Cytogenetic Subgroups Research Articles

Increased prevalence of bicuspid aortic valve in Turner syndrome links with karyotype: the crucial importance of detailed cardiovascular screening.

Bicuspid aortic valve (BAV) represents one of the strongest risk factors for aortic dissection in Turner syndrome (TS). An exact relation between the occurrence of BAV and a particular karyotype has not been established yet. The aim of this study was to determine the association between karyotype and prevalence of BAV. Sixty-seven TS patients aged between 6.6 and 32.5 years underwent cardiac magnetic resonance imaging (MRI) study. They were divided into four cytogenetic subgroups-45,X karyotype (n=27); 45,X/46,XX mosaicism (n=17); structural abnormalities of the X chromosome (n=10); and 45,X/structural abnormality of the X chromosome mosaicism (n=13). Prevalence of BAV and odds ratio (OR) compared with the general population in the whole study group, and statistical comparison of prevalences of BAV among the individual subgroups were determined. Prevalence of BAV in the whole study group was established as 28.4% [OR 208.3 (95% CI - 103.8-418.0); p-value<0.0001]. Individuals with 45,X karyotype had the highest prevalence of BAV - 40.7%, p-value<0.0001. Presence of any 45,X cell line in karyotype significantly predisposed to BAV (p-value=0.05). The 45,X karyotype is associated with the highest prevalence of BAV. Also, the presence of the 45,X cell line in any mosaic karyotype increases the probability of BAV.

The Msi2 Protein Expression Positive Correlation with Favorable Cytogenetics Findings in AML

Acute myeloid leukaemia (AML) is the most common subtype of acute leukaemias with a poor outcome. Msi2 protein is a newly discovered prognostic marker and it has been considered as a new target for therapy in AML. The study of Msi2 protein expression in AML cases has not been performed in Malaysia, to date. The main aim of the present study was to observe the expression of Msi2 protein in AML patients by immunohistochemistry (IHC) and to correlate its expression with the well-established prognostic and clinical parameters in AML as well as the overall survival (OS). Sixty four bone marrow trephine biopsy sections were immunostained for Msi2 protein. The percentage of blasts with positive reaction and the intensity of the cytoplasmic and nuclear staining were evaluated. The expression of Msi2 protein was found in 95.3% cases with Msi2 pattern varying between the cases. In 71.9% of cases, the blasts showed total cellular positivity and 23.4% cases showed only cytoplasmic positivity. Majority showed high expression of Msi2 for cytoplasmic staining. Interestingly, there was significant correlation between total cellular staining and the intermediate cytogenetic subgroup (P= 0.04). In conclusion, the results showed that the majority of the patients had high expression of Msi2 but this did not correlate to OS. However, the Msi2 expression correlated to the cytogenetic findings. The results suggest future extensive research to be conducted in order to ascertain the exact role of Msi2 positive blast cells in AML in our population and their association with prognosis and outcome.

Intrachromosomal amplification of chromosome 21 in Korean pediatric patients with B-cell precursor acute lymphoblastic leukemia in a single institution.

BackgroundIntrachromosomal amplification of chromosome 21 (iAMP21), defined as the presence of three or more RUNX1 signals on one marker chromosome, is a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) that is known to have a poor prognosis when treated with standard therapy. The aim of this study was to evaluate the clinical characteristics of Korean children with iAMP21.MethodsThe cytogenetic data from BCP-ALL children were reviewed. The ETV6/RUNX1 ES Dual Color Probe was used for fluorescence in situ hybridization (FISH).ResultsIn total, 295 children were included. Of these, 10 patients (3.4%) had iAMP21. The median age of iAMP21 patients was 9 years, and the median value of white blood cell count was 5.09×109/L. Slow early treatment response was observed more in iAMP21 patients. Patients with iAMP21 had a higher incidence of relapse and worse survival rates. In patients with iAMP21, the estimated 10-year cumulative incidence of relapse was 53.3%. The estimated 10-year event-free survival and overall survival rate were 46.7% and 64.8%, respectively. Most cases of leukemic relapse developed in the late period (median, 43 mo). In multivariate analysis, high risk group was the only factor that had a significant impact on death.ConclusionThe existence of iAMP21 was related to delayed treatment response and was likely to affect increased relapse and death in the late period. Further studies are needed to reveal its effect on BCP-ALL treatment outcomes and its role as an independent prognostic factor.

