Susanne Isfort

Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

BackgroundSomatic calreticulin (CALR), Janus kinase 2 (JAK2), and thrombopoietin receptor (MPL) mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN), suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated.MethodsThe murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment.ResultsThe present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation.ConclusionsThese findings demonstrate the potency of CALR mutants to drive expression of megakaryocytic differentiation markers such as NF-E2 and CD41 as well as Mpl. Furthermore, CALR mutants undergo accelerated protein degradation that involves the secretory pathway and/or protein glycosylation.

Bosutinib: A Novel Second-Generation Tyrosine Kinase Inhibitor

Bosutinib (SKI-606) is a 4-anilino-3-quinoline carbonitrile, which acts as a dual inhibitor of Src and ABL kinases. In addition, the BCR-ABL fusion gene product, a constitutively activated tyrosine kinase which is crucial for the development of chronic myeloid leukemia (CML), is highly sensitive to bosutinib. Interestingly, distinctly lower concentrations of bosutinib are required to ablate BCR-ABL phosphorylation when compared to the first-generation tyrosine kinase inhibitor imatinib (IM). Bosutinib is a potent inhibitor of CML cell proliferation in vitro and has demonstrated promising activity in CML patients resistant or intolerant to IM as well as in newly diagnosed patients with chronic phase CML (CML-CP). Remarkably, bosutinib has been found to be capable of overcoming the majority of IM-resistant BCR-ABL mutations. Bosutinib has the potency to induce deep and fast responses in second- and third-/fourth-line treatment, and as a consequence, the drug has recently been licensed for patients previously treated with one or more tyrosine kinase inhibitor(s) and for whom imatinib, nilotinib, and dasatinib are not considered appropriate treatment options. Due to its potency and differing toxicity profile, it promises to be a good therapeutic option for a defined cohort of patients. The most common side effects are gastrointestinal with most of the patients suffering from nausea, vomiting, or diarrhea. For the most part, these gastrointestinal symptoms occur early after treatment initiation, are manageable, and often self-limiting. Continuous monitoring of liver enzymes upon treatment initiation is necessary during bosutinib treatment. In addition to CML treatment, bosutinib has shown some efficacy in selected patients suffering from advanced-stage solid tumors. In conclusion, bosutinib is a promising novel small molecule inhibitor approved now for targeted therapy of CML and in clinical development for other malignancies.

Dissecting Genomic Aberrations in Myeloproliferative Neoplasms by Multiplex-PCR and Next Generation Sequencing

In order to assess the feasibility of amplicon-based parallel next generation sequencing (NGS) for the diagnosis of myeloproliferative neoplasms (MPN), we investigated multiplex-PCR of 212 amplicons covering genomic mutational hotspots in 48 cancer-related genes. Samples from 64 patients with MPN and five controls as well as seven (myeloid) cell lines were analyzed. Healthy donor and reactive erythrocytosis samples showed several frequent single-nucleotide polymorphisms (SNPs) but no known pathogenic mutation. Sequencing of the cell lines confirmed the presence of the known mutations. In the patient samples, JAK2 V617F was present in all PV, 4 of 10 ET, and 16 of 19 MF patients. The JAK2 V617F allele burden was different in the three groups (ET, 33+/-22%; PV 48+/-28% and MF 68+/- 29%). Further analysis detected both previously described and undescribed mutations (i.e., G12V NRAS, IDH1 R132H, E255G ABL, and V125G IDH1 mutations). One patient with lymphoid BC/Ph+ ALL who harbored a T315I ABL mutation and was treated with ponatinib was found to have developed a newly acquired V216M TP53 mutation (12% of transcripts) when becoming resistant to ponatinib. Ponatinib led to a decrease of ABL T315I positive transcripts from 47% before ponatinib treatment to 16% at the time of ponatinib resistance in this patient, suggesting that both TP53 and ABL mutations were present in the same clone and that the newly acquired TP53 mutation might have caused ponatinib resistance in this patient. In conclusion, amplicon-sequencing-based NGS allows simultaneous analysis of multiple MPN associated genes for diagnosis and during treatment and measurement of the mutant allele burden.

Health care setting and severity, symptom burden, and complications in patients with Philadelphia-negative myeloproliferative neoplasms (MPN): a comparison between university hospitals, community hospitals, and office-based physicians.

