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

0892
INTEGRATION OF GENOME WIDE SNP ARRAYS AND GENE EXPRESSION DATA
OF CHILDHOOD ALL WITHOUT KNOWN GENETIC ABERRATIONS IDENTIFY SUBGROUPS
WITH SPECIFIC GENETIC HALLMARKS
S. Bungaro, 1 M. Campo dellOrto, 2 D. Basso, 3 A. Zangrando, 4
T.A. Gorletta, 5 M. Raghavan, 6 A. Leszl, 4 B.D. Young, 6 G. Basso, 2
S. Bicciato, 3 G. Te Kronnie, 2 A. Biondi, 1 G. Cazzaniga 1
1 M.Tettamanti Research Center, MONZA, Italy; 2 Laboratorio Emato-Oncologia
Pediatrica, PADOVA, Italy; 3 Dip.Processi Chimici dell’Ingegneria, PADOVA,
Italy; 4 Laboratorio di Emato-Oncologia Pediatric, PADOVA, Italy; 5 Genopolis
Consortium, MILANO, Italy; 6 Barts & the London School of Medicine, LON-
DON, United Kingdom
Background. Approximately 25% of childhood ALL patients cannot be
classified according to cytogenetic detectable hallmarks. Gene-expression
(GE) and SNP-arrays represents powerful tools for the identification
of hidden genetic abnormalities in cancer. Genotype and gene expression
data can be integrated to directly infer the variation of transcript levels in
deleted or amplified regions, and to identify variation in gene expression
of genes indirectly affected by genomic alterations. Aim of the study was
to identify cryptic abnormalities in unclassified childhood ALL patients
by performing an integrative analysis of GE and SNP arrays data. Methods.
29 patients followed the inclusion criteria: a) B-cell precursor childhood
ALL b) DNA index 1; c) negativity at t(4;11), t(12;21), t(9;22), t(1;19)
RT-PCR screening; d) normal karyotype when cytogenetics available. All
analyzed patients were part of a 125 B-ALL patients cohort analyzed by
GE profiling in the context of international Microarray Innovation in
Leukemia (MILE) study. Affymetrix GeneChip Mapping 100K SNP arrays
and HG-U133Plus2 Probe Arrays were used. Results. The presence of
del(9)(p21) was found in 10/29 (30%) patients, with an homozygous
commonly deleted region involving CDKN2A in 7/29. Hemizygous losses
of 9p13 including the PAX5 gene were found in 7/29 (24%) patients.
Three patients (10%) showed specific deletions involving the TEL gene
on chromosome 12p13.2. Three patients (10%), including two with TEL
deletion, showed the presence of the intrachromosomal amplification of
chromosome 21 (iAMP21). Five patients did not show copy number
change, and random microdeletions were found in single cases. We found
a statistically significant reduction of CDKN2A probe sets expression in
9p21 deleted cases. Due to the retention of the non deleted allele, no significant
down regulation of TEL and PAX5 specific probes was found in
12p13 and 9p13 deleted cases, respectively. More interestingly, a list of
differentially expressed genes was generated by supervised clustering
analyses comparing patients with and without the TEL, CDKN2A, and
PAX5 gene deletions, respectively. Although a list of down regulated
genes was generated for the CDKN2A and PAX5 deleted cases, likely
due to heterogeneity and large extension of both deletions, a specific signature
was not confirmed. Conversely, the JAG1 gene encoding for Jagged
1 ligand of the Notch receptor was among a list of differentially expressed
(upregulated) genes in TEL deleted cases. The expression level of the
JAG1 specific probes was evaluated in the GE dataset of 76/125 B-ALL
patients of the MILE study, including t(4;11), t(9;22), t(12;21) and 24
unclassified patients analyzed by SNP arrays, indicating statistical significant
JAG1 upregulation in TEL deleted and TEL/AML1 positive subgroups
relative to BP-ALL with other genetic lesions, also confirmed by
RQ-PCR. Conclusions. The integration of genomic variation and gene
expression profiling allowed us not only to identify hidden genetic lesions
but also to discover genes differentially expressed in new genotype-specific
subgroups. Larger studies are mandatory to extend and further confirm
the observation of JAG1 over expression in TEL-related leukemia;
moreover, functional studies are required to understand the role of Notch
pathway in this context.
12 th Congress of the European Hematology Association
Myeloproliferative disorders - Clinical
0893
HIGH TROUGHPUT SEQUENOM BASED ASSAY FOR MONITORING JAK2 V617F POST
ALLOGENEIC STEM CELL TRANSPLANTATION FOR CLASSIC
MYELOPROLIFERATIVE DISORDERS
M. Koren-Michowitz, Y. Cohen, A. Shimoni, N. Amariglio, A. Nagler
Chaim Sheba Medical Center, RAMAT GAN, Israel
Background. AlloSCT is currently the only curative treatment in classic
myeloproliferative disorders (MPD) and is often performed in advanced
disease stages. JAK2 V617F mutation is thought to play a causative role
in the pathogenesis of MPD. There is limited data on patients (pts) with
JAK2 V617F undergoing alloSCT and on the course of this mutation in
the post transplantation period. Methods. To investigate the course of
JAK2 V617F mutation in pts undergoing alloSCT we developed a sensitive
and quantitative high throughput assay. JAK2 mutation detection
was carried out using a chip-based matrix-assisted laser desorption-timeof-flight
mass spectrometer (Sequenom, San Diego, CA) with a specific
primer extension assay designed to detect the JAK2 V617F mutation. The
level of mutation was calculated by the following formula:% T allele =
(AUC T allele) x 100 / (AUC T allele +AUC G allele). T allele proportions
were compared with routine methods for chimerism detection. Results.
Fifteen pts underwent alloSCT in the study period (1.2001-6.2006), mean
age 59 (34-72) years, 9 males and 7 females. Their diagnoses included idiopathic
myelofibrosis -5, post polycythemia myelofibrosis -2, post essential
thrombocythemia myelofibrosis -2 and sAML -6. Nine pts underwent
SCT with reduced intensity conditioning regimen and 6 with myeloablative
conditioning, donors were matched siblings-11 and MUD-4.
JAK2 V617F was found in 9/15 (56%) pts. Compared to pts without JAK2
V617F, more pts with JAK2 V617F underwent alloSCT due to sAML (4/9
vs. 1/6) and more have undergone splenectomy (4/9 vs. 1/6) (ns). Disease
duration was 180 and 131 months in pts with and without JAK2 V617F,
respectively (ns). Before the SCT, all but one pt had more than 50% of
the mutated allele (mean T allele 74%, range 16-98%), suggesting the
existence of a homozygous clone for this mutation in most pts at the
time of alloSCT. After alloSCT, level of the mutant T allele decreased in
a timely manner in 5 pts, there was a limited decrease in 1 pt, it remained
unchanged in 1 pt and it was not evaluable in 2. There was a very high
correlation between the decrease in the mutant T allele and the decrease
in host cells as detected by I-FISH analysis for XY chromosomes in sex
mismatched SCT (n=4) and PCR for STR (n=3) (r=0.97, p