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Complex karyotype (CK) identified by chromosome-banding analysis (CBA) has shown prognostic value in chronic lymphocytic leukemia (CLL). Genomic arrays offer high-resolution genome-wide detection of copy-number alterations (CNAs) and could therefore be well equipped to detect the presence of a CK. Current knowledge on genomic arrays in CLL is based on outcomes of single center studies, in which different cutoffs for CNA calling were used. To further determine the clinical utility of genomic arrays for CNA assessment in CLL diagnostics, we retrospectively analyzed 2293 arrays from 13 diagnostic laboratories according to established standards. CNAs were found outside regions captured by CLL FISH probes in 34% of patients, and several of them including gains of 8q, deletions of 9p and 18p (p, This study was partly funded by an unrestricted contribution from Janssen Pharmaceuticals and from GILEAD Sciences SA. A.C.L. is supported by the Peters van der Laan foundation. J.C.S. was funded by Bloodwise (11052, 12036), the Kay Kendall Leukaemia Fund (873), Cancer Research UK (C34999/A18087, ECMC C24563/A15581), Wessex Medical Research and the Bournemouth Leukaemia Fund. K.P., M.J., and S.P. are supported by the project MHCR DRO no. 65269705, the research infrastructures NCMG LM2015091, and EATRIS-CZ LM2015064, and the project CEITEC2020 LQ1601, funded by MEYS CR. R.R. is supported by Swedish Cancer Society, the Swedish Research Council, the Knut and Alice Wallenberg Foundation, Karolinska Institutet, Karolinska University Hospital, and Radiumhemmets Forskningsfonder, Stockholm.

Introduction. Pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) is the most common pediatric hematological malignancy and it remains an important cause of morbidity and mortality in children. In this study, we performed an allele-specific expression (ASE) analysis of pediatric BCP ALL with the aim to investigate the relationship between cis-regulatory mutations and gene expression patterns. Materials and methods. Twenty-two high hyperdiploid ALL, twenty ETV6/RUNX1-positive ALL, seven TCF3/PBX1-positive ALL and twenty-eight genetically unclassified BCP ALL ("B-other") were subjected to whole genome sequencing, SNP array analysis and RNA sequencing. The binomial test was applied to estimate the allelic bias of heterozygous exonic single nucleotide variants (SNVs) in the RNA sequencing data against the genomic data. Allelic ratios >2 or Results. We identified 12,693 expressed genes, of which 9,672 (76%) had heterozygous exonic SNVs (informative genes), in multiple BCP ALL samples (n>2) in 77 of the investigated samples. Genes with ASE were distributed evenly across the autosomal chromosomes in the different subtypes with a range of 30 - 165 ASE genes per case (median number, 86). We found that 630 (6.5%) genes displayed ASE in multiple BCP ALL samples (n>2), of which only eight autosomal genes had monoallelic expression in more than two investigated samples. This suggests that ASE and monoallelic expression are relatively rare in BCP ALL. Gene enrichment analyses showed that pathways involving negative regulation of natural killer cell-mediated cytotoxicity and cell proliferation were enriched, indicating that ASE events possibly were associated with the cell proliferation and leukemia progression in BCP ALL. Furthermore, the hematopoiesis pathway was also enriched in ASE genes that showed high allelic expression bias (allelic ratios >2.5), suggesting that ASE genes might be associated with leukemia development. Somatic genomic aberrations that could cause ASE were also investigated in this study. All informative cases with TCF3/PBX1 rearrangement (n=4) showed monoallelic expression of the PBX1 gene, likely associated with the PBX1 truncation caused by the fusion. Additionally, CHP1, located in 15q15.1, displayed ASE in one case with an inversion involving that chromosome band, indicating a potential cis-acting element in the inversion region that regulated the CHP1 gene expression. Notably, PAX5 displayed various patterns of ASE in BCP ALL. One of three cases with PAX5/ZCCHC7 gene rearrangements displayed PAX5 ASE while the other two did not, indicating a potential uncovered cis-regulatory element around the PAX5/ZCCHC7 breakpoints. Furthermore, two cases with no PAX5 gene rearrangement displayed monoallelic expression of the PAX5 gene, suggesting that there are additional epigenetic alterations were also involved in the regulation of PAX5 gene expression in BCP ALL. Conclusions. In this study, we have characterized genes displaying ASE in childhood BCP ALL. Our data provide new insight into pathogenesis of BCP ALL and may be used to identify novel targets for treatment. Disclosures No relevant conflicts of interest to declare.

