Regions Of Uniparental Disomy Research Articles

Dissecting Childhood B-Other Acute Lymphoblastic Leukemia: Lower Frequency of Druggable Gene Fusions in European Population?

Novel biological subtypes within B-other (B-o) acute lymphoblastic leukemia (ALL) have been described recently, based on the presence of genomic rearrangements (r.) of specific genes (DUX4 , ZNF384 and MEF2D ) or gene expression signatures (Ph-like and ETV6-RUNX1 -like ALL). Genomic profiling not only helped to dissect heterogeneity of B-o phenotype and outcome, but it also identified novel druggable aberrations such as kinase and cytokine receptor gene fusions, that were shown to occur mainly in Ph-like ALL. Unfortunately, only very limited population-based data are available to estimate an unbiased frequency of these novel genetic aberrations and subtypes, especially in European countries.We have analyzed a consecutive cohort of pediatric B-o ALL (negative for ETV6-RUNX1 , TCF3-PBX1 , BCR-ABL1 and KMT2A -involving fusions, hyper- and hypo-diploidy) diagnosed in the Czech Republic between 11/2012 and 5/2017. Transcriptome sequencing (RNAseq) was performed in 69/75 cases. High-density SNP array and whole exome sequencing were performed in 65 and 48 of those, respectively. Fusion transcripts, SNVs and indels were called from sequencing data using in-house pipeline based on publicly available software tools. Targeted analysis was performed to identify DUX4- r. Hierarchical clustering based on published gene sets defining DUX4 -r., Ph-like and ETV6-RUNX1 -like ALL was performed using RNAseq data. Somatic copy number aberrations and regions of uniparental disomy were called from SNP array data.Seventy five B-o cases represented 24% of all pediatric ALL (1-18 years) diagnosed within this period. According to hierarchical clustering, 23/69 cases were classified as DUX4 -r. ALL; all harbored DUX4 -r. They accounted for 33% of B-o ALL, representing a larger proportion than reported by others (Liu et al., EBioMedicine 2016; Lilljebjörn et al., Nat Commun 2016). Three cases (4%) and one case (1%) were positive for ZNF384-r. and MEF2D-r., respectively. Eleven cases (16%) were classified as Ph-like ALL; this subgroup included 7/9 cases with CRLF2 -r. and a single case with ETV6-ABL1 fusion. No other kinase/cytokine receptor gene fusion was detected. Four patients (6%) were classified as ETV6-RUNX1 -like ALL; all harbored aberrations of ETV6 . Strikingly, all 4 ETV6-RUNX1 -like cases displayed concurrently also Ph-like signature. The remaining 27 cases (39%) were not assigned to any novel subtype. Other lesions activating kinase signaling [mutations of NRAS (n=14), KRAS (n=7), FLT3 (n=4), PTPN11 (n=3), JAK2 (n=3), JAK1 (n=1), NF1 (n=1) and BRAF (n=1)] were found in 36 cases and were not enriched in Ph-like or other subtype. Of note, 9/27 of “unassigned” (but 0/42 of “assigned”) B-o harbored in-frame “non-kinase” PAX5 gene-involving fusions, both already described and novel, with largely unknown biological and clinical significance.Our data show that frequency of ZNF384 -r., MEF2D -r. and, importantly, also of kinase/cytokine receptor gene fusions (apart from CRLF2 -r.) might be lower in European population than estimated from data based on non-consecutive non-European cohorts (Liu et al.; Gu et al., Nat Commun 2016). To that extent our findings correspond with the recently published Swedish study (Lilljebjörn et al.); however, higher frequency of DUX4-r. ALL in our cohort indicates that differences in genetic composition of pediatric ALL may still occur even within Europe. Our findings highlight one of the flaws of Ph-like testing - non-specificity of Ph-like signature. Of note, the overlap of Ph-like and ETV6-RUNX1 -like signatures is also evidenced by positivity of Ph-like signature in ETV6-RUNX1 + ALL in study by Reshmi et al. (Blood 2017). Very low frequency of “kinase fusions” (apart from CRLF2 -r. which can be indirectly, yet reliably, diagnosed by flow cytometry) questions the use of Ph-like testing as a tool to pre-select ALL with high probability of having these lesions. Despite the use of complex genomics techniques a significant proportion of B-o ALL remains unclassified; interestingly, PAX5 fusions seem to be present in a significant proportion of them.To conclude, our population-based data confirm and specify novel genetic subtypes of pediatric B-o ALL; however, the low incidence of druggable kinase-activating fusions questions the extent of applicability of recently promoted individually “tailored” treatment.Support: AZV 15-30626A; AZV 16-30186A; GACR 15-06049Y; Primus/MED/28 DisclosuresNo relevant conflicts of interest to declare.

Limited Therapeutical Impact of Extensive Genomic Profiling in Relapsed Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia: Population-Based Four-Year Consecutive Cohort

