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Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood and adolescence. In more than 60% of cases of this heterogeneous disease, a genetic marker is identified via cytogenetic or molecular analyses. TCF3 gene fusions occur in 5-11% of ALL patients. In

NGS-based multiple gene panel resequencing in combination with a high resolution CGH-array was used to identify genetic risk factors for hereditary breast and/or ovarian cancer in 237 high risk patients who were previously tested negative for pathogenic BRCA1/2 variants. All patients were screened for pathogenic variants in 94 different cancer predisposing genes. We identified 32 pathogenic variants in 14 different genes (ATM, BLM, BRCA1, CDH1, CHEK2, FANCG, FANCM, FH, HRAS, PALB2, PMS2, PTEN, RAD51C and NBN) in 30 patients (12.7%). Two pathogenic BRCA1 variants that were previously undetected due to less comprehensive and sensitive methods were found. Five pathogenic variants are novel, three of which occur in genes yet unrelated to hereditary breast and/or ovarian cancer (FANCG, FH and HRAS). In our cohort we discovered a remarkably high frequency of truncating variants in FANCM (2.1%), which has recently been suggested as a susceptibility gene for hereditary breast cancer. Two patients of our cohort carried two different pathogenic variants each and 10 other patients in whom a pathogenic variant was confirmed also harbored a variant of unknown significance in a breast and ovarian cancer susceptibility gene. We were able to identify pathogenic variants predisposing for tumor formation in 12.3% of BRCA1/2 negative breast and/or ovarian cancer patients.

PAX5 is a member of the paired box (PAX) family of transcription factors involved in B‐cell development. PAX5 P80R has recently been described as a distinct genetic B‐cell precursor (BCP) acute lymphoblastic leukemia (ALL) subtype with a favorable prognosis in adults. In contrast, an unfavorable outcome has been observed in children. Our aim was to determine the frequency of PAX5 P80R in childhood BCP‐ALL treated according to the Associazione Italiana Ematologia ed Oncologia Pediatrica‐Berlin‐Frankfurt‐Muenster (AIEOP‐BFM) ALL 2000 protocol and to evaluate its clinical significance within this study cohort. The analyses included 1237 patients with ALL treated in the AIEOP‐BFM ALL 2000 trial with complete information for copy number variations (CNVs) of IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, and ERG. A customized TaqMan genotyping assay was used to screen for PAX5 P80R. Sanger sequencing was used to confirm PAX5 P80R‐positive results as well as to screen for second variants in PAX5. Agilent CGH + SNP arrays (e‐Array design 85 320; Agilent Technologies) were performed in PAX5 P80R‐positive patients to verify additional CNVs. Almost 2% (20/1028) of our BCP‐ALL cohort were PAX5 P80R‐positive. White blood cell counts higher than 50 000/μl as well as male sex were significantly (P < .05) associated with PAX5 P80R. Most of the PAX5 P80R‐positive cases were 10 years of age or older. PAX5 P80R‐positive samples were enriched for deletions affecting PAX5, IKZF1, CDKN2A, and CDKN2B. Compared to PAX5 P80R ‐wildtype BCP‐ALL, PAX5 P80R‐positive patients showed a significantly reduced 5‐year overall survival (P = .042). Further studies should evaluate the interaction of PAX5 P80R with other genetic aberrations to further stratify intermediate risk pediatric BCP‐ALL.

Hereditary mutations in polynucleotide kinasephosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie- Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA singlestrand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity. Universidad de Costa Rica/[111-B8-372]/UCR/Costa Rica European Research Council/[694996]/ERC/Unión Europea Medical Research Council/[MR/P010121/1]/MRC/Unión Europea Ministry of Education, Youth and Sports/[LM2015062 Czech-BioImaging]/MEYS/República Checa UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biología