High-throughput sequencing was applied to investigate the mutation/methylation patterns on 1q and gene expression profiles in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) with/without (w/wo) dup(1q). Sequencing of the breakpoint regions and all exons on 1q in seven dup(1q)-positive cases revealed non-synonymous somatic single nucleotide variants (SNVs) in BLZF1, FMN2, KCNT2, LCE1C, NES, and PARP1. Deep sequencing of these in a validation cohort w (n = 17)/wo (n = 94) dup(1q) revealed similar SNV frequencies in the two groups (47% vs. 35%; P = 0.42). Only 0.6% of the 36,259 CpGs on 1q were differentially methylated between cases w (n = 14)/wo (n = 13) dup(1q). RNA sequencing of high hyperdiploid (HeH) and t(1;19)(q23;p13)-positive cases w (n = 14)/wo (n = 52) dup(1q) identified 252 and 424 differentially expressed genes, respectively; only seven overlapped. Of the overexpressed genes in the HeH and t(1;19) groups, 23 and 31%, respectively, mapped to 1q; 60-80% of these encode nucleic acid/protein binding factors or proteins with catalytic activity. We conclude that the pathogenetically important consequence of dup(1q) in BCP ALL is a gene-dosage effect, with the deregulated genes differing between genetic subtypes, but involving similar molecular functions, biological processes, and protein classes.

Most previous studies on telomere length (TL) in chronic lymphocytic leukemia (CLL) are based on referral cohorts including a high proportion of aggressive cases. Here, the impact of TL was analyzed in a population-based cohort of newly diagnosed CLL (n = 265) and in relation to other prognostic markers. Short telomeres were particularly associated with high-risk genetic markers, such as NOTCH1, SF3B1, or TP53 aberrations, and predicted a short time to treatment (TTT) and overall survival (OS) (both P0.0001). TL was an independent prognostic factor and subdivided patients with otherwise good-prognostic features (e.g., mutated IGHV genes, favorable cytogenetics) into subgroups with different outcome. Furthermore, in follow-up samples (n = 119) taken 5-8 years after diagnosis, TL correlated well with TL at diagnosis and remained unaffected by treatment. Altogether, these novel data indicate that short TL already at diagnosis is associated with poor outcome in CLL and that TL can be measured at later stages of the disease.

Chronic lymphocytic leukemia (CLL) is a clinically and biologically heterogeneous disease where the majority of patients have an indolent disease course, while others may experience a far more aggressive disease, treatment failure and poor overall survival. During the last two decades, there has been an intense search to find novel biomarkers that can predict prognosis as well as guide treatment decisions. Two of the most reliable molecular prognostic markers, both of which are offered in routine diagnostics, are the immunoglobulin heavy chain variable (IGHV) gene mutational status and fluorescence in situ hybridization (FISH) detection of prognostically relevant genomic aberrations (e.g. 11q-, 13q-, +12 and 17p-). In addition to these markers, a myriad of additional biomarkers have been postulated as potential prognosticators in CLL, on the protein (e.g. CD38, ZAP70, TCL1), the RNA (e.g. LPL, CLLU1, micro-RNAs) and the genomic (e.g. TP53, NOTCH1, SF3B1 and BIRC3 mutations) level. Efforts are now being made to test these novel markers in larger patient cohorts as well as in prospective trials, with the ultimate goal to combine the "best" markers in a "CLL prognostic index" applicable for the individual patient. Although it is clear that these studies have significantly improved our knowledge regarding both prognostication and the biology of the disease, there is still an immediate need for recognizing biomarkers that can predict therapy response, and efforts should now focus on addressing this pertinent issue. In the present article, we review the extensive literature in the field of prognostic markers in CLL, focus on the most clinically relevant markers and discuss future directions regarding biomarkers in CLL.