Philadelphia-negative myeloproliferative neoplasms (MPN) comprise a heterogeneous group of chronic hematological malignancies with significant variations in clinical characteristics. Due to the long survival and the feasibility of oral or subcutaneous therapy, these patients are frequently treated outside of larger academic centers. This analysis was performed to elucidate differences in MPN patients in three different health care settings: university hospitals (UH), community hospitals (CH), and office-based physicians (OBP). The MPN registry of the Study Alliance Leukemia is a non-interventional prospective study including adult patients with an MPN according to WHO criteria (2008). For statistical analysis, descriptive methods and tests for significant differences were used. Besides a different distribution of MPN subtypes between the settings, patients contributed by UH showed an impaired medical condition, a higher comorbidity burden, and more vascular complications. In the risk group analyses, the majority of polycythemia vera (PV) and essential thrombocythemia (ET) patients from UH were classified into the high-risk category due to previous vascular events, while for PV and ET patients in the CH and OBP settings, age was the major parameter for a high-risk categorization. Regarding MPN-directed therapy, PV patients from the UH setting were more likely to receive ruxolitinib within the framework of a clinical trial. In summary, the characteristics and management of patients differed significantly between the three health care settings with a higher burden of vascular events and comorbidities in patients contributed by UH. These differences need to be taken into account for further analyses and design of clinical trials.

Bosutinib in chronic myeloid leukemia: patient selection and perspectives

During recent years, the therapeutic landscape in chronic myeloid leukemia (CML) has changed significantly. Since the clinical introduction of tyrosine kinase inhibitors (TKIs) approximately 15 years ago, patients’ concerns have shifted from reduced life expectancy toward long-term toxicities of TKI, depth of remission, and the probability of successful treatment discontinuation. Patients with newly diagnosed CML in chronic phase (at least with a Sokal score not exceeding intermediate) may now expect an almost normal life expectancy. However, even if almost 30% of all newly diagnosed chronic-phase patients might eventually be facing the prospect of a life without CML-specific treatment, based on current knowledge, most, if not all, patients would have to undergo an expected minimum of 5–8 years of TKI treatment and the majority would face a life-long exposure to the side-effects of TKIs. At present, 5 different TKIs are licensed for the treatment of CML, that is, imatinib, which is a first-generation TKI (including its generic derivatives); nilotinib, dasatinib, and bosutinib, which are second-generation TKIs; as well as ponatinib, which is a so-called third-generation TKI and is supposed to be used for patients harboring the T315I-mutation. One of the important, yet unanswered questions is the choice of the best possible TKI upfront for each individual patient. Bosutinib is currently licensed for patients with CML after failure or intolerance of at least 2 other TKIs. It can also be prescribed according to label if after failure of the first TKI therapy, another option does not seem feasible. This review focuses on the existing data on clinical efficacy, tolerability, and side effects of bosutinib treatment in CML patients with the aim to identify patient characteristics and treatment scenarios most suitable for treatment with bosutinib.

Leukocyte counts based on site-specific DNA methylation analysis

The composition of white blood cells is usually assessed by histomorphological parameters or flow cytometric measurements. Alternatively, leukocyte differential counts (LDCs) can be estimated by deconvolution algorithms for genome-wide DNA methylation (DNAm) profiles. We identified cell-type specific CG dinucleotides (CpGs) that facilitate relative quantification of leukocyte subsets. Site-specific analysis of DNAm levels by pyrosequencing provides similar precision of LDCs as conventional methods, whereas it is also applicable to frozen samples and requires only very small volumes of blood. Furthermore, we describe a new approach for absolute quantification of cell numbers based on a non-methylated reference DNA. Our “Epi-Blood-Count” facilitates robust and cost effective analysis of blood counts for clinical application.

Serum of myeloproliferative neoplasms stimulates hematopoietic stem and progenitor cells

Background Myeloproliferative neoplasms (MPN)—such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF)—are typically diseases of the elderly caused by acquired somatic mutations. However, it is largely unknown how the malignant clone interferes with normal hematopoiesis. In this study, we analyzed if serum of MPN patients comprises soluble factors that impact on hematopoietic stem and progenitor cells (HPCs). Methods CD34+ HPCs were cultured in medium supplemented with serum samples of PV, ET, or MF patients, or healthy controls. The impact on proliferation, maintenance of immature hematopoietic surface markers, and colony forming unit (CFU) potential was systematically analyzed. In addition, we compared serum of healthy young (<25 years) and elderly donors (>50 years) to determine how normal aging impacts on the hematopoiesis-supportive function of serum. Results Serum from MF, PV and ET patients significantly increased proliferation as compared to controls. In addition, serum from MF and ET patients attenuated the loss of a primitive immunophenotype during in vitro culture. The CFU counts were significantly higher if HPCs were cultured with serum of MPN patients as compared to controls. Furthermore, serum of healthy young versus old donors did not evoke significant differences in proliferation or immunophenotype of HPCs, whereas the CFU frequency was significantly increased by serum from elderly patients. Conclusion Our results indicate that serum derived from patients with MPN comprises activating feedback signals that stimulate the HPCs–and this stimulatory signal may result in a viscous circle that further accelerates development of the disease.

