A pharmacokinetics is concomitantly performed to determine dose linearity and pharmacokinetic parameters of increasing 90Y-Ab and Ab. The second dose level will be completed in the third quarter 2007 and available data on immunoscintigraphy, safety, and efficacy of included HL patients will be provided for the 49th ASH congress.
Low Expression of hOCT1 May Be a Key Point Mediating Low Intracellular Imatinib Accumulation in Imatinib Mesylate Resistance K562 Cell Line.
Authors: Zhu Huanling , Liu Ting , Jia Yongqian
Published: October 2019

Objective To establish an imatinib resistance cell line and to study its resistant principia.

Methods

K562 cells were cultured in imatinib at gradually increased concentrations to generate their resistance cell line. Clone imatinib resistance cell lines by limited dilution culture. MTT assay, real time PCR and Semi-quantity PCR, flow cytometry and HPLC were used to clarify the possible mechanisms of the resistance.

Results

Imatinib resistance cell line K562R was successfully induced by continuous culture in the presence of gradually increasing doses of imatinib up to 5μmol/L. K562R cells were maintained in the media containing 5μmol/L imatinib. Proliferation data showed that cell growth of K562R was not inhibited in 5 μmol/L imatinib, whereas the parental sensitive cell was significantly inhibited by up to 2μM imatinib. The IC50 of K562R was about 7.5μmol/L which was ten times higher than that of the parental cell. HPLC revealed that the intracellular imatinib concentration of K562R was strikingly lower than that of the parental cells (up to 27.8-fold).

MDR1 were not detected in mRNA (by RT-PCR)and protein(by flow cytometry) levels on K562R cell, whereas hOCT1 level measured by semi-quantity PCR showed lower expression in K562R cell lines than that of parental sensitive cell, indicating that low intracellular imatinib concentration may be due to lower affluence of imatinib by low level of hOCT1. (5) Real time PCR analysis showed no BCR-ABL/G6PD gene amplification and sequence analysis of the 374bp ABL kinase domain showed no mutation in K562R cell lines.

Conclusion An imatinib resistance cell line K562R has been successfully established. Low expression of hOCT1 may be a key point mediating low intracellular imaitnib accumulation in K562R cell lines.
Multicenter Experience with Imatinib Mesylate in Newly Diagnosed Chronic Myeloid Leukemia (CML) Patients.
Authors: Carella A.M. , Lerma E. , Orlandi E. , Lazzarino M. , Annunziata M. , Ferrara F. , Pungolino E. , Morra E. , Miglino M. , Gobbi M.
Published: October 2019

Imatinib mesylate (IM) is a powerful and selective p210 Bcr-Abl tyrosine kinase inhibitor that has been demonstrated to be effective for the treatment of CML. An Italian multicenter study was performed to investigate the safety, efficacy, tolerance and compliance of IM in newly diagnosed CML patients. From February 2000 through February 2007, we collected 339 CML patients in different phases of disease and previously treated with Interferon-alpha and/or other therapies. One hundred fifty-two untreated Ph+ CML patients received Imatinib at diagnosis at a dose of 400 mg orally per day; 88 were male and 64 female with a median age of 50 (range, 17–80) years. Complete hematological remission was achieved in 94% of patients. The estimated rate of complete cytogenetic remission (CCR) was 69% after 12 months; with a median follow up of 38 months the rate of CCR raised to 86,8%. Levels of Bcr-Abl transcripts had fallen by at least 3 log in 59% of cytogenetic remitters patients at 12 months; after a median follow up of 38 months, 26% of these patients achieved complete molecular remission (CMR). The most commonly reported adverse events after IM were edema (including peripheral and periorbital edema) (50%), muscle cramps (37,7%), diarrhea (40,7%), fatigue (38,8%); moreover in 8 (5,2%) patients grade 3–4 events were observed consisting of pancitopenia and/or elevated liver enzymes. Eight (5,2%) patients died: 2 patients for progressive disease after allografting, 2 patients of cardiac infarction, 1 patient of severe necrotic fascitis, 1 patient of metastatic colon cancer and 2 patients of blastic crisis. In summary, at 84 months 144/152 (94,7%) patients are alive and 125 (86,8%) of them are still receiving IM. Nineteen (13%) patients discontinued Imatinib: 7 patients for adverse events grade 3–4 and 12 patients for progressive disease. These data are comparable to those of IRIS study and confirm the safety and efficacy of IM in newly diagnosed patients.
BRAF Mutations in Juvenile Myelomonocytic Leukemia.
Authors: de Vries Andrica C.H. , Stam Ronald W. , Kratz Christian , Zenker Martin , Haas Oskar A. , van Wering Elisabeth , Zwaan Christian M. , Locatelli Franco , Zecca Marco , Hasle Henrik , Stary Jan , Niemeyer Charlotte Marie , Van Den Heuvel M.M.
Published: October 2019