The IGHV3-21 gene has been shown to be overrepresented in Scandinavian patients with chronic lymphocytic leukemia (CLL). By investigating a population-based cohort of 337 Swedish patients with CLL, a lower (6.5%) IGHV3-21 frequency was determined relative to our previous hospital-based studies (10.1%-12.7%), yet this frequency remained higher compared to other Western CLL cohorts (2.6%-4.1%). Furthermore, we confirmed the poor outcome for patients with IGHV3-21 to be independent of mutational and stereotypy status. Background: Scandinavian patients with CLL have shown an overrepresentation of the poor-prognostic IGHV3-21 gene. Furthermore, approximately 50% of patients with IGHV3-21 carry stereotyped B-cell receptors, which implicate antigen selection in leukemogenesis. These patients have also been reported to have shorter time to progression than patients with nonstereotyped IGHV3-21. Materials and Methods: To investigate the IGHV3-21 frequency and the clinical impact of IGHV3-21 stereotypy, 337 newly diagnosed Swedish CLL patients from a population-based cohort were analyzed. Results: Interestingly, the IGHV3-21 frequency was indeed lower (6.5%) in this indolent patient cohort than in our previous hospital-based cohort studies (10.1%-12.7%). Hence, a selection bias of more-aggressive cases rendered a higher proportion of IGHV3-21 cases in our original studies. Nevertheless, the Swedish IGHV3-21 frequency still remained higher when compared with other larger European or American studies (2.6%-4.1%). Finally, we confirmed the poor outcome for IGHV3-21 patients to be independent of mutational status and found stereotypy to have no impact on survival or time to treatment. Conclusion: The Swedish geographic bias in IGHV3-21 gene frequency was validated albeit at a lower frequency than previously reported. Moreover, no prognostic value could be attributed to IGHV3-21 stereotype status.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a varying clinical outcome; however, the pathogenic mechanisms involved in disease development have remained largely unknown. In recent years, novel biomarkers, such as certain recurrent genomic alterations and the immunoglobulin heavy variable gene mutational status, have significantly improved the subdivision of the disease along with the prognostic assessment of individual patients. Advanced molecular studies have also revealed important genetic/epigenetic events and potential susceptibility loci for CLL, as well as implicating antigens in CLL development. Furthermore, the presence of monoclonal B cell lymphocytosis (MBL) has been demonstrated to precede CLL and appears to be a pre-leukemic condition. In this review, we will not only focus on recent developments made in the fields of genetics and immunogenetics in CLL, but also provide a brief overview of MBL, since we believe that advancements in these areas will have a major impact on our understanding of CLL pathobiology.

The expression levels of LPL, ZAP70, TCL1A, CLLU1 and MCL1 have recently been proposed as prognostic factors in chronic lymphocytic leukemia. However, few studies have systematically compared these different RNA-based markers.Using real-time quantitative PCR, we measured the mRNA expression levels of these genes in unsorted samples from 252 newly diagnosed chronic lymphocytic leukemia patients and correlated our data with established prognostic markers (for example Binet stage, CD38, IGHV gene mutational status and genomic aberrations) and clinical outcome.High expression levels of all RNA-based markers, except MCL1, predicted shorter overall survival and time to treatment, with LPL being the most significant. In multivariate analysis including the RNA-based markers, LPL expression was the only independent prognostic marker for overall survival and time to treatment. When studying LPL expression and the established markers, LPL expression retained its independent prognostic strength for overall survival. All of the RNA-based markers, albeit with varying ability, added prognostic information to established markers, with LPL expression giving the most significant results. Notably, high LPL expression predicted a worse outcome in good-prognosis subgroups, such as patients with mutated IGHV genes, Binet stage A, CD38 negativity or favorable cytogenetics. In particular, the combination of LPL expression and CD38 could further stratify Binet stage A patients.LPL expression is the strongest RNA-based prognostic marker in chronic lymphocytic leukemia that could potentially be applied to predict outcome in the clinical setting, particularly in the large group of patients with favorable prognosis.