High throughput genomic technologies, such as high-density SNP arrays and whole exome and transcriptome sequencing (WES, RNAseq), significantly deepened our insight into the genomic background and biology of acute lymphoblastic leukemia (ALL) during the past decade. Novel genetic aberrations and genomic and gene expression patterns have been described which led to the definition of several novel ALL subtypes and risk-predictive factors and to the identification of novel druggable and/or resistance-associated lesions potentially impacting therapy tailoring. Anecdotal evidence of therapeutical interventions based on the up-to-date genetic data has resulted into a great demand for these investigations. However, it is unclear what proportion of pediatric ALL patients, especially the cases of therapy-resistant leukemia, would really benefit from extensive genomic profiling.Between V/2013 and VIII/2017, relapse of Ph-negative B-cell precursor (BCP) ALL was diagnosed in 20 children (1-18 years) within the Czech Republic. In addition to the routine genetics, 18/20 cases were profiled by high density SNP array (18/18), WES (18/18) and RNAseq (17/18). The examined cohort comprised 10 B-other, 4 hyperdiploid, 3 ETV6-RUNX1 -positive and 1 KMT2A -rearranged ALL. At primary leukemia manifestation, all children were treated according to the ALL-BFM protocols [BFM 95 (n=1), BFM 2000 (n=1) and BFM 2009 (n=16)] and suffered very early (n=1), early (n=4) or late relapses (n=13). Upon disease relapse, 5, 11 and 2 cases were treated according to ALL-REZ BFM 2002 and IntReALL 2010 SR and HR protocols, respectively.Bulk bone marrow (BM) samples or sorted leukemic blasts were analyzed in isolated BM (n=15) and combined BM+CNS (n=2) relapses, while tissue from uterus infiltrated with blasts was analyzed in a single case of isolated extramedullary relapse. In addition to leukemic blasts, remission samples were analyzed by WES in all and by SNP array in selected cases to distinguish germline genetic variants. Somatic copy number aberrations and regions of uniparental disomy were called from SNP array data using commercial softwares. WES and RNAseq data were processed and somatic single nucleotide variants, indels and fusion transcripts were called using in-house pipeline based on publicly available software tools. Targeted analysis was performed to identify DUX4 gene rearrangements (r.). Hierarchical clustering based on gene expression profiles (unsupervised clustering and clustering with gene sets defining DUX4 -r., Ph-like and ETV6-RUNX1 -like ALL) was performed using RNAseq data. To facilitate this analysis, gene expression data from relapse cohort were combined with primary diagnosis data from >50 B-other ALL cases. Clinically important findings were reported to national study centre.Two B-other cases were classified as DUX4 -r. and 2 other cases as Ph-like ALL. Of note, 2 cases (both treated as standard risk in primary diagnosis) had iAMP21. Apart from 3 CRLF2 -rearrangements (1 present in Ph-like case), 6 KRAS and 2 NRAS genes mutations, no potentially druggable lesions were found. While the frequency of CRLF2 -rearrangements and RAS mutations found in relapse was comparable to that seen in primary disease, relapse cohort was enriched for lesions associated with unfavorable prognosis and therapy resistance. Three and four cases harbored deletion of TP53 gene and IKZF1 plus deletion pattern; however, in 1/2 cases with available data from primary diagnosis the IKZF1 plus pattern was identified only in relapse. Deletion of CREBBP and TBL1XR1 genes, potentially associated with resistance to glucocorticoids, were found in 2 cases and 1 case, respectively. Mutations of NT5C2 gene resulting in resistance to mercaptopurine were found in 2 cases. Three out of 18 analyzed cases did not harbor any of the above mentioned lesions. None of the genetic findings directly impacted patients' treatment.To conclude, we successfully implemented advanced genomics into centralized diagnostics of resistant ALL at the national level. Although it improved our understanding of disease relapse (yet, not in 100% of cases) the results were not translated to therapy tailoring in patients treated according to the large international protocols, thus, questioning the cost/benefit ratio of such extensive genetic diagnostics.Support: AZV 15-30626A and 16-30186A, GACR 15-06049Y, Primus/MED/28 DisclosuresNo relevant conflicts of interest to declare.

Early infantile-onset epileptic encephalopathy 28 due to a homozygous microdeletion involving the WWOX gene in a region of uniparental disomy.

The genetic etiologies of many rare disorders, including early infantile epileptic encephalopathies, are largely undiagnosed. A 6-year-old girl was admitted to the National Institutes of Health Undiagnosed Diseases Program with profound intellectual disability, infantile-onset seizures, chronic respiratory failure, facial dysmorphisms, skeletal abnormalities, and atrial septum defect. A large region of homozygosity was discovered on chromosome 16, spanning 16q22.1-16q24.3' caused by uniparental disomy (UPD) that included a maternally inherited homozygous microdeletion covering exon 6 of WWOX (NM_016373.3). mRNA expression analysis revealed that the deletion led to nonsense-mediated decay of the NM_016373.3 transcript; the exon 6 of an alternative transcript (NM_130791.3), lacking the short-chain dehydrogenase, was utilized. The microdeletion in WWOX explains the seizures and intellectual disability, while pathogenic variants in another gene, HSPG2, are likely responsible for the patient's skeletal abnormalities. This report describes a rare autosomal recessive disorder with multiple genetic etiologies, one of which involves UPD.

Genome-Wide Uniparental Disomy and Copy Number Variations in Renal Cell Carcinomas Associated with Birt-Hogg-Dubé Syndrome

Birt-Hogg-Dubé syndrome is an inherited disorder caused by germline mutations of the folliculin gene (FLCN). The affected patients are prone to developing renal cell carcinomas (RCCs). Most mutant FLCN-associated RCCs (mFLCN-RCCs) are histologically chromophobe RCCs and hybrid oncocytic/chromophobe tumors. It is incompletely understood whether mFLCN-RCCs have different chromosomal abnormalities compared with their sporadic histological counterparts. Herein, we describe somatic mutations of FLCNand DNA-copy number abnormalities using a high-density, whole-genome, single-nucleotide polymorphism array. The histological types included chromophobe RCC (n = 12), hybrid oncocytic/chromophobe tumor (n = 5), and clear-cell RCC (n = 2). Of 19 tumors, 8 had pathological somatic mutations of FLCN. Among 11 mFLCN-RCCs investigated by single-nucleotide polymorphism array, 8showed balanced genomic profiles, 2 had gains in chromosome 3q, and 1 had gains in chromosomes 1q and 7. All had copious numbers of loss of heterozygosity in a wide range of chromosomes. The common loss-of-heterozygosity regions were chromosomes 3p24, 8q11, 16q11, Xp22-21, Xp11, Xq11, Xq13, and Xq23. Most of the loss of heterozygosity was because of uniparental disomy. Common uniparental disomy patterns in chromophobe RCCs and hybrid oncocytic/chromophobe tumors indicated that these types were relatively similar in cytogenetic events. Two clear-cell RCCs also shared several uniparental disomy regions with chromophobe RCCs and hybrid oncocytic/chromophobe tumors. mFLCN-RCCs may have common therapeutic targets among different histological types.

Monoallelic and biallelic deletions of 13q14 in a group of CLL/SLL patients investigated by CGH Haematological Cancer and SNP array (8x60K).

BackgroundDeletion of 13q14 is the most common cytogenetic change in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and is detected in about 50 % of patients by fluorescence in situ hybridization (FISH), which can reveal presence of del(13)(q14) and mono- or biallelic deletion status without information about the size of the lost region. Array-comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) can detect submicroscopic copy number changes, loss of heterozygosity (LOH) and uniparental disomy (UPD) regions. The purpose of this study was detection of the size of del(13)(q14) deletion in our group of patients, comparing the size of the monoallelic and biallelic deletions, detection of LOH and UPD regions.ResultsWe have investigated 40 CLL/SLL patients by karyotype, FISH and CGH and SNP array. Mutational status was of immunoglobulin heavy-chain variable-region (IGVH) was also examined. The size of deletion ranged from 348,12 Kb to 38.97 Mb. Detected minimal deleted region comprised genes: TRIM13, miR-3613, KCNRG, DLEU2, miR-16-1, miR-15a, DLEU1. The RB1 deletions were detected in 41 % of cases. The average size in monoallelic 13q14 deletion group was 7,2 Mb while in biallelic group was 4,8 Mb. In two cases 13q14 deletions were located in the bigger UPD regions.ConclusionsOur results indicate that bigger deletion including RB1 or presence of biallelic 13q14 deletion is not sufficient to be considered as adverse prognostic factor in CLL/SLL. CytoSure Haematological Cancer and SNP array (8x60k) can precisely detect recurrent copy number changes with known prognostic significance in CLL/SLL as well as other chromosomal imbalances. The big advantage of this array is simultaneous detection of LOH and UPD regions during the same test.

