Lipoprotein lipase SNPs rs13702 and rs301 correlate with clinical outcome in chronic lymphocytic leukemia patients

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and is characterized by a heterogeneous clinical course. This variability in clinical course has spiked the search for prognostic markers able to predict patient evolution at the moment of diagnosis. Markers demonstrated to be of value are the mutation status of the immunoglobulin heavy chain variable region genes (IGHV) and lipoprotein lipase (LPL) expression. High LPL mRNA expression has been associated with short treatment free (TFS) and decreased overall survival (OS) in CLL. The LPL SNPs rs301 (T<C), rs328 (C<G) and rs13702 (T<C) have been associated with various metabolic disorders, but the association with CLL evolution is unknown. Here, in a cohort of 248 patients, we show that patients with the LPL SNP rs13702 wild-type T/T genotype had significantly shorter OS than patients with C/C and T/C genotypes (median time until CLL related death: 90 and 156 months respectively, p=0.008). The same was observed for LPL SNP rs301 (median time until CLL related death T/T: 102 and C/C, T/C: 144 months, p=0.03). Both SNPs rs301 and rs13702 were significantly associated with each other and notably, no association was found between IGHV status and presence of the SNP genotypes, indicating that these LPL SNPs are reliable prognostic markers that could add extra prognostic and predictive information to classical markers and help to improve the management of CLL

Functional exploration of ZAP70 and LPL in chronic lymphocytic leukemia

A typical hallmark of chronic lymphocytic leukemia (CLL) is its highly variable clinical course, with life expectancies ranging from months to decades. Although the majority of patients presents with low-grade CLL at diagnosis, a significant subset will eventually develop a more aggressive and life-threatening disease. Identification of these patients with an elevated risk of progression is of utmost importance in the context of individual risk-adapted clinical management. Over the last decades many markers have been identified that allow predicting a patient’s prognosis in an early disease stage, among them zeta-chain- associated protein of 70kDa (ZAP70) and lipoprotein lipase (LPL). Although their prognostic value has repeatedly been reported before, whether and how ZAP70 and LPL contribute to an aggressive phenotype is not elucidated yet. Unraveling the functional role of these and other prognostic markers in CLL disease biology might provide more insights into oncogenic pathways and could unravel new therapeutic targets. By performing whole genome expression profiling in CLL cells overexpressing the protein of interest, being either ZAP70 or LPL, we aimed at further unraveling the role of these prognostic markers in the pathogenesis of CLL. In summary, we were able to demonstrate that ZAP70 induces enhanced nuclear factor-kappa B (NF-κB) signaling upon B cell receptor (BCR) stimulation. Given that NF-κB is involved in protecting cells from spontaneous and drug induced apoptosis, this could represent a mechanism by which ZAP70 contributes to an aggressive disease course. Our data concerning the functional implications of LPL overexpression in CLL require further validation, but point to involvement of LPL in epigenetic gene regulation. As such, LPL could modulate disease-promoting pathways and contribute to an aggressive disease course. Besides this, we studied the prognostic implications of three single nucleotide polymorphisms (SNPs) in the LPL gene and found that the presence of LPL SNPs rs301 and rs13702 was correlated with prolonged overall survival. Interestingly, several prognostic markers in CLL, including ZAP70 and probably also LPL, relate to the BCR, which has been demonstrated to exert a critical role in the crosstalk between the leukemic clone and the surrounding tissue in the lymphoid organs. Accumulating evidence suggests that activation of the BCR within this tissue microenvironment, along with other co-stimulatory signals, promotes the survival and expansion of the leukemic clone. Exactly how this is achieved and which downstream pathways are involved is not entirely clear yet. To gain more insight in the role of BCR signaling in CLL, we analyzed whole genome mRNA and microRNA (miRNA) expression profiles obtained in CLL cells upon BCR stimulation in vitro. Both the BCR driven mRNA and miRNA signatures are involved in cell cycle entry and progression and point to a MYC driven proliferative response. Next, we re-evaluated our in vitro BCR stimulation method and compared it to other protocols in an attempt to provide a standardized way to perform CLL BCR stimulation in vitro. We found a dramatic difference in respect to immobilized versus soluble triggers of the BCR and show that both IGHV mutated and unmutated CLL cells respond equally efficient to BCR triggering

