TP53 Mutation in Waldenstrom Macroglobulinemia

Abstract Background. Waldenstrom macroglobulinemia (WM) is a B-cell malignancy characterized by bone marrow (BM) infiltration of clonal lymphoplasmacytic cells, which produce a monoclonal immunoglobulin M. MYD88L265P mutation may be considered as a founder event because of it high frequency in WM. WM cells may acquire additional genetic hits that may potentially promote disease progression: CXCR4 or CD79B mutations, copy number variation,…TP53 is a tumor suppressor gene that functions as regulator influencing cellular responses to DNA damage. Little is known regarding TP53 alteration in WM. Our aim was to screen TP53 mutation in a large cohort of WM at diagnosis to analyze the genomic landscape of WM using targeted next generation sequencing (NGS) and genome wide single nucleotide polymorphism array (SNPa) and to identify clinical and biological characteristics. Method. BM samples of 125 WM (mean age: 67 years) were analyzed at diagnosis. Tumoral DNA was extracted following CD19 B cell selection. TP53 mutations were analyzed by targeted NGS to scan the coding exons of TP53. MYD88L265P, CD79A, CD79B, and CXCR4mutations were analyzed by sanger sequencing and/or NGS. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was performed in 62 cases. CN-LOH (copy neutral- loss of heterozygosity) and CNA (copy number aberration) were mapped using console 3.02 software (Affymetrix). Flow cytometry was performed to assess P53 and p21 expression after nutlin3a exposition to characterize functional mutant of TP53. Viability and cell growth of treated cells were determined using the MTS assay. Results. We have identified TP53 mutations using NGS in 7.3 % of WM (6 non-sense, 3 frameshift mutations located in the DNA binding domain) (TP53mut WM). The mutation load of TP53 varied from 13% to 98.9% (mean: 62.0%) using the variant allele frequency in NGS. We next examined the effects of nutlin-3a which is an mdm2 inhibitor on WM patients CD19+ cells genotyped for TP53 mutation. Nutlin-3a increased the expression of p53 and p21 in TP53Wild WM patients using flow cytometry (n=6). In contrast, in TP53MutWM cells, no significant variation of p53, p 21 and viability using MTS assay was observed suggesting the presence of functional mutation of TP53. The minimal deleted region of 17p in 17p deleted (TP53Del) samples was mapped using SNP array and contained 79 genes, among which was systematically comprised the loss of TP53. A high correlation between TP53 mutation and deletion 17p (p<106) was observed. One case of CN-LOH was observed at TP53 locus (1,6% of cases). Overall, we have identified alteration of TP53 locus including mutation, deletion and copy neutral loss of heterozigosity in 11, 2% of WM. Using SNP array, we found a relationship between deletion 17p, alteration of TP53 locus including mutation, UPD or del17p (TP53Alt) and TP53Mutand a greater frequency of genomic aberrations in WM compared toTP53wild (p=0.01, p=0.024 and p=0.06 respectively). A higher frequency of WM patients with more than 3 CNA identified by SNPa was observed in TP35Mut group (p=0.03) and del17p group (p<0.00001). No association was observed between TP53Mut and CXCR4 and MYD88mutations. We thoughtto identify clinical and biological characteristics of WM according to TP53Mutand/or Del17pfeatures. With a median follow-up of 5 years, 33 (26%) patients had died. 69% of cases were treated. Front line therapy included rituximab-based regimens in 76%, alkylating agent in 78%, fludarabine in 6%. The WM with TP53alteration, irrespective of TP53Mut or del17p, displayed features of adverse prognosis in regards to higher serum levels of b2m (89% versus 40%, p=0.012), and also greater IPSSWM score 2 and 3 (50% versus 30%, and 43% versus 30%, p=0.041, respectively). Importantly, the presence of TP53alteration, irrespective of TP5Mut or del17p, was associated to poor outcome in overall survival in our series, TP53alteration (p=0.003), del17p (p=0.002), and TP53Mut(p=0.015). TP53 alteration prognostic value was independent of CXCR4 or MYD88L265Pmutations. Conclusion: A low frequency of TP53 mutation was observed in WM at diagnosis. We identified a genomic signature associated to their presence. In addition, a pejorative prognostic value of TP53 mutation was observed in WM highlighted the need of new therapeutic in this sub group of WM. Disclosures Leleu: TEVA: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; LeoPharma: Honoraria; Pierre Fabre: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria.

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Quantitative Whole Genome Analysis of Sequential Samples From Patients with B-CLL Identifies Novel Recurrent Copy Number Alterations Involving Critical B-Cell Transcription Factors

Blood ◽

10.1182/blood.v116.21.3590.3590 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3590-3590

Author(s):

Adele Timbs ◽

Sam Knight ◽

Elham SadighiAkha ◽

Adam Burns ◽

Helene Dreau ◽

...

Keyword(s):