The UBC-40 Urothelial Bladder Cancer cell line index: a genomic resource for functional studies.

BackgroundUrothelial bladder cancer is a highly heterogeneous disease. Cancer cell lines are useful tools for its study. This is a comprehensive genomic characterization of 40 urothelial bladder carcinoma (UBC) cell lines including information on origin, mutation status of genes implicated in bladder cancer (FGFR3, PIK3CA, TP53, and RAS), copy number alterations assessed using high density SNP arrays, uniparental disomy (UPD) events, and gene expression.ResultsBased on gene mutation patterns and genomic changes we identify lines representative of the FGFR3-driven tumor pathway and of the TP53/RB tumor suppressor-driven pathway. High-density array copy number analysis identified significant focal gains (1q32, 5p13.1-12, 7q11, and 7q33) and losses (i.e. 6p22.1) in regions altered in tumors but not previously described as affected in bladder cell lines. We also identify new evidence for frequent regions of UPD, often coinciding with regions reported to be lost in tumors. Previously undescribed chromosome X losses found in UBC lines also point to potential tumor suppressor genes. Cell lines representative of the FGFR3-driven pathway showed a lower number of UPD events.ConclusionsOverall, there is a predominance of more aggressive tumor subtypes among the cell lines. We provide a cell line classification that establishes their relatedness to the major molecularly-defined bladder tumor subtypes. The compiled information should serve as a useful reference to the bladder cancer research community and should help to select cell lines appropriate for the functional analysis of bladder cancer genes, for example those being identified through massive parallel sequencing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1450-3) contains supplementary material, which is available to authorized users.

An atypical human induced pluripotent stem cell line with a complex, stable, and balanced genomic rearrangement including a large de novo 1q uniparental disomy.

Human induced pluripotent stem cells (hiPSCs) hold great promise for cell therapy through their use as vital tools for regenerative and personalized medicine. However, the genomic integrity of hiPSCs still raises some concern and is one of the barriers limiting their use in clinical applications. Numerous articles have reported the occurrence of aneuploidies, copy number variations, or single point mutations in hiPSCs, and nonintegrative reprogramming strategies have been developed to minimize the impact of the reprogramming process on the hiPSC genome. Here, we report the characterization of an hiPSC line generated by daily transfections of modified messenger RNAs, displaying several genomic abnormalities. Karyotype analysis showed a complex genomic rearrangement, which remained stable during long-term culture. Fluorescent in situ hybridization analyses were performed on the hiPSC line showing that this karyotype is balanced. Interestingly, single-nucleotide polymorphism analysis revealed the presence of a large 1q region of uniparental disomy (UPD), demonstrating for the first time that UPD can occur in a noncompensatory context during nonintegrative reprogramming of normal fibroblasts.

Specific and Recurrent Cytogenomic Abnormalities Originate in the Spleens of Myelofibrosis Patients and Contribute to Clonal Diversity and Disease Progression

Hematopoietic stem cells located in spleens (SP) of patients with myelofibrosis (MF) have functional properties that differ from those present in peripheral blood (PB) (Wang et al, JCI,122: 3888, 2012 ). We hypothesized that the spleen might be a source of malignant stem cells in MF which ultimately lead to disease progression and leukemic transformation. To investigate the genetic diversity of MF SP cells, cytogenetic analyses of SP and peripheral blood (PB )/bone marrow (BM) cells from 13 MF patients were performed. Nine of 13 patients (69%) had concordant normal (n=5) and abnormal (n=4) cytogenetic and FISH analyses when comparing SP and PB/BM cells. Four (30%) patients had discordant results with specific chromosomal abnormalities present in either SP or PB cells. One patient had 3 cytogenetic abnormalities associated with an unfavorable prognosis: (1;7), del(12p), and i(17q) in SP cells that were not observed in PB cells. A second patient who had progressed to acute leukemia had pentasomy 21 in 45% of SP cells but not in PB cells and had del (20q) in PB cells but not SP cells, suggesting that del (20q) had arisen in the BM while pentasomy 21 had originated in the SP . In a third patent 20% of PB cells had +8 while only 1.6% of SP cells had +8, indicating that the +8 clone originated in the BM. Studies of BM and PB of the 4 th patient showed a normal karyotype in 2008 and 2011 while 2% of SP cells in 2011, at the time of progression to MF, possessed del 20q while the BM remained normal. In 2014, when the patient developed an accelerated phase of MF, 85% of BM cells possessed del (20q), indicating that del(20q) originated in the SP and migrated to the BM. We next hybridized SP DNA from 12 of the 13 patients to the Agilent 400k platform [355515 (CGH) + 59646 (SNP)] and performed CGH and SNP analysis. These studies revealed that all 12 patients had additional gains and losses of chromosomal regions as well as uniparental disomy (UPD) in SP cells. Two groups of acquired genomic changes were observed in SP cells: 1) four acquired regions were present in over 60% of pts and 2) three regions of acquired changes were observed in 25- 33% of patients. Irrespective of the patient’s karyotype, gains of 4 chromosomal regions: 1p13.2 (RHOC), 12q24.31 (NCOR2), 13q34 (RASA3) and 17q12 (TAF15) were detected in 83%, 83%, 75% and 67%, of patient’s SP cells, respectively. All four genes are known to be involved in leukemogenesis. Gains of these 4 chromosomal regions have not been observed in normal controls or PB CD34+ cells from 437 patients with MPNs including MF (Klampfl et al, Blood 167, 2011, Rumi et al Am J Hematol 974, 2011). Gains of an additional three regions, 18q21.31 (NEDD4L), 16q23.2 (WWOX) and 17q21.31 (WNT3) were detected in the SP cells of 25-33% of patients. These genes have also been implicated in leukemogenesis. The greater the complexity of the karyotype of the SP cells the greater the number of copy number genomic changes (up to 92) were observed. Although SNP analyses demonstrated 28 acquired UPD regions in 12 patients 9p13-p24 was the most common occurring in 25% of patients. SNP analyses also demonstrated triplication of 9q and quadruplication of 9p, suggesting that UPD of the entire chromosome 9 in SP cells can be associated with disease progression. Transplantation of SP CD34+ cells with a normal karyotype or with one chromosomal abnormality into NOD/SCID/IL2Rγ(null) mice resulted in a modest degree of donor cell chimerism (0.2%-4%), while transplantation of SP CD34+ cells with UPD of 9p, the entire chromosome 9 UPD, del20q originating in SP cells, complex karyotypes and gains of 4 chromosomal regions (1p,13, 12q24, 13q34 and 17q12 ) resulted in a higher degree of donor cell chimerism (18%-25%) indicating the association of these genetic events with altered stem cell function. Our findings indicate that some chromosomal abnormalities are acquired in MPNs initially in the SP sometimes occurring years prior to their appearance in the BM/PB. Other abnormalities, such as del(20q) may originate in either the BM or SP. All MF SP cells were characterized by additional diverse cytogenomic changes involving at least four regions, containing genes associated with leukemogenesis, leading to recurrent copy number gains in 67% to 83% of MF patients. We hypothesize that the SP provides a microenvironment in a subpopulation of MF patients which is associated with increased genomic diversity resulting in disease progression and leukemic transformation. DisclosuresMascarenhas:Incyte Corporation: Consultancy.

