ALBERT

Beschreibung: Diffuse large B-cell lymphomas (DLBCLs) can be divided into germinal-center B cell–like (GCB) and activated-B cell–like (ABC) subtypes by gene-expression profiling (GEP), with the latter showing a poorer outcome. Although this classification can be mimicked by different immunostaining algorithms, their reliability is the object of controversy. We constructed tissue microarrays with samples of 157 DLBCL patients homogeneously treated with immunochemotherapy to apply the following algorithms: Colomo (MUM1/IRF4, CD10, and BCL6 antigens), Hans (CD10, BCL6, and MUM1/IRF4), Muris (CD10 and MUM1/IRF4 plus BCL2), Choi (GCET1, MUM1/IRF4, CD10, FOXP1, and BCL6), and Tally (CD10, GCET1, MUM1/IRF4, FOXP1, and LMO2). GEP information was available in 62 cases. The proportion of misclassified cases by immunohistochemistry compared with GEP was higher when defining the GCB subset: 41%, 48%, 30%, 60%, and 40% for Colomo, Hans, Muris, Choi, and Tally, respectively. Whereas the GEP groups showed significantly different 5-year progression-free survival (76% vs 31% for GCB and activated DLBCL) and overall survival (80% vs 45%), none of the immunostaining algorithms was able to retain the prognostic impact of the groups (GCB vs non-GCB). In conclusion, stratification based on immunostaining algorithms should be used with caution in guiding therapy, even in clinical trials.

Beschreibung: The balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural β-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and β-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (KD, 107.9 ± 3nM) was restored in the β-L99F glycoform (KD, 53.9 ± 5nM) to values close to the activity of α-wild type (KD, 43.9 ± 0.4nM). Accordingly, the β-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional β-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin mutations.

Beschreibung: Patients with multiple myeloma (MM) carrying high-risk cytogenetic abnormalities (CA) have inferior outcome despite achieving similar complete response (CR) rates when compared to cases with standard-risk CA. This questions the legitimacy of CR as treatment endpoint for high-risk MM, and represents a biological conundrum regarding the nature of tumor reservoirs persisting after therapy in patients with standard- and high-risk CA. Here, we used next-generation flow (NGF) to evaluate measurable residual disease (MRD) in MM patients with standard- (N=300) vs high-risk CA (N=90) enrolled in the PETHEMA/GEM2012MENOS65 trial (NCT01916252), and to identify mechanisms determining MRD resistance in both patient subgroups (N=40). The 36-month progression-free and overall survival rates were higher than 90% in patients with undetectable MRD, with no significant differences (P≥0.202) between cases having standard- vs high-risk CA. Persistent MRD resulted in median progression-free survival of approximately three and two years in patients with standard- and high-risk CA, respectively (P

Beschreibung: Background FLT3, PIM1, PIM2 and CXCR4 are proteins implicated in the signal transduction of the regulation of proliferation, differentiation and survival of hematopoietic stem cells, at different levels. The impairing of these three processes, regulated by these molecules, constitutes the main hallmark of Acute Myeloid Leukemia (AML). The objectives of this work were to evaluate the expression level of the genes that codify such proteins in patients with AML; as well as correlate this level of expression with biological variables at diagnosis and survival. Methods peripheral blood or bone marrow samples from 31 healthy subject (HS) and 92 AML patients at diagnosis between 2004 and 2012 were studied. The median follow up was 14 months (69% of patients had died). We quantified the expression of FLT3, PIM1 and 2, and CXCR4 by real time PCR, employing primer pairs designed in our laboratory to amplify all known protein-coding transcripts; highlighting the presence of a 30.4% FLT3 ITD, 5.4% of other FLT3 mutations and 26% of NMP1 mutations. Results were then analyzed with the statistical package IBM“ SPSS” Statistics, v20 (IBM“, Nueva York). Results FLT3 was overexpressed in AML patients (FLT3 expression: controls 0.99 vs patients 36.97; p

Beschreibung: Abstract 2924 Background: Clonal composition and clone dynamic changes of neoplasms are a controversial issue, whose investigation is now facilitated by the development of massive parallel sequencing. Here we have analyzed the changes in the mutational spectrum associated with progression, treatment response and relapse in a multiple myeloma patient. We sequenced exomes for the primary quiescent-tumor, the progression and the relapsed samples. M&M: Patient and samples description Samples from asymptomatic, progression and relapse phases were compared by FISH and Whole exome massive parallel sequencing in a multiple myeloma patient carrying the t(4;14)(p16.3;q32) alteration. At relapse the cytogenetic study identified the presence of two major clones, 13q14 deletion and t(4;14)(p16.3;q32) in the 60% of the cells, and 17p13 deletion in the 12% of the cells. Whole exome sequencing was performed at CNAG (Barcelona, Spain) following standard protocols for high-throughput paired-end 76pb sequencing on the Illumina HiSeq2000 instruments (Illumina Inc., San Diego, CA). The variant calling was performed using an in house written software calling potential mutations showing a minimum independent multi-aligner evidence. Results: We performed whole exome sequencing on 3 tumor samples from the same patient: the first one at the time of diagnosis correspond to bone marrow infiltrated by 7% of plasma cells. The two additional samples, at progression and relapse, were done in CD138+ bone marrow cells, at this moment the percentage of infiltration was of 84% and 64% respectively. The germinal DNA from the same patient was used as reference. The mean coverage obtained for the four samples were 93x, with around 85% of bases with at least 20X coverage. After filtering, a total of 104 single nucleotide variations (SNV) were identified, some of them in more than one sample. The variations were classified into silent (25), missense (71), nonsense (6), and essential splice (2), according to their potential functional effect. In addition to t(4;14) and del13q14, progression and relapse samples shared 36 common SNVs. There were also some variants gained and/or loss in the different time points, suggesting the presence of at least five different clones, independent but related in their evolution. The two main clones were present in progression and relapse samples, but the ratio of the mutant alleles decreased in parallel to the decrement in the percentage of cells carrying on the described cytogenetic alterations Conclusions: There is a coupling between the cytogenetic and tumor sequence changes indicating that tumor at progression was composed by a dominant clone, together with multiple minor clones. Relapse after treatment was associated with multiple changes in the clone dynamics, progressive reduction of the main clone, emerging of new subclones and lost of minor clones. Dynamic changes along progression could be facilitated/induced by the therapy received. Disclosures: No relevant conflicts of interest to declare.