B Cell ◽

Allele Frequency ◽

Neutral Loss ◽

Clonal Evolution ◽

Snp Array ◽

Chromosome 8 ◽

Copy Number Alterations ◽

Genome Wide ◽

Sequential Samples ◽

Pre Treatment

Abstract Abstract 3590 Genome-wide array or sequencing analyses are powerful tools for identifying genetic aberrations in cancers including leukaemias. However, the majority of these aberrations are likely to be random passenger events that do not drive clonal expansion. Currently, it is unknown whether cancers are maintained by a finite set of recurrent mutations similar for each patient or whether and to what extent malignancies are ‘personalised’, and also how molecular disease drivers evolve over time in the context of clinical intervention. The answers to these questions will determine whether future treatment modalities must be tailored according to individual and dynamic cancer characteristics. We hypothesized that differential quantitative high-resolution genome-wide array analysis of sequential samples from the same patients before treatment and at subsequent relapse would have the potential to identify emerging structural abnormalities with relevance to disease progression and/or response to treatment in a given patient. In order to test this hypothesis, we chose B-cell chronic lymphocytic leukaemia (CLL), because of its unique clinical characteristics, as our model to begin to evaluate the potential role of ‘companion diagnostics’ for this condition. We analysed DNA samples of 80 patients with CLL using a 1 million high resolution SNP array. On 34 of them, sequential pre-treatment and relapse samples were available. The raw data was analysed using the OncoSNP analysis tool designed in-house specifically for cancer samples as it enables quantification of copy number alterations (CNA) and copy neutral loss of heterozygosity (cnLOH) based on B-allele frequency plots in complex mixtures. This allows low levels of aberrations to be detected and for mosaic samples to be identified. Results were compared against the data from the Wellcome Trust Case Control Consortium, the DGA and germline DNA in selected cases. Large CNAs (>1Mb) and cnLOH (>5Mb) without deletions of 11q22.3 or 17p13.1 were identified in a third of patients. These patients had an intermediate clinical risk score that increased with the number of large CNAs. SNP array demonstrated clonal evolution in 32% of patients in the sequential sample cohort. These consisted of extension of the 13q abnormality (2), loss of the 13q deletion (1), a 10q23.1-q25.1 deletion (1), gain of 2pter-p14 (1), deletion of 2q33.1-q36.3, (1) a heterozygous deletion of 2q37.1 (1), gain of 8q22.2-qter (1), deletion of 8p (1), amplification of 8q (1), deletion of 8q (1), loss of 16p13.3 (2), mosaic deletion of 17q11.2 (1), an expansion of chromosomes carrying a 19p13.2-p13.11 gain and a 19p13.11 loss (1), deletions within 3p (1), conversion of a gain of 12p12.2-q21.31 to a copy neutral loss of heterozygosity (cnLOH) (1), deletion of 17pter-13.1 (1), increased proportion of chromosomes with the 7q33–34 deletion (1), expansion of cnLOH for 20q11.22-qter (1) and an increased number of cells with a deletion of 2q22.2-q24.1 (1) found at relapse. Importantly, most CNAs occurring at relapse were recurrent in the entire cohort implying that these are non-random events that are important in disease progression. Analysis of the minimal deleted region (MDR) of these recurrent and relapse associated CNAs revealed genes important in lymphoid development, such as NFκB2 and TRAP1 found in the alternative NFκB pathway and BLIMP1 involved in B-cell differentiation. There were also recurrent abnormalities in the region coding for SP140 which has been implicated in familial CLL. Known cancer genes were also affected by these recurrent and relapse-associated CNAs such as RND3, RIF1, RFXANK, and RHOT1, which are all members of the RAS pathway family. Using the OncoSNP program it was possible to determine that most of these emerging abnormalities were present in low numbers at diagnosis (fig. 1) suggesting that treatment does not induce the genetic alterations but may select for them. Fig .1 CNA and B-allele frequency plots of pre-treatment and relapse samples of a patient demonstrating clonal evolution. A low level of cnLOH can be seen in the B-allele frequency plot at diagnosis (a) at the end of chromosome 8 (dashed box) which becomes more apparent at relapse (b). Fig .1. CNA and B-allele frequency plots of pre-treatment and relapse samples of a patient demonstrating clonal evolution. A low level of cnLOH can be seen in the B-allele frequency plot at diagnosis (a) at the end of chromosome 8 (dashed box) which becomes more apparent at relapse (b). In conclusion, this is the first attempt to quantify CNAs in sequential leukaemia samples. The results demonstrate that recurrent and relapse associated CNAs affect genes important in B-cell development and cancer progression. Disclosures: No relevant conflicts of interest to declare.

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Genome-Wide DNA Copy Number Analysis by SNP Arrays of B-Cell Chronic Lymphocytic Leukemia: Correlation with Known Biological and Molecular Prognostic Markers.

Blood ◽

10.1182/blood.v112.11.1061.1061 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1061-1061

Author(s):

Laura Mosca ◽

Sonia Fabris ◽

Giovanna Cutrona ◽

Luca Agnelli ◽

Serena Matis ◽

...

Keyword(s):