Hypoglycaemia following diabetes remission in patients with 6q24 methylation defects: expanding the clinical phenotype

Hypoglycaemia following diabetes remission in patients with 6q24 methylation defects: expanding the clinical phenotype

A high resolution genomic portrait of bladder cancer: correlation between genomic aberrations and the DNA damage response

One major challenge in cancer research is to understand the complex interplay between the DNA damage response (DDR), genomic integrity, and tumor development. To address these issues, we analyzed 43 bladder tumor genomes from 22 patients using single nucleotide polymorphism (SNP) arrays, and tissue expression of multiple DDR proteins, including Timeless and its interaction partner Tipin. The SNP profiles confirmed and extended known copy number alterations (CNAs) at high resolution, showed clustering of CNAs at nine common fragile sites, and revealed that most metachronous tumors were clonally related. The occurrence of many novel uniparental disomy regions (UPDs) was of potential functional importance in some tumors because UPDs spanned mutated FGFR3 and PIK3CA alleles, and also homozygous deletion of the CDKN2A tumor suppressor locus. The DDR signaling as evaluated by phospho-epitope-specific antibodies against Ser139-phosphorylated H2A histone family member X (γH2AX), ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR) was commonly activated in tumors with both moderate and high extent of accumulated genomic aberrations, the latter tumors showing a more frequent loss of ATM expression. Strikingly, the tumor genomes exhibiting the most complex alterations were associated with a high Ki67-proliferation index, abundant Timeless but not Tipin expression, aberrant p53 expression, and homozygous CDKN2A deletions. Of clinical relevance, evaluation of a tissue microarray (TMA; n=319) showed that abundant Timeless expression was associated with risk of progression to muscle-invasive disease (P<0.0005; hazard ratio, 2.4; 95% confidence interval, 1.6-3.8) and higher T stage (P<0.05). Univariate analysis confirmed this association (P=0.006) in an independent cohort (n=241) but statistical significance was not reached in a multivariate model. Overall, our results are consistent with DDR activation preceding the accumulation of genomic aberrations. Tumors with extensive genomic rearrangements were associated with inactivation of CDKN2A, excessive proliferation, and robust Timeless expression, the latter also correlating with the risk of disease progression. Moreover, we provide evidence to suggest that UPDs likely contribute to bladder tumorigenesis.

Array-Based Cytogenetic Approaches in Acute Myeloid Leukemia: Clinical Impact and Biological Insights

Conventional cytogenetic studies have shown that the clinical and biological diversity of acute myeloid leukemia (AML) can be attributed, in part, to distinct chromosome aberrations, several of which are now routinely used for diagnosis, risk stratification, and outcome prediction. Although chromosome banding analysis has recently been complemented by the identification of point mutations in a growing number of hematopoiesis-associated genes, current genetic categories do not fully reflect the heterogeneity of AML. To close the gap between standard karyotyping and molecular analyses at the single-base-pair level and gain additional insight into the genetics underlying myeloid leukemogenesis, AML is increasingly being studied using genome-wide, microarray-based cytogenetic methods. These investigations have revealed that AML genomes commonly harbor acquired submicroscopic copy number alterations, even though the prevalence of most of these cryptic lesions appears to be low, as well as regions of uniparental disomy that are often associated with homozygosity of functionally relevant gene mutations. Current efforts are focusing on the application of this expanded knowledge to improve the classification of AML, develop new tools for prognostication and prediction of response to therapy, and accelerate the discovery of genes that are likely to contribute to AML pathogenesis.