Genomic Landscape and Prognosis in Pediatric Acute Myeloid Leukemia: A Study on the French ELAM02 Trial

Background. Acute Myeloid Leukemia (AML) is a rare and genetically heterogeneous disease that constitutes 15 to 20% of childhood leukemia. Despite major treatment improvement over the past decades pediatric AML remains a challenging disease with poor outcome compared to acute lymphoid leukemia (ALL). About 50% of these patients relapse after standard intensive chemotherapy. Molecular analysis pointed out the prognostic impact of gene mutation such as FLT3-ITD, NPM1 or CEBPA; and new categories of regulators like epigenetic modifiers. More recently mutational profiling studies revealed distinct molecular subgroups with prognostic significant and stratification in adult AML. Nevertheless cytogenetic and mutational profiles are quite different between adult and pediatric AML. Extensive genomic studies have not been reported to date in pediatric AML. In this context it is of importance to identify additional genetic or molecular abnormalities to better understand leukemogenesis and also to predict outcome and serve as novel therapeutic targets.Methods. We performed a mutational analysis on diagnostic samples from patients enrolled in the French National Multicenter ELAM02 trial. 438 patients with de novo AML (except AML3) were enrolled between march 2005 and December 2011 (median age: 8,22yrs [0-18.61]; median WBC: 15.4G/l [0.4-575]; cytogenetic subgroups: CBF-AML[n=97], NK-AML [n=109], MLL-AML[n=95], MRC2 other[n=77], MRC3 [n=55], failure [n=5]). Diagnostic samples were prospectively collected and 386 of the 438 patients (88%) were studied by next-generation sequencing (Miseq, Illumina with haloplex librairy and ion Proton, thermofischer with ampliseq librairy) including 36 genes frequently reported in myeloid malignancy. Two different technologies of next generation sequencing (NGS) were used, allowing direct validation. FLT3-ITD was detected and quantified by Genescan analysis.Results. We identified 579 driver mutations involving 36 genes or regions in 386 patients (mean 1.5 per case), with at least 1 driver mutation in 291 patients (75%) and 2 or more driver mutations in 44% of samples. The number of mutation identified at diagnosis in cytogenetic subgroup is significantly lower in MLL-AML (0.44 mutation/patient; p<10-4). Mutations involving genes from the tyrosine kinase pathways (i.e RAS, FLT3, KIT, PTPN11, JAK2, MPL, CBL) were the most frequent and represent 56.3% of all aberrations. Among them N-RAS was detected in 26.4% of all cases, followed by FLT3-ITD, KIT and K-RAS in 14.8%, 12.4% and 12.2% respectively. We identified 64 driver mutations in the group of transcription factors (CEBPA, RUNX1, GATA, ETV6), 60 in the combined group of chromatin modifier (ASXL1, EZH2, BCOR) and DNA methylation (DNMT3A, IDH, TET2), 59 in the group of tumor suppressor genes (WT1, PHF6, TP53) 36 mutations in NPM1 gene, and few mutations in cohesion and spliceosome sub-groups. Identified mutations are indicated in the figure according cytogenetic subgroups. Among the 438 patients, 398 (91%) were in complete remission (CR) after two courses (induction and first consolidation), the 5-year overall survival (OS) is 71.5% [65-78] and the 5-year leukemia free survival (LFS) is 56.6% [49.7-63.5]. In univariate analysis, we found that FLT3-ITD, mutations in RUNX1, WT1 and PHF6 were associated with reduced LFS (p=0.0003 for FLT3-ITD, p=0.01 for RUNX1, p=0.02 for WT1 and p=0.025 for PHF6) and reduced OS (p=0.0003 for FLT3-ITD, p=0.0003 for RUNX1, p=0.015 for WT1 and p=0.04 for PHF6). Mutations in NPM1 is associated with an improved 5-yr LFS (p=0.014) and 5-yr OS (p=0.005). Multivariate analysis revealed that FLT3-ITD, RUNX1 and PHF6 were independently associated with an adverse outcome and NPM1 with an improved outcome.Conclusions. We performed an extensive mutational study in de novo pediatric AML enrolled in the ELAM02 trial. We described the genomic landscape of 386 patients and showed the frequency of different mutations according cytogenetics. Interestingly we found mutations in genes involved in constitutional or pre-leukemic disease such as PTPN11, RUNX1, MPL or ETV6. We found that FLT3-ITD, RUNX1 and PHF6 mutations predict poor outcome although NPM1 mutations predict a better outcome. Mutational profiling reveals useful information for risk stratification and therapeutic decisions. [Display omitted] DisclosuresBaruchel:Amgen: Consultancy.