Approximately 75% of patients with juvenile myelomonocytic leukemia (JMML) harbour mutations in PTPN11, NF1 and RAS genes. The remaining cases presumably carry somatic mutations in other genes in the RAS pathway. BRAF plays a central role in this pathway between RAS and downstream molecules including MEK and ERK. BRAF mutations frequently occur in cancer. Recently, BRAF mutations were found in leukemia. Besides that, germline BRAF mutations cause cardio-facio-cutaneous syndrome, which shares many features with Noonan syndrome (NS). NS predisposes to a myeloproliferative disease resembling JMML. In 65 JMML patients screening for V600E mutations in exon 15 of the BRAF gene was performed from mononuclear cells. In a subset of patients, without RAS or PTPN11 mutations, and no clinical signs of NF1, the entire coding sequence of BRAF was analyzed. Sequence analysis was performed by direct, bidirectional sequencing of purified polymerase chain reaction products. In none of the 65 cases a V600E mutation of the BRAF gene was found. In a subset of patients in which the entire coding sequence of BRAF was analyzed, no mutations were identified either. Mutant proteins of the RAS-RAF-MEK-ERK pathway play an important role in the pathogenesis of JMML, resulting in GM-CSF hypersensitivity. In about 75% of the JMML cases these mutations affect RAS, NF1 or PTPN11 genes. The hypothesis for this study was that BRAF might play an important role in JMML as it is an important downstream effector of RAS. Our data show that apparently BRAF mutations do not play a role in JMML. Therefore, additional analysis of genes of the RAS pathway will be necessary to identify genetic aberrations in cases without known mutations.
T Cell Mediated Autologous Cytotoxicity Against Hematopoietic Precursor Cells in Low and Intermediate-1 Risk Myelodysplastic Syndrome.
Authors: Chamuleau Martine E.D. , Ing Theresia M. Westers , van Dreunen Linda , Groenland Judith , Ing Adri Zevenbergen , Eeltink Corien , Ossenkoppele Gert J. , Van de Loosdrecht Arjan A.
Published: October 2019

The immune system plays a role in the pathogenesis of myelodysplastic syndrome (MDS) but its precise contribution to disease development and control is not fully clarified. T cell activation could reflect undesired autoimmune reactions against normal hematopoietic precursor cells as well as effective immune-surveillance against dysplastic clones. We have investigated lymphocyte subsets and activation markers of 42 low risk and intermediate-1 risk MDS patients and compared them to those of intermediate-2 risk, high risk MDS patients, and healthy donors. In low and intermediate-1 risk MDS patients, we have found an activated state of lymphocytes, determined by increased percentages of effector T cells with cytotoxic profile, increased number of clonal expansions, increased frequencies of Wilms’ Tumor 1 (WT1) specific lymphocytes and decreased regulatory T cell activation, as compared to healthy donors. Moreover, we demonstrate autologous T cell mediated cytotoxicity against aberrant hematopoietic precursor cells. These findings provide evidence for the existence of immune-surveillance in the pathogenesis of low and intermediate-1 risk MDS patients. Our data are important for adequate evaluation of the role of immune-modulatory drugs in the future and justify the reconsideration of the role of immune-suppressing therapy for low and intermediate-1 risk MDS patients.
Leukemogenesis by Truncated CBFβ-SMMHC Defective in Repressing RUNX1.
Authors: Kamikubo Yasuhiko , Garrett-Beal Lisa , Liu Pu Paul
Published: October 2019