Mimicking the tumour microenvironment of chronic lymphocytic leukaemia in vitro critically depends on the type of B-cell receptor stimulation

Background: The B-cell receptor (BCR) has a key role in the cross-talk between chronic lymphocytic leukaemia (CLL) cells and the tissue microenvironment, which favours disease progression by promoting proliferation and drug resistance. In vitro studies on downstream signalling and functional effects of CLL BCR ligation often report contradictory results, in part owing to the lack of a standardised stimulation protocol. Our aim was to define a biologically relevant and robust in vitro stimulation method with regard to cellular phenotypic and transcriptional responses. Methods: We evaluated mRNA (FOS, MYC, LPL) and protein (CD54, CD19, CD62L, CD184) expression of genes modulated by BCR triggering in immunoglobulin heavy-chain variable region genes (IGHV)-mutated and -unmutated CLL cells, after stimulation using soluble or immobilised anti-IgM antibodies from different suppliers. Results: The effect of BCR stimulation on gene and protein expression was comparable in all CLL patients, irrespective of IGHV mutation status. However, immobilised anti-IgM stimulation elicited clear and robust changes in gene and protein expression, whereas the response to soluble anti-IgM was far less obvious. Conclusions: These data indicate that the method of BCR stimulation is of major importance regarding responsiveness of CLL cells in the context of the tumour microenvironment, whereas genetic differences in the BCR pathway are less critical

CLL cells respond to B-Cell receptor stimulation with a microRNA/mRNA signature associated with MYC activation and cell cycle progression.

Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation

Clinical and biological characteristics among <i>LPL</i> rs301, rs328 and rs13702 Genotypes.

<p>^a Cross-tabulations of prognostic markers versus <i>LPL</i> SNP genotypes. <i>p</i> values of Pearson χ² statistics (with the Yates continuity correction for 2x2 tables)</p><p>^b<i>p</i> value of Mann-Whitney non parametric test comparing median age at diagnosis between <i>LPL</i> SNP genotypes</p><p>^c HWE, Hardy-Weinberg equilibrium</p><p>Clinical and biological characteristics among <i>LPL</i> rs301, rs328 and rs13702 Genotypes.</p

Overview of examined genetic variants in the <i>LPL</i> gene.

<p>Both expected (exp) and observed (obs) minor allele frequencies (MAF) are shown for Caucasians. All variants are in accordance with Hardy Weinberg law. rs indicates referenced SNP id number; T</p><p>Overview of examined genetic variants in the <i>LPL</i> gene.</p

Correlation between LPL protein expression and <i>LPL</i> mRNA expression (A) and between <i>miRNA-410</i> expression and <i>LPL</i> mRNA (B) or LPL protein (C) expression.

<p><i>MiRNA-410</i> mRNA (n = 25) and <i>LPL</i> mRNA (n = 92) expression levels were determined by qPCR analysis, LPL protein levels were determined by ELISA (n = 44). No correlation was found between LPL protein and mRNA levels (Spearman’s rank correlation coefficient = 0.29) (A). No significant correlation between <i>miRNA-410</i> expression and LPL mRNA (B) or protein levels (C) could be observed.</p

Kaplan Meier survival curves for TFS with regard to <i>IGHV</i> mutation status (A) and LPL mRNA expression (B).

<p><i>IGHV</i> gene mutation status was based on a 98% cut-off value (n = 207; M, mutated; U, unmutated). Differentiation between <i>LPL</i> positive and negative cases was based on the optimal cut-off value determined by ROC curve analysis (n = 192). Log-rank tests showed significantly different TFS curves for <i>IGHV</i> mutation status (<i>p</i><0.0001) and <i>LPL</i> mRNA expression (<i>p</i> = 0.001).</p