Pathogenesis of MONOSOMY 7 In BONE MARROW FAILURE SYNDROMES

Abstract 2411One of the most serious complications of aplastic anemia (AA) is the clonal evolution to myelodysplastic syndrome (MDS). Monosomy 7 (del[7]) is the most common chromosomal aberration encountered in this setting, but it can also occur in primary MDS along with deletion 7q (del[7q]). In our whole cohort of AA, we detected del[7] in 24 cases of clonal evolution; however, unlike in MDS no cases of del[7q] were observed. In contrast, in MDS or myelodysplastic/myeloproliferative neoplasms, del[7]/[7q] were present in 12% and 11%, respectively. Based on an initial observation of an index AA case with a mutation of CBL, we speculated that certain molecular events such as point mutations can promote or are at least associated with the evolution of del[7]. Given the distinct distribution of del[7] and del[7q] in primary and secondary MDS (derived from AA), we stipulate that the associated mutational pattern will be distinct. This may be due to different driving mechanisms, such as immune escape or a growth factor-rich milieu in AA.Pursuant to this theory, we performed a screen for CBL, RUNX1, TET2, UTX, EZH2, DNMT3A, ASXL1 and IDH family mutations in patients who developed del[7] after AA (N=24). 14 cases were adults and 10 were pediatric cases. In addition, we analyzed 15 del[7], 13 del[7q] and 10 UPD[7q] abnormalities in patients with MDS without antecedent AA. We identified 3 CBL (13%), 5 RUNX1 (20%), 1 TET2 and 1 DNMT3A mutation in patients with del[7] after AA; all CBL mutations are observed in adult cases of AA (21%). However, 0, 1, and 2 CBL mutations were found in primary MDS with del[7], del[7q] and UPD[7q], respectively. In contrast, RUNX1 mutations were seen in both AA sub-groups (14% and 30% in adults and children, respectively). Also, no differences were seen with regard to the distribution of RUNX1 mutations in either post-AA MDS with del[7] or primary MDS with del[7], del[7q] and UPD[7q]. Most significantly, ASXL1 was found to be the most frequently mutated gene in the cohort without AA (37%), although no mutations were seen in patients with del[7] after AA. Additionally, UTX and EZH2, both involved in the trimethylation of histone H3 lysine 27, were found to be mutated only in cases of MDS with UPD[7q] and del[7q], but no mutations were seen in primary and AA-derived MDS with del[7]. When studied serially, none of the tested mutations was seen in the initial AA phase, but the mutated clone increased gradually during clonal evolution of del[7].We also investigated the presence of additional chromosomal abnormalities using single nucleotide polymorphism array (SNP-A)-based karyotyping in the cases with del[7] after AA. In addition to del[7], 22 gains, 20 losses and 4 regions of somatic uniparental disomy (UPD) were identified. These abnormalities, including microdeletions, were found in 50% and 60% of adult and pediatric cases, respectively. Recurrent lesions were detected on chromosome 6, 8 and 21. A microdeletion on chromosome X involved the tumor-associated gene PHF6 in a male case. In another patient, trisomy 21 was accompanied by a RUNX1 mutation. Regions of UPD included 6p resulted in loss of heterozygosity of MHC class I and 11q was associated with homozygous CBL mutations in 2 cases.In sum, our analysis supports the theory that distinct mechanisms of molecular progression during MDS evolution may be operative in AA and primary MDS. Moreover, del[7], del[7q] and UPD[7q] have distinct molecular profiles with frequent TET2 mutations in del[7q] and UPD[7q]. ASXL1 mutations appear to be most ubiquitous promoting event in MDS with LOH [7/7q], suggestive of their “type II' nature. In contrast, other mutations such as those in CBL may be selected for e.g., through cytokine-rich milieu and activation of CBL-regulated receptor tyrosine kinases. Disclosures:No relevant conflicts of interest to declare.

The consequences of uniparental disomy and copy number neutral loss-of-heterozygosity during human development and cancer

UPD (uniparental disomy) describes the inheritance of a pair of chromosomes from only one parent. Mechanisms that lead to UPD include trisomy rescue, gamete complementation, monosomy rescue and somatic recombination. Most of these mechanisms can involve aberrant chromosomes, particularly isochromosomes and Robertsonian translocations. In the last decade, the number of UPD cases reported in the literature has increased exponentially. This is partly due to the advances in genomic technologies that have allowed for high-resolution SNP (single nucleotide polymorphism) studies, which have complemented traditional methods relying on polymorphic microsatellite markers. In this review, we discuss aberrant cellular mechanisms leading to UPD and their impact on gene expression. Special emphasis is placed on the unmasking of mutant recessive alleles and the disruption of imprinted gene dosage, which give rise to specific and recurrent imprinting phenotypes. Finally, we discuss how copy number maps determined from SNP array datasets have helped identify not only deletions and duplications but also recurrent copy number neutral regions of loss-of-heterozygosity, which have been reported in many cancer typesand that may constitute an important driving force in cancer. These tiny regions of UPD also alter imprinted gene dosage, which may have cumulative tumourgenic effects in addition to that of unmasking homozygous cancer-associated mutations.

Gene dosage effects in chronic lymphocytic leukemia

To understand the influence of chromosomal alterations on gene expression in a genome-wide view, chromosomal imbalances detected by single nucleotide polymorphism (SNP) chips were compared with global gene expression in 16 cases of chronic lymphocytic leukemia (CLL). A strong concordance between chromosomal gain or loss and increased or reduced expression of genes in the affected regions was found, respectively. Regions of uniparental disomy (UPD) were rare and had usually no consistent influence on gene expression, but in one instance, a large UPD was associated with a downregulation of most genes in the affected chromosome. The frequently deleted miRNAs, MIRN15A and MIRN16-1, did not show a reduced expression in cases with monoallelic deletions. The BCL2 protein, considered to be downregulated by these miRNAs, was upregulated not only in CLL with biallelic deletion of MIRN15A and MIRN16-1, but also in cases with monoallelic deletion. This suggests a complex regulation of BCL2 levels in CLL cells. Taken together, in CLL, a global gene dosage effect exists for chromosomal gains and deletions and in some instances for UPDs. We did not confirm a consistent correlation between MIRN15A and MIRN16-1 expression levels and BCL2 protein levels, indicating a complex regulation of BCL2 expression.

Prognostic Significance of Cryptic Genomic Aberrations In AML with Normal Karyotype