High Resolution ◽

Copy Number ◽

Prognostic Markers ◽

Snp Array ◽

Lymphocytic Leukemia ◽

Array Analysis ◽

Genome Wide ◽

Trisomy 12 ◽

Snp Array Analysis ◽

Cell Chronic Lymphocytic Leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is a genetically heterogeneous disease with a variable clinical course. Chromosomal changes have been identified by FISH in approximately 80% of patients, and the presence of specific lesions, such as trisomy 12 and 13q14, 11q23, 17p13.1 and 6q23 deletions represent prognostic markers for disease progression and survival. In order to characterize further the complexity of B-CLL genomic lesions, we performed high density, single nucleotide polymorphism (SNP) array analysis in highly purified neoplastic cells (>92%) from a panel of 100 untreated, newly diagnosed patients (57 males and 43 females; age, median 63 years, range 30–87) in Binet stage A. All patients were investigated by FISH for the presence of trisomy 12 (21 cases); 13q14 deletion (44 cases, 34 as the sole abnormality); 11q22.3, 17p13.1 and 6q23 (15, 7 and 2 patients, respectively). In addition, ZAP-70 and CD38 expression resulted positive in 42 and 46 patients, whereas IgVH genes were mutated in 45 patients. Genome-wide DNA profiling data were generated on GeneChip® Human Mapping 250K NspI arrays (Affymetrix); copy number alterations (CNA) were calculated using the DNA copy Bioconductor package, which looks for optimal breakpoints using circular binary segmentation (CBS) (Olshen et al, 2004). A total of 782 CNAs (ranging from 1 to 31 per sample, mean and median values 7.82 and 7, respectively) were detected; DNA losses (365/782=46.67% loss; 194/782=24.81% biallelic deletion) were found to be more frequent than gains (148/782=18.93% gain; 75/782=9.59% amplification). The most recurrent alterations detected by FISH were all confirmed by SNP array analysis, strengthening further the good reliability of such high-resolution technology. We identified 12 minimally altered regions (MARs) larger than 100 kb with a frequency higher than 5%. Among well known alterations, the largest was represented by chromosome 12 trisomy, followed by 6q, 17p and 11q23 deletions (32.87, 19.09 and 10.43 Mb, respectively) and 13q14 deletion (635 kb). Gain of 2p25.3 involves a common region of 4.39 Mb region in 7 patients, although it was extended to the whole short arm of chromosome 2 in 3 cases. Among those alterations previously described in B-CLL, we found losses at 14q32.33 (12 pts) and 22q11.2 (5 pts) involving the IGH and IGLλ loci, respectively. With regard to novel regions, we identified losses at 4q35.2 (5 pts) and 11q25 (6 pts). In addition we found a high frequency of losses/gains at 14q11.2 (42 pts) and 15q11.2 (33 pts), two genomic regions reported to be affected by DNA copy number variations in normal individuals. As regards correlations between CNAs and biological markers, we found that the number of CNAs is significantly higher in cases with unmutated IgVH (9.4; range 2–31) as compared with mutated IgVH (6; range 1–13) (p=0.002), while neither CD38 nor ZAP-70 expression showed significant correlation. In addition, a significant higher number of either CNAs (p=0.001), total MARs (p<0.0001) or even only novel MARs (p=0.009) was significantly associated with cases with 17p deletion or multiple cytogenetic aberrations as evaluated by FISH analysis. Our data indicate that genetic abnormalities involving chromosomal gains and losses are very common in early-stage B-CLL and further support the application of high resolution SNP array platforms in the characterization of genetic changes in the disease. In addition, we detected novel altered chromosomal regions that warrant further investigations to better define their pathogenetic and prognostic role in B-CLL.

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Identification of Novel Recurrent Copy Number Variations and Regions of Copy-Neutral Loss of Heterozygosity by High Resolution Genomic Array in Pre-Treatment and Relapsed B-CLL.

Blood ◽

10.1182/blood.v114.22.1098.1098 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1098-1098

Author(s):

Samantha JL Knight ◽

Elham Sadighi Akha ◽

Adele Timbs ◽

Tariq Enver ◽

Andrew R Pettitt ◽

...

Keyword(s):

High Resolution ◽

Copy Number Variation ◽

B Cell ◽

Genomic Instability ◽

Loss Of Heterozygosity ◽

Copy Number ◽

Neutral Loss ◽

Single Gene ◽

Number Variation ◽

Pre Treatment

Abstract Abstract 1098 Poster Board I-120 Background B-cell chronic lymphocytic leukaemia (B-CLL) is the most common form of adult leukaemia in the Western World. It is a heterogeneous disease and important biological and clinical differences have been identified. However, the molecular mechanisms underlying emergence and maintenance of B-CLL after treatment remain elusive. Array based comparative genomic hybridization (aCGH) has revolutionized our ability to perform genome wide analyses of copy number variation (CNV) within cancer genomes. Single Nucleotide Polymorphism arrays (aSNP) provide genotyping and copy number variation data and detect regions of copy neutral Loss of Heterozygosity (cnLOH) with the potential to indicate genes involved in leukaemia pathogenesis. Both technologies are evolving rapidly and emerging platforms are thought to allow high resolution (HR) of abnormalities down to a single gene level. Aim The aim of the current study was therefore to test a HR-aCGH and a HR-aSNP platform for their ability to detect large and small CNVs and regions of cnLOH in B-CLL. More specifically, we wanted to: Method We used a high resolution 244K aCGH platform and a 1Mio SNP array in parallel to test and characterize enriched B-CLL peripheral blood samples (>80% CD19+;CD5+) from 44 clinically annotated patients collected at our institution. To distinguish CNVs seen commonly in the general population the results were compared with ‘in house’ control data sets and the Database of Genomic Variants (http://projects.tcag.ca/variation/). Results Our results show that large abnormalities, already noted by FISH, were reliably identified and the boundaries of abnormalities at 11q22.3, 13q14.2 and 17p could be defined more precisely. In addition, novel and recurrent CNVs within the sample set were identified (1p33; 3p24.3; 3p14.2; 4q12; 4q13.3; 6q21; 6q27; 8p22; 10q24; 11p15.4; 11q12; 11q13.4; 11q14.1; 11q22.1; 11q23.3; 13q14.11; 14q21.1; 15q15.1; 15q25.3; 17p13.3; 17q22; 18p11.32; 18p23; 19p13.13; 19p13.12; 19p13.32; 22q11.21; 22q11.22). Interestingly, some of these abnormalities contain single gene alterations involving oncogenes, chemokine receptors, kinases and transcription factors important in B cell development and differentiation. Assessment of smaller CNVs (less then 10 consecutive oligonucleotides) also revealed recurrent CNVs involving single genes that were clustered according to function and pathways. Comparison of paired pre-treatment and relapse samples showed differences in large CNVs in 6 out of the 14 pairs with the majority being losses within the relapse sample. In particular, relapse samples contained new losses within 2q33.1-2q37.1; 4q13.2-4q13.3; 5q31.3-5q34; 7q36.3; 10q23.1-10q25.1 11q12.3 and multiple losses within 13q14.1-13q14.3. Taken together, these data indicates that genomic instability plays a role in clonal evolution and selection after treatment in at least some patients. Analysis of a bigger cohort of matched pre-treatment and relapse samples is on-going. The importance of copy neutral LOH in B-CLL has been a subject of debate. Using the 1Mio HR-aSNP, we were able to detect multiple regions of cnLOH throughout the genome. Examination of the four regions that are known to have prognostic significance when deleted identified cnLOH involving 13q11-13q34(ter) and cnLOH of 13q21.1-q34(ter) outside the FISH region. Deletions of the 17p13.1 locus including the p53 gene confer poor prognosis in B-CLL and direct treatment decisions. Interestingly, we were able to identify cnLOH involving this region in 5% of samples. In addition, we also noticed cnLOH in 17p13.2 containing genes previously implicated in cancer. The exact pathogenetic and prognostic implications of these findings remain to be established. Conclusion Using HR-aCGH and HR-aSNP we have identified novel recurrent CNVs and regions of cnLOH in patients with B-CLL. Sequential analysis of the same patients over time suggests that at least in some patients, clonal complexity and dynamics are driven by genomic instability. Disclosures No relevant conflicts of interest to declare.

