Frauke Ringel

Selective internalization of monoclonal antibodies by B‐cell chronic lymphocytic leukaemia cells

Summary. B‐cell chronic lymphocytic leukaemia (B‐CLL) cannot be cured by conventional chemotherapy, therefore, toxin‐linked therapeutic monoclonal antibodies (mAbs) are increasingly examined for their potential to improve clinical outcome. The current study aimed to identify mAbs that were internalized by the B‐CLL cells of 14 patients, using both flow cytometry and confocal laser scanning microscopy. Anti‐CD5, CD22 and CD40 mAbs were effectively taken up by B‐CLL cells, whereas mAbs against CD19, CD20, CD23 and CD45 were not. This study may form a basis for further research to identify antibodies that may serve as carriers for toxins to treat B‐CLL.

Clinical relevance of CD95 (Fas/Apo-1) on T cells of patients with B-cell chronic lymphocytic leukemia

OBJECTIVE Apoptosis mediated via CD95 (Fas/Apo-1) is a key regulator for the biology of normal and malignant lymphocytes. Although the function of CD95 on B-cell chronic lymphocytic leukemia cells (B-CLL cells) has been studied intensively, the clinical importance of CD95 expression on normal T cells in B-CLL has not been clarified. This study aimed to investigate whether expression of CD95 on peripheral blood T cells correlates with clinically relevant parameters of B-CLL disease. MATERIALS AND METHODS Expression of CD95 (Fas/Apo-1) on peripheral blood T lymphocytes of patients with B-CLL was determined using flow cytometry and was correlated with expression of activation markers, sensitivity to apoptosis by anti-CD95, and clinical data, such as blood count, Binet stage, therapy, progression-free probability, and survival probability. RESULTS Differential CD95 expression did not correlate with activation markers or with levels of apoptosis through anti-CD95. However, high levels of CD95 on T cells from B-CLL patients correlated significantly with low lymphocyte doubling time, increased Binet stages, and requirement for chemotherapeutic treatment. Furthermore, increased cell-surface CD95 on T cells was associated with reduced progression-free probability and poorer survival. CONCLUSIONS CD95 levels on T cells correlate with the clinical course of B-CLL. Prospective studies appear warranted to investigate whether CD95 on T cells has a direct influence on B-CLL disease progression.

Thrombopoietin Receptor Agonists Are Often Ineffective in Immune Thrombocytopenia and/or Cause Adverse Reactions: Results from One Hand

Background: Eltrombopag and romiplostim are thrombopoietin receptor agonists (TPOs) that have been increasingly used for the treatment of immune thrombocytopenia (ITP). Based on our experience, the incidence of abortive treatment with these drugs and the occurrence of adverse reactions that lead to therapy break-off despite response are higher than has been previously suggested. Methods: During the last 8 years, a total of 65 patients were treated with eltrombopag and/or romiplostim at our institute. Results: 36 of a total of 58 patients responded well to eltrombopag. In 12 patients that responded, treatment with eltrombopag was discontinued due to the development of adverse reactions. Eltrombopag was replaced by romiplostim in 23 cases (14 non-responders, 9 patients with adverse reactions). Of these patients, 83% responded to romiplostim. Among all patients treated with romiplostim (n = 32), 75% initially responded; however, 8 of these patients developed adverse reactions. Romiplostim was replaced by eltrombopag in 5 cases (4 due to adverse reactions, 1 non-responsive patient), and only 3 (60%) of these patients were observed to respond to eltrombopag. Conclusion: TPOs often remain ineffective in ITP or result in adverse reactions, which lead to treatment stop or to drug switch. Therefore, alternative treatment options are required.

Identification of novel biomarkers in chronic immune thrombocytopenia (ITP) by microarray-based serum protein profiling.