Background The existence of multiple subsets of chronic lymphocytic leukemia expressing ‘stereotyped’ Bcell receptors implies the involvement of antigen(s) in leukemogenesis. Studies also indicate that ‘stereotypy’ may influence the clinical course of patients with chronic lymphocytic leukemia, for example, in subsets with stereotyped IGHV3-21 and IGHV4-34 B-cell receptors; however, little is known regarding the genomic profile of patients in these subsets. Design and Methods We applied 250K single nucleotide polymorphism-arrays to study copy-number aberrations and copy-number neutral loss-of-heterozygosity in patients with stereotyped IGHV3-21 (subset #2, n=29), stereotyped IGHV4-34 (subset #4, n=17; subset #16, n=8) and non-subset #2 IGHV3-21 (n=13) and non-subset #4/16 IGHV4-34 (n=34) patients. Results Over 90% of patients in subset #2 and non-subset #2 carried copy-number aberrations, whereas 75-76% of patients in subset #4 and subset #16 showed copy-number aberrations. Subset #2 and non-subset #2 patients also displayed a higher average number of aberrations compared to patients in subset #4. Deletion of 13q was the only known recurrent aberration detected in subset #4 (35%); this aberration was even more frequent in subset #2 (79%). del(11q) was more frequent in subset #2 and non-subset #2 (31% and 23%) patients than in subset #4 and nonsubset #4/16 patients. Recurrent copy-number neutral loss-of-heterozygosity was mainly detected on chromosome 13q, independently of B-cell receptor stereotypy. Conclusions Genomic aberrations were more common in subset #2 and non-subset #2 than in subset #4. The particularly high frequency of del(11q) in subset #2 may be linked to the adverse outcome reported for patients in this subset. Conversely, the lower prevalence of copy-number aberrations and the absence of poor-prognostic aberrations in subset #4 may reflect an inherently low-proliferative disease, which would prevent accumulation of genomic alterations.

Large but not small copy-number alterations correlate to high-risk genomic aberrations and survival in chronic lymphocytic leukemia: a high-resolution genomic screening of newly diagnosed patients

Mansouri et al. applied targeted deep sequencing to identify mutations within NF-κB core complex genes in CLL. NFKBIE, the gene encoding the inhibitory IκBε molecule, was most frequently mutated, especially in poor-prognostic subgroups of CLL. The authors show that NFKBIE mutations were associated with significantly reduced IkBε expression and p65 inhibition, ultimately leading to NF-κB activation and a more aggressive disease., NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.

On the way towards a 'CLL prognostic index' : focus on TP53, BIRC3, SF3B1, NOTCH1 and MYD88 in a population-based cohort.

Feasibility of targeted next-generation sequencing of the TP53 and ATM genes in chronic lymphocytic leukemia

Background: NOTCH1 PEST domain mutations in chronic lymphocytic leukemia have recently been shown to be of prognostic relevance. Both NOTCH1 and NOTCH2 are constitutively activated in B-cell CLL but not expressed in normal B cells and may be involved in survival and resistance to apoptosis in CLL. We screened for mutations in different parts of both NOTCH1 and NOTCH2 genes and related the changes to survival and other known risk factors. Methods: In a cohort of 209 CLL patients, we used single strand conformation analysis to determine which of the samples carrying the NOTCH mutations and direct dideoxy sequencing was used to determine the exact nucleotide changes. Kaplan-Meier curves and log rank test were used to determine overall survival for NOTCH1 mutated cases and Cox regression analysis was used to calculate hazardous ratios. Results: In the present study, we found NOTCH1 PEST domain mutations in 6.7% of the cases. A shorter overall survival was found in patients with NOTCH1 mutations compared to wildtype (p = 0.049). Further, we also examined the extracellular and the heterodimerisation domains of the NOTCH1 gene and the PEST domain and heterodimerisation domain of the NOTCH2 gene, but no mutations were found in these regions. NOTCH1 mutations were most commonly observed in patients with unmutated IGHV gene (10/14), and associated with a more aggressive disease course. In addition, NOTCH1 mutations were almost mutually exclusive with TP53 mutations. In the combined group of NOTCH1 (6.7%) or TP53 (6.2%) mutations, a significant difference in overall survival compared to the wildtype NOTCH1 and TP53 was found (p = 0.002). Conclusions: Both NOTCH1 and TP53 mutations seem to be independent predictive markers for worse outcome in CLL-patients and this study emphasizes the contention that NOTCH1 mutations is a novel risk marker.