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B-Cell-Specific Transcription Factor BACH2 Involved in the Clinical Behavior Heterogeneity of Waldenström Macroglobulinemia

Blood ◽

10.1182/blood.v120.21.1288.1288 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1288-1288

Author(s):

Charles Herbaux ◽

Guillemette Marot ◽

Elisabeth Bertrand ◽

Natacha Broucqsault ◽

Sylvie Zouitna-Galiègue ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

B Cell ◽

Candidate Genes ◽

Gene Expression Profile ◽

Supervised Classification ◽

Snp Array ◽

Qrt Pcr ◽

Genome Wide ◽

Underlying Mechanisms

Abstract Abstract 1288 Background. Approximately 30% of the patients who fulfil the criteria of Waldenström macroglobulinemia (WM) are diagnosed while asymptomatic, and will not require immediate therapy; these cases are called indolent WM (IWM). However, patients with a disease-related event will be considered for therapy, these cases are called symptomatic or aggressive WM (AWM). The physiopathology of these 2 groups remains unclear, and the mechanisms of progression have not been fully understood so far. We hypothesized that a gene signature that differentiates these two categories could be identified to better understand the underlying mechanisms of progression of WM. Methods. Seventeen patients diagnosed with WM (8 IWM and 9 AWM) were included in this study. We selected tumour cells from the bone marrow (BM) using mononuclear cell isolation, then B cell enrichment (B cell isolation kit, Myltenyi-Biotec, USA). The purity was confirmed by flow cytometry. Total RNA was extracted using the Trizol method. Gene expression profiling was performed using U133A arrays (Affymetrix, USA). Gene expression was normalized using the RMA algorithm. We ranked genes by fold-change of expression levels on a first series of 11 patients (5 IWM and 6 AWM) calculated with the ‘limma’ package in R. Next, we used a supervised classification to establish a gene expression profile to distinguish IWM from AWM. Therewith, we validated this profile on an independent set of 6 patients (3 IWM and 3 AWM). We then performed a pathway analysis using Ingenuity® analysis software. We confirmed gene expression deregulation with qRT-PCR on 3 candidate genes in the first series of patients. Genome-wide detection of copy number alteration and loss of heterozygosity were performed on 13 of the 17 WM cases, using the Genome-Wide Human SNP Array 6.0 (Affymetrix, USA). Finally, we investigated the functional consequences of the deregulation of these candidate genes in BCWM1 and MWCL1, both B cell lines originated from WM. Survival was studied using a colorimetric method with MTS (Promega, USA). Proliferation was analyzed using incorporation of a nucleoside analog (EdU) into DNA during active DNA synthesis (Invitrogen, USA). Results. The differential analysis has identified 82 probes, corresponding to 48 genes, significantly deregulated and capable of differentiating samples from IWM and AWM in an unsupervised classification. Moreover, with a supervised classification, this gene expression profile accurately classified 94% of the 17 WM samples, including the 6 WM of the independent validation set. The two molecular networks that appeared to play a major role in the physiopathology of IWM versus AWM were the plasma cell differentiation pathway and the AKT pathway. We have then identified 3 key genes in those 2 pathways, BACH2 and CIITA on the one hand and PTEN, respectively. We have then confirmed the deregulation of these gene expression levels by qRT-PCR in 3 IWM and 4 AWM; these 3 genes were over-expressed in IMW relatively to AMW. BACH2 is a B-cell-specific transcription factor known to be a tumour suppressor gene. It was shown that BACH2 reduces proliferation and induces cell death when over-expressed in B lymphoma tumour cells. We have thus pharmacologically over-expressed BACH2 in BCWM1 and MWCL1 and significantly reduced the proliferation and the survival of the two cell-lines. Further studies using BACH2 specific overexpression with lentiviral infection are underway, in vitro. The data will be presented at ASH. In order to further study the mechanisms of deregulation of BACH2 in IWM and AWM, we have conducted a genome wide SNP array study of 13 patients. Among those, 7 patients (4 IWM and 3 AWM) demonstrate a deletion of long arm of chromosome 6 (del6q), the most frequent chromosomal abnormality in WM. BACH2 gene is located on the 6q15 locus. Interestingly, we found that 3 out of the 3 AWM had a del6q that took in the 6q15 region, whereas 3 out of 4 of the IWM had a del6q preserving the 6q15 region. Therefore, haploinsufficiency could participate in the under-expression of BACH2 in aggressive WM; this hypothesis will be verified by using DNA qRT-PCR of BACH2. Conclusion. To the best of our knowledge, we have identified for the first time a specific gene expression signature that differentiates IWM and AWM. We have exposed several genes from this dataset, including BACH2, which is a candidate to better understand the underlying mechanisms of progression of WM. Disclosures: No relevant conflicts of interest to declare.