GT198, located 470 kb downstream of BRCA1, encodes for the nuclear PSMC3-interacting protein, which functions as co-activator of steroid hormone-mediated gene expression, and is involved in RAD51 and DMC1-mediated homologous recombination during DNA repair of double-strand breaks. Recently, germline variants in GT198 have been identified in hereditary breast and ovarian cancer (HBOC) patients, mainly in cases with early-onset. We screened a cohort of 166 BRCA1/2 mutation-negative HBOC patients, of which 56 developed early-onset breast cancer before the age of 36 years, for GT198 variants. We identified 7 novel or rare GT198 variants in 8 out of 166 index patients: c.-115G>A (rs191843707); c.-70T>A (rs752276800); c.-37A>T (rs199620968); c.-24C>G (rs200359709); c.519G>A p.(Trp173*); c.537+51G>C (rs375509656); c.*24G>A. Three out of 7 identified variants (c.-115G>A, c.519G>A and c.*24G>A) with putative pathogenic impact were found in HBOC patients with breast cancer onset at ≤ 36 years. The nonsense mutation c.519G>A p.(Trp173*) was located within the DNA binding domain of GT198 and is predicted to induce nonsense-mediated mRNA decay. Functional analyses of c.-115G>A, and c.*24A>G indicated an influence of these variants on gene expression. This is the second study that gives evidence for an association between pathogenic GT198 germline variants and early-onset breast cancer in HBOC.

Suv39h1 mediates heterochromatin formation in pericentric and telomeric regions by trimethylation of lysine 9 of histone 3 (H3K9me3). Yet, its role in the induction of chromosomal instability is poorly understood. We established a leukemia model by retrovirally expressing Myc in wild-type and histone methyltransferase Suv39h1-deficient hematopoietic cells and characterized the resulting leukemias for chromosomal instability. All mice that received cells overexpressing Myc developed myeloid leukemia with a median survival of 44 days posttransplantation. Myc-overexpressing wild-type leukemias demonstrated clones with numerical chromosomal aberrations (5/16). In secondary transplantations of these leukemic cells, structural changes, mostly end-to-end fusions of chromosomes, appeared (10/12). In contrast, leukemic cells overexpressing Myc with reduced or no Suv39h1 expression had a normal karyotype in primary, secondary, and tertiary transplantations (16/16). Myc-transduced Suv39h1-deficient cells showed less critically short telomeres (P < 0.05) compared with Myc-transduced wild-type bone marrow cells. Gene expression analysis showed upregulation of genes involved in the alternative lengthening of telomeres (ALT) mechanism. Thus, we hypothesize that loss of Suv39h1 implies activation of the ALT mechanism, in turn ensuring telomere length and stability. Our data show for the first time that Suv39h1 deficiency may prevent chromosomal instability by more efficient telomere stabilization in hematopoietic bone marrow cells overexpressing Myc.

Telomere shortening, clonal evolution and disease progression in myelodysplastic syndrome patients with 5q deletion treated with lenalidomide

Telomere biology is frequently associated with disease evolution in human cancer and dysfunctional telomeres have been demonstrated to contribute to genetic instability. In BCR-ABL(+) chronic myeloid leukemia (CML), accelerated telomere shortening has been shown to correlate with leukemia progression, risk score and response to treatment. Here, we demonstrate that proliferation of murine CML-like bone marrow cells strongly depends on telomere maintenance. CML-like cells of telomerase knockout mice with critically short telomeres (CML-iG4) are growth retarded and proliferation is terminally stalled by a robust senescent cell cycle arrest. In sharp contrast, CML-like cells with pre-shortened, but not critically short telomere lengths (CML-G2) grew most rapidly and were found to express a specific 'telomere-associated secretory phenotype', comprising secretion of chemokines, interleukins and other growth factors, thereby potentiating oncogene-driven growth. Moreover, conditioned supernatant of CML-G2 cells markedly enhanced proliferation of CML-WT and pre-senescent CML-iG4 cells. Strikingly, a similar inflammatory mRNA expression pattern was found with disease progression from chronic phase to accelerated phase in CML patients. These findings demonstrate that telomere-induced senescence needs to be bypassed by leukemic cells in order to progress to blast crisis and provide a novel mechanism by which telomere shortening may contribute to disease evolution in CML.