During recent years, microarray-based technologies and next-generation sequencing (NGS) have been applied in chronic lymphocytic leukemia (CLL) in order to identify novel genomic aberrations that may contribute to the pathogenesis of the disease. Even though high-resolution microarray studies have confirmed the importance of the known recurrent aberrations, i.e. del(11q), trisomy 12, del(13q) and del(17p), and have more precisely delineated the genomic borders of these aberrations, only a few novel aberrations, found at a low frequency, have been detected with these techniques. In contrast to this, the application of NGS technology of the coding genome (exome sequencing) or the entire genome (whole-genome sequencing) has unveiled a number of novel recurrent mutations in e.g. the NOTCH1, SF3B1 and BIRC3 genes. Importantly, mutations in these latter genes were reported to be associated with a particularly poor outcome, similar to TP53 aberrations, and may play key roles in tumor development, treatment resistance and prognosis. In this review, we not only summarize the latest achievements using array-based or NGS technologies, but also point to new directions for research aiming to unravel the complex genetic "map" in CLL and its prognostic subsets.

Advances in next-generation RNA-sequencing have revealed the complexity of transcriptomes by allowing both coding and noncoding (nc) RNAs to be analyzed. However, limited data exist regarding the whole transcriptional landscape of chronic lymphocytic leukemia (CLL). In this pilot-study, we evaluated RNA-sequencing in CLL by comparing two subsets which carry almost identical or `` stereotyped'' B-cell receptors with distinct clinical outcome, that is the poor-prognostic subset # 1 (n = 4) and the more favorable-prognostic subset # 4 (n = 4). Our analysis revealed that 156 genes (e.g. LPL, WNT9A) and 76 ncRNAs, (e. g. SNORD48, SNORD115) were differentially expressed between the subsets. This technology also enabled us to identify numerous subset-specific splice variants (n = 406), which were predominantly expressed in subset # 1, including a splice-isoform of MSI2 with a novel start exon. A further important application of RNA-sequencing was for mutation detection and revealed 16-30 missense mutations per sample; notably many of these changes were found in genes with a strong potential for involvement in CLL pathogenesis, e. g., ATM and NOTCH2. This study not only demonstrates the effectiveness of RNA-sequencing for identifying mutations, quantifying gene expression and detecting splicing events, but also highlights the potential such global approaches have to significantly advance our understanding of the molecular mechanisms behind CLL development.

Coexistence of trisomies of chromosomes 12 and 19 in chronic lymphocytic leukemia occurs exclusively in the rare IgG-positive variant

Background High-resolution genomic microarrays enable simultaneous detection of copy-number aberrations such as the known recurrent aberrations in chronic lymphocytic leukemia [del(11q), del(13q), del(17p) and trisomy 12], and copy-number neutral loss of heterozygosity. Moreover, comparison of genomic profiles from sequential patients' samples allows detection of clonal evolution. Design and Methods We screened samples from 369 patients with newly diagnosed chronic lymphocytic leukemia from a population-based cohort using 250K single nucleotide polymorphism-arrays. Clonal evolution was evaluated in 59 follow-up samples obtained after 5-9 years. Results At diagnosis, copy-number aberrations were identified in 90% of patients; 70% carried known recurrent alterations, including del(13q) (55%), trisomy 12 (10.5%), del(11q) (10%), and del(17p) (4%). Additional recurrent aberrations were detected on chromosomes 2 (1.9%), 4 (1.4%), 8 (1.6%) and 14 (1.6%). Thirteen patients (3.5%) displayed recurrent copy-number neutral loss of heterozygosity on 13q, of whom 11 had concurrent homozygous del(13q). Genomic complexity and large 13q deletions correlated with inferior outcome, while the former was linked to poor-prognostic aberrations. In the follow-up study, clonal evolution developed in 8/24 (33%) patients with unmutated IGHV, and in 4/25 (16%) IGHV-mutated and treated patients. In contrast, untreated patients with mutated IGHV (n=10) did not acquire additional aberrations. The most common secondary event, del(13q), was detected in 6/12 (50%) of all patients with acquired alterations. Interestingly, aberrations on, for example, chromosome 6q, 8p, 9p and 10q developed exclusively in patients with unmutated IGHV. Conclusions Whole-genome screening revealed a high frequency of genomic aberrations in newly diagnosed chronic lymphocytic leukemia. Clonal evolution was associated with other markers of aggressive disease and commonly included the known recurrent aberrations.