Chromosome 16 inversion, inv(16)(p13q22), is one of the most frequent chromosome abnormalities in human acute myeloid leukemia (AML), comprising almost 100% of subtype M4Eo and up to 15% of all AML. This inv(16) leads to an in-frame fusion of CBFB and MYH11 genes. CBFB-MYH11 encodes a fusion protein between CBFβ, which is an obligate partner of RUNX1 or AML1, and smooth muscle myosin heavy chain (SMMHC). Using knock-in mouse models we have previously demonstrated that Cbfb-MYH11 dominantly blocks Runx1/Cbfb function in hematopoiesis and predisposes mice to AML (requiring a second hit from ENU or retroviral mutagenesis). However, the molecular mechanism underlying these findings remains unclear. Current hypotheses, which are based on previous in vitro studies, focus on the ability of CBFβ-SMMHC to dominantly inhibit RUNX1/CBFβ and include

CBFβ-SMMHC binding RUNX1 with higher affinity than CBFβ; CBFβ-SMMHC sequestration of RUNX1 in the cytoplasm; CBFβ-SMMHC stabilization of RUNX1 by decreasing RUNX1 ubiquitination; and CBFβ-SMMHC repression of RUNX1 transactivation, which is dependent on SMMHC multimerization and repressor recruitment.

To test these hypotheses in vivo, we generated knock in chimeric and F1 heterozygous mice expressing CBFβ-SMMHC with C-terminal and internal deletions. One of the deletions removes only the domain responsible for high affinity binding of RUNX1 (HABD). The CBFβ-SMMHC protein with HABD deletion did not bind RUNX1 with higher affinity than CBFβ and was hypothesized to be unable to dominantly repress RUNX1. Consistent with this hypothesis, the HABD-deleted protein was less efficient in sequestering RUNX1 and caused less severe hematopoietic defects in F1 embryos than full length CBFβ-SMMHC. In contrast to mice expressing full length CBFβ-SMMHC, which develop AML only after ENU or retroviral mutagenesis, most HABD-deleted chimeric and all HABD-deleted F1 mice developed AML spontaneously shortly after birth. A larger deletion removed both the HABD and the RUNX1 stabilization domain (RSD). CBFβ-SMMHC with the HABD-RSD double deletion did not bind RUNX1 with high affinity and could not sequester RUNX1 in the cytoplasm. Mice expressing HABD-RSD double deleted CBFβ-SMMHC had normal hematopoiesis, did not develop leukemia spontaneously and developed T cell (not myeloid) malignancies after ENU treatment. These data suggest that HABD and RSD are important for CBFβ-SMMHC to block RUNX1 function and to dominantly impair hematopoiesis. The accelerated leukemogenesis associated with HABD deletion and induction of T cell malignancies in mice expressing HABD-RSD double deleted CBFβ-SMMHC strongly support the hypothesis that CBFβ-SMMHC can induce leukemia through RUNX1-inhibition-independent pathways.
Novel, 2ND Generation Histone Deacetylase (HDAC) Inhibitor JNJ-26481585 Exhibits Potent Antitumor Activity Against Chronic Lymphocytic Leukemia Cells In-Vitro and Ex-Vivo.
Authors: Bommert Kurt , Knop Stefan , Arts Janine , King Peter , Page Martin , Leo Eugen , Bargou Ralf
Published: October 2019