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Somatic and Germline Copy Neutral Loss of Heterozygosity Are Common in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v120.21.4567.4567 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4567-4567

Author(s):

Megan Hanna ◽

Bethany Tesar ◽

Kristen E. Stevenson ◽

Alexander R. Vartanov ◽

Stacey M. Fernandes ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Loss Of Heterozygosity ◽

Copy Number ◽

Neutral Loss ◽

Snp Array ◽

Lymphocytic Leukemia ◽

Alternative Mechanism ◽

Large Set ◽

The Impact ◽

13Q Deletion

Abstract Abstract 4567 Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults, and prognosis is still difficult to predict, although cytogenetic abnormalities identified by FISH are most helpful. Isolated reports have suggested that copy neutral loss of heterozygosity (cnLOH) can involve 13q and 17p in CLL, but the extent and the impact on clinical outcome is not well established. We therefore embarked upon characterization of cnLOH in a large set of 230 CLLs with matched normal DNA. The median age at diagnosis of CLL in this patient population was 54 (33–79). 87% of patients were Rai 0–1 at diagnosis, and 79% were chemotherapy naive at sampling. 121 of 230 patients were treated, with a median TTFT of 42 months. The median follow-up for surviving patients is 74 months. 44% of patients carried one somatic copy number abnormality (CNA) by SNP array, 20% two, 7% three, 5% four and 4% more than five. cnLOH was called by the Affymetrix Genotyping Console Software, which evaluates each SNP for copy number and then subtracts the A allele value from the B allele value within an individual sample, thereby allowing independent evaluation of tumor (somatic) and normal (germline). All calls were manually reviewed. A size cut-off of 1.0 Mb was used to determine significant cnLOH events. In total, of 230 patients, we found 26 events of somatic cnLOH (11%) and 36 events of germline cnLOH (16%), affecting 56 separate patients (24%). This frequency of cnLOH was surprisingly high and suggested that cnLOH might be an alternative mechanism affecting known loci in CLL. This was the case, as the most common events overall involved 13q in 25 patients, the X chromosome in 9 patients, chromosomes 17 and 18 in five patients each, and chromosomes 9, 11 and 12 in four patients each. Interestingly, germline events were quite common. Six patients had small regions of germline LOH with much more extensive adjacent somatic LOH, two on chr 13, one on chr 17, two on chr X and one on chr 20; these were coded as germline in the analysis. In addition, of the 25 patients with cnLOH on chromosome 13, 18 of these were in the germline and 7 were somatic. The region(s) of cnLOH were typically adjacent to a 13q deletion, and often involved the entire chromosome arm. Somatic cnLOH at 13q was associated with intermediate sized deletions including the RB gene (p=0.002). Of the 18 patients with germline cnLOH at 13q, 7 of them had no 13q deletion, while 7 had monoallelic deletion and 4 biallelic deletion. Thus 7 patients (3%) had cnLOH events at 13q, in the absence of 13q deletion, again suggesting an alternative mechanism affecting this locus. Germline cnLOH was associated with treatment prior to sampling (44% vs 17%, p<0.001), possibly due to its association with unmutated IGHV(58% vs 32%, p=0.008), and ZAP70 positivity (59% vs 36%, p=0.024). Somatic cnLOH was not associated with any patient characteristics. Neither somatic nor germline cnLOH was associated with >= 1 somatic CNA, but an association between both LOH types and >= 2 somatic CNAs was observed (p=0.053 germline and p=0.030 somatic). TTFT was reduced in patients with either germline cnLOH (61 mos vs 103, p=0.004) or somatic cnLOH (53 mos vs 107, p=0.008). Presence of two or more CNAs was also associated with short TTFT (48 mos vs 115, p<0.001). In order to assess the impact of cnLOH and CNAs on outcome independent of prior therapy, we evaluated TTFT in the 181 chemotherapy naive patients. In this subgroup, germline cnLOH was not associated with short TTFT, while somatic cnLOH (80 mos vs 125, p=0.018) and two or more somatic CNAs (80 mos vs 125, p=0.009) were. In multivariable Cox modeling including germline cnLOH, IGHV, and del 11q or 17p by FISH, the only significant predictor of TTFT was unmutated IGHV (hazard ratio (HR) 4.48, p<0.001). In multivariable Cox modeling including somatic cnLOH and the variables above, the only significant predictor of TTFT was again unmutated IGHV (HR 4.41, p<0.001). When the presence of two or more somatic CNAs was added to these models, this variable was significant along with IGHV (HR 2.04, p=0.009 in germline model; HR 1.84, p=0.033 in somatic model). We conclude that both somatic and germline cnLOH are common in CLL, affecting one quarter of patients in this dataset, and frequently involve chromosomal regions known to be important in CLL. cnLOH is associated with increased somatic CNAs and unmutated IGHV, and therefore poor prognosis. Disclosures: No relevant conflicts of interest to declare.

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Genetic Basis of Primary Central Nervous System Lymphoma

Blood ◽

10.1182/blood.v126.23.2687.2687 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2687-2687

Author(s):