To evaluate whether copy number alterations (CNAs) are present that may contribute to disease development and/or progression of childhood myelodysplastic syndromes (MDS), 36 pediatric MDS patients were analyzed using array-based comparative genome hybridization (aCGH). In addition to monosomy 7, the most frequent chromosome aberration in childhood MDS, novel recurrent CNAs were detected. They included a loss of 3p14.3–p12.3, which contains the putative tumor suppressor gene FHIT, a loss of 7p21.3–p15.3, a loss of 9q33.3–q34.3 (D184) and microdeletions in 17p11.2, 6q23 containing MYB, and 17p13 containing TP53. In this small patient cohort, patients without CNA, patients with monosomy 7 only and patients with one CNA in addition to monosomy 7 did not differ in their survival. As expected, all patients with complex karyotypes, including two patients with deletions of TP53, died. A challenge inherent to aCGH analysis of MDS is the low percentage of tumor cells. We evaluated several approaches to overcome this limitation. Genomic profiles from isolated granulocytes were of higher quality than those from bone marrow mononuclear cells. Decreased breakpoint calling stringency increased recognition of CNAs present in small clonal populations. However, further analysis using a custom-designed array showed that these CNAs often did not confirm the findings from 244k arrays. In contrast, constitutional CNVs were reliably detected on both arrays. Moreover, aCGH on amplified DNA from distinct myeloid clusters is a new approach to determine CNAs in small subpopulations. Our results clearly emphasize the need to verify array-CGH results by independent methods like FISH or quantitative PCR. © 2010 Wiley-Liss, Inc.

Telomere shortening and chromosomal instability are believed to play an important role in the development of myeloid neoplasia. So far, published data are only available on the average telomere length in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), but not on the telomere length of individual chromosomes. We used a new technique, telomere/centromere-fluorescence in situ hybridization (T/C-FISH), which combines fluorescence R-banding and FISH using a probe against the telomere repeats to measure the telomere length of each chromosome arm in 78 patients with MDS. In line with the previous results, patients with MDS showed significantly shorter telomeres than those of healthy controls. Telomere lengths did not differ significantly between distinct morphological subtypes of MDS. However, there was a significant difference in telomere length between patients with an isolated monosomy 7 and patients with a normal karyotype (P < 0.05). Notably, patients with an isolated monosomy 7 showed significantly longer telomeres than patients with a normal karyotype in many chromosome arms, among them 7p and 7q. Neo-telomeres were found in two patients with a complex karyotype, in one case at the fusion site of a dic(14;20). Normal and aberrant metaphases of the same patient did not differ in telomere length, thus indicating to telomere shortening as a basic mechanism affecting all hematopoietic cells in patients with MDS. In some MDS subtypes, like MDS with isolated monosomy 7, telomeres may be stabilized and even increase in length because of the activation of telomerase or alternative mechanisms.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell (HSC) disease caused by the reciprocal translocation t(9;22). Although there is clear evidence that the resulting oncogenic tyrosine kinase BCR-ABL is the key event of leukemia initiation which drives stem cell proliferation and expansion of myeloid progenitors in early chronic phase (CP), the mechanism leading to advanced phases remains elusive. Recently, we could show that telomere attrition correlates with disease stages due to increased leukemic stem cell turnover. Here, we could provide first time evidence that this can functionally contribute to disease progression in CML. In our study we made use of the well-described telomerase knockout mouse model (mTR-/-), lacking the RNA subunit of telomerase and resulting in significant telomere shortening with each generation, and retrovirally introduced BCR-ABL into primary bone marrow cells of different generation. Although all CML-like cultures (hereafter referred to as “CML”) grew exponentially and growth factor independently in vitro, they showed remarkable differences in cellular growth kinetics depending on the generation of mTR-/-mice the cells were derived from. CML-HSCs of generation iG4 (CML-iG4) are functionally impaired with respect to their growth properties and ceased to proliferate due to a robust senescent-like cell cycle arrest. Interestingly, they did not show overt genomic instability, but and are less susceptible to Imatinib-induced apoptosis compared to wildtype cells (CML-WT). In sharp contrast, CML-G2 cells with only pre-shortened telomere lengths grew most rapidly and presented with an impressive proliferation advantage compared to CML-WT and -iG4 cells, while they still retain Imatinib sensitivity. Notably, we uncovered that this growth advantage is related to a “telomere-associated secretory phenotype” (TASP), comprising the upregulation and secretion of chemokines, interleukins and other growth factors, thereby potentiating oncogene-driven growth in an autocrine fashion. In line with those observations, we found that conditioned supernatant of CML-G2 cells markedly enhanced proliferation of CML-WT and pre-senescent CML-iG4 HSCs. To investigate if a TPE (telomere position effect)-related mechanism is responsible for inducing inflammatory gene expression in BCR-ABL positive cells, we mapped selected TASP genes for their chromosomal location. However, although they are frequently found in well-known cluster (e.g. chemokines), TASP genes are not preferentially located close to the (sub-) telomere. This suggests that a yet unknown mechanism controls TASP gene expression upon telomere shortening. Most importantly, a similar inflammatory mRNA expression pattern was found in CML patients of accelerated phase (AP), but not in blast crisis (BC). Taken together, those data support the hypothesis that accelerated telomere shortening contributes to disease progression in BCR-ABL-driven leukemogenesis by the expression of an inflammatory signature, while telomere-induced senescence needs to be bypassed (e.g. by upregulation of telomerase) in order for leukemic cells to be able to progress to blast crisis (BC) CML. Disclosures: Brümmendorf: Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Ariad: Consultancy.