The Prognostic Impact of Cytogenetics and Karyotype Changes in Pediatric Patients with Relapsed Acute Myeloid Leukemia: A Retrospective Cohort Study within the Relapsed AML 2001/01 Study

IntroductionAfter treatment response, cytogenetics and molecular aberrations are the most important prognostic factors in children with de novo acute myeloid leukemia (AML). However, little is known about the impact of cytogenetics at relapse. This international retrospective study aimed to provide insight into the prognostic impact of cytogenetic profiles and the role of karyotype changes from diagnosis to relapse in pediatric patients with relapsed AML.MethodsCytogenetic reports from patients registered to the Relapsed AML 2001/01 Study and diagnosed between 2001 and 2010 were centrally reviewed and classified by two independent researchers plus a cytogenetic expert. Patients with refractory or relapsed AML and available cytogenetics at relapse were included in order to assess the prognostic impact of different cytogenetic subgroups at relapse. Patients with karyotypes available at both diagnosis and relapse were included in order to study the impact of karyotype changes. Recurrent cytogenetic aberrations present in ≥5 patients defined the subgroups. Changes at relapse were categorized as: no change, gain, loss, both gain and loss, or structural other aberration(s). Primary endpoints were the probabilities of event-free survival (pEFS) and overall survival (pOS). Univariate analyses were conducted using chi-square tests, binary univariate logistic regression or Kaplan Meier estimates with a log-rank test. Multivariable Cox regression analyses were conducted to evaluate the independent impact of cytogenetic profiles and karyotype changes at relapse. For these analyses, cytogenetic subgroups were regrouped into good risk (GR) cytogenetics [t(8;21)(q22;q22) or inv(16)/t(16;16)(p13.1;q22)] or "other".ResultsOf the 569 registered patients, 402 patients (71%) had available cytogenetic information at relapse. Frequently detected aberrations at relapse were t(8;21) (n=60, 15%) and inv(16)/t(16;16) (n=24, 6%), both indicating a relatively good prognosis. Although patient numbers were small (n=5), t(6;9)(p23;q34) also had a relatively good outcome. Monosomy 7/7q-, t(9;11)(p22;q23), t(10;11)(p12;q23) and complex karyotypes had a relatively poor outcome. Figure 1 shows the Kaplan Meier curves of the investigated cytogenetic subgroups with corresponding patient numbers.In total, 306 patients (54%) with available karyotypes at both diagnosis and relapse were included to study cytogenetic changes. Patients with any change (n=148, 48%) had inferior outcome compared to patients without changes (3-year pEFS 21% [SE, 3.4%] versus 39% [SE, 3.9%]; overall P<0.01). Patients with both loss and gain or structural other aberrations had the worst outcome at the univariate level.After multivariable adjustment (final models including cytogenetics at relapse, time to relapse </≥ 1 year and type of change), having both gain and loss of aberrations remained associated with inferior pEFS and pOS (Hazard Ratio [HR] 2.10 [95% confidence interval (CI) 1.17-3.76] and HR 2.18 [95% CI 1.19-3.99] respectively). Having structural other aberrations at relapse was also associated with inferior pEFS (HR 1.81 [95% CI 1.03-3.18]).GR cytogenetics at relapse were significantly associated with better early treatment response. Subsequently, response to treatment at day 28 (Creutzig et al. Haematologica 2014) was an important mediator. If this prognostic parameter was included in the models, the effect of changes diminished, but GR cytogenetics at relapse remained an important prognostic factor associated with superior outcome (pEFS: HR 0.53 [95% CI 0.35-0.81], pOS: HR 0.51 [95% CI 0.32-0.80]).ConclusionTogether with early treatment response, the cytogenetic profile at relapse is an important prognostic factor. In particular t(8;21) and inv(16)/t(16;16) at relapse were associated with a favorable outcome. Furthermore, cytogenetic changes between diagnosis and relapse were associated with inferior outcome. Future studies should explore the mechanism(s) of these changes, being either clonal evolution or clonal selection.Interpretation of our results is hampered by the retrospective design of the study, small subgroup numbers and missing cytogenetic and molecular data. Nonetheless, these findings suggest that assessing cytogenetics at time of relapse is of high importance, and can be used for risk group adapted treatment. [Display omitted] [Display omitted] [Display omitted] DisclosuresReinhardt:Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding.