Kenichi Yoshida ◽

Rie Nakamoto-Matsubara ◽

Kenichi Chiba ◽

Yusuke Okuno ◽

Nobuyuki Kakiuchi ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

B Cell ◽

Copy Number ◽

Genetic Basis ◽

Snp Array ◽

Central Nervous System Lymphoma ◽

Genetic Alterations ◽

Targeted Sequencing ◽

Systemic Dlbcl

Abstract Introduction Primary central nervous system lymphoma (PCNSL) is a rare subtype of non-Hodgkin lymphoma, of which approximately 95% are diffuse large B-cell lymphomas (DLBCLs). Despite the substantial development of intensive chemotherapy during the past two decades, overall clinical outcome of PCNSL has been poorly improved especially in elderly and so has been our knowledge about the molecular pathogenesis of PCNSL, in terms of driver alterations that are relevant to the development of PCNSL. Method To delineate the genetic basis of PCNSL pathogenesis, we performed a comprehensive genetic study. We first analyzed paired tumor/normal DNA from 35 PCNSL cases by whole-exome sequencing (WES). Significantly mutated genes identified by WES and previously known mutational targets in PCNSL and systemic DLBCL were further screened for mutations using SureSelect-based targeted deep sequencing (Agilent) in an extended cohort of PCNSL cases (N = 90). Copy number alterations (CNAs) have been also investigated using SNP array-karyotyping (N =54). We also analyzed WES and SNP array data of systemic DLBCL cases (N = 49) generated by the Cancer Genome Atlas Network (TCGA) to unravel the genetic difference between PCNSL and systemic DLBCL. Results The mean number of nonsynonymous mutations identified by WES was 183 per sample, which was comparable to the figure in systemic DLBCL and characterized by frequent somatic hypermutations (SHMs) involving non-Ig genes. A higher representation of C>T transition involving CpG dinucleotides and hotspot mutations within the WRCY motif targeted by SHM further suggested the involvement of activation-induced cytidine deaminase (AID) in the pathogenesis of PCNSL. We found 12 genes significantly mutated in PCNSL (q < 0.1), including MYD88, PIM1, HLA-A, TMEM30A, B2M, PRDM1, UBE2A, HIST1H1C, as well as several previously unreported mutational targets in systemic DLBCL or PCNSL, such as SETD1B, GRB2, ITPKB, EIF4A2. Copy number analysis identified recurrent genomic segments affected by focal deletions (N = 27) and amplifications (N = 10), most of which included driver genes targeted by recurrent somatic mutations or known targets of focal CNAs such as CDKN2A and FHIT. Subsequent targeted sequencing finally identified a total of 107 significantly mutated genes, of which 43 were thought to be targeted by SHM according to their mutational signature and genomic distribution. Most cases with PCNSL (98%) had mutations and CNAs involving genes that are relevant to constitutive NF-KB/Toll-like receptor (TLR)/BCR activity, including those in MYD88 (80%), CD79B/A (60%), CARD11 (18%), TNFAIP3 (26%), GRB2 (24%) and ITPKB (23%). Genetic alterations implicated in escape from immunosurveillance were also frequently identified in as many as 76% of cases. Mutations of HLA-B (64%), HLA-A (36%), HLA-C (28%), B2M (14%) and CD58 (12%) were commonly detected in addition to CNAs in 6p21.32 (HLA class II), 1p13.1 (CD58) and 15q15.2 (B2M), suggesting the importance of immune escape in the pathogenesis of PCNSL. SHMs were also seen in most cases (98%), which affected not only known targets of AID including PIM1, IGLL5 and BTG2 but also previously unreported genes involved in cell proliferation, apoptosis, or B cell development. The pattern of frequently mutated genes in PNCSL was more uniform compared with that in systemic DLBCL, and similar to that found in the activated B cell subtype of DLBCL (ABC-DLBCL), which was in accordance with the previous report of immunophenotypic analysis of PCNSL. On the other hand, mutations of HLA class I genes (HLA-B, HLA-A) were more frequently mutated in PCNSL compared with ABC-type DLBCL. Conclusion WES, SNP array karyotyping and follow-up targeted sequencing of a large cohort of PCNSL cases revealed the genetic landscape of PCNSL, which were more homogeneous than that of systemic DLBCL, and thought to be involved in activation of constitutive NF-KB/TLR/BCR signaling, escape from immunosurveillance, as well as highly frequent SHMs. Disclosures No relevant conflicts of interest to declare.

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Distinctive Genomic Alterations in Testicular Diffuse Large B Cell Lymphoma

Blood ◽

10.1182/blood.v126.23.3655.3655 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3655-3655

Author(s):

Connie Batlevi ◽

Franck Rapaport ◽

Lu Wang ◽

Andrew M. Intlekofer ◽

Amanda R. Copeland ◽

...

Keyword(s):

