2016) Complex karyotypes and KRAS and POT1 mutations impact outcome in CLL after chlorambucilbased chemotherapy or chemoimmunotherapy. Blood, 128, 395-404.

arrays identify high-risk chronic lymphocytic leukemia with genomic complexity: a multi-center study. AbstractComplex 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<0.01) were linked to poor outcome after correction for multiple testing. Patients (n=972) could be divided in three distinct prognostic subgroups based on the number of CNAs. Only high genomic complexity (high-GC), defined as ≥ 5 CNAs emerged as an independent adverse prognosticator on multivariable analysis for time to first treatment (Hazard ratio: 2.15, 95% CI: 1.36-3.41; p=0.001) and overall survival (Hazard ratio: 2.54, 95% CI: 1. 54-4.17; p<0.001; n=528).Lowering the size cutoff to 1 Mb in 647 patients did not significantly improve risk assessment.Genomic arrays detected more chromosomal abnormalities and performed at least as well in terms of risk stratification compared to simultaneous chromosome banding analysis as determined in 122 patients. Our findings highlight genomic array as an accurate tool for CLL risk stratification.

Genomic analyses of chronic lymphocytic leukemia (CLL) identified somatic mutations and associations of clonal diversity with adverse outcomes. Clonal evolution likely has therapeutic implications but its dynamic is less well studied. We studied clonal composition and prognostic value of seven recurrently mutated driver genes using targeted next-generation sequencing in 643 CLL patients and found higher frequencies of mutations in TP53 (35 vs. 12%, p < 0.001) and SF3B1 (20 vs. 11%, p < 0.05) and increased number of (sub)clonal (p < 0.0001) mutations in treated patients. We next performed an in-depth evaluation of clonal evolution on untreated CLL patients (50 "progressors" and 17 matched "non-progressors") using a 404 gene-sequencing panel and identified novel mutated genes such as AXIN1, SDHA, SUZ12, and FOXO3. Progressors carried more mutations at initial presentation (2.5 vs. 1, p < 0.0001). Mutations in specific genes were associated with increased (SF3B1, ATM, and FBXW7) or decreased progression risk (AXIN1 and MYD88). Mutations affecting specific signaling pathways, such as Notch and MAP kinase pathway were enriched in progressive relative to non-progressive patients. These data extend earlier findings that specific genomic alterations and diversity of subclones are associated with disease progression and persistence of disease in CLL and identify novel recurrently mutated genes and associated outcomes.

Recurrent mutations in splicing factor 3B subunit 1 (SF3B1) have been identified in several malignancies and are associated with an increased expression of 3’ cryptic transcripts as a result of alternative branchpoint recognition. A large fraction of cryptic transcripts associated with SF3B1 mutations is expected to be sensitive for RNA degradation via nonsense-mediated mRNA decay (NMD). Several studies indicated alterations in various signaling pathways in SF3B1-mutated cells, including an impaired DNA damage response (DDR) in chronic lymphocytic leukemia (CLL). In this study, we investigated isogenic cell lines and treatment naïve primary CLL samples without any TP53 and/or ATM defect, and found no significant effects of SF3B1 mutations on the ATM/p53 response, phosphorylation of H2AX and sensitivity to fludarabine. Cryptic transcripts associated with SF3B1 mutation status were observed at relatively low levels compared to the canonical transcripts and were validated as target for mRNA degradation via NMD. Expression of cryptic transcripts increased after NMD inhibition. In conclusion, our results confirm involvement of NMD in the biological effects of SF3B1 mutations. Further studies may elucidate whether SF3B1-mutant patients could benefit from NMD modulatory agents.

The ATM-p53 DNA-damage response (DDR) pathway has a crucial role in chemoresistance in CLL, as indicated by the adverse prognostic impact of genetic aberrations of TP53 and ATM. Identifying and distinguishing TP53 and ATM functional defects has become relevant as epigenetic and posttranscriptional dysregulation of the ATM/p53 axis is increasingly being recognized as the underlying cause of chemoresistance. Also, specific treatments sensitizing TP53- or ATM-deficient CLL cells are emerging. We therefore developed a new ATM-p53 functional assay with the aim to (i) identify and (ii) distinguish abnormalities of TP53 versus ATM and (iii) enable the identification of additional defects in the ATM-p53 pathway. Reversed transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) was used to measure ATM and/or p53-dependent genes at the RNA level following DNA damage using irradiation. Here, we showed that this assay is able to identify and distinguish three subgroups of CLL tumors (i.e., TP53-defective, ATM-defective and WT) and is also able to detect additional samples with a defective DDR, without molecular aberrations in TP53 and/or ATM. These findings make the ATM-p53 RT-MLPA functional assay a promising prognostic tool for predicting treatment responses in CLL.

Early data suggested that CC-115, a clinical molecule, already known to inhibit the mammalian target of rapamycin kinase (TORK) and DNAdependent protein kinase (DNA-PK) may have additional targets beyond TORK and DNA-PK. Therefore, we aimed to identify such target(s) and investigate a potential therapeutic applicability. Functional profiling of 141 cancer cell lines revealed inhibition of kinase suppressor of morphogenesis in genitalia 1 (SMG1), a key regulator of the RNA degradation mechanism nonsense-mediated mRNA decay (NMD), as an additional target of CC-115. CC-115 treatment showed a dose-dependent increase of SMG1mediated NMD transcripts. A subset of cell lines, including multiple myeloma (MM) cell lines sensitive to the endoplasmic reticulum stress-inducing compound thapsigargin, were highly susceptible to SMG1 inhibition. CC-115 caused the induction of UPR transcripts and cell death by mitochondrial apoptosis, requiring the presence of BAX/BAK and caspase activity. Superior antitumor activity of CC-115 over TORK inhibitors in primary human MM cells and three xenograft mouse models appeared to be via inhibition of SMG1. Our data support further development of SMG1 inhibitors as possible therapeutics in MM.