The Mutational Patterns Associated with Cytogenetic Subsets of De Novo Acute Myeloid Leukemia (AML): A Study of 1603 Adult Patients (Pts)

The classification of AML is based on the morphology of leukemic blasts, their immunophenotypes, and the presence of specific chromosomal abnormalities and selected gene mutations. It is widely acknowledged that some gene mutations are enriched in certain cytogenetic subsets, in which they may serve as therapeutic targets. We created a comprehensive oncoprint of mutations associated with centrally reviewed cytogenetic findings in 1603 adults (1080 aged <60 years, and 523 aged ≥60 years) with de novo AML (excluding acute promyelocytic leukemia) who were treated on CALGB/ALLIANCE protocols. The pts were classified into 5 cytogenetic subgroups: (1) cytogenetically normal (CN); (2) with complex karyotype (CK); (3) with t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22), i.e., core-binding factor (CBF)-AML; (4) with balanced rearrangements other than t(8;21) and inv(16)/t(16;16); (5) with unbalanced chromosomal abnormalities in non-complex karyotype.Using a targeted next-generation sequencing panel, we analyzed the mutational status of 80 cancer- and leukemia-associated genes. The mutated genes were assigned to 9 previously described by the Cancer Genome Atlas Research Network (N Engl J Med. 2013;368:2059) functional groups: chromatin remodeling, cohesin complex, kinases, methylation-related, NPM1, RAS pathway, spliceosome, transcription factors, and tumor suppressors. The mutational oncoprint revealed major differences in gene mutation frequencies among the 5 major cytogenetic groups.CN-AML pts had a broad mutational spectrum, involving all functional groups. Mutations in methylation-related genes (61% of pts), NPM1 (57%), and kinases (46%) dominated the mutation pattern.TP53 mutations were most common in pts with CK (38%). The second most common mutation, TET2, was detected only half as often (17%). The mutation patterns associated with "typical" (containing abnormalities resulting in loss of chromosome material from 5q, 7q and/or 17p), and "atypical" (without the aforementioned abnormalities) CKs differed. TP53 mutations were present in 52% of pts with typical but only in 5% of pts with atypical CKs (P<0.001). Conversely, the latter pts harbored mutations in another tumor suppressor gene, PHF6, more often (15% vs. 2%, P=0.03). Furthermore, atypical CKs had a broader spectrum of recurrent mutations, with 6 mutations occurring in ≥10% of pts (IDH2, NPM1, PHF6, TET2 and ZRSR2 mutations and FLT3-TKD), compared with only 2 genes, TP53 and TET2, mutated in ≥10% of pts with typical CKs, suggesting major biological differences between typical and atypical CKs.CBF-AML pts with had few detectable mutations, with a median of 1 (range, 0-5) versus 3 mutations (range, 0-9) detected in the remaining AML pts (P<0.001).In AML with balanced rearrangements, AML pts with MLL-rearrangements often had mutations in RAS pathway genes, whereas they rarely harbored mutations in other functional groups compared with pts with non-MLL-rearranged balanced translocations [MLL-rearranged vs. non-MLL-rearranged: chromatin remodeling, 9% vs. 26%; methylation-related, 12% vs. 23%; spliceosome, 12% vs. 24%]. Some cytogenetic subgroups with particular recurrent chromosome abnormalities presented with specific molecular features, supporting claims that they represent separate AML entities. For instance, 51% of pts with t(6;11)(q27;q23) harbored KRAS mutations, compared with only 3% of all remaining pts. Among t(9;22)(q34;q11.2)-positive pts, 47% had mutations in RUNX1,and 27% mutations in ZRSR2. Pts with inv(3)(q21q26.2/t(3;3)(q21q26.2) had frequent mutations in SF3B1 and BCOR (both 38%).Pts with gains or losses of chromosome material in non-complex karyotypes (unbalanced chromosomal abnormalities) were characterized by high mutation frequencies in methylation-related (55%) and spliceosome genes (34%). Strikingly, spliceosome mutations were enriched in pts with chromosome gains, with almost half of these pts harboring one or more mutations. Except for pts with sole +11, who most often harbored U2AF1 mutations (43%), SRSF2 was the most frequently mutated spliceosome gene in pts with unbalanced chromosomal abnormalities.Our comprehensive depiction of frequencies of single gene mutations and functional groups in recurrent cytogenetic subsets may be useful in guiding mutation testing in AML pts, and result in more focused application of targeted therapy of AML. DisclosuresWang:Immunogen: Research Funding; Incyte: Speakers Bureau. Stone:Pfizer: Consultancy; Sunesis Pharmaceuticals: Consultancy; Novartis: Consultancy; Merck: Consultancy; Karyopharm: Consultancy; Agios: Consultancy; Juno Therapeutics: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; ONO: Consultancy; Celator: Consultancy; Amgen: Consultancy; Jansen: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Xenetic Biosciences: Consultancy; Roche: Consultancy.