B Cell ◽

Copy Number ◽

Research Funding ◽

Cell Lymphoma ◽

Snp Array ◽

B Cell Lymphoma ◽

Immune Recognition ◽

Variant Allele Frequency ◽

Large B Cell Lymphoma ◽

Myd88 L265p

Abstract Background: Primary testicular diffuse large B cell lymphoma (DLBCL) is an uncommon malignancy portending a poor prognosis with increased risk of central nervous system disease. Phenotypically, most primary testicular lymphomas have a non-germinal center B-cell like (non-GCB) origin. To identify the genetic characteristics of testicular DLBCL, we evaluated DNA copy number and mutational profiling using SNP array and a next generation targeted sequencing platform. Methods: Twelve cases of testicular DLBCL with patient consent for tissue specimens and sufficient tumor tissue were retrospectively identified. Cell of origin was determined by Hans immunohistochemistry (IHC) model. We performed a custom, targeted deep-sequencing assay of 585 cancer genes (HemePACT) on matched tumor and normal pairs. Barcoded pools were sequenced on Illumina HiSeq 2500 to 500-1000x coverage per sample Sequencing was compared to a matched normal tissue control (N=10) if available or alternatively a pooled normal tissue control. We excluded all mutations either present at a high variant allele frequency in the matching germline samples, present in two databases of inherited variants (DBSNP and 1000 genomes) or present in one databases of inherited variants and absent from COSMIC. We evaluated copy number and allelic imbalance with an Affymetrix OncoScan SNP-array. IHC was performed for select genes. Results were compared to a panel of non-testicular DLBCL previously described (N=78). Results: The median age of the patients was 55.1 years (range 21.9-77.9). Patients had clinical stage IE (50%) and IV (50%) disease. All samples were sequenced from pre-treatment biopsies. Eleven of 12 patients were initially treated with R-CHOP chemotherapy, intrathecal methotrexate and radiation. Treatment history for one patient was unknown. We identified 124 mutations in 12 cases of testicular DLBCL. The most common mutation was MYD88 occurring in 10/12 patients (83%) with 6 mutations in non-GCB and 2 mutations in GCB (Fig 1A). The MYD88 L265P allele was most frequent and occurred in 9/12 patients (75%). The median MYD88 L265P variant allele frequency was 0.36 (range 0.07-0.51) with normal copy number status at that loci. In contrast, MYD88 mutations were less frequent in DLBCL without testicular involvement, 12/37 (32%) non-GCB and 3/41 (7%) GCB DLBCL, p<0.05 by Fisher's t-test (Fig 1B). Furthermore, mutations in CD79B were significantly more common in testicular DLBCL (5/12, 42%) versus non-testicular DLBCL (7/78, 9%). Concurrent mutations affecting the BCR receptor pathway was noted in 10/12 patients (83%): CD79B (5/12, 42%), TNFAIP3 (1/12, 8%), CARD11 (1/12, 8%) (Fig 1A). Frequency of TNFAIP3, CARD11 was not statistically significant between testicular versus non-testicular DLBCL. Other commonly mutated pathways include epigenetics (10/12, 83%) and immune recognition (6/12, 50%). Deregulation of immune recognition was noted by HLA-A (3/12, 25%) and beta-2-microglobulin (2/12, 17%) mutations as well as loss of HLA locus in 8/10 samples (80%) (Fig 1C). IHC revealed 6/10 (60%) cases with no MHC Class I expression of the tumor cells. MHC Class I negative cases also lacked B2M expression (3/6) or displayed mislocalization of B2M to the cytosol (3/6). PD-L1 was expressed by lymphoma cells as assayed by IHC in 1/10 (10%) cases but no amplification or mutation was identified. No copy gains of the 9p24.1, PD-1, PD-L1, PD-L2 locus were identified via HemePACT or SNP array. Although mutations in CDKN2A/B were not identified in HemePACT, SNP array confirmed loss of CDKN2A/B at the 9p21 loci in 3/10 cases (30%). Conclusion: Targeted genomic sequencing and SNP array analysis have identified a distinctive genetic pattern with alterations of MYD88, BCR pathway mutations, and immune recognition deficiency in testicular DLBCL compared to non-testicular DLBCL. These findings may have implications in guiding the design of future treatment strategies for testicular DLBCL. Disclosures Younes: Novartis: Research Funding; Janssen: Honoraria; Johnson and Johnson: Research Funding; Takeda Millenium: Honoraria; Seattle Genetics: Honoraria, Research Funding; Sanofi-Aventis: Honoraria; Bayer: Honoraria; Bristol Meyer Squibb: Honoraria; Curis: Research Funding; Celgene: Honoraria; Incyte: Honoraria.

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Copy number variation analysis in Chinese children with complete atrioventricular canal and single ventricle

BMC Medical Genomics ◽

10.1186/s12920-021-01090-y ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Xingyu Zhang ◽

Bo Wang ◽

Guoling You ◽

Ying Xiang ◽

Qihua Fu ◽

...

Keyword(s):

Copy Number ◽

Trisomy 21 ◽

Single Ventricle ◽

Snp Array ◽

Copy Number Variations ◽

Chinese Children ◽

Atrioventricular Canal ◽

Genome Wide ◽

Number Variation ◽

Complete Atrioventricular

Abstract Background Congenital heart disease (CHD) is one of the most common birth defects. Copy number variations (CNVs) have been proved to be important genetic factors that contribute to CHD. Here we screened genome-wide CNVs in Chinese children with complete atrioventricular canal (CAVC) and single ventricle (SV), since there were scarce researches dedicated to these two types of CHD. Methods We screened CNVs in 262 sporadic CAVC cases and 259 sporadic SV cases respectively, using a customized SNP array. The detected CNVs were annotated and filtered using available databases. Results Among 262 CAVC patients, we identified 6 potentially-causative CNVs in 43 individuals (16.41%, 43/262), including 2 syndrome-related CNVs (7q11.23 and 8q24.3 deletion). Surprisingly, 90.70% CAVC patients with detected CNVs (39/43) were found to carry duplications of 21q11.2–21q22.3, which were recognized as trisomy 21 (Down syndrome, DS). In CAVC with DS patients, the female to male ratio was 1.6:1.0 (24:15), and the rate of pulmonary hypertension (PH) was 41.03% (16/39). Additionally, 6 potentially-causative CNVs were identified in the SV patients (2.32%, 6/259), and none of them was trisomy 21. Most CNVs identified in our cohort were classified as rare (< 1%), occurring just once among CAVC or SV individuals except the 21q11.2–21q22.3 duplication (14.89%) in CAVC cohort. Conclusions Our study identified 12 potentially-causative CNVs in 262 CAVC and 259 SV patients, representing the largest cohort of these two CHD types in Chinese population. The results provided strong correlation between CAVC and DS, which also showed sex difference and high incidence of PH. The presence of potentially-causative CNVs suggests the etiology of complex CHD is incredibly diverse, and CHD candidate genes remain to be discovered.

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Infant ALL Patients Carrying t(4;11) Have a Different Genotypic Profile Than Older ALL Children.

Blood ◽

10.1182/blood.v110.11.1436.1436 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1436-1436

Author(s):

Michela Bardini ◽

Lilia Corral ◽

Eleonora Mangano ◽

Roberta Spinelli ◽

Grazia Fazio ◽

...

Keyword(s):

