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Next-generation sequencing (NGS) is becoming increasingly used for diagnostic mutation analysis in myeloid neoplasms and may also represent a feasible technique in mastocytosis. However, detection of the KIT D816V mutation requires a highly sensitive method in most patients due to the typically low mutation levels. In this study, we established an NGS-based KIT mutation analysis and analyzed the sensitivity of D816V detection using the Ion Torrent platform. Eighty-two individual NGS analyses were included in the study. All samples were also analyzed using highly sensitive KIT D816V mutation-specific qPCR. Measurements of the background level in D816V-negative samples supported a cutoff for positivity of 0.2% in three different NGS panels. Clinical samples from patients with SM that tested positive using qPCR with a D816V allele burden >0.2% also tested positive using NGS. Samples that tested positive using qPCR with an allele burden

Mutation in the NPM1 gene occurs in 60% of acute myeloid leukaemia (AML) patients with normal karyotype. NPM1 mutation is potentially a superior minimal residual disease (MRD) marker compared to WT1 gene overexpression by being specific to the malignant clone, although experimental evidence published so far includes very limited numbers of relapsed cases. Also, the stability of the NPM1 mutation has been questioned by reports of the mutation being lost at relapse. In the present study we compared NPM1 mutation and WT1 overexpression as MRD markers in 20 cases of relapsed AML. The 20 patients experienced a total of 28 morphological relapses. Karyotypic evolution was detected in 56% of relapses. All relapses were accompanied by high levels of NPM1 mutation, along with high WT1 mRNA levels, thus demonstrating complete stability of both markers during relapse. Detectable NPM1 mutation following a period of morphological remission was accompanied by a morphological relapse in all cases. In contrast, WT1 expression was detected in 33% of the NPM1 mutation negative samples. This background WT1 expression produced by non-leukaemia cells was highly variable, both between and within patients, and limited the de facto sensitivity of the WT1 expression analysis. The present study therefore provides important experimental evidence demonstrating that NPM1 mutation is superior to WT1 overexpression as marker of MRD in NPM1-mutated AML, even in the presence of extensive karyotypic evolution.

Chromosomal aberrations have significant prognostic importance in multiple myeloma (MM). However, proteasome inhibitors (PI) and IMiDs may partly overcome the poor prognostic impact of some of them. In this study, we investigated a population-based consecutive cohort newly diagnosed patients with MM admitted during a defined time period to hospitals in Denmark, Norway, and Sweden. The impact of treatment modality on the prognostic importance of specific chromosomal aberration was investigated, with special reference to gain 1q21. The median follow-up of patients still alive at analysis was 40 months for the high-dose (HDT)-treated ones and 29 months for the whole population. Three hundred forty-seven patients with a known 1q21 status were included in this study. The 347 patients were divided into three groups, that is, 119 patients with the 1q21 gain, 105 patients with other aberrations (OA), that is, del(13q), del(17p), t(4,14), and/or (14;16), and 123 patients with no aberrations (NA). The groups were compared in terms of overall survival (OS), time to progression (TTP), and response. The 3-yr OS for patients with gain 1q21 was 60% compared to patients with OA 74% and NO 82% (gain 1q21 vs. NO P < 0.001; gain 1q21 vs. OA P = 0.095). If treated with PI or IMiDs, the 3-yr OS was 58% for patients with gain 1q21 compared to patients with OA 78% and NO 78%, respectively (P = 0.041, P = 0.140). In HDT patients, the 3-yr OS was 69% for patients with gain 1q21 compared to patients with OA 84% and NO 88%, respectively (P < 0.008, P = 0.600). Thus, neither HDT nor using PI or IMiDs could overcome the poor prognostic impact of gain 1q21, while these drugs and HDT seemed to improve OS in patients with OA, approaching the survival in NO. Further, gain 1q21 appears to be one of the most important poor prognostic chromosomal aberrations in multiple myeloma with current treatments. Trials using new drugs or allogeneic transplantation are warranted.

Udgivelsesdato: 2005-Jun OBJECTIVE: Hierarchical clusterings of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures have previously been used to classify DLBCL into Germinal Center B-cell (GCB) and Activated B-cell (ABC) types. To examine if it was feasible to perform a cross-platform validation on the Affymetrix HG-U133A oligonucleotide arrays and improve the classification, we determined the expression profiles of pretreatment, diagnostic samples from 52 primary nodal DLBCL. METHODS AND RESULTS: First, three previously published gene lists were converted to the HG-U133A probe sets and used for hierarchical clustering. In this way, three subtypes, including the GCB type (n = 20), the ABC type (n = 25) and an intermediate group, Type-3 (n = 5), were distinguished. The CD10 and Bcl-6 expression as well as t(14;18) translocation were prevalent, but not exclusive to the GCB type. By contrast, MUM1 was only expressed in the ABC and in Type-3 samples. The 5-year survival was similar between the groups, but GCB patients showed a better initial response to CHOP or CHOP-like regimens than the remaining patients and tended to have less advanced disease and lower IPI scores. As a next step, an improved set of classifier genes was generated by analysis of 34 patients that were consistently classified as GCB or ABC in the above analyses. Seventy-eight genes were selected and demonstrated on two previously published data sets (Shipp et al. Nat Med 2002;8:68-74 and Houldsworth et al. Blood 2004;103:1862-1868) to exhibit a higher specificity than the original gene lists. CONCLUSION: We conclude that gene expression profiling with Affymetrix Genechips is efficient to distinguish between GCB and ABC types of DLBCL and that these are likely to represent separate biological entities. The Genechip platform is highly standardised and therefore useful for future prospective investigations to establish the value of gene expression profiling in the clinical management of DLBCL.