The pathological mechanisms underlying the development of immune thrombocytopenia (ITP) are unclear and its diagnosis remains a process of exclusion. Currently, there are no known specific biomarkers for ITP to support differential diagnosis and treatment decisions. Profiling of serum proteins may be valuable for identifying such biomarkers. Sera from 46 patients with primary chronic ITP and 34 healthy blood donors were analysed using a microarray of 755 antibodies. We identified 161 differentially expressed proteins. In addition to oncoproteins and tumour‐suppressor proteins, including apoptosis regulator BCL2, breast cancer type 1 susceptibility protein (BRCA1), Fanconi anaemia complementation group C (FANCC) and vascular endothelial growth factor A (VEGFA), we detected six anti‐nuclear autoantibodies in a subset of ITP patients: anti‐PCNA, anti‐SmD, anti‐Ro/SSA60, anti‐Ro/SSA52, anti‐La/SSB and anti‐RNPC antibodies. This finding may provide a rational explanation for the association of ITP with malignancies and other autoimmune diseases. While RUNX1mRNA expression in the peripheral blood mononuclear cells (PBMC) of patients was significantly downregulated, an accumulation of RUNX1 protein was observed in the platelets of ITP patients. This may indicate dysregulation of RUNX1 expression in PBMC and megakaryocytes and may lead to an imbalanced immune response and impaired thrombopoiesis. In conclusion, we provide novel insights into the pathogenic mechanisms of ITP that warrant further exploration.

Up-regulated MSI2 is associated with more aggressive chronic myeloid leukemia

Abstract A better understanding of events triggering chronic myeloid leukemia progression is critical for optimized clinical management of chronic myeloid leukemia (CML). We sought to validate that increased expression of Musashi 2 (MSI2), a post-transcription regulator, is associated with progression and prognosis. Screening of 152 patients with CML showed that MSI2 was significantly decreased among patients with CML in chronic phase (CP) at diagnosis (p < 0.0001), but found no significant difference between the normal control group and treated patients with CML in CP. Moreover MSI2 was significantly increased (p < 0.0001) in patients with advance disease (AD) CML. Furthermore, our human hematopoietic cell line data imply that MSI2 and BCR-ABL1 mRNA expression are correlated. However, these data cast a doubt on earlier reports that MSI2 effects HES1 expression via NUMB–NOTCH signaling.

New aspects on the efficacy of high-dose intravenous immunoglobulins in patients with autoimmune thrombocytopenia

The clinical significance of autoimmune thrombocytopenia (ITP) is reflected by bleeding and/or an increased bleeding risk due to low platelet counts. In most cases, treatment with high‐dose (1–2 g/kg body weight) intravenous immunoglobulin has been demonstrated to result in an increase in platelet counts after one day of treatment. Until now, there is little information on the true beginning of therapy effect in patients treated with high‐dose intravenous immunoglobulin. In this study, we focused on the kinetic of platelet counts and cessation of bleeding within the first 24 h of treatment.

Differential gene expression in peripheral blood lymphocytes of cancer patients treated with whole body hyperthermia and chemotherapy: A pilot study

Purpose: The effect of whole body hyperthermia (WBH) at 41.8–42°C on the cellular immune system is still poorly investigated. The aim of this study was to identify genes that become upregulated in peripheral blood lymphocytes (PBLs) of cancer patients during a combined treatment with WBH and chemotherapy by generating complex arrays of cDNA. Methods: PBLs were obtained from four patients with different malignancies treated with WBH and varying cytostatic schedules before treatment and immediately thereafter. After constructing subtracted cDNA libraries, clones were screened for cDNA induction by dot-blot and semi-quantitative RT-PCR (sq-RT-PCR). Results: Among 192 clones, 39 cDNAs were significantly upregulated. Sequencing revealed three groups of genes for which upregulation of mRNA was confirmed by sq-RT-PCR. The first group consisted of genes encoding for various heat shock proteins (HSP 60, 90a, 90b, 105). Further sq-RT-PCR demonstrated differential expression of HSP27 and HSP70 as well. The second group (calcyclin-binding-protein, haemoglobin-beta-chain) comprised genes without pre-specified association to hyperthermia. The cDNA encoding macrophage-inflammatory-protein-1-beta was also observed and may be associated with the pre-described activation of lymphocyte sub-populations during WBH. Conclusion: Treatment with WBH and chemotherapy elicits significant short-term effects on the expression of a variety of genes responsible for cellular integrity, stimulation and migration of immune effector cells. Further investigation is warranted to more clearly define the role of those genes for the clinical effect of WBH.

