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Complex karyotypes are associated with a poor prognosis in chronic lymphocytic leukemia (CLL). Using mFISH, iFISHand T/C FISH we thoroughly characterized 59 CLL patients regarding parameters known to be involved in chromosomal instability: status of the genes ATM and TP53 and telomere length (10 patients with a normal karyotype, 10 patients with an isolated deletion 11q, 19 patients with a complex karyotype without a deletion 11q and 20 patients with a complex karyotype including a deletion 11q). Among the patients with a complex karyotype, 29 of 39 (74%) showed a karyotypic evolution or a composite karyotype expressing great karyotypic heterogeneity and high chromosomal instability. Deletion of TP53 and ATM, two master regulators of DNA repair, was mutually exclusive in all but one patient. Interestingly, a deletion in 11q, either isolated or in the complex context of a complex karyotype, was associated with a significantly diminished risk (p Figure 1 a-b) Results of the Cytogenetic Data Analysis System (CyDAS) evaluation of all integratedkaryotypicdata from R-banding,iFISHandmFISHanalyses for recurrent gains and losses (a) as well as for recurrent breakpoints (b) c-f) T/C FISH on metaphases (d,e) and T/C FISHkaryograms(f,g) of patient 46 with a complex karyotype. Depicted are a normal cell (d and f) and an aberrant cell (e and f) The fluorescence intensity correlating with telomere length is higher in the normal cell than in the aberrant cell indicating telomere shortening in the aberrant cell. g)Karyogramof patient 46 aftermulticolorfluorescence in situ hybridization (mFISH) demonstrating a complex karyotype with cryptic aberrations. h) Median telomere lengths (kilobases) of normal and aberrant metaphases in three patients with CLL.*statistically significant difference (p Disclosures No relevant conflicts of interest to declare.

Introduction: Acute myeloid leukemia (AML) is one of the most frequent forms of leukemia in children younger than 15 years. The detection of several mutations in a blast population of pediatric AML (pAML) is supposed to be caused by a clonal evolution from a leukemic stem cell (LSC) to leukemic blasts. LSC are believed to be more resistant to chemotherapy, to be able to survive during treatment and to be responsible for the emergence of a relapse due to the persistence in the bone marrow (BM) niche. Since LSC and potential leukemic subclones are only present in small subpopulations, it has been a major technical challenge to particular analyse only the specific population. To acquire a better understanding of the underlying mechanisms of mutagenesis, clonal evolution and leukemogenesis, the aim of this study was to establish methods that allow the analysis and detection of mutations in single cells of a subpopulation known to contain HSC as well as LSC (CD34+CD38-). We especially focused on a pAML subgroup with mutations in Nucleophosmin (NPM1) and/or fms related tyrosine kinase 3 (Flt3). Methods and Results: We established methods to perform single cell sorting, whole genome amplification (WGA) using multiple displacement amplification (MDA) technology (Qiagen) and subsequent whole exome sequencing. The sorting efficiency was checked as Hoechst stained cells were sorted onto glas slides with 48 defined spots and the presence of single cells was checked under an inverse fluorescent microscope. Subsequently, single CD34+CD38- patient derived cells were sorted into 0,5ml low binding tubes containing 4µl PBS followed by WGA and whole exome sequencing. The mutational status of the sorted single cells from three patients suffering from pAML was analysed and compared to mutations detected at initial diagnosis in DNA from a bulk of BM cells. WGA from single CD34+CD38-PI- cells resulted in an amount of 29 to 31.7µg DNA from each of five single cells. The quality of the amplified DNA was sufficient for whole exome sequencing. A 4bp insertion in exon 12 of NPM1 reflecting a common NPM1 mutation (MutA) initially detected from a bulk of cells was identified in amplified DNA from single cells using whole exome sequencing in 2/3 patients. Internal tandem duplications in Flt3 indicated by mismatches in the alignment could be detected in amplified DNA from single cells of two patients. The detected ITD resemble those initially detected in DNA from a bulk of BM cells. Discussion and Conclusion: Single cell sequencing provides a useful tool to amend the detection of genetic aberrations from a bulk of cells and to confirm the presence of specific mutations in single cells from small subpopulations. It therefore helps to get further insights into the clonal evolution in pAML. Disclosures No relevant conflicts of interest to declare.

