Protein and RNA Expression of Genetic Subgroups in CLL

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COMPREHENSIVE PROTEIN AND RNA EXPRESSION ANALYSIS OF GENETIC

SUBGROUPS OF CHRONIC LYMPHOCYTIC LEUKAEMIA

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Author Winkler, Dirk , Ulm, University of Ulm Germany

Co-author(s) Schneider, Christof MD, Ulm, University of Ulm

Kienle, Dirk MD, Ulm, University of Ulm

Döhner, Hartmut Prof., Ulm, University of Ulm

Stilgenbauer, Stephan Prof., Ulm, University of Ulm

Chronic lymphocytic leukemia is a disorder with a highly variable

clinical course. The VH mutation status and genomic aberrations such

as deletion 11q, trisomy 12, deletion 13q, and deletion 17p identify

subgroups with different survival times.

Protein expression levels of candidate genes involved in cell cycle

and apoptosis (AKT1, Apaf-1, ATM, BAX, BCL-2, CDK2, CDK4, cIAP2,

Cyclin-D1/D2/D3, MCL-1, MDM-2, p21, p27, p53, PI3K, RB, SMAC,

Survivin, XIAP) were studied by immunoblotting. 97 CLL cases divided

into subgroups with 11q- (n=23), +12q (n=24), 13q- (n=19), 17p-/p53

mutation (n=15) or a normal karyotype (n=16) were examined and

compared to the cell lines EHEB, JVM-2 and Granta-519. In 25 cases,

mRNA expression data (studied by RQ-PCR) was compared for AKT1, ATM,

BAX, BCL-2, CDK4, Cyclin-D1/D2/D3, MCL-1, p21, p27, p53, PI3-K to

assess if protein expression and mRNA levels are correlated.

Expression levels were related to EHEB for standardization. VH-

mutation status was available for 91 cases. 28 cases had mutated VH

genes.

In the normal karyotype subgroup a highly homogenous expression

pattern for all proteins studied was observed independently of the

VH-status. CLL samples with +12q, 13q- or normal karyotype had equal

levels of ATM protein, whereas the 11q-subgroup showed reduced

levels in 5 cases and absent ATM protein in 1 case (among 15

evaluable cases). The 17p-subgroup included 13 cases without

deletion 11q showing ATM protein levels comparable to the normal

karyotype group.

17p-cases showed stronger expression of p53 compared to all other

cases, except for 4 cases with normal karyotype or deletion 11q. In

all of these a p53 mutation could be detected in exons 2-11 by

sequencing. Survivin protein and mRNA was absent or low in all

cases. CDK4 protein expression was high in cases with 17p-, 11q- and

13q- but low in the subgroup with +12q and normal karyotype. MCL-1

expression was highest in the 17p-subgroup. Regarding PI3K, MDM-2,

p21, p27, CDK2, Cyclin-D1, -D2, -D3, BAX, BCL-2, Apaf-1, SMAC, XIAP,

cIAP2, RB, E2F, AKT1, ARF and STAT6 no variation in protein

expression levels were observed across the subgroups. P27-expression

was higher in all CLL cases compared to the cell lines contrary to

stronger levels of p21 in the cell lines. Clear concordance between

RNA and protein expression was seen for BCL-2, Cyclin-D2, p21 and

p27 regardless of the subgroup affiliation. However, the exact

levels of RNA expression could not be retrieved by immunoblotting.

In conclusion, the 17p-subgroup was the only group with a high level

of p53 and MCL-1 protein expression suggesting that p53 is the

affected gene in this subgroup. In contrast, the ATM protein levels

are reduced only in a part of 11q- cases indicating a possible role

of additional candidate genes. Cases with trisomy 12 and normal

karyotype showed weak expression of CDK4 pointing to a functional

relevance in these subgroups. Protein levels and mRNA levels

correlated only for a subset of the genes investigated. This may be

due to posttranscriptional mechanisms regulating gene expression or

a lower sensitivity of immunoblotting to detect subtle expression

differences.