Chronic lymphocytic leukemia (CLL), the most frequent lymphoproliferative disease is characterized by the insidious accumulation of mature-appearing lymphocytes predominantely in bone marrow, blood and lymphatic tissues. Despite a generally indolent course the vast majority of subjects diagnosed with CLL eventually require treatment. Apart from cytostatic drugs recently antibodies have been added to the therapeutic armamentarium. However, only a minority of patients will enjoy long-term disease-free intervals thus making further intervention of therapy highly desirable. Histone deacetylase inhibitors (HDACi) constitute a new class of epigenetic anti-cancer agents that inhibit growth, induce differentiation and, as recently shown, apoptosis in neoplastic cells. Antitumor acitivity of 1st generation compounds such as SAHA/Zolinza™ is encouraging, yet approval has been limited to treatment of cutaneous T-cell lymphoma so far, a hematologic niche indication. Novel compounds are investigated to assess for more potent antitumor activity, hopefully allowing to broaden the therapeutic spectrum of HDACi. Therefore, we investigated the apoptosis-inducing capacity of JNJ-26481585, a novel “second generation” hydroxamic acid-based oral pan-HDACi in a CLL cell line and primary, patient-derived CLL cells. In the MEC1 cell line JNJ-26481585 induced apoptosis very effectively within 72hrs at an EC50 of approx. 0,01 μM. In comparison, SAHA exhibited an EC50 of approx. 3 μM in the same cell line and the EC50 for Fludarabine is approx. 4 μM. In primary, patient derived CLL samples [n= 12 pts] the mean EC50 for JNJ-26481585 was 0,005 μM [range: 0.14 nM to 25 nM; 5 days of incubation] compared to SAHA which had an EC50 of around 7 μM in primary CLL cells. Strong histone H3 acetylation and HSP70 upregulation was observed as assessed by Western blot, potentially related to HSP90 acetylation. The induction of apoptosis was paralleled by a down-regulation of the bcl-2 protein in western blot. Initial combination therapy explorations showed strong synergism with the proteasome inhibitor bortezomib (Velcade™) shifting the EC50 to the sub nM range both in MEC1 cells as well as in primary CLL samples. In summary, the 2nd generation HDACi JNJ-26481585 induces potent histone acetylation as well as HSP70 upregulation and bcl-2 downregulation in MEC1 cells and primary, patient-derived CLL cells, resulting in an apoptosis-inducing capacity at low nano-molar ranges, far superior to the 1st generation HDACi SAHA/Zolinza™.
Feasiblity and Implication of Bidirectional Sequencing Using a Multiplex Framework 2 Region Primer for Somatic Mutation Analysis of the Immunoglobulin (IgH) Heavy Chain in Chronic Lymphocytic Leukemia (CLL).
Authors: Nelson Adin , Rijo Ivelise , Zhang Zhigang , Zelenetz Andrew D. , Noy Ariela
Published: October 2019

Background: A somatic mutation rate of >2% of the immunoglobulin (IgH) heavy chain gene, in comparison to germline DNA, is a positive prognostic indicator in chronic lymphocytic leukemia (CLL). Genomic DNA is amplified using the Biomed-2 multiplex PCR primer set, sequenced in the reverse direction only using a 3′ JH primer, and compared to the germline sequence in VBase, a comprehensive directory of all human germline variable region sequences compiled from over a thousand published sequences. It is unknown whether forward sequencing using the multiplex 5′ Biomed-2 primers in a single reaction is feasible or whether doing so improves the accuracy of the mutational analysis.

Methods: DNA was extracted from peripheral blood or bone marrow mononuclear cells from patients with CLL using the QIAGEN QIAspin DNA mini-kit. The FR2 region of the IgH gene was amplified using tube B of the Biomed-2 multiplex PCR primer set, separated in 2% agarose gel, excised, and prepared for sequencing using the QIAGEN QIAquick gel extraction kit. Bidirectional DNA sequencing was performed with the full set of multiplex 5′ FR2 primers combined in one reaction for the forward sequence and in a second reaction with the Biomed-2 3′ JH primer for the reverse sequence. A consensus sequence was obtained and mutation rates were calculated based on the consensus sequence and the reverse sequence separately.

Results: The FR2 region was amplified from 30 CLL patients. 20 samples produced bidirectional consensus DNA sequences, 2 sequenced only with multiplex FR2 primers, 6 sequenced only with a JH primer, and 2 produced no sequences. Compared with unidirectional JH sequencing, bidirectional sequencing did not reassign any patient in the germline IgH group to the mutated category. In 15 patients, the uni- and bi-directional sequencing were identical. In 4 patients, a single basepair difference was noted. A random permutation test yields a two-sided p-value as 12.5%, indicating that no significant difference was detected between the uni- and bi-directional sequencing. Multiplex bidirectional sequencing did provide sequence information within the CDR3 region at the 3′ terminus of the amplimer which is partially truncated when using the JH primer alone.