AbstractAbstract 2729Acute myeloid leukaemia with a normal karyotype (AML-NK) is a heterogeneous clonal disorder of haematopoietic progenitor cells with an intermediate prognosis. Acquired mutations of FLT3, NPM1 and CEBPA as well as gene expression profiles have recently been used as biomarkers to further stratify prognostic subgroups, especially in AML-NK.We have previously shown that cryptic genomic aberrations in MDS cases are prognostically significant and regions of uniparental disomy (UPD), detectable by SNP array karyotyping, can harbour mutations in specific gene(s). We analysed 83 AML patients (pts), median age 52 (range 8–78) with normal cytogenetics, excluding pts who died within 30 days of entry, who had been treated on one of the UK MRC/NCRI AML clinical trial protocols, using high resolution 250K SNP microarrays to seek genomic aberrations. In addition, we sequenced the ‘mutation hotspot’ for IDH1/2 as well as screened for the presence of FLT3 ITD and NPM1 mutations. Genomic aberrations and mutational lesions were correlated with outcome data.To circumvent the lack of constitutional DNA, all aberrations overlapping by >50% with copy number variants from the Database of Genomic Variants as well variations observed in a cohort of 91 healthy subjects were excluded from further analysis. We demonstrate the presence of 69 cryptic genomic aberrations in 43 pts consisting of 12 deletions (del) (8 pts), 10 gains (10 pts) and 47 regions of UPD (35 pts). The median size of del was 1.6Mb (0.18Mb-3.4Mb) with chromosome 4 (q24) and 17 (q11.2 and q22.1) being the most affected. One pt had micro del on chromosome 12p13.2-p12.3 and 21q22.11-q22.12 affecting the ETV6 and AML1 genes respectively, suggestive of a t(12:21) aberration. Gains (median 0.37Mb (0.26–0.71Mb)) were identified predominantly on chromosome 8 (4 pts) with a common amplified region, 8q21.11. The median size of UPD's was 47Mb (2.3Mb-158Mb). Chromosome 13 was the most frequently affected with UPD of the entire chromosome observed in 17 pts. Interestingly, one pt had an interstitial region of UPD on q21.32-q21.33 (4.8Mb). Additional regions affected by UPD included chromosomes 6, 2, 5 in 5 pts and chromosome 11 in 4 pts.NPM1 mutations occurred in 55 pts (65%) and FLT3 ITD's occurred in 44 pts (53%). Of 39 pts with wild type FLT3, 22 had NPM1 mutations; of 28 pts with wild type NPM1, 11 had FLT3 ITD mutations. Conversely of the 44 pts with mutant FLT3, 33 had NPM1 mutations. Six pts had mutations of FLT3, NPM1 and IDH. Of note, only 2 of 17 pts with UPD of chromosome 13 did not have FLT3 mutations, suggesting the presence of additional pathological genes in this region. IDH1 (R130H) and IDH2 (R140Q and R172K) mutations occurred in 3 and 16 pts, respectively and were mutually exclusive. Interestingly, the R172K mutation occurred in only 3 pts. Of the 19 pts with IDH1/2 mutations, 9 and 12 pts respectively, had co-existing FLT3 ITD and NPM1 mutations.The median duration of follow-up from time of diagnosis of the study group was 29 months (range 2–160). There were no significant correlations between the presence of genomic aberrations and age, sex, WBC, secondary disease or IDH1/2 mutations; however, a significant difference was observed in the frequency of UPD in pts with mutant or wild type FLT3 (p=0.01) and NPM1 (p=0.008). A difference in overall survival (OS) was seen on comparison of pts with or without genomic aberrations, (5 year OS 10% vs 46%, hazard ratio (HR) 1.86 (1.01–3.44), p=0.05): significant differences were seen on univariate analyses when analysing gains (11% vs 27%, HR 3.07 (1.09–8.68) p=0.03), or del (0% vs 28%) HR 3.46 (1.02–11.8) p=0.05) separately, with some evidence that UPD also produced worse results (0% v 34% HR 1.82 (0.97–3.45) p=0.06). In multivariate analyses, adjusted for age, WBC, sex, secondary disease, performance status, IDH1, IDH2, FLT3 ITD and NPM1 all SNP array detected aberrations were independently prognostic for overall survival. Analysis combining all aberrations gave a HR of 2.99(1.33–0.75) p=0.006. When a model-building approach was used, after adjustment for baseline covariates, del p=0.005, gains p=0.002 and UPD p=0.02 all entered the model. We show, for the first time, that SNP microarray analysis in AML-NK pts reveals cryptic UPD and CN changes that may allow risk stratification and alter therapeutic strategies for this poor prognostic subgroup of patients. Disclosures:No relevant conflicts of interest to declare.

Reduction of Tregs with Expansion of Th1 Cells and Lack of IFN-γ Secretion by GPI Negative T-Cells In PNH

AbstractAbstract 2240 Introduction and Aim:Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haematopoietic stem cell disorder characterized by intravascular haemolysis and thrombosis. The pathogenetic link with bone marrow failure syndromes is well recognized, however the process of clonal expansion of the glycosylphosphatidylinositol (GPI)-deficient cells over normal haemotopoiesis remains unclear.To further elucidate mechanisms leading to clonal expansion in PNH, we investigated the immunological profile and performed high-resolution genome-wide karyotyping using Affymetrix SNP6 microarrays. Patients and Methods:The percentage and absolute numbers of CD4+ and CD8+ T-cell subsets, NK cells and B cells in peripheral blood were assessed in 8 patients with PNH prior to any therapy and 8 healthy age matched controls. High resolution SNP6 karyotyping was performed on bone marrow (n=15) and peripheral blood (n=12) of these patients. Bone marrow from an additional 8 patients was enriched for CD34+59- and CD34+59+ cell fractions for SNP array karyotyping. Abberations that overlapped by >50% with variations found in the Database of Genomic Variants, as well as an internal series of 91 normal subjects were excluded from further analysis.T-cells were stimulated and then stained intracellularly for TNF-α and IFN-γ (Th1), IL-4 (Th2) and IL-17 (Th17). NK cells were defined as CD3– CD56+. B cells were defined as CD3-CD19+. CD3+ CD4+ T-cell subsets were defined as CD45RO–CD27+ naïve, CD45RO+ CD27+ CD62L+ central memory, CD45RO+ CD27+ CD62L– effector memory, CD45RO+CD27– effectors and CD45RO–CD27– terminal effectors. CD4+ Tregs were defined as CD3+CD4+ CD25high CD27+Foxp3+. Results:There were no significant differences in the number or percentage of different CD8+ and CD4+ T-cells compared to healthy controls except for the number of Tregs and Th1 cells. In our cohort of patients, the number of Th1 cells was significantly higher than healthy controls (4.1×107/L v 0.93 × 107/L, p=0.039), whereas the number of Tregs cells was lower (0.75 × 107/L v 1.36 × 107/L, p=0.028). There was no significant difference in the number of Th2 and Th17 cells between patient and healthy subjects.Within CD4+ T-cells two distinct CD59+ and CD59- populations were identified, of which the CD59- cells were unable to secrete IFN-γ in response to stimulation compared to CD59+ population. On average 48% of CD4+ CD59+ T-cells secrete IFN-γ compared to 2% in the CD59- population. There was no significant difference in IL17 and IL4 secretion between CD59+ and CD59- T-cells.SNP karyotyping revealed three regions of uniparental disomy (UPD); UPD1p26.11-p34.3, UPD1p13.3-p13.1in one peripheral blood sample and UPD7q32.1-q34 in one bone marrow sample. There were no additional somatic genomic aberrations detected in any of the samples. Of note, purified CD34+59- cells did not reveal any clonal copy number changes or regions of UPD. Conclusion:Specific analysis of Xp22.1 did not reveal any aberrations of the PIGA gene, suggesting aberrations of the PIGA gene may be restricted to mutations or epigenetic abnormalities. Our immunological profiling revealed an expansion of Th1 cells and diminished Tregs in the peripheral blood, which is in contrast to our published data from both MDS and AA patients. The lack of IFN-γ secretion by GPI deficient T-cells also suggests an additional immunological defect in these patients, which may contribute in disease pathogenesis. Disclosures:No relevant conflicts of interest to declare.