Copy Number ◽

Critical Role ◽

Snp Array ◽

Point Mutations ◽

Latency Period ◽

Older Children ◽

Available N ◽

Childhood All ◽

Genome Wide ◽

The Individual

Abstract Mice models and prenatal studies indicate that in childhood ALL the individual genetic lesions alone are insufficient to generate a full leukemic phenotype, and cooperating oncogenic lesions are required. Recently, multiple genome-wide studies on childhood ALL (1–18 years) identified deletions at several loci, mainly affecting genes that play a critical role in regulating B cell development and differentiation. By contrast, the prenatal and postnatal steps in the pathogenesis of Infant ALL (less than 1 year at diagnosis) are not defined. Infant ALL is a very aggressive disease, with t(4;11)/MLL-AF4 fusion representing the major subgroup. Although the very short latency period suggests that leukemogenic events occur prenatally, mice models indicates that MLL-AF4 alone is not sufficient to induce leukemia, and additional mutations may occur. Also unclear is whether the molecular pathways needed for lymphoid cell differentiation are altered in cases with an MLL rearrangement and, if so, whether these alterations differ between the leukemia of infants and older children. Aim of this study was to detect MLL-cooperating aberrations, undetectable by conventional techniques, by using genome-wide single nucleotide polymorphism (SNP) genome wide analysis (100K SNP human mapping, Affymetrix). More specifically, we searched for Loss of Heterozygosity (LOH) associated or not to copy number alteration. The identification of these lesions could help identifying leukemia pathogenesis, as well as providing the basis for targeted therapy. We have analyzed 28 cases of Infant ALL with t(4;11) at diagnosis and their corresponding samples at remission, when available (n=18). SNP data were analyzed by using dChip software, and confirmed by CNAG 2.0. A more dense SNP array analysis (250K) has been applied in selected cases to confirm LOH and precisely dissect the affected chromosomal regions. Compared to older childhood ALL patients, a far limited number of deletions/amplifications has been found; only 2/28 patients showed deletions, namely 1p36.33-p36.31 in 1 patient and 3p11.1-p12.2 plus 7q22.1-q22.2 in another patient, while 26/28 Infant ALL did not present any visible structural variation. Different from older children, several segmental copy-number neutral (CNN) LOH have been detected by dChip. The extension and prevalence of the affected regions was variable; among them 6p21.32 (4/28 cases), 7q31.33-q32.1 (3/28), 8q21.12-q21.3 (2/28), 8q24.11 (2/28) and 14q21.2 (2/28). Overall, these results confirm that Infant ALL with t(4;11)/MLL-AF4 fusion represents a biologically unique disease, different from other type of leukemia occurring in older children. While in older children a multistep mechanism (with the involvement of several genes) is required for the full leukemic phenotype, MLL rearrangements per se might play a major role on the leukemogenesis. By this approach it could not be excluded that different mechanisms could cooperate with MLL in transforming cells, including point mutations. The functional role of CNN-LOH still needs to be understood: they could either reflect the duplication of oncogenic mutations, or be related to epigenetic mechanisms.

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SNP Array Karyotyping Improves Detection Rate of Clonal Chromosomal Abnormalities in Refractory Anemia with Ringed Sideroblasts.

Blood ◽

10.1182/blood.v110.11.4132.4132 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4132-4132

Author(s):

Theodore Ghazal ◽

Lukasz P. Gondek ◽

Abdo S. Haddad ◽

Karl S. Theil ◽

Mikkael A. Sekeres ◽

...

Keyword(s):

Copy Number ◽

Chromosomal Abnormalities ◽

Neutral Loss ◽

Snp Array ◽

Uniparental Disomy ◽

Blood Analysis ◽

Refractory Anemia ◽

Ringed Sideroblasts ◽

Normal Copy Number

Abstract Among WHO low-risk categories of MDS, refractory anemia with ringed sideroblasts (RARS) can be more accurately diagnosed by characteristic pathomorphology. Clonal hematopoiesis and chromosomal abnormalities exemplify a close pathogenetic relationship to other forms of MDS. RARS shows considerable clinical variability even for patients (pts) with identical cytogenetic defects. Due to the low resolution of metaphase cytogenetics (MC) and its dependence on cell growth in vitro, this test is often non-informative in MDS. High-density SNP arrays (SNP-A) allow for a precise identification of unbalanced genomic lesions and copy-neutral loss of heterozygozity. We hypothesize that cryptic chromosomal (chr) aberrations exist in most, if not all, pts with RARS. Their detection may help to improve prognostication, distinguish distinct phenotypes and point towards unifying pathogenic defects. Initially, we analyzed the results of MC in pts with MDS and MDS/MPD (N=455) and in a sub-cohort of RARS, RCMD-RS, RARSt and other MDS subtypes with >15% RS. When we compared pts with/without RS, chr defects were found at comparable frequencies (∼50%). The most commonly occurring defects associated with RS, compared to other forms of MDS, included those of chr 5 (9% vs. 16%, 7 (8% vs. 12%) and 20 (3% vs. 8%). DNA was available for 36 pts with RS and was subjected to 250K SNP-A karyotyping. Pathologic lesions were defined upon exclusion of normal copy number polymorphisms identified in 81 controls (O’Keefe at al ASH 2007), as well as the Database of Genomic Variants (http://projects.tcag.ca/variation). By MC, a defective karyotype was present in 16/36 pts (44%). Deletions involving chr 5, 7 and complex MC were found in 3, 5, and 2pts, respectively. However, when SNP-A was applied as a karyotyping tool (copy number and LOH analysis), all aberrations found by MC were confirmed, but also new lesions were detected so that an abnormal karyotype was established in 62% of pts. Several previously cryptic/recurrent lesions included losses of a portion of chr. 2 (N=2; 2p16.2, 2p16.3), and deletions (N=4; 7p11.1–14.1, 7p21.3, 7q11.23–21.11, 7q21.12-qter) as well as gains (N=1; 7q33) on chr 7. We have also detected segmental uniparental disomy (UPD) in chr 1 (N=2; 1p21.3–22.2, 1p). This type of lesion cannot be detected using MC and provides an additional mechanism leading to LOH. When both bone marrow and blood of 5 RARS patient were tested using SNP-A, blood analysis had 100% accuracy rate as compared to marrow; all defects seen in the marrow were also found in blood. We conclude that chromosomal defects are present in a majority of RARS patients and arrays with higher resolution will identify defects in most, if not all of the patients. Our study also demonstrates testing of peripheral blood by SNP-A can complement marrow MC, especially in cases in which marrow is not available. Detection of clonal marker aberrations in blood of RARS patients suggests that mostly clonal dysplastic progenitor cells contribute to blood production rather than residual “normal” progenitors.

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