Conclusion: Bidirectional sequencing does not provide a somatic IgH mutation rate that differs significantly from that obtained from the reverse sequence alone and does not likely influence the IgH mutational prognostic assignment. Nonetheless, the bidirectional consensus sequence adds bases in the CDR3 region which can be used for research applications such as the generation of patient-specific primers for PCR. It is also noteworthy that multiplex PCR using the Biomed-2 primers is feasible.
Protein Expression Profiling of Cytokines and Cytokine Receptors on Purified Chronic Lymphocytic Leukemia Cells.
Authors: Yu Zhifeng , Sun Baohua , Kantarjian Hagop M. , Amin Hesham M. , Sun Xiaoping
Published: October 2019

Chronic lymphocytic leukemia (CLL) B-cells rapidly undergo apoptosis when cultured in vitro, which contrasts with their prolonged survival in vivo. Multiple cytokines and cytokine receptors are believed to work together to regulate the survival of CLL cells. The literature is conflicting as to whether the CLL cells themselves produce significant amounts of cytokines compared with normal B-cells and how the CLL cells respond to these cytokines. This discrepancy is largely due to the different experimental conditions that have been used whereby various amounts of exogenous cytokines were introduced into the experimental system from, for example, the serum used to supplement the culture medium and the lysate or conditioned medium of CLL cells where other types of mononuclear cells were not removed. The aim of the current study is to reveal the intrinsic production and secretion of cytokines and cytokine receptors in CLL cells when exogenous sources are minimized. We purified CD19+ cells by magnetic beads from peripheral blood mononuclear cells of five CLL patients who had stage I or II disease and had not received any therapy. CD19+ cells from healthy donors were used as control. We used a cytokine antibody array approach that simultaneously measured 174 cytokines and cytokine receptors. We determined both intracellular levels in purified CLL cells and secreted levels in serum-free conditioned medium. The intracellular levels of cytokines and cytokine receptors of the purified CLL cells and the normal B-cells were not significantly different. However, the secretion of interleukin-6 (IL-6) was 3.0 times lower (p = 0.038) and that of eotaxin was 2.2 times higher (p = 0.028) in CLL-conditioned medium than in normal B-cell-conditioned medium. We further studied the effect of IL-6 and anti-IL-6 antibody on the apoptosis of purified CLL B-cells in serum-free culture, but no significant change was found in the presence or absence of IL-6 or IL-6 antibody. Except for IL-6 and eotaxin, our results suggest that CLL cells and their normal counterparts produce and secrete similar amounts of cytokines and cytokine receptors in vitro and that the in vivo longevity of CLL cells may be due to the concerted effects of various molecules and cellular interactions in the microenvironment.
What Have We Learned from Measuring Health-Related Quality of Life in Patients with Chronic Lymphocytic Leukemia? A Systematic Review of Published Studies from 1990 to 2007.
Authors: Molica Stefano , Mauro Francesca , Vignetti Marco , Foà Robin , Efficace Fabio
Published: October 2019