TP53 mutations in the absence of 17p-deletion correlate with rapid disease progression and poor survival in chronic lymphocytic leukemia (CLL). Herein, we determined the TP53 mutation frequency in 268 newly diagnosed CLL patients from a population-based material. Overall, we detected TP53 mutations in 3.7% of patients (n = 10), where 7/10 cases showed a concomitant 17p-deletion, confirming the high prevalence of TP53 mutation in 17p-deleted patients. Only 3 (1.1%) of the newly diagnosed patients in our cohort thereby carried TP53 mutations without 17p-deletion, a frequency that is much lower than previous reports on referral cohorts (3-6%). Our findings imply that TP53 mutations are rare at CLL onset and instead may arise during disease progression. (C) 2010 Elsevier Ltd. All rights reserved. (Less)

The 309T>G polymorphism in the promoter region of the MDM2gene, known as SNP309, has recently been suggested as an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL) although this has been questioned. To investigate this further, we analyzed the MDM2 SNP309 genotypes in 418 CLL patients and correlated the results with established CLL prognostic factors, time to treatment and overall survival. In this Swedish cohort, no association existed between any particular MDM2 SNP309 genotype, overall survival and time to treatment. Furthermore, no correlation was shown between the MDM2 SNP309 genotypes and Binet stage, IGHV mutational status and recurrent genomic aberrations. In summary, this study argues against the use of the MDM2 SNP309 as a prognostic marker in CLL.

Screening for gene copy-number alterations (CNAs) has improved by applying genome-wide microarrays, where SNP arrays also allow analysis of loss of heterozygozity (LOH). We here analyzed 10 chronic lymphocytic leukemia (CLL) samples using four different high-resolution platforms: BAC arrays (32K), oligonucleotide arrays (185K, Agilent), and two SNP arrays (250K, Affymetrix and 317K, Illumina). Cross-platform comparison revealed 29 concordantly detected CNAs, including known recurrent alterations, which confirmed that all platforms are powerful tools when screening for large aberrations. However, detection of 32 additional regions present in 2-3 platforms illustrated a discrepancy in detection of small CNAs, which often involved reported copy-number variations. LOH analysis using dChip revealed concordance of mainly large regions, but showed numerous, small nonoverlapping regions and LOH escaping detection. Evaluation of baseline variation and copy-number ratio response showed the best performance for the Agilent platform and confirmed the robustness of BAC arrays. Accordingly, these platforms demonstrated a higher degree of platform-specific CNAs. The SNP arrays displayed higher technical variation, although this was compensated by high density of elements. Affymetrix detected a higher degree of CNAs compared to Illumina, while the latter showed a lower noise level and higher detection rate in the LOH analysis. Large-scale studies of genomic aberrations are now feasible, but new tools for LOH analysis are requested.

Dysregulated NF-κB signaling appears to be particularly important in B-cell malignancies, with recurrent mutations identified within both the canonical and non-canonical NF-κB pathways, as well as in components of the B-cell receptor (BcR) and Toll-like receptor (TLR) signaling pathways. In chronic lymphocytic leukemia (CLL), although recurrent mutations have been identified in MYD88 (TLR signaling) and BIRC3 (non-canonical NF-κB pathway), their frequency is low (10% for the mutant allele, we identified mutations (n=35) within 30/124 (24%) patients in 14/18 NF-κB genes analyzed. IκB genes, which encode for cytoplasmic inhibitor proteins, accounted for 20/35 (57%) mutations, with IκBε (encoded by NFKBIE) mutated in 8 patients; notably, 3/8 cases carried an identical 4bp deletion within exon 1 of NFKBIE. Prompted by these findings, we proceeded to validate our findings in an independent CLL cohort (n=168) using the same methodology as above and primarily focusing on cases with poor-prognostic features. We identified 30 mutations within 28 CLL patients in 11/18 NF-κB genes analyzed. Strikingly, 13/30 mutations were found within IκBε, with 10/13 patients carrying the same 4bp NFKBIE deletion. Notably, investigations into whether additional cases (within both the discovery and validation cohort) may harbor mutations of low clonal abundance ( Disclosures No relevant conflicts of interest to declare.