Predictive Value of Minimal Residual Disease in WHO2016-Defined Mixed Phenotype Acute Leukemia (MPAL)

▪BACKGROUND: Mixed phenotype acute leukemia (MPAL) as defined according to the World Health Organization 2016 guidelines (WHO2016) comprises approximately 2-3% of acute leukemia. In other acute leukemias, minimal residual disease (MRD) has emerged as the predominant predictor of relapse. MRD at end of Induction (EOI) is therefore a key risk-stratifying feature for lymphoid and myeloid disease regimens. To our knowledge, no studies to date have reported on the predictive value of MRD for treatment of MPAL. Although the absence of randomized trials for MPAL precludes a clear determination of optimal regimen, acute lymphoblastic leukemia (ALL) directed-therapy is increasingly considered frontline treatment internationally. The primary objective for this study was to evaluate the association of EOI MRD with event free survival (EFS) from ALL regimens used for treatment of MPAL.METHODS: Flow cytometry data for all acute leukemias diagnosed 2008-2015 and treated with Children’s Oncology Group regimens for ALL were examined by two flow cytometrists blinded to clinical outcomes (MO, SL). Data was analyzed using CellQuestPro and FACSDiva software (BD Biosciences, San Jose, CA). A comprehensive panel of 21 B-, T-, and myeloid markers were evaluated in all cases. A diagnosis of MPAL was strictly established according to the WHO2016 definition. EOI MRD was performed routinely by flow cytometry using a "difference from normal" approach. Cases with insufficient flow cytometry to either (1) establish the diagnosis of MPAL or (2) evaluate the presence and quantity of MRD were excluded. Due to the rarity of the T/myeloid phenotype in the dataset (n=1), the analysis was restricted to B/Myeloid. The primary endpoint was EFS; secondary outcomes explored predictors of EOI MRD positivity and the prevalence of recurrent cytogenetic abnormalities. MRD was categorized as a binomial (negative <0.01%) or ordinal (<0.01%, <0.1%, ≥0.1%) variable in separate models. EFS was calculated by Kaplan-Meier analysis. Step-wise multivariable Cox proportional hazard models included MRD and demographic/disease candidate predictors (age [years], sex, initial white blood cell count [WBC], central nervous system disease, cytogenetic abnormality, isolated myeloperoxidase expression). As some patients were classified as MPAL retroactively, category of treatment regimen (ALL high-risk [36/53] versus standard-risk [17/53]) was "forced" into the final multivariable model. Due to the non-normal distribution of MRD, predictors of MRD positivity were assessed by rank-sum test.Results: Of 59 cases identified as MPAL, 53 met inclusion criteria for analysis (1 Induction death, 1 insufficient MRD for analysis, 2 received AML therapy, 1 T/myeloid, 1 MPAL lymphoma). At a mean follow-up time of 3.5±1.7 years, EFS for the overall cohort was 88.0±4.6%. However, EFS was significantly greater for those MRD- versus those MRD+ (94.4±3.9% vs 67.5±12.1%, p=0.0005; Figure 1). Multivariable analysis confirmed MRD as a significant predictor of EFS even after accounting for treatment regimen (hazard ratio 5.0, 95%CI 1.7-15.3, p=0.0036). No other candidate predictors remained significant in the final multivariable models. Although different levels of MRD positivity were significantly associated with poorer EFS, visual examination of the dataset showed the 0.01% threshold to be most representative. Recurrent cytogenetic abnormalities were found in 15/53 children (Trisomy 4+10 n=11/53, TELAML n=1/53, iAMP21 n=2/53, BCRABL n=1/53). No MLL rearrangements were identified. The single patient with BCRABL received continuous imatinib during therapy (no event). No differences at diagnosis were present in those EOI MRD+ versus MRD-, although a WBC count ≥50K/uL was borderline significant (p=0.051).Conclusion: Using a "difference from normal" approach, EOI MRD at a threshold of 0.01% significantly predicts risk for strikingly poorer EFS in children with B/Myeloid MPAL. Children who achieve an EOI MRD-negative remission demonstrate excellent survival with ALL chemotherapy alone. Future investigation is necessary to prospectively validate these findings, examine MRD in T/myeloid and cytogenetic subgroups, and to explore the success of myeloid salvage therapy ±SCT to "rescue" those with a poor response to ALL therapy. [Display omitted] DisclosuresO'Gorman:Becton, Dickinson and Company: Consultancy, Research Funding.