Genomic aberrations in CLL are important independent predictors of disease progression and survival in chronic lymphocytic leukemia (CLL). The most frequent changes are a deletion in 13q with a median survival of 133 months, a deletion in 11q (median survival 79 months), trisomy of chromosome 12 (114 months) and a deletion in 17p (32 months) (Döhner et al., NEJM, 2000; Hallek et al., Lancet, 2010). Between 2001 and 2012 we performed cytogenetic analyses of 2360 patients with CLL. We detected clonal chromosome aberrations in 1298/2360 (55%). Besides typical recurrent aberrations associated with CLL, we observed in 72/2360 (3%) clonal numerical aberrations involving the X chromosome. In 50/72 patients (69%) a loss of chromosome X and in 22/72 patients (31%) a gain of an X chromosome was detected. So far, numerical alterations of the X chromosome have not been described as recurrent aberrations detected in CLL. In order to understand their significance, we analysed those cases in greater detail. Median age of patients was 69 years (33-89 years) and the sex distribution was 64 female and 8 male (ratio 8:1). The patient cohort with the loss of the X chromosome consisted of 48 female and two male patients between 35 and 89 years of age (median age 69 years). In 52% (26/50) loss of chromosome X was detected as a sole abnormality and 22% (11/50) showed the loss of X within a complex karyotype. There was an association between loss of the X chromosome and trisomy 12 in 10% (5/50) and with deletion in 13q in 22% (11/50). In two cases, a clonal evolution was observed with the loss of the X chromosome in the main clone. Four independent clones (8%) were identified, three with trisomy 12 (6%) and one with a deletion in 11q (2%). Further, we analysed cases with a gain of the X chromosome (22 patients with CLL (16 female,6 male) between 33 and 81 years of age (median age 63 years)). In 15/22 patients (68%) a gain of the X chromosome was identified as a sole abnormality and in 6 of 22 within a complex aberrant karyotype. In 3/22 patients (14%) with an isolated gain of the X chromosome, we detected an independent clone. The independent clone was a trisomy 12 (two patients) and a monosomy X (one patient). In two cases, a clonal evolution was identified with gain of chromosome X in the subclone as a secondary event. Interestingly, the clone size was rather small. The median size of clones with loss of the X chromosome was 6/20 metaphases (30%) and with gain of the X chromosome was 4/20 metaphases (20%). In 8 patients, a loss of chromosome X could be detected in two metaphases only or a gain in one metaphase only, not fulfilling the criteria for clonality. In these cases, fluorescence in situ hybridization never confirmed the presence of a clone with X chromosome abnormalities. Even though a monosomy X has been reported as a recurrent somatic sole abnormality in MDS, it is not a characteristic aberration specific for any subtype of leukemia, lymphoma or a solid tumor (Abruzzese et al., Cancer Genet Cytogenet, 1997). The constitutional loss of the X chromosome is associated with Turner syndrome, a genetic disorder affecting 1/2500-3000 live-born. Notably, in a mosaic Turner syndrome patient with CLL, abnormal clones were restricted to the monosomic cells, indicating that loss of the X chromosome provides an advantage for leukemia cells (Manola et al., Leuk Res., 2008). However, CLL is not associated with Turner syndrome. A gain of chromosome X was observed by Aamot et al. (J Cancer Res Clin Oncol., 2007) in 17% of patients with follicular lymphomas as a secondary aberration with the characteristic translocation t(14;18) not affecting the overall survival. This is in accordance with our findings that a gain of the X chromosome could be a characteristic aberration in lymphatic neoplasms. Since we detected the aberrations involving chromosome X mostly in CLL, a constitutional mosaicism is unlikely. Otherwise, a constitutional mosaic would possibly be a predisposing factor for CLL. Putative candidate genes on chromosome X are not known. BRWD3, located on Xq13, encodes a bromodomain and WD repeat domain-containing protein and could be a putative novel transcription factor, since it is involved in translocations and is slightly down-regulated in CLL patients (Kalla et al., Genes Chromosomes Cancer, 2005). In summary, we identified numerical aberrations of the X chromosomes as new recurrent aberrations in CLL. The impact is still unknown and has to be analysed in further studies. Disclosures: No relevant conflicts of interest to declare.