Abstract 1254 Poster Board I-276 Introduction Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with varying clinical outcome, where many patients have an indolent course for many years, whereas others show a more aggressive disease despite treatment. This has prompted the search for biomarkers that can predict outcome in this disease. Recent studies have proposed the RNA expression levels of certain genes, i.e. LPL, CLLU1, TCL1, MCL1 and ZAP70 to be novel predictors of clinical outcome in CLL. However, a comprehensive assessment of these RNA-based markers is still lacking. The current study aimed to investigate the potential of these markers in CLL prognostication, either as single markers or in combination with established markers. Patients and Methods By applying real-time quantitative PCR, we measured the RNA expression levels of LPL, CLLU1, TCL1, MCL1 and ZAP70 in 256 newly diagnosed CLL samples from a Scandinavian population-based cohort collected from 1999 to 2002 (median follow-up, 89 months) and correlated with clinical outcome. The expression cut-offs for each RNA marker was determined by constructing ROC curves. Additionally, Binet stage, IGHV mutation status, CD38 expression (cut-off 7%) and the presence of recurrent genomic aberrations (i.e. 11q-, 17p-, 13q- and +12) were evaluated for all cases. Results High expression of all RNA-based markers except MCL1 predicted significantly shorter overall survival (OS) and time to treatment (TTT), with LPL being the most significant prognostic marker in both log-rank (Table 1) and Cox univariate regression analyses. In multivariate analysis including the RNA markers, LPL expression was the only independent prognostic factor for OS, whereas both LPL and CLLU1 could predict TTT. When including all established markers, LPL lost its significance in the model, due to its close association to the IGHV mutation status. Once the mutation status was excluded from the analysis, LPL regained its prognostic power in addition to genomic aberrations and CD38. Interestingly, all of the RNA-based markers added further prognostic information to established markers in subgroups of patients, with LPL expression status giving the most significant results. Notably, high LPL expression predicted a worse outcome in favorable prognostic subgroups such as patients with Binet stage A, CD38 negativity or favorable genomic aberrations (Table 2). Conclusions Altogether, we conclude that LPL expression appear to be the strongest among the RNA-based markers for prediction of clinical outcome in CLL and thus could potentially be applied in the clinical laboratory to predict outcome, particularly in combination with established markers. Disclosures No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia (CLL) is a biologically heterogeneous disease where no common genetic aberration so far has been described. Although recurrent genomic aberrations of prognostic significance are well established (e.g. deletions of 11q, 13q, 17p and trisomy 12), less is known about the overall genetic complexity. Recent development of high-resolution single nucleotide polymorphism (SNP)-arrays will allow screening of genetic complexity, which includes detection of smaller copy-number alterations (CNAs) in addition to the known, recurrent aberrations. We here applied the Affymetrix GeneChip® Mapping 250K Nsp arrays and screened peripheral blood samples from 203 newly diagnosed CLL patients (≥70% tumor cells) from a population-based Scandinavian cohort. The male:female ratio was 2:1, the majority of patients was in stage A (70%) and the median age at diagnosis 61 years. Sixty percentage of cases displayed mutated IGHV genes whereas 35% showed unmutated IGHV genes. The Nexus copy-number software (Biodiscovery, Inc.) was applied and CNAs were identified in all CLL samples investigated, where deletions were more commonly detected than gains (in average, 3.5 vs. 2.2 per sample, respectively). The average length of deletions was 3.5 Mb while gains had an average length of 9.5 Mb (3.6 Mb when excluding trisomy 12). More than 50% of deletions and gains had a size ranging between 0.1–1 Mb, whereas 22% of CNAs were less than 0.1 Mb and 25% larger than 1 Mb. When investigating known, recurrent alterations, 105 samples (52%) carried del(13)(q14); 82 samples displayed a mono-allelic deletion, 15 samples a bi-allelic deletion and 8 samples carried 2 mono-allelic deletions of different sizes. The minimal overlapping region was 0.44 Mb and most of the 13q14 deletions covered the genomic region of miR-15, miR-16 and/or DLEU7. Twenty-one samples (10%) showed trisomy 12 and del(11q) was detected in 27 samples (13%), all covering the ATM gene. del(17p) was detected in 7 patients (3.4%) with one patient also carrying several gains and losses of 17q. Moreover, large alterations involving chromosome arms or entire chromosomes were recurrently observed among the samples, for instance, 3 large 8p losses and 4 large 8q gains. Interestingly, 5 samples, which all carried del(11q), displayed a gain covering whole or large parts of the p-arm of chromosome 2, a region which covers MYCN, REL and BCL11A. Furthermore, comparison of samples with different IGHV mutation status (M vs. UM) illustrated a significantly higher frequency of del(11q) (32% of UM vs. 4% of M), trisomy 12 (22% of UM vs. 5% of M) and gain of 2p (12% vs. 0% in M) in samples with unmutated IGHV genes. As expected, patients with mutated IGHV genes showed a higher frequency of del(13q) (61% of M vs. 31% of UM). In addition, large aberrations (>1Mb) were more common in UM samples which in average displayed 0.57 gains and 1.22 deletions compared to 0.26 gains and 0.67 deletions in M samples. In conclusion, with whole-genome screening using high resolution SNP-arrays, higher complexity was revealed in CLL, including a higher number of gains and losses and a higher frequency of larger aberrations in UM compared to M samples. A novel, recurrent combination of del(11q) and gain of 2p was demonstrated which deserves further investigation.

