Sequenzierung des NUMB-Gens in Patienten mit Chronischer Myeloischer Leukämie

Better understanding of the transformation of chronic myeloid leukemia (CML) from chronic phase (CP) to the invariably fatal blast phase (BP) is of critical importance for the clinical management of patients with CML. However, the mechanisms responsible for triggering disease progression have eluded investigators’ efforts. Recently, our group verified reported data showing increased levels of Musashi-2 (MSI2) transcripts in patients with CML in BP compared to those in CP, implying a role for MSI2 in CML transformation1-3. The Musashi gene family is reported to control critical cell fate decisions by binding to target mRNAs, including the NUMB mRNA, thereby inhibiting translation4,5. Unregulated increased expression of MSI2 results in the dysfunction of NUMB-Notch signalling, leading to haematopoietic stem cell (HSC) proliferation, impaired myeloid differentiation and worse clinical prognosis in CML2,3. Therefore, we hypothesized that mutations mapping to the NUMB gene may perturb this signalling pathway and thereby influence CML transformation. I tested this notion by directly sequencing the entire NUMB transcript in 22 patients with CML of whom 10 were in CP and 12 were in BP. Archived RNA extracted from peripheral blood from subjects with CML was reverse transcribed to cDNA and the entire NUMB gene transcript was amplified. The amplified products were subjected to Sanger sequencing. For the 22 patients with CML, the NUMB gene transcript sequence was determined to be identical to the published wild type sequence, apart from two previously reported single nucleotide polymorphisms (SNP) mapping to the 3’-UTR: rs11625196 (C/G) and rs7202 (C/T) 6,7. I observed no significant difference in the distribution of the genotypes of the two SNP between that reported for normal healthy individuals and the CML patients, nor between the different disease phases. However, rs7202 genotype had significant influence on the mortality rate of patients in BP – an observation which was fortuitously biased by different treatment modalities. The software tools Mfold and SNPfold predicted a negligible effect of the two SNP on the secondary structure of NUMB mRNA. In a summary, these observations suggest that NUMB, which regulates Notch, Hedgehog and p53 signalling, is not the primary cause of CML evolution8-11. However, it would be prudent to confirm this finding in a study with greater number of CML CP and BP patients and/or using a sequencing method with higher sensitivity, such as deep-gene sequencing.Ein besseres Verständnis der molekularbiologischen Vorgänge, die zur Transformation der chronischen myeloischen Leukämie (CML) von der relativ indolenten chronischen Phase (CP) zur fatalen Blastenkrise führen, ist von entscheidender Bedeutung für das klinische Management von CML-Patienten. Unsere Arbeitsgruppe konnte vorher publizierte Daten bestätigen, die eine höhere Expression von Musashi-2 (MSI2) in der Blastenkrise im Vergleich zur CP zeigten. Eine Funktion von MSI2 im Rahmen der Transformation der CML wird diskutiert1-3. Die Mitglieder der Musashi-Genfamilie gelten als Regulatoren von Zellteilung und Zelldifferenzierung unreifer Zellen und agieren, indem sie die Translation bestimmter mRNAs wie der NUMB mRNA inhibieren4,5. Eine Dysregulation von MSI2 führt zu einer Dysfunktion des NUMB-Notch-Signalweges und daraufhin zu einer verstärkten Proliferation hämatopoietischer Stammzellen (HSC), einer eingeschränkten myeloischen Differenzierung und einer schlechteren Prognose2,3. Wir vermuteten, dass Mutationen im NUMB-Gen den NUMB-Notch-Signalweg deregulieren und zur CML-Transformation beitragen können. Diese Vermutung testete ich, indem ich NUMB cDNA von 22 CML-Patienten, davon 10 in CP und 12 in BP, nach der Sanger-Kettenabbruchmethode sequenzierte. Dafür wurde RNA aus dem peripheren Blut von CML-Patienten extrahiert und revers transkribiert zu cDNA. Das gesamte Transkript des NUMB-Genes wurde mittels PCR amplifiziert und daraufhin sequenziert. Unter den 22 CML-Patienten fanden sich keine Abweichungen der NUMB cDNA-Basensequenz im Vergleich zur publizierten Wildtyp-Sequenz, abgesehen von zwei Einzelnukleotid- polymorphismen (SNP) in der 3‘-untranslatierten Region: rs11625196 (C/G) und rs7202 (C/T) 6,7. Ich konnte keine signifikanten Unterschiede im Auftreten der SNP-Genotypen zwischen gesunden Kontrollpersonen und CML-Patienten oder zwischen CP- und Blastenkrise-Patienten beobachten. Allerdings zeigte sich ein signifikanter Einfluss des rs7202-Genotyps auf die Mortalität von Blastenkrise-Patienten. Diese Beobachtung ist jedoch am ehesten auf ungleiche Therapieregime zurückzuführen. Mfold- und SNPfold-Software sagen einen vernachlässigbaren Effekt der beiden SNPs auf die räumliche NUMB-mRNA-Struktur vorher. Zusammengefasst weisen die Beobachtungen dieser Studie daraufhin, dass NUMB-Mutationen nicht als die primäre Ursache der CML-Transformation anzusehen sind. Dennoch wäre es gerechtfertigt, die Erkenntnisse dieser Studie mit einer größeren Anzahl an CML-Patienten oder auch einer sensitiveren Next-Generation- Sequenzierungsmethode zu überprüfen

Clinical Lessons to Be Learned from Patients Developing Chronic Myeloid Leukemia While on Immunosuppressive Therapy after Solid Organ Transplantation: Yet Another Case after Orthotopic Heart Transplantation