While a large amount of health-related quality of life (HRQOL) studies has been conducted in patients with solid tumors, no data are available for patients with CLL. The aim of the present work was therefore to quantify and evaluate the quality of studies investigating patient’s self-reported HRQOL in CLL patients. A systematic literature search for studies meeting the criteria, published from 1990 to 2007, was undertaken on a number of databases. Candidate articles were mainly searched in PubMed and the Cochrane Central Register of Controlled Trials (CENTRAL). Meeting abstracts at ASCO and ASH conferences were also assessed. We used the following key terms to identify candidate articles “quality of life” and “CLL” or “lymphocytic leukemia” or “chronic leukemia”. All articles included in the review were independently evaluated based on a pre-defined set of methodological issues and criteria by two reviewers (SM and FE). These included a number of methodological and statistical quality criteria such as HRQOL missing data documentation, timing of assessment, rationale for instrument selection and discussion of outcomes in terms of clinical significance. In addition, HRQOL and traditional clinical outcomes were extracted and analyzed. More than 200 candidate articles were evaluated, but only 5 studies met the inclusion criteria for the review. The majority of articles were excluded as HRQOL was not measured by patient’s reported tools. Overall, some 1019 patients were involved in these studies. HRQOL was assessed in 3 prospective randomized controlled trials (RCTs) and 2 longitudinal non-randomized studies. Two of these 5 studies were only published as abstracts. The EORTC QLQ-C30 was the most frequently used measure being utilized in 4 out of 5 studies. Some methodological constraints were identified, mainly in terms of handling of HRQOL missing data and possible lack of sensitivity of the measure being used. The most robust evidence stemmed from a prospective RCT comparing Fludarabine (F) vs. Fludarabine + Cyclophopsphamide (FC) where HRQOL was assessed as a secondary endpoint. While there was no significant HRQOL difference between treatment arms over time, this study showed nonetheless that the newer FC combination had no detrimental effect on patient’s HRQOL compared with the standard F arm. This information definitely reinforces concomitant clinical findings which favor the more intensive FC treatment (e.g. in terms of clinical response and progression-free survival). Taken together, very limited conclusions can to date be drawn with regard to HRQOL issues in patients with CLL in view of the paucity of data in this area. There is urgent need of implementing HRQOL research in patients with CLL and of providing the scientific community with robust evidence-based data on the impact of new treatment approaches in these patients.
The Non-Death Domain Containing TNFR Member HVEM Induces Apoptosis and Chemokine Production on B-Chronic Lymphocytic Leukemia Cells.
Authors: Olive Daniel R. , Pasero Christine , Barbarat Bernadette , Truneh Alem , Just Sylvaine , Aurrand Therese , Rey Jerome , Costello Regis T.
Published: October 2019

B-cell chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia and it still remains incurable. New therapies are required for this disease characterized by failure of mature lymphocytes to undergo apoptosis. Members of tumor necrosis factor receptors (TNFR) family play important roles in cell activation, proliferation, differentiation and apoptosis. We focused our study on HVEM (Herpes virus entry mediator), devoid of intracytoplasmic death domain, which is known to costimulate T- and B-cell activation, and its ligand LIGHT. Surprisingly, we found that LIGHT, as well as some anti-HVEM antibodies, induced apoptosis of B-CLL cells. This apoptosis was associated with activation of caspase-3, -8 and -9, decrease in mitochondrial membrane potential, and upregulation of the pro-apoptotic protein Bax. Importantly, the efficiency of HVEM-cell killing compared favorably with that of the pan-B cell therapeutic monoclonal antibody Rituximab. In addition, HVEM induced upregulation of various cytokines and chemokines, and a major increase in IL-8 secretion. Thus, HVEM stimulation induces apoptosis of B-CLL cells and could potentially also participate in the recruitment of immune effectors, and may therefore be useful for clinical treatment of this malignancy.
Biologic Sequelae of CDC2 Inhibition in MM.
Authors: Fulciniti Mariateresa , Tassone Pierfrancesco , Hideshima Teru , Anderson Kenneth C. , Munshi Nikhil C.
Published: October 2019

Multiple Myeloma (MM) is a malignant proliferation of plasma cells characterized by disruption of cell cycle checkpoint controls which maintain G2M transition and/or mitosis. CDC2 is the cyclin-dependent kinase that normally drives cells into mitosis and is universally expressed in MM. To examine the biologic role of CDC2 in MM, we evaluated cellular and molecular effects of Terameprocol (M4N, tetra-O-methyl nordihydroguaiaretic acid) that has been shown to inhibit cell cycle progression at the G2/M phase by inhibiting the transcription of sp-1 dependent expression of CDC2. We observed that Terameprocol downregulated the expression of cdc2 in a time-dependent manner, with a maximal effect at 24h. This was associated with induction of G2/M growth arrest in a panel of MM cell lines (INA6, OPM1, OPM2, MM1S, RPMI-8226, U266), as determined by PI staining. Interestingly, Terameprocol treatment led to increase in p21waf1 protein levels. Importantly, we observed inhibition of DNA synthesis by Terameprocol in a dose- and time-dependent manner, with IC50 range from 1–20 uM for a 24 hours period of treatment, as assessed by 3H-thymidine uptake. Longer exposure of MM cells to Terameprocol resulted in cytoxicity, as assessed by MTT assay, via induction of apoptosis, evidenced by Annexin V+ /PI staining, in all the MM cell lines tested. Terameprocol -induced apoptosis is predominantly associated with caspase-9 and caspase-3, but not caspase-8 activation, suggesting that Terameprocol triggers intrinsic apoptotic pathway in MM cells. Our results show that genes that control entry and progression of G2/M phase, especially cdc2, may be an attractive target for MM therapy and Terameprocol represents a prototypic agent for the control of unregulated cellular proliferation in MM.
An Anti-VEGF Antibody, in Combination with Bortezomib, Markedly Inhibits Tumor Growth in SCID-hu Models of Human Multiple Myeloma.
Authors: Campbell Richard A. , Sanchez Eric , Steinberg Jeffrey , Share Michael , Wang Joseph , Li Mingjie , Leuchter Richard , Chen Haiming , Shalitin Dror , Pang Shen , Said Jonathan , Plowman Gregory D. , Berenson James R.
Published: October 2019