Survival Improvement in Therapy Related Myeloid Neosplasm ? a Single Center Analysis of 428 Patients

Therapy-related acute myeloid leukemia/myelodysplastic syndrome (t-AML/MDS) arise after cytotoxic chemotherapy and/or radiotherapy administered for a prior neoplasm and have a dismal outcome (median survival of 8 months in the largest series published including 306 patients (pts), Smith, Blood 2003). Recent registry data suggested a continued increase in survival in AML (Derolf, Blood 2009) and we wondered whether this was also observed in the setting of t-AML/ MDS.All pts with a t-AML/MDS diagnosed and/or treated for their prior neoplasm at Gustave Roussy Cancer Center between July 1986 and 2016 were included in this retrospective study. Data regarding pts' demographics, primary diagnosis and treatment, latency time, cytogenetic, treatment and outcome were collected. The diagnosis of t-AML/MDS was based on the WHO 2016 classification. t-AML were classified based on cytogenetic results as favorable, intermediate and adverse according to international classification, t-MDS based on IPSS score as favorable (Low, Int-1) and adverse (Int-2 and High).428 pts were analyzed. The median age at diagnosis of t-AML/MDS was 56.4 years with a female predominance (60%). 224/428 (52.3%) pts had t-AML, 204/428 (47.7%) t-MDS. The most common primary malignancies were breast cancer (24%), non-Hodgkin lymphoma (15%), Hodgkin lymphoma (HL) (9%) and ovarian cancer (9%). Occurrence of t-AML/MDS following HL represented 26.6% of t-AML/MDS cases between 1986-96 comparing to 4 % between 2006-16 whereas breast cancer rose from 16% to 45%. Prior treatments included chemotherapy alone in 137/428 pts (32%), radiotherapy alone in 61 pts (14%) and both in 230 pts (54%).At diagnosis of t-AML/MDS, 295 pts (69%) were in complete remission (CR) of their prior neoplasm, 29 (7%) had a stable and 104 (24%) a progressive disease. Median interval between primary cancer and t-AML/MDS was 5 years (4.3 and 5.7 years for t-AML and t-MDS respectively, (p=0.03)). Furthermore, delay to develop t-AML/MDS after radiotherapy alone was longer compare to chemotherapy or both (6.1, 5.1 and 4.3 years, respectively (p=0.0087)). In the t-AML subgroup, 47% of pts presented unfavorable cytogenetic (including complex karyotype (20%) and 11q23 abnormalities (16.9%)), 26% intermediate and 26% favorable cytogenetic (core binding factor mutations (12.7%), t(15;17) (13.3%)). In the t-MDS subgroup, 78% of pts were considered adverse; complex karyotype, chromosome 7 and 5 abnormalities were found in 40.8%, 46.7% and 28.9% respectively. Pts received intensive chemotherapy (including 41 allografts), low dose chemotherapy (including 74 treatments with hypomethylating agents) and best supportive care in 42%, 24% and 34% respectively.The median overall survival (OS) was 10.6 months and the 5-year survival was 19.1% (Figure 1A). The 5-year OS of patients in CR of their prior neoplasm was 25.5% compared to 3.65 and 0% for pts with progressive and stable disease, respectively (p<0.001). 