repair and cell cycle genes. In the discovery phase, a total of 285 Spanish patients and 5,608 ancestry-matched controls were evaluated. In the validation stage, an independent cohort of 514 European patients and 27,173 ancestry-matched controls were analyzed. An FDR correction was applied to both datasets and genes with a q-value <0.2 in both cohorts were considered significant. Results: Our joint analysis of 799 CLL patients from 2 genetically distinct cohorts and 32,781 ancestry-matched cancer-free controls identified ATM and CHEK2 as significantly enriched in both CLL datasets. First, our analysis recaptured the previously reported finding of ATM variant enrichment in CLL patients. Carriers of pathogenic ATM mutations in our cohorts (n = 9 patients, discovery: 1.05%, validation: 1.17%) were 2.8-3.7 times more likely to develop CLL compared to cancer-free individuals (discovery: OR = 2.8, 95%CI = 0.7-9.0, q-value = 0.181; validation: OR = 3.7, 95%CI = 1.6-8.3, q-value = 0.0454). In addition, our analysis identified 21 CLL patients carrying pathogenic CHEK2 alterations (discovery: 1.40%, validation: 3.31%), making CLL patients 4.4-8.0 times more likely to carry such alterations compared to controls (discovery: OR = 8.0, 95%CI = 2.3-27.0, q-value = 0.026; validation: OR = 4.4, 95%CI = 2.5-7.3, q-value <0.001). Summary/Conclusion: Our analysis of genetically distinct CLL cohorts, using a high-sensitivity variant calling algorithm, supports CHEK2 as a potentially novel CLL predisposition gene that may explain a portion of the missing monogenic heritability of CLL. In addition, this study highlights the DNA repair and cell cycle regulation pathways as potential drivers of CLL susceptibility.

The ATM-p53 DNA damage response pathway plays a crucial role in chemoresistance in CLL, as indicated by the adverse prognostic impact of genetic aberrations of TP53 and ATM. Identifying and distinguishing TP53 and ATM functional defects has become relevant as epigenetic and posttranscriptional dysregulation of the ATM/p53 axis is increasingly being recognized as underlying cause of chemoresistance. Also, specific treatments sensitizing TP53-or ATM-deficient CLL cells are emerging. We therefore developed a new ATM-p53 functional assay with the aim to (i) identify and (ii) distinguish abnormalities of TP53 versus ATM and (iii) enable the identification of additional defects in the ATM-p53 pathway. Previously we showed that a reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) procedure which quantifies expression levels of the p53-targets, CDKN1A, BBC3 and Bax, in CLL cells following irradiation is able to determine p53 functionality. With the aim of identifying and distinguishing abnormalities of TP53 versus ATM and enabling the identification of additional defects in the DDR, we developed a new RT-MLPA based functional assay. Reversed transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) was used to measure ATM and/or p53-dependent genes at the RNA level following DNA-damage using irradiation. A new RT-MLPA probe set (R016-X2, MRC-Holland), which included several p53 and ATM target genes was designed. Genes were selected based on the results of an earlier microarray study [Stankovic et al. Blood 2004;103:291-300]. A cohort of 30 CLL patients was utilized to set-up the RT-MLPA functional assay (training cohort) and an independent second cohort including 67 CLL patients included in the HOVON68 clinical trial was used as a validation cohort. From all these patients genetic analysis of ATM and TP53 was available using FISH and sequencing. First, the assay was performed on the training cohort and was able to identify and distinguish three subgroups of CLL tumors (i.e. TP53-defective, ATM-defective and WT). Next, based on the fold induction factors (i.e. gene expression upon irradiation versus non-irradiation) of 10 selected genes, a support vector machine (SVM) classifier was constructed to enable the classification of CLL samples into 3 different types of response, i.e. ATM/p53 functional, p53-dysfunctional, or ATM-dysfunctional. The RT-MLPA assay and SVM classifier were validated in a separate validation cohort. CLL samples with clear genotypic characteristics (i.e. biallelic defects) were assigned with a high degree of confidence to one of the three categories with sensitivities of 93%, 100% and 78% for TP53/ATM WT, biallelic TP53-defective and biallelic ATM-defective samples, respectively. Interestingly the assay was also able to detect additional samples with a defective DNA-damage response, without molecular aberrations in TP53 and/or ATM (22%; 6 out of 27 WT patients). Additional analysis of these samples showed significantly reduced apoptosis to fludarabine, doxorubicin and irradiation in comparison to the ATM/p53 functional samples, indicating that defects in the DNA-damage response other than TP53/ATM aberrations are indeed present and probably responsible for the observed defective DNA-damage induced apoptotic responses. In conclusion, the newly designed ATM-p53 RT-MLPA assay is able to distinguish three subgroups of CLL tumors (i.e. TP53-defective, ATM-defective and WT) and was also able to detect additional samples with a functional defective DNA-damage response, without molecular defects of TP53 and/or ATM. This indicates that the ATM-p53 RT-MLPA might not only be of additional clinical value over FISH to screen for mutations of TP53 and ATM instead of sequencing, but might also be useful for screening of other defects in the DNA-damage response pathway in addition to ATM and/or TP53 aberrations. These findings make the ATM-p53 RT-MLPA functional assay a promising prognostic tool for predicting treatment responses in CLL. Disclosures Hoogendoorn: Novartis: Consultancy; Gilead: Consultancy.