Differential expression of SHP-1 in chronic myeloid leukemia

DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication. Just Accepted by Leukemia & Lymphoma

JAK2 V617F allele burden quantified by real time quantitative polymerase chain reaction and competitive polymerase chain reaction in patients with chronic myeloproliferative neoplasia.

Abstract Assessing the clinical significance of JAK2 V617F mutant allele burden is complicated by a myriad of techniques reported to detect and quantify the mutation. As a consequence, the level of sensitivity and how the data is reported vary. Harmonization of well-defined molecular studies would permit evaluation of the clinical significance of measuring allele burden and rapid determination of the efficacy of novel agents for the treatment of chronic myeloproliferative neoplasia via multicenter clinical trials, at the subclinical level. Here we report a comparison between the widely available TaqMan quantitative real time polymerase chain reaction (Q-PCR) and competitive PCR (C-PCR) assays. We found that the tumor load was invariably greater when measured by C-PCR compared to that recorded by Q-PCR. Furthermore, none of the samples converted from undetectable to detectable when the enriched granulocyte (GR) fraction was tested. While a difference in the V617F allele levels was detected between GR fraction and whole blood, this was not statistically significant.

Longitudinal Study to Assess the Clinical Significance of MSI2 Expression in Chronic Myeloid Leukemia Patients

by undertaking a retrospective longitudinal study in CML patients in BC. We identified CML patients in BC from our previous study for whom we had 5 or more serial samples in total prior to and/or following progression to the blastic disease phase [1] . The 5 CML patients (2 female, 3 male) had a median age of 62 years (range 46–76), and 4 were treated with one or more of the tyrosine kinase inhibitors (TKIs) ( table 1 ). The other had undergone stem cell transplant (SCT). The study was performed using previously described standard molecular biology methods [1] . The MSI2 expression in 4 of the 5 patients appeared to mirror the BCR-ABL1 kinetics ( fig. 1 ). With the exception of the post-SCT samples of patient 5, the MSI2 expressions were lower than the BCR-ABL1 transcript numbers. BCR-ABL1 was undetectable in the post-SCT samples. The notion that regular and close monitoring of MSI2 mRNA levels in serial samples might identify patients at risk of progressing to BC was not supported by this study. Moreover, the data presented here show that an increase in MSI2 transcripts does not precede an increase in BCRABL1 mRNA levels. More importantly, this raises the question as to whether increased MSI2 expression in BC is the cause or effect. The early identification of chronic myeloid leukemia (CML) patients in the chronic phase (CP) who are at risk of progression to blast crisis (BC) would enhance the clinical management of these individuals. Recently, we verified reported data showing increased levels of Musashi 2 (MSI2) transcripts in CML patients in BC compared to those in CP, implying a role for MSI2 in CML transformation [1–3] . MSI2 , a posttranscriptional regulator, is reported to control hematopoietic stem cell self-renewal and differentiation [2, 3] . MSI2 achieves this control by binding to mRNA and thereby impeding translation of the target RNA [2, 3] . Interestingly, we found the MSI2 transcript numbers were significantly lower (p < 0.0001) in CML patients in CP at diagnosis than normal adult blood control samples [1] . Based on these observations and knowing that MSI2 expression is restricted to early hematopoietic progenitors, we reasoned that regular close monitoring of its mRNA levels might identify patients at risk of transformation prior to detecting increasing levels of BCR-ABL1 . If confirmed, it would provide a window in which to intervene early with alternative therapy with a view to reversing the progression, before the patient becomes refractory to further treatment. We addressed this notion Received: January 14, 2016 Accepted: March 1, 2016 Published online: May 10, 2016