Single Nucleotide Polymorphism Array (SNP-A) Genomic Profiling of Mantle Cell Lymphoma (MCL) Against a Large Control Database Reveals Recurring Copy Number Alterations (CNAs) and Copy Neutral Loss of Heterozygosity (CN-LOH)

AbstractAbstract 2001 Background:MCL is characterized by extreme genomic instability. Conventional techniques such as metaphase cytogenetics are unable to detect small deletions, amplifications, or uniparental disomy (UPD) in the MCL tumor genome. SNP-A analysis permits high resolution karyotyping and detection of unbalanced DNA defects, including somatic UPD. We performed SNP-A analysis on MCL tumor samples, excluded CNA present in normal controls, and assessed our results in context of clinical outcome and Ki-67 index. Methods:With IRB approval, available frozen tissue from 18 patients diagnosed with cyclin D1-positive MCL between 1997–2006 was analyzed using high-resolution genome-wide human SNP Array 6.0 (Affymetrix). Signal intensity and SNP calls were determined using the Gene Chip Genotyping Analysis Software Version 4.0 (GTYPE) (Affymetrix). Copy number was also determined. Somatic MCL CNAs and CN-LOH were discerned from germline variants (CNV) by comparing to a database of 1535 normal controls subjected to 250K and/or SNP Array 6.0 analysis.Clinical data was available for all patients, and 15/18 samples were subject to immunohistochemistry (IHC) for Ki67 using an automated immunostainer (Discovery; Ventana Medical Systems). Kaplan-Meier survival analysis was performed. Results:Analysis of 18 MCL patient samples revealed an average 13 CNAs (6.2 gains, 6.4 losses) and 0.4 CN-LOH per patient (Figure 1). Gains were frequently observed in 3q (33%), 8q (22%), 12q (17%), and 18q (11%). A unique 12q micro-gain in one patient narrowed the minimal common region (MCR) to linear region 130.4 – 131.3 Mbp, overlapping with an area of CN-LOH, and including candidate genes MMP17 (upregulated in invasive breast cancer), ULK1 (regulator of autophagy), and EP400 (regulator of chromatin remodeling, proliferation and apoptosis).Recurring deletions were observed at 11q (50%), 1p, 6q (39%), 9p (33%), 13 q (28%), 9q (22%), 7q, and 17p (17%). Similar to prior studies, losses at 1p encompassing CDKN2C and FAF were seen though we further narrowed a common MCR, spanning 93.1–99.7 Mpb and including ARHGAP29 (PARG1)—previously identified in MCL by aCGH/gene expression, whose promoter is a frequent target of MCL methylation. As previously reported, losses in components of the Hippo tumor suppressor pathway were frequently affected by these recurring deletions (6q: LATS1, 9p: MOBKL2B) and by one deletion on 19p (MOBK2LA). Other high-frequency losses encompassed CDKN2A, CDKN2B, and MTAP (on 9p), RB1 and DLEU1/2/miR15a/16-1 (13q), and TP53 (17p).Unique homozygous losses were detected at 9p (3.2-3.3 Mbp; involving only RFX3), 11q (94.5-111.7 Mbp; spanning the ATM region), and 13q (82.7-99.5 Mbp; including the miR17-92 region), and micro-deletions at 6q (121.1-121.9; GJA1/Cx43), 12p (7.5-7.6; CD163), and 13q (73.3-73.4; KLF12, and 75.2–75.3; LMO7).CN-LOH was observed at 6p, similar to prior studies, though we found novel regions of UPD at 4p, 8q, 18q, 19q, and 22q.An overall survival of 3.6 years and relapse-free survival of 1.3 years was observed. Survival was significantly worse among 8 pts with Ki67 >75% (OS 1.4 years, p=.003), but was unaffected by del 11q, del 9p, gain 3q, or gain 8q. Conclusions:SNP-A analysis of 18 primary samples confirms that gains in 3q and 8q and losses in 11q, 6q, and 9p represent common secondary genetic lesions in MCL, and are not frequent in normal controls. We narrowed the MCR of several deletions, potential targets for gene sequencing, and confirm the presence of deletions of potential relevance to the Hippo pathway. Further analysis of our findings in light of tissue micro-array and fluorescence in-situ hybridization studies is underway, to assess pathobiologic consequences of genomic lesions as well as potential therapeutic targets. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

High-density single nucleotide polymorphism array analysis and ASXL1 gene mutation screening in chronic myeloid leukemia during disease progression

We have undertaken a genome-wide single nucleotide polymorphism (SNP) array analysis of 41 chronic myeloid leukemia (CML) patients. In total, 44 regions of uniparental disomy (UPD) >3 Mb were identified in 24 of 32 patients in chronic phase (CP), and 21 regions of UPD >3 Mb were identified in 13 of 21 patients in blast crisis (BC). Chromosome 8 had the highest frequency of UPD regions in both CP and BC samples. Eight recurrent regions of UPD were observed among the 41 patients, with chromosome 8 showing the highest frequency. Ten regions of copy number change (CNC) >3 Mb were observed in 4 of 21 patients in BC, whereas none were observed in CP. We have identified several recurrent regions of UPD and CNC in CML that may be of pathogenetic importance. Overrepresentation of genomic aberrations (UPD and copy number gain) mapping to chromosome 8 was observed. Selected candidate genes mapping within the aberrant genomic regions were sequenced and mutation of the TP53 gene was observed in one case in BC and of the ASXL1 gene in 6 of 41 cases in CP or BC. Mutation of ASXL1 represents an important new molecular abnormality in CML.

Recurrent Novel Regions of Uniparental Disomy and Homozygous Deletions in Mycosis Fungoides Identified Using High Resolution Single Nucleotide Polymorphism Analysis.