Screening for copy number alterations (CNA) has improved by applying genome wide microarrays, where SNP-arrays also allow analysis of loss of heterozygosity (LOH). Currently, comparisons of high resolution microarray platforms are few, thus we performed a study to evaluate the power of differently designed microarrays for copy number analysis and LOH. We here analyzed 10 diagnostic chronic lymphocytic leukemia (CLL) samples (five IGVH mutated and five IGVH unmutated) using four different high-resolution platforms: BAC-arrays (32K), oligonucleotide-arrays (185K, Agilent), and two SNP-arrays (250K, Affymetrix and 317K, Illumina). Comparison of copy number data showed that the platforms are concordant in terms of detecting large CNA, including the known recurrent alterations. Mono-allelic and bi-allelic loss of 13q14 (3 and 1 sample, respectively), mono-allelic loss of 11q (1 sample), trisomy 12 (2 samples) and mono-allelic loss of 17p (2 samples) were concordant in all platforms. These aberrations were validated with FISH, which in addition identified subclones with mono-allelic loss of 13q14 in two cases, only detected with the BAC platform, rendering a cut-off for the power of detecting subclones to approximately 25% of investigated cells. As expected, all poor prognostic aberrations were detected in patients carrying unmutated IGHV genes whereas four of five mutated samples were detected with mono-allelic loss of 13q14, as the only recurrent alteration. Furthermore, detection of small CNA were in many cases discordant between platforms. Therefore, we defined alterations identified by at least two platforms and identified 47 losses and 31 gains using this criterion. We are currently validating the presence of a number of these alterations using other techniques. Evaluation of LOH showed concordance for 86 regions between the Illumina and Affymetrix platforms. Of these regions 12 LOH coincided with CNA, leaving the remaining 74 as copy-neutral LOH. In conclusion, all platforms investigated are powerful tools for screening of CNA, however, since non-overlapping CNA were detected by individual platforms, we emphasize the importance of validating findings. Also, there is a cut-off for detecting subclones, here estimated to 25%. Genomic arrays will improve the detection of new recurrent aberrations, which may potentially refine the prognostic hierarchy established by FISH.