PRIMARY AIM
1. CHARACTERIZATION OF A NEW RECURRENT GAIN AT 11q24.3 GENOMIC LOCUS OF DLBCL
PATIENTS CONTAINING TWO INTERESTING ETS FACTORS
1.1. 11q24.3 GAINS A RECURRENT LESION IN A SUBSET OF DLBCL PATIENTS
A previous work of our group had analyzed DNA genome profiles of a series of 166 DLBCL patients [186]. The analysis of genomic profile by high-density genome-wide SNP-based array [186] had identified a minimal common region (MCR) of gain at 11q24.3 not previously functionally characterized, in 23% (31/166) of the cases.
Figure 15: upper panel: copy number frequency plot of whole genome of 166 DLBCL samples.
Gains are represented in red, losses in blue. X-axis, represents all chromosomes and their physical mapping; Y-axis, represents the frequency of patients affected by alterations. Lower panel:
Frequency of gains on chromosome 11 alone in 166 DLBCL samples. X-axis, physical mapping; Y-axis, frequency of patients with gains. 11q24.3 gain is indicated by the black arrow [186].
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Figure 16: Genes localized in 11q.24.3 locus according to the UCSC data base (NCBI Build 36.1).
Figure 15 (upper panel) represents the frequency plot of detected DNA gains and DNA losses.
The genome of DLBCL patients is characterized by a high complexity with a lot of aberrations that vary from gains and amplifications to losses and deletions that confirms the difficulty in classification and treatment of DLBCL cases. This analysis is not used to identify translocations.
It was decided to study deeper the region at chromosome locus 11q24.3 (GRCh37/hg19chr:128 137,4000-128 994,334, Fig.15 lower panel) not only for the frequency of the gain, but also because chromosome 11q is very genes rich and it is the target of a variety of chromosomal aberrations in different hematological cancers. This aberration contain 6 transcripts: ETS1, FLI1, KCNJ1, KCNJ5, P53AIP1, and RICS (Fig.16).
1.2. ETS1 AND FLI1 ARE THE TWO TARGETS OF 11q24.3 GAIN
Integration of genomic profiles with gene expression analysis done with the Affymetrix U133 plus 2.0 on a set of 54 cases (16 bearing 11q24.3 gain) showed that, among the six genes previously described, only the ETS1 and FLI1 genes were expressed in DLBCL patients (Fig.17). Moreover, cases with 11q24.3 gain presented significantly higher RNA levels of ETS1 (fold change: 1.4; P=0.0129) and FLI1 (fold change: 1.5;
P=0.0082) than wild type cases suggesting that in these cases the gain affects the expression of the two factors (Fig.17). Correlation analysis with gene expression data revealed that high levels of ETS1 mRNA correspond to high FLI1 mRNA levels pointing out that patients carrying the 11q24.3 gain express concomitantly high levels of ETS1 and FLI1 (correlation coefficient 0.73; Fig.18).
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Figure 17: 11q24.3 gain is a recurrent event in DLBCL and it is associated with high levels of ETS1 and FLI1. Patients with the 11q24.3 gain express high levels of ETS1 and FLI1 mRNA. RNA levels of ETS1, FLI1, KCNJ1, KCNJ5, TP53AIP1, and RICS in patients with 11q24.3 gain compared with patients without the gain (wt). *P value <0.05.
Figure 18: Correlation between ETS1 and FLI1 mRNA levels in DLBCL samples. Values of mRNA major then 7 correspond to the expression status of the gene. Here just one samples does not expresses ETS1 and FLI1.
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Figure 19: Immunohistochemical analysis of ETS1 and FLI1 in three representative DLBCL cases carrying the 11q24.3 gain (40X).
Using immunohistochemical analysis, expression of ETS1 and FLI1 was also confirmed at protein level on 12 DLBCL cases, 4 bearing the gain (Fig.19). The same analysis was used to detect the expression of ETS1 and FLI1 in normal B cells from human tonsil (Fig.20).
Here, the Ki-67 marker, a protein strictly associated with cell proliferation, was used to distinguish dark proliferative zone of centroblasts to the light non-proliferative zone of centrocytes. We observed that ETS1 and FLI1 are both expressed with an expression gradient from dark to light zone, with a stronger difference for FLI1. This finding confirms previous knowledge from the literature in which ETS1 and FLI1 are essential factors for early phases of GC. As we can see, other important lymphatic cells such as macrophages, dendritic cells or T cells around the GC, also express ETS1 and FLI1.
Because DLBCL molecular subtypes are characterized by specific chromosomal aberrations, I try to understand if 11q24.3 gain is associated with one DLBCL subtypes, but no significant associations were revealed by this analysis. A correlation emerged by GEP analysis using GSE10846 dataset [161], in which the mRNA levels of both ETS factors derived from ABC- and GCB-DLBCL patients (n=167; n= 183,respectively) and from hyperplasia samples (9 cases). ETS1 levels were higher in ABC- and GCB-DLBCL cases than in benign hyperplasia (P=.013 and P =.035, respectively) and in ABC- than in
GCB-90
DLBCL (P<.001), suggesting that their expression can have a role in lymphomagenesis, in particular, in maintaining the early phase conditions of GC (Fig.21 left panel). FLI1 expression levels did not show any differences between the two DLBCL subtypes or in respect to the benign hyperplasia. ETS1 was more expressed in normal B cells compared to FLI1 (Fig.21 right panel), thus, maybe, a variation in ETS1 expression is more detectable than variations in FLI1 mRNA levels.
Figure 20: Immunohistochemistry on normal human tonsil using antibodies anti- Ki67, ETS1 and FLI1.
CB=centroblasts, CC=centrocytes.
Figure 21: Left panel: GEP analysis on ABC-/GCB- DLBCL samples and hyperplasia samples to detect ETS1 and FLI1 expression differences. GEP data from LLMPP DLBCL series vs Hyperplasia.
Right panel: qRT-PCR for ETS1 and FLI1 detection on a pool of cDNA from naïve B cells, centroblasts, centrocytes, memory B cells. mRNAs from B cells of different origin were join together during qRT-PCR.
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