Telomere shortening correlates with leukemic stem cell burden at diagnosis of chronic myeloid leukemia

Telomere length (TL) in peripheral blood (PB) cells of patients with chronic myeloid leukemia (CML) has been shown to correlate with disease stage, prognostic scores, response to therapy, and disease progression. However, due to considerable genetic interindividual variability, TL varies substantially between individuals, limiting its use as a robust prognostic marker in individual patients. Here, we compared TL of BCR-ABL-, nonleukemic CD34+CD38- hematopoietic stem cells (HSC) in the bone marrow of CML patients at diagnosis to their individual BCR-ABL+ leukemic stem cell (LSC) counterparts. We observed significantly accelerated telomere shortening in LSC compared with nonleukemic HSC. Interestingly, the degree of LSC telomere shortening was found to correlate significantly with the leukemic clone size. To validate the diagnostic value of nonleukemic cells as internal controls and to rule out effects of tyrosine kinase inhibitor (TKI) treatment on these nontarget cells, we prospectively assessed TL in 134 PB samples collected in deep molecular remission after TKI treatment within the EURO-SKI study (NCT01596114). Here, no significant telomere shortening was observed in granulocytes compared with an age-adjusted control cohort. In conclusion, this study provides proof of principle for accelerated telomere shortening in LSC as opposed to HSC in CML patients at diagnosis. The fact that the degree of telomere shortening correlates with leukemic clones size supports the use of TL in leukemic cells as a prognostic parameter pending prospective validation. TL in nonleukemic myeloid cells seems unaffected even by long-term TKI treatment arguing against a reduction of telomere-mediated replicative reserve in normal hematopoiesis under TKI treatment.

Improve hip fracture outcome in the elderly patient (iHOPE): a study protocol for a pragmatic, multicentre randomised controlled trial to test the efficacy of spinal versus general anaesthesia

Introduction Hip fracture surgery is associated with high in-hospital and 30-day mortality rates and serious adverse patient outcomes. Evidence from randomised controlled trials regarding effectiveness of spinal versus general anaesthesia on patient-centred outcomes after hip fracture surgery is sparse. Methods and analysis The iHOPE study is a pragmatic national, multicentre, randomised controlled, open-label clinical trial with a two-arm parallel group design. In total, 1032 patients with hip fracture (>65 years) will be randomised in an intended 1:1 allocation ratio to receive spinal anaesthesia (n=516) or general anaesthesia (n=516). Outcome assessment will occur in a blinded manner after hospital discharge and inhospital. The primary endpoint will be assessed by telephone interview and comprises the time to the first occurring event of the binary composite outcome of all-cause mortality or new-onset serious cardiac and pulmonary complications within 30 postoperative days. In-hospital secondary endpoints, assessed via in-person interviews and medical record review, include mortality, perioperative adverse events, delirium, satisfaction, walking independently, length of hospital stay and discharge destination. Telephone interviews will be performed for long-term endpoints (all-cause mortality, independence in walking, chronic pain, ability to return home cognitive function and overall health and disability) at postoperative day 30±3, 180±45 and 365±60. Ethics and dissemination iHOPE has been approved by the leading Ethics Committee of the Medical Faculty of the RWTH Aachen University on 14 March 2018 (EK 022/18). Approval from all other involved local Ethical Committees was subsequently requested and obtained. Study started in April 2018 with a total recruitment period of 24 months. iHOPE will be disseminated via presentations at national and international scientific meetings or conferences and publication in peer-reviewed international scientific journals. Trial registration number DRKS00013644; Pre-results

Myeloproliferative Neoplasien: Aktueller Stand zu (molekularer) Diagnostik und (zielgerichteter) Therapie

ZusammenfassungMit dem zunehmendem Erkenntnisgewinn der letzten Jahre ist nunmehr nicht nur für die chronische myeloische Leukämie (CML) ein klonaler Marker (Philadelphia-Chromosom bzw. BCR-ABL) bekannt, sondern auch die sog. Philadelphia-Chromosom-negativen myeloproliferativen Neoplasien (Ph-MPN) können seit Entdeckung der erworbenen JAK2-, CALR- und MPL-Mutationen sowie weiterer seltener molekularer Marker als klonal identifiziert und somit von reaktiven Zuständen besser abgegrenzt werden. Durch die Nutzung der molekularen Marker als Therapieansatzpunkt konnten neue Substanzen entwickelt werden, sodass sowohl in der Therapie der CML als auch der Ph-MPN individuelle Therapiekonzepte ermöglicht werden können. Dieser Artikel gibt einen Überblick über die CML und die Ph-MPN als sog. klassische myeloproliferative Neoplasien. Der Schwerpunkt liegt auf den Empfehlungen zum Einsatz molekularer Diagnostik und Prinzipien der leitliniengerechten Therapie.