Vascular endothelial growth factor (VEGF) is an important signaling protein that plays a critical role in vasculogenesis and angiogenesis, and serves as one of the contributors to physiological or pathological conditions that can stimulate the formation of new blood vessels. The uncontrolled growth of new blood vessels is an important contributor to a number of pathological conditions, including multiple myeloma (MM). In support of this, bone marrow angiogenesis has been shown to correlate with disease status and poor prognosis in MM. VEGF also directly induces myeloma cell proliferation. We previously evaluated the effects of single agent mouse/human anti-VEGF antibody G6.31 (Campbell et al, Blood (ASH Annual Meeting Abstracts), Nov 2006) in several of our mouse models of human MM. In this study, we evaluated the effects of the same anti-VEGF antibody in combination with bortezomib or lenalidomide. Severe combined immunodeficient (SCID) mice were implanted into the left superficial gluteal muscle with either a 2.0 – 4.0 mm3 fragment from a patient when she was bortezomib-sensitive, LAGκ-1A, or resistant, LAGκ-1B. The tumors were allowed to grow for 21 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into treatment groups (n=10 mice/group). Treatment groups consisted of a control IgG antibody or anti-VEGF antibody administered via i.p. injection twice weekly at a dose of 2 mg/kg, bortezomib administered via intravenous injection at a dose of 0.25 or 0.5 mg/kg twice weekly, lenalidomide administered via i.p. injection at a dose of 50 mg/kg daily × 5 per week, anti-VEGF antibody (2 mg/kg) + bortezomib (0.25 or 0.5 mg/kg), and anti-VEGF (2 mg/kg) + lenalidomide (50 mg/kg). Mice receiving the combination therapy of anti-VEGF + bortezomib (0.5 mg//kg) antibody showed marked inhibition of tumor growth and reduction of paraprotein levels compared to mice receiving control antibody. Notably, this combination also produced much more marked anti-MM effects compared to bortezomib treatment alone, anti-VEGF antibody alone, or vehicle alone. This combination was well tolerated. In contrast, mice receiving the combination of anti-VEGF antibody + lenalidomide showed no significant differences in tumor volume or hIgG levels compared to single agent treatment or vehicle alone. The markedly improved anti-MM effects of the combination of bortezomib and anti-VEGF antibody compared to single agent treatment in this in vivo study of human MM is promising, and these results have provided the preclinical rationale for an ongoing randomized multi-center Phase II trial.
Elevated Pretransplant Serum Ferritin in Autologous Stem Cell Transplant.
Authors: Mahindra Anuj , Bolwell Brian , Sobecks Ronald , Rybicki Lisa , Brown Stacey , Pohlman Brad , Dean Robert , Andresen Steve , Kalaycio Matt , Sweetenham John , Copelan Edward
Published: October 2019