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 522 Introduction: A complex karyotype, detected in approximately 10%-15% of patients with myelodysplastic syndromes (MDS), is associated with a very short median survival and a high risk of transformation into AML. The most frequent chromosome aberration in complex karyotypes is a deletion of 5q (del(5q)). It is still unclear, how complex karyotypes develop. One possibility is via stepwise accumulation of chromosome aberrations according to the so-called Vogelstein model (Fearon ER, Vogelstein B, Cell 1990; 61:759–67). Another possibility is a one-step catastrophic event called chromothripsis that seems to be associated with TP53 inactivation (Rausch T et al., Cell 2012;148:59–71). We recently described that leukemic progression in low-grade MDS with isolated del(5q) is associated with clonal evolution (Tehranchi R et al., N Engl J Med 2010;363:1025–37, Göhring G et al., Ann Hematol 2010; 89:365–74) and identified TP53 mutations and excessive telomere shortening as driving forces for clonal evolution and leukemic progression in MDS with del(5q) (Jädersten M et al., J Clin Oncol 2011; 29:1971–9, Göhring G et al., Leukemia 2012; 26:356–8). Yet, the modes of clonal evolution and the mechanisms responsible for the induction of chromosomal instability in MDS with isolated del(5q) remain largely unclear. Patients and Methods: Among 1645 patients with MDS and del(5q) investigated cytogenetically in our institution, 157 patients (9.5%) acquired additional aberrations and thus underwent clonal evolution. We reviewed the cytogenetic follow-up data of the 157 patients and carefully evaluated all additional aberrations, particularly those of complex karyotypes, which are defined as at least 3 aberrations, i.e. del(5q) and two additional chromosome abnormalities. Moreover, we investigated the clonal heterogeneity and the presence of independent clones, defined as clones that do not contain a del(5q). Results: During follow-up, 76 of 157 patients (48%) acquired two or more aberrations, thus evolving into a complex karyotype. Eighty-nine of 157 patients (57%) underwent a stepwise accumulation of additional aberrations (range 1–8, median: 1), while 38 patients (24%) developed highly complex clones (no of aberrations: 3–35, median: 7.5) at one time point during follow-up. This “catastrophic” route led to the development of a complex karyotype significantly more frequently than the stepwise accumulation of chromosome aberrations (38 of 38 cases compared to 38 of 89 patients; p Conclusions: Although MDS with del(5q) is assumed to be a genetically stable hematologic neoplasm, clonal evolution, even into complex karyotypes, occurs in a significant proportion of patients. There are two routes of clonal evolution. One route of stepwise acquisition of additional aberrations resulted in clonal selection of clones that had accumulated mostly only one or two additional aberrations. In contrast, the other route led to an immediate development of highly complex clones. In some of these cases, this catastrophic event was preceded by the acquisition of one aberration, repeatedly by loss of 12p or loss of 17p harboring the ETV6/TEL and TP53 genes, respectively. These data provide further evidence that the inactivation of TP53 seems to play an important role in clonal evolution and leukemic progression. Disclosures: No relevant conflicts of interest to declare.

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.

Abstract 30 Lenalidomide has been shown to induce transfusion independence and cytogenetic response in a high proportion of patients with MDS and isolated 5q deletion (del5q). However, some of these patients progress into acute myeloid leukemia, particularly those who do not show an erythroid or cytogenetic response. Yet the mechanisms inducing the leukemic transformation in patients with MDS and del(5q) are mainly unclear. Since dysfunctional telomeres play an important role in the development of genetic instability and shorter telomeres are associated with advanced MDS and AML, we reasoned whether telomere shortening may contribute to leukemic progression in patients with MDS and del5q. This study included 14 patients with transfusion-dependent anemia and low- or intermediate-1-risk MDS enrolled in the studies MDS-003 (n=42) or MDS-004 (n=260) who were treated with lenalidomide according to the study protocols. Written informed consent was provided according to the Declaration of Helsinki. Seven patients progressed into RAEB-1 or AML while on study and 7 patients did not. Time span between initial diagnosis and study entry was similar (median 51.3 and 44.4 months respectively in the patients with and without leukemic progression). Combined fluorescence R-banding and T/C-FISH analysis to determine the telomere length of each individual chromosome was performed as described earlier (K Lange et al, Genes Chromosomes Cancer, 2010). Telomere length at study entry was 6.1 kb (range 4.8 to 7.9 kb) in patients with MDS and del5q who later underwent leukemic transformation. Patients without later disease progression had a median telomere length of 9.3 kb (range 8.4 to 10.2 kb). Thus, patients who progressed after a median of 29 months (range 5 to 51 months) had significantly shorter telomeres than patients who had a cytogenetic response or stable disease (p Disclosures: Göhring: Celgene Corp.: Consultancy. Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.