Abstract 1761 Chronic lymphocytic leukemia (CLL) is a neoplastic disorder of B-lymphocytes, typically with a high number of peripheral B-lymphocytes and small mature lymphocytes (M Hallek et al, Ann Oncol. 16, Suppl 1:i50-1 (2005). Clinically, some patients have a mild, largely asymptomatic course of the disease and a normal life expectancy, while others suffer from fulminant progression and have a very short survival. An important predictive factor is the presence of typical chromosome aberrations (H Doehner et al, N Engl J Med343, 1910–1916 (2000)). Due to a low proliferative rate of the cells, the gold standard for cytogenetic diagnostics in CLL is fluorescence in situ hybridization (FISH). Therefore, not much is known about the incidence of complex karyotypes, although they are strong predictors of a very poor prognosis in CLL (C Mayr et al, Blood107, 742–751 (2007)). By stimulating the cells with different interleukins and CpG-oligodeoxynucleotides, we were able to detect complex karyotypes in about 10% of investigated cases of CLL by classical banding analysis. In this study, we characterized 24 patients with CLL and complex karyotype by performing multicolor fluorescence in situ hybridization (mFISH). Hereby, we could identify cryptic aberrations and describe the karyotype in greater detail. In addition to typical aberrations involving 6q, 11q, 13q and 17p and trisomy 12, (iso)dicentric chromosomes and whole-arm translocations of chromosomes Y, 1, 3, 4, 5, 13, 15, 17, 18, 21 and 22 were detected. These chromosome aberrations were mostly generated by breaks in heterochromatic and telomeric regions indicating an increased breakage of these regions. This may indicate that epigenetic alterations and critically short telomeres predispose for the generation of chromosome aberrations in CLL. Telomere shortening and chromosomal instability are believed to play an important role in the development of neoplasia. Recently, it was shown that short telomeres in CLL are associated with a poor survival and increased genetic complexity (G Roos et al, Blood111, 2246–2252 (2008)). So far, published data are only available on the average telomere length in CLL, but not on the telomere length of individual chromosomes. We used a new technique, telomere/centromere-fluorescence in situ hybridization (T/C-FISH), which combines fluorescence R-banding and FISH using a probe against the telomere repeats to measure the telomere length of each chromosome arm. In line with previous results, patients with CLL showed significantly shorter telomeres than those of healthy controls. Comparing the telomere lengths of distinct chromosome arms with specific aberrations, there was no significant association. In addition, we could compare the telomere lengths of cells with aberrations and cells without aberrations within one patient. Aberrant metaphases of the same patient showed significantly shorter telomeres than metaphases with a normal karyotype (p Thus, telomere shortening is not a basic mechanism affecting all hematopoietic cells in CLL patients, e.g. due to aging, but affects only the malignant cells, indicating that telomere attrition is involved in the pathogenesis of CLL with complex karyotypes. Disclosures: No relevant conflicts of interest to declare.