Iron overload is an adverse prognostic factor in patients who undergo allogeneic hematopoietic stem-cell transplantation (HSCT) for thalassemia and apparently in patients with acute leukemia and myelodysplastic syndrome as well. Iron overload has also been associated with susceptibility to infection following autotransplantation and with impaired immunity in other settings. We report here the results of a large study of consecutive patients undergoing autologous hematopoietic stem-cell transplantation (ASCT) for various hematologic malignancies to assess the influence of pretransplantation ferritin on outcomes following transplant. Pretransplantation ferritin, obtained within 100 days preceding transplant, was available on 397 patients undergoing autologous HSCT for various disorders (NHL=257, MM= 61, HD=58, AML=21) from 11/2/2000 to 12/28/2006.The median ferritin was 529.3 ng/ml (range, 12.8–4330). The median patient age was 52 (range, 19–77). Recursive partitioning analysis identified baseline ferritin >685 ng/ml as an adverse prognostic factor for survival as shown: ESTIMATED SURVIVAL BY PRETRANSPLANT FERRITIN LEVEL

Figure Figure

Age, gender, disease, disease status at transplant, interval from diagnosis to transplant, number of prior chemotherapy regimens, prior radiation therapy, ferritin, albumin, AST, Alkaline phosphatase, LDH, preparative regimen and CD 34+ dose were analyzed in a multivariable analysis. Elevated ferritin was an independent, adverse, significant prognostic factor for survival (p<0.001, HR=2.29),relapse-free survival (P<.001, HR=1.79) and relapse (p=0.006, HR=1.68). The addition of albumin, a negative acute phase reactant, did not change the prognostic impact of ferritin. Elevated ferritin was also significantly predictive of a higher incidence of relapse mortality (p<0.001) as shown below: RELAPSE MORTALITY BY FERRITIN LEVEL

Figure Figure

Conclusions: Elevated ferritin adversely influences survival, relapse-free survival, and relapse mortality following autologous transplantation. Whether the increased number of deaths due to relapse is related to the established immunosuppressive affect of iron overload is unknown. Iron chelators have been shown to inhibit the growth of tumor cells in vitro and in vivo. Trials designed to analyze the benefit of iron chelation therapy prior to ASCT in patients with iron overload are warranted.
Reactive Oxygen Species Target the Degradation of the EVI1 Oncoprotein.
Authors: Shackelford David , Wang You , Waxman Samuel , Ren Ruibao
Published: October 2019

Aberrant expression of EVI1 has been frequently found in myeloid malignancies as well as in cancers of the ovary, lung, head and neck, cervix, and breast, and is associated with a poor patient survival. Targeted degradation of oncoproteins is an effective strategy for cancer therapy. The AML1/MDS1/EVI1 (AME) transcription factor fusion protein is a product of the human t(3;21)(q26;q22) translocation found as a secondary mutation in some cases of CML during the blast phase (CML-BP) and in patients with de novo and therapy-related myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Expression of AME in mouse bone marrow cells by retroviral transduction impairs hematopoiesis and eventually induces an acute myeloid leukemia (AML)-like disease in mice. Arsenic Trioxide (ATO) has been found to be an effective treatment for patients suffering from acute promyelocytic leukemia (APL). This is, at least in part, mediated by degradation of the PML/RARα oncoprotein that is associated with over 90% of APL. We have recently shown that ATO used at therapeutic levels also degrades AME. The ATO treatment induces differentiation and apoptosis in AME leukemic cells in vitro and causes decrease in peripheral leukemic cells and splenomegaly in vivo. ATO appears to target AME at both the EVI1 and MDS moieties of the protein for degradation via the ubiquitin-proteasome pathway and proteasome-independent mechanism, respectively. To investigate the mechanism of ATO induced degradation of EVI1 oncoproteins, we examined the effect of reactive oxygen species (ROS), on EVII expression, one of the downstream effectors of ATO-induced apoptosis. We found that EVI1 degradation correlates with the amount of ROS generated in cells. EVI1 can also be targeted for degradation by doxorubicin, a chemotherapeutic agent that is effective in treating AML and ovarian cancer and a strong ROS inducer. Interestingly, the antioxidant N-acetyl cysteine (NAC) abrogates the degradation of the EVI1 protein in the presence of doxorubicin. These results demonstrate that EVI1 can be targeted for degradation by ROS. ROS inducing agents could be used as a part of targeted therapy for EVI1-positive malignancies.