12th Congress of the European Hematology ... - Haematologica

12 th Congress of the European Hematology Association
0172
SNP ARRAY PROFILING OF FOLLICULAR LYMPHOMA REVEALS NOVEL REGIONS OF
ACQUIRED UPD
D. O'Shea, 1 S. Iqbal, 2 E. Carlotti, 2 A. Davies, 2 T. Chaplin, 2
J. Matthews, 2 M. Manoj, 2 A. Norton, 3 J. Fitzgibbon, 2 T.A. Lister, 2
B.D. Young 2
1 CRUK, LONDON; 2 CRUK Medical Oncology, LONDON; 3 Barts and the
London School of Medicine, LONDON, United Kingdom
Background. The use of Single Nucleotide Polymorphism (SNP) array
profiling has uncovered extensive regions of acquired uniparental disomy
(aUPD) in the cancer genome undetected using cytogenetics or
array-CGH platforms. These usually arise by mitotic recombination and
can render a cell homozygous for a pre-existing abnormality. Methods.
SNP array analysis was performed using the Affymetrix 10K Gene-chip
mapping array on DNA extracted from a series of Follicular Lymphoma
(FL) lymph nodes biopsies from 52 patients, taken at time of diagnosis
(n=20), progression (n=26) and transformation (n=34), and the t(14;18)
positive lymphoma cell lines SCI-1, DoHH2 and RL 2261. Analysis was
performed using the genome oriented laboratory file system (GOLF), a
software package designed to interpret SNP data. In the absence of available
germline DNA from the majority of these patients; an algorithm
was devised to define significant regions of homozygosity. The criteria
of > 96% homozygosity in at least 50 contiguous SNPs was found to
detect no abnormalities in 23 normal remission bone marrows and was
therefore adopted. Results. Abnormalities were detected in 63/80 patient
specimens; these were non-random with recurring sites of aUPD on
several chromosomes. In the patient samples aUPD was observed most
frequently at 6p (n=12; 23%) and 12q (n=10; 19%); all 3 cell lines had
aUPD 12q and aUPD 6p was seen in SCI-1. Additionally, loss of heterozygosity
at 1p was seen in 7 patients (13%); this was copy neutral in
4/7 cases with reduced copy number in the other 3 cases. Rearrangements
of the distal end of chromosome 1p have been described in >10%
of cases of Non-Hodgkin’s Lymphoma and in all 7 cases the overlapping
region included 1p36 the location of the known tumour suppressor
genes CHD5, ID3 and p73. Twelve out of 52 patients studied (23%)
have either loss of the whole of chromosome 6 (n=2) or aUPD of chromosome
6p (n=10). This appears an early event in lymphomagenesis given
that it was identified at disease presentation, progression and transformation.
The sites of mitotic recombination cluster in a region immediately
proximal to the MHC complex at 6p21-12 in 9/12 cases. Mutations
in CCND3, CDKN1A, HLA-DQB1 and two translocation partners
of BCL6; SRP20 and HIST1H4I, which are all located within or just distal
of this cluster, were excluded by direct sequencing. Ten patients had
aUPD of 12q, which ran from varying points on the long arm to the
telomere in 9/10 cases; 1 case had an interstitial UPD of 17 Mb (95-112
Mb). Conclusions. This study highlights a high frequency of novel areas
of aUPD in FL and the selective basis of aUPD at these locations in the
pathogenesis of lymphoma continues to be investigated.
62 | haematologica/the hematology journal | 2007; 92(s1)
0173
INCREASED LEVEL OF B-CATENIN MRNA AND MUTATIONAL ALTERATIONS IN APC GENE
ARE PRESENT IN ACUTE LEUKEMIA
F. Atalar, 1 M. Sayitoglu, 1 O. Hatirnaz, 2 Y. Erbilgin, 1 U. Ozbek2 1 Istanbul University, DETAE Genetics Dept, ISTANBUL; 2 Istanbul
University,DETAE Genetics Dept, ISTANBUL, Turkey
Background.WNT proteins initiate B-catenin-regulated canonical signaling
cascade by binding to seven-transmembrane spanning receptors
of the Frizzled (FZ) family,leading to phosphorylation of the disheveled
protein forming a complex with the axin and APC (adenomatous polyposis
coli) proteins that blocks the kinase activity of GSK3B. A functional
APC protein is necessary for the proper regulation of the Wnt signaling
pathway. Loss of APC expression interferes with the phosphorylation
and degradation of B-catenin, allowing a B-catenin/TCF/LEF transcription
factor complex to act in the nucleus, where it stimulates cell
growth by targeting genes such as cyclin D1 and c-myc. Aims.Our aim
was to determine the role of the WNT pathways in the development of
acute leukemias. Method. We studied the gene expression levels of several
components of WNT signaling pathway; WNT5A, WNT10B, FZ5,
B-catenin, APC, TCF1 (T-cell factor), LEF1 (lymphoid enhancer factor)
and their important targets c-MYC and CCND1 (cyclin D1), in 34 AML
patient and 118 ALL patient (T-ALL, n=42, B-ALL, n=46 and preB-ALL,
n=30) bone marrow and/or blood samples and normal hematopoietic
cells by quantitative real time polymerase chain reaction. APC and Bcatenin
mutations were studied with dHPLC and sequencing methods.
Results.Results were compared by peripheral blood samples from healthy
donors by using Ct values. WNT5A, WNT10B, FZ5, B-catenin and LEF-
1 showed high level of mRNA in B-ALL patients. APC expression levels
in pre B-ALL samples were higher than B-ALL (p>0.001) and T-ALL
(p>0.001). LEF-1 expression was found to be increased in B-cell and in
preB-ALL samples and significantly different in T-ALL patients (p=0.01).
Highest level of TCF-1 mRNA was observed in the T-ALL patients. c-
MYC expression levels were found to be increased both in preB-
ALL(p=0.02) and T-ALL (p=0.02) patients. B-ALL patients did not significantly
express c-MYC gene compared to normal hematopoietic controls
(p=0.3). CCND1 mRNA level was observed to be slightly increased in
preB-ALL (p=0.04) but not in T-ALL and B-ALL groups . In AML FZ5
gene expression (10 fold) and LEF1gene expression (3 fold) showed a significant
increase when compared to controls (p>0.01 and p=0.02 respectively).
B -catenin was 5 fold higher in AML patients than normal peripheral
blood samples. The WNT10b expression was slightly lower comparing
to other genes. TCF1 expression level also seemed to be higher
in AML patients (p=0.06). c-MYC gene expression in AML patients
showed five fold increase compare to CCND1 gene expression
(p>0.001). The decreased APC mRNA levels led us to investigate APC
and B-catenin mutations with dHPLC and sequencing methods. Sequential
alterations in APC gene were determined in 65% of AML patients
and 47% of ALL patients. B-catenin mutation was detected in one ALL
patients and no mutation was observed in AML patients. Summary.These
results provide the evidence of WNT signal activation existing in acute
leukemia patients and the different functions of WNT signaling through
TCF/LEF between acute lymphoid and myeloid leukemias. We also
believe that APC function could be the key to the pathogenesis of acute
leukemias therefore we are currently studying its methylation status in
our patient group .