Abstract 2644Poster Board II-620Mycosis Fungoides (MF) is the commonest form of primary cutaneous T-cell lymphoma but our understanding of the underlying molecular pathogenesis remains limited. Cytogenetic studies remain limited due to difficulties in obtaining analysable mitotic chromosomes for metaphase G-band analysis from MF patients. We have utilised high resolution 250k Affymetrix single nucleotide polymorphism (SNP) arrays to analyse tumour samples from 34 MF patients including 12 paired skin samples using and identified 156 chromosomal aberrations in ten patients (29%). Compared with previous studies using comparative genomic hybridization (CGH) in both MF and Sezary syndrome (SS), SNP array analysis detected a greater frequency of deletions and amplifications on chromosomes 1, 2, 3, 4, 5, 6, 11, and 16. Heterozygous deletions of chromosome 8 were also detected in 3 patients which have been undetected by CGH studies. A particular feature of SNP array technology is the detection of uniparental disomy (UPD) which are copy neutral regions of homozygosity. UPD (median size 32.3Mb (range: 6.6Mb – 159Mb)) was detected in 21% of our MF patients affecting either chromosome 7 (whole chromosome), 8p, 9p/9q, 10q, 12p, 14q, 16p/16q, 17q and 19q. Of note, two patients shared a common UPD region on chromosome 9p that specifically encompassed the p14 and p16 tumour suppressor genes. Thirteen regions of homozygous deletions were also observed in six patients (18%) on chromosomes 1p21.1, 7q34, 9p22.1-21.3, 10p12.1/p12.2/p14/q23.31/q23.1 and 16q23.1-q23.2. Common minimal homozygously deleted regions at 9p21-p22.3 and 16q23.1, suggest the presence of tumour suppressor gene in these regions. Interestingly, all the homozygous regions appeared within a larger region of the UPD or heterozygous deletion. Grouping patients with UPD/deletions or UPD/amplification identified eleven common minimal regions of genomic aberrations occurring in at least 3 patients; 3p24.1-14.2, 7p22.3-12.2, 7q31.1-31.2, 9p21.3-22.1, 10q21.2-26.13, 12p13.33-13.31, 13q14.2-21.1, 16p13.13, 16q23.1, 17p13.3-13.1 and 17q21.32-25.1. The detection of these recurrent abnormalities associated with UPD suggests a series of candidate genes selected for dysregulation through mutational or epigenetic modulation such as MLH, CDKN2A/B, RB1 and TP53. Paired plaque samples were also analysed from 12 patients. Of note, SNP analysis did not always show identical aberrations between these samples. Six patients had multiple aberrant regions in at least one of their samples. Of these, three patients had unique aberrations which were present in only one. This discordance in the distribution of genomic aberrations between the samples may reflect clonal evolution of the disease. To our knowledge, this is the first high resolution SNP array study in MF that has provided additional insight into the pathogenesis of MF. We have identified distinct aberrations not detected by previous studies as well as frequent and widespread UPD harbouring important oncogenes and tumour suppressor genes likely to play key roles in the dysregulation of MF cells. Disclosures:No relevant conflicts of interest to declare.

Genome Wide SNP Analysis Reveals Frequent Cryptic Clonal Chromosomal Aberrations Including Uniparental Disomy (UPD) in Waldenstrom's Macroglobulinemia.

Abstract 3932Poster Board III-868 BackgroundWaldenstrom's macroglobulinemia (WM) is a rare lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells that secrete monoclonal IgM antibody. Approximately 50% of patients (pts) with WM exhibit a normal karyotype using either conventional chromosome banding analysis (CBA) or FISH approach. However, CBA is a low resolution method and FISH only target previously described abnormalities. Comparative genomic hybridization (CGH) array delineated the minimal deleted region on 6q deletion, the most frequent aberration in WM, and pointed out the role of NFKB pathway key regulators genes. However, tumour suppressor gene or oncogene involved in WM physiopathology is not known, to date. Partial uniparental disomy (UPD) induced by copy neutral loss of heterozygosity (LOH) are important mechanisms for tumour suppressor gene inactivation or oncogene activation in cancer. Recently, Single Nucleotide Polymorphism (SNP) based arrays had been described as a powerful high resolution method allowing both the detection of LOH and copy number alteration (CNA) analysis in the same experiment, a major advantage over CGH array. Our aim was to identify new CNA and LOH involved in WM pathogenesis using SNP arrays analysis. Material and Methods. BM samples of 12 pts with WM (7 males, mean age: 67 years, 7 symptomatic pts) were analysed. DNA was extracted following CD19 B cells selection. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was used to detect both LOH and CNA. In 6 pts, paired samples (tumor / normal T lymphocytes) were used as an intra-individual reference to identify germline polymorphisms. In the 6 left pts, CN polymorphisms were excluded using copy-number variants database (http://projects.tcag.ca/variation) and the reference genotyping data from the HapMap project. Size, position and location of genes were identified with UCSC Genome Browser HG18 assembly, LOH and CNA using genotyping console 3.02 software (Affymetrix) and Partek genomic suite. FISH analysis was performed to detect deletion 6q; 13q14, 11q22, TP53, trisomy 4 and 12 chromosomal aberrations using Vysis probes. ResultsSNP array detected 35 CNA (23 gains, 12 losses) in 9/12 pts, with a mean of 2.9 abnormalities per patient (range 0 to 10). 91% of CNA were observed in symptomatic pts. 50% of CNA were < 5 mb in size, the lower limit of detection by CBA. 15 cryptic aberrations were identified among the somatic CNA spread over 7 pts. These 11 gains and 4 losses are identified by *. Deletions were observed on chromosomal segments 1p36*, 3p21.*, 6q16-q27 (2pts), 8p21*, 8p23-q24, 9p21*, 11q22 (2pts), 16p13*, 19p13*. One cryptic homozygous deletion in 13q14 was identified. Gains were observed on chromosomal segments 3p26, 4p16-q35 (3pts), 4q24*, 5p15 (2pts)*, 5p14, 7p22, 12p13-q24, 16p13*, 17q11*, 18p11-q23, 19p13-q13, Xq21* (5pts), Xq22*, Xq25*, Xp22-q28, Xq27-q28*(2pts).We also identified 42 LOH in 9/12 pts (mean of 3.5 per genome, range 0 to 7) dispersed on 17/23 chromosomes. The LOH observed in the absence of CNA loss are consistent with UPD in 35 cases (83%). 59% of LOH were observed in symptomatic pts. The interstitial or telomeric UPD regions varied in size, from 0.4 to 154mb. Two recurrent regions were identified on chromosome 4 (1.2mb involving genes DCLK2 and LRBA) and 13 (0.8mb involving genes KPNA3, ARL11 and SETDB2), probably subsequent to mitotic recombination. No UPD was observed on chromosome 6q.SNP arrays detected all FISH and CBA findings, except in one pt with subclonal deletion of TP53. Of the 5 pts with normal CBA or FISH, SNP array detected a CNA and 4 UPD in one patient and 7 UPD in an other patient. The remaining 3 cases (25%) had no detected CNA or LOH.Overall, SNP array detected a total of 77 genetic aberrations (CNA + LOH) (6.4 per genome, range 0 -16) in this cohort allowing the selection of 976 relevant genes (159 implicated in cellular growth and proliferation, 165 in cancer, 69 in cell cycle). ConclusionNew cryptic clonal chromosomal lesions were detected using high resolution SNP array in this study. Several abnormalities were recurrent. We described a high frequency of UPD in WM, that might contribute to the inactivation of tumour suppressor genes by mutations or epigenetic alterations and subsequently to the regulation of tumor progression in WM. Further confirmation of the role of these candidate genes is underway. Disclosures:Leleu:Celgene: Research Funding; Janssen Cilag: Research Funding; Chugai: Research Funding.