In western countries, the age distribution of Hodgkin lymphoma (HL) follows a characteristic bimodal curve showing an early and a late peak at approximately 35 and 70 years, respectively. Furthermore, the presence of latent Epstein-Barr Virus (EBV) genome in the Hodgkin Reed-Sternberg cells, the tumour cell population of classical HL (cHL), has been found to have adverse prognostic impact in elderly, but not in younger cHL patients. We have characterized the protein expression in tumour tissue samples from younger (≤55yrs) and elderly (>55yrs) cHL patients and correlated the findings with EBV status. Differentially expressed proteins according to patients' age as well as tumoral EBV status belonged to different biological functional domains, such as apoptosis, cytoskeletal organization, response to oxygen levels and regulation of catabolic/metabolic processes. The differential expression of selected proteins, cytosolic aminopeptidase, heterogeneous nuclear ribonucleoprotein K, serotransferrin, and alpha-1-antitrypsin, was further validated by western blot analysis. Discovery based proteomics characterising biological features distinctive for subsets of cHL patients may be useful for the identification of novel biomarkers with potential therapeutic relevance. An evaluation of the prognostic impact of protein expression pattern in general and individually expressed proteins in particular is warranted. This article is protected by copyright. All rights reserved.

It is presently accepted that the KIT D816V mutation is detectable in tissues with neoplastic mast cells in most patients with indolent systemic mastocytosis. In this study, neoplastic mast cells were detected in bone marrow, but not in peripheral blood, by flow cytometry in all 25 included cases of indolent systemic mastocytosis. However, the KIT D816V mutation was detected using mutation-specific qPCR in both bone marrow and peripheral blood in all 25 cases, demonstrating for the first time that the KIT D816V mutation is consistently present in non-mast cells in indolent systemic mastocytosis and that these cells are circulating in peripheral blood.

Udgivelsesdato: 2007-Dec Diffuse large B-cell lymphoma (DLBCL) is a frequent lymphoma subtype with a heterogeneous behavior and a variable response to conventional chemotherapy. This clinical diversity is believed to reflect differences in the molecular pathways leading to lymphomagenesis. In this study, we have analyzed pretreatment, diagnostic samples from 108 DLBCL by immunohistology for expression of four markers linked to germinal center B-cells (CD10, Bcl-6), postgerminal center B-cells (MUM1) and apoptosis (Bcl-2). The results indicate that both CD10 and Bcl-6 are favorable prognostic indicators, in contrast to Bcl-2, which is an adverse parameter. Furthermore, using two algorithms for distinction between low- and high-risk patients proposed by Hans et al. (Blood, 2004; 103:275) and Muris et al. (Journal of Pathology, 2006; 208:714), it is shown that both are useful for predicting outcome in DLBCL. However, in this report, the algorithm of Hans et al. was superior to that of Muris et al. These findings confirm and extend other studies and indicate that different prognostic subgroups of DLBCL can be distinguished by simple immunohistological investigations for a limited number of markers. Whether these groups are also relevant for individual treatment decisions will be important to investigate in prospective studies.

Udgivelsesdato: 2007-Dec BACKGROUND AND OBJECTIVES: The JAK2 V617F tyrosine kinase mutation is present in the great majority of patients with polycythemia vera (PV), and approximately half of the patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). The three distinct disease entities may be considered as three phenotypic presentations of the same JAK2 V617F positive chronic myeloproliferative disorder. Together with physiological and genetic modifiers the phenotype may be determined by the JAK2 V617F allele burden. In the present study, we aimed to asses the JAK2 mutational load and its impact on phenotype. METHODS: A highly sensitive real-time quantitative PCR (qPCR) assay was used for quantification of the JAK2 V617F mutational load in 165 patients with Philadelphia chromosome negative chronic myeloproliferative disorders (ET = 40, PV = 95, PMF = 30). RESULTS: We provide evidence of increasing JAK2 V617F allele burden from ET, over PV to PMF (P = 0.001 and P < 0.00001 respectively). The present data suggests the JAK2 V617F allele burden as a key determinant of the degree of myeloproliferation and myeloid metaplasia reflected by significantly higher levels of white blood cell counts (WBC) (P = 0.03), CD34 counts (P = 0.03), lactate dehydrogenase and Polycythemia Rubra Vera gene 1 levels (P = 0.03 and P < 0.00001 respectively), as well as lower platelet counts (P = 0.02) and more cases of splenomegaly (P = 0.001) in homozygous PV patients compared to their heterozygous counterparts. CONCLUSION: The present study support the concept of the JAK2 V617F positive chronic myeloproliferative disorders as a biological continuum with phenotypic presentation in part influenced by JAK2 V617F mutational load.