Chronic myeloid leukemia developing after transplantation of solid organs and concomitant immunosuppression is a rare but still significant clinical phenomenon. We here describe an additional case of a 62-year-old male patient developing CML after orthotopic heart transplantation and medication with cyclosporine A, mofetil-mycophenolate, and steroids. Initial antileukemic therapy was imatinib at a standard dose and within 15 months of therapy a complete cytogenetic response was noted. In this report we discuss the clinical implications of these rare but biologically important cases

Uncertainty from sampling: workshop to launch a Nordtest handbook on sampling uncertainty estimation and control. Accreditation and Quality Assurance

A workshop on uncertainty in sampling was held in Hillerød, Denmark, on 12¿13 April 2007 to launch a new handbook on sampling quality assurance and uncertainty estimation. The participants of the workshop were approximately 60 delegates from 15 European countries, representing institutions performing sampling, users of the data, research institutions, as well as accreditation bodies. Materials from the workshop, including examples, tools, and calculation aids for the work can be found at http://www.samplersguide.com. The Nordtest handbook Uncertainty from sampling will be made available on the Nordtest web site at http://www.nordicinnovation.net/nordtest.cfm under NT technical reports, report number NT tec 604. Until the final report is available on the Nordtest web site, an advance draft of the Nordtest handbook is available from http://www.samplersguide.com

ENCORT-CDW : Evaluation of the European recovery target for construction and demolition waste

The present EU target for recovery of construction and demolition waste does not promote sustainability. Interpretations of waste and recovery definitions rather than resource efficiency and safe handling affect the monitoring. The general weight-based target has to be altered to favour recycling of resource-rich materials, not only of high weight materials. High grade recycling should be distinguished and prioritized to ensure safe recovery. This requires improved knowledge on waste generation and handling as well as on emissions of dangerous substances. A common Nordic approach is advocated

Effects of the JAK2-selective inhibitors NVP-BSK805 and NVP-BVB808 in BCR-ABL or JAK2 mutation-positive cell lines

Janus kinases are critical components of cytokine signaling pathways that regulate hematopoiesis, growth, immunity, inflammation, and development. Oncogenic mutations of the non-receptor tyrosine kinase JAK2 are found in many Philadelphia chromosome negative myeloproliferative neoplasms. Preclinical results strongly support the concept that JAK2 inhibitors could be effectively usedshow efficacy in treating patients with chronic myeloproliferative neoplasms (CMPN). JAK2 has also been postulated to play an important role in BCR-ABL signal transduction. Therefore, inhibitors of the tyrosine kinase activity of JAK2 are under investigation as new therapeutic strategies for treatment of CML. In this study the effects of two novel JAK2 inhibitors, NVP-BSK805 and NVP-BVB808, have been investigated in cell lines expressing either BCR-ABL or mutant JAK2. Possible synergistic effects between NVP-BSK805 / NVP-BVB808 and the already established tyrosine kinase inhibitors imatinib and nilotinib were assessed. Proliferation and apoptosis tests with both substances showed an intense response in the JAK2 mutated cell lines CHRF-288-11, SET-2 and UKE-1. Interestingly, the JAK2 V617F-positive HEL cells showed only a weak response. All BCR-ABL positive cell lines showed some reduction of proliferation, but with GI50 values higher than 1 µM. Combination of the JAK2 inhibitors with imatinib and nilotinib showed no significant additive or synergistic effects, although all BCR-ABL positive cell lines responded well to both CML therapeutic agents. Interestingly, it seemed that the combination of imatinib with NVP-BSK805 had a protective effect on the cells. Combination with nilotinib did not show this effect

VEXAS and Myelodysplastic Syndrome: An Interdisciplinary Challenge

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a recently recognized systemic autoinflammatory disease caused by somatic mutations in hematopoietic progenitor cells. This case series of four patients with VEXAS syndrome and comorbid myelodysplastic syndrome (MDS) aims to describe clinical, imaging, and hematologic disease presentations as well as response to therapy. Four patients with VEXAS syndrome and MDS are described. A detailed analysis of imaging features, hemato-oncological presentation including bone marrow microscopy and clinical–rheumatological disease features and treatment outcomes is given. All patients were male; ages ranged between 64 and 81 years; all were diagnosed with MDS. CT imaging was available for three patients, all of whom exhibited pulmonary infiltrates of varying severity, resembling COVID-19 or hypersensitivity pneumonitis without traces of scarring. Bone marrow microscopy showed maturation-disordered erythropoiesis and pathognomonic vacuolation. Somatic mutation in the UBA1 codon 41 were found in all patients by next-generation sequencing. Therapy regimes included glucocorticoids, JAK1/2-inhibitors, nucleoside analogues, as well as IL-1 and IL-6 receptor antagonists. No fatalities occurred (observation period from symptom onset: 18–68 months). Given the potential underreporting of VEXAS syndrome, we highly recommend contemporary screening for UBA1 mutations in patients presenting with ambiguous signs of systemic autoinflammatory symptoms which persist over 18 months despite treatment. The emergence of cytopenia, especially macrocytic hyperchromic anemia, should prompt early testing for UBA1 mutations. Notably conspicuous, pulmonary alterations in CT imaging of patients with therapy-resistant systemic autoinflammatory symptoms should be discussed in interdisciplinary medical teams (Rheumatology, Hematology, Radiology and further specialist departments) to facilitate timely diagnosis during the clinical course of the disease