Jitka Kabáthová

High Expression of Lymphocyte-Activation Gene 3 (LAG3) in Chronic Lymphocytic Leukemia Cells Is Associated with Unmutated Immunoglobulin Variable Heavy Chain Region (IGHV) Gene and Reduced Treatment-Free Survival

Chronic lymphocytic leukemia (CLL) is characterized by a monoclonal expansion of mature B-lymphocytes. Mutational status of the immunoglobulin variable heavy chain region (IGHV) gene stratifies CLL patients into two prognostic groups. We performed microarray analysis of CLL cells using the Agilent platform to detect the most important gene expression differences regarding IGHV status in CLL cells. We analyzed a cohort of 118 CLL patients with different IGHV mutational status and completely characterized all described prognostic markers using expression microarrays and quantitative real-time RT-PCR (reverse transcription PCR). We detected lymphocyte-activation gene 3 (LAG3) as a novel prognostic marker: LAG3 high expression in CLL cells correlates with unmutated IGHV (P < 0.0001) and reduced treatment-free survival (P = 0.0087). Furthermore, quantitative real-time RT-PCR analysis identified a gene-set (LAG3, LPL, ZAP70) whose overexpression is assigned to unmutated IGHV with 90% specificity (P < 0.0001). Moreover, high expression of tested gene-set and unmutated IGHV equally correlated with reduced treatment-free survival (P = 7.7 * 10(-11) vs. P = 1.8 * 10(-11)). Our results suggest that IGHV status can be precisely assessed using the expression analysis of LAG3, LPL, and ZAP70 genes. Expression data of tested markers provides a similar statistical concordance with treatment-free survival as that of the IGHV status itself. Our findings contribute to the elucidation of CLL pathogenesis and provide novel prognostic markers for possible application in routine diagnostics.

Analysis of the DNA-binding activity of p53 mutants using functional protein microarrays and its relationship to transcriptional activation.

Abstract Sequence-specific DNA binding is the key function through which tumor suppressor p53 exerts transactivation of the downstream target genes, often being impaired in cancer cells by mutations in the TP53 gene. Functional protein microarray technology enables a high-throughput parallel analysis of protein properties within one experiment under the same conditions. Using an array approach, we analyzed the DNA binding activity of wild type p53 protein and of 49 variants. Our results show significant differences in the binding properties between the p53 mutants. The C-terminal mutant R337C displayed the highest DNA binding activity on the array. However, the same mutant showed only a partial activation in the reporter gene assay and almost no activation of downstream target genes after transfection of expression vector into cells lacking endogenous p53. These observations demonstrate that DNA binding itself is not sufficient for activating the p53 target genes in at least some of the p53 mutants and, therefore, in vitro studies might not always reflect in vivo conditions.

Low-burden TP53 mutations in chronic phase of myeloproliferative neoplasms: association with age, hydroxyurea administration, disease type and JAK2 mutational status

The multistep process of TP53 mutation expansion during myeloproliferative neoplasm (MPN) transformation into acute myeloid leukemia (AML) has been documented retrospectively. It is currently unknown how common TP53 mutations with low variant allele frequency (VAF) are, whether they are linked to hydroxyurea (HU) cytoreduction, and what disease progression risk they carry. Using ultra-deep next-generation sequencing, we examined 254 MPN patients treated with HU, interferon alpha-2a or anagrelide and 85 untreated patients. We found TP53 mutations in 50 cases (0.2–16.3% VAF), regardless of disease subtype, driver gene status and cytoreduction. Both therapy and TP53 mutations were strongly associated with older age. Over-time analysis showed that the mutations may be undetectable at diagnosis and slowly increase during disease course. Although three patients with TP53 mutations progressed to TP53-mutated or TP53-wild-type AML, we did not observe a significant age-independent impact on overall survival during the follow-up. Further, we showed that complete p53 inactivation alone led to neither blast transformation nor HU resistance. Altogether, we revealed patients age as the strongest factor affecting low-burden TP53 mutation incidence in MPN and found no significant age-independent association between TP53 mutations and hydroxyurea. Mutations may persist at low levels for years without an immediate risk of progression.

Gene expression profiling of acute graft-vs-host disease after hematopoietic stem cell transplantation

Acute graft-vs-host disease (aGVHD) is a frequent, life-threatening complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Despite that, there are no reliable molecular markers reflecting the onset or clinical course of aGVHD. We performed a pilot study on gene expression profiling in peripheral blood mononuclear cells taken from 15 patients with hematological malignancies who underwent allo-HSCT and developed aGVHD. Based on survival rates after aGVHD, patients were divided into two groups-favorable (all patients alive; median follow-up 40 months) vs unfavorable group (all patients died; median survival 2 months). Two-hundred and eighty genes differentially expressed between these two groups were identified; among them, genes responsible for cytokine signaling, inflammatory response, and regulation of cell cycle were over-represented; interleukin-8, G0S2, ANXA3, and NR4A2 were upregulated in the unfavorable group, CDKN1C was downregulated in the same group. Interestingly, the same genes were also described as overexpressed in connection with autoimmune diseases. This indicates an involvement of similar immune regulatory pathways also in aGVHD. Our data support use of gene expression profiling at aGVHD onset for a prediction of its outcomes.

Fragment analysis represents a suitable approach for the detection of hotspot c.7541_7542delCT NOTCH1 mutation in chronic lymphocytic leukemia

The hotspot c.7541_7542delCT NOTCH1 mutation has been proven to have a negative clinical impact in chronic lymphocytic leukemia (CLL). However, an optimal method for its detection has not yet been specified. The aim of our study was to examine the presence of the NOTCH1 mutation in CLL using three commonly used molecular methods. Sanger sequencing, fragment analysis and allele-specific PCR were compared in the detection of the c.7541_7542delCT NOTCH1 mutation in 201 CLL patients. In 7 patients with inconclusive mutational analysis results, the presence of the NOTCH1 mutation was also confirmed using ultra-deep next generation sequencing. The NOTCH1 mutation was detected in 15% (30/201) of examined patients. Only fragment analysis was able to identify all 30 NOTCH1-mutated patients. Sanger sequencing and allele-specific PCR showed a lower detection efficiency, determining 93% (28/30) and 80% (24/30) of the present NOTCH1 mutations, respectively. Considering these three most commonly used methodologies for c.7541_7542delCT NOTCH1 mutation screening in CLL, we defined fragment analysis as the most suitable approach for detecting the hotspot NOTCH1 mutation.