5-year OS was not statistically different between t-AML and t-MDS subgroups (23.3% vs 13.5%) and between hematologic or oncologic malignancies as primary diagnosis (12.6% vs 21.1%). Pts with favorable risk t-AML had better 5-year survival compared with patients with intermediate or unfavorable risk disease (55.5% vs 20% vs 12.1% respectively, p<0.001). In addition, favorable t-MDS was associated with better 5-year OS compared to adverse t-MDS (34.9% vs 6.6%, p<0.001). We next compared OS of pts diagnosed for their t-AML/MDS after or before July 2001. A trend for better OS for pts diagnosed in the last 15 years was observed (21.5% vs 15.1%, p=0.39). Interestingly outcome of t-AML patients with favorable subtype significantly improved over the last 15 years (68.8% vs 25%, p=0.03, Figure 1B) which was not the case for other cytogenetic subgroups and for t-MDS, especially pts with adverse prognosis (4.6% vs 3.9%, p=0.92). Pts who received allograft had a trend for better OS in the last 15 years (52.8% vs 21.5% p=0.2).t-AML/MDS are still associated with a low 5-year OS (19.1%) but our results are upper than previous publications. However, a significant improvement of survival in favorable t-AML was observed during the past 30-years, with a trend for pts who benefit from allograft. In addition to improve treatment for t-MDS pts, detection of the disease at the earliest stage is a real challenge to improve their survival. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interdependency Between TP53 Mutations, Cytogenetics, Genetic Instability and Prognosis in MDS and Secondary AML

Introduction: Alterations of TP53 (cytogenetic 17p13.1 deletions and molecular TP53 mutations) were reported to be frequent in pts with myelodysplastic syndromes (MDS) and complex abnormalities (≥3 clonal cytogenetic aberrations, CA) that represent around 15% of all MDS cases. It was suggested that pts with MDS and complex abnormalities may be further prognostically subdivided by the molecular TP53 mutation status (Bejar et al, ASH, 2014, abstract #532). In this study we investigated the frequency of different types of TP53 alterations, their cytogenetic profile and their clinical impact in the adverse cytogenetic MDS subgroup of complex abnormalities. We performed comprehensive cytogenetic and molecular genetic analysis focusing as well on the extent of cytogenetic instability.Methods: We included 105 pts (57 m/48 f; median 71 yrs, range, 47-95 yrs) with MDS (n=86) and sAML after MDS (n=19) with complex abnormalities in our study. A total of 56/89 (62.9%) pts had received azacitidine. Survival was censored at allogeneic stem cell transplantation (26/83; 31.3%). Pts were characterized by chromosome banding analysis, interphasefluorescence in situ hybridization (FISH) with a panel including a 17p13/TP53-covering probe, multicolor FISH (mFISH), Sanger sequencing of TP53 and SNP-array analysis (SNP-A). The extent of genetic imbalances was objectified by counting the number of CA, the number of cytogenetic fusions as shown by mFISH and the size of total genomic aberrations (TGA) measured by SNP-A in megabases (Mb).Results: A molecular TP53 mutation was found in 46/105 (43.8%) pts; a cytogenetic TP53 deletion in 38/105 (36.2%) pts. TP53 was not affected by a molecular mutation or a cytogenetic deletion in 44/105 (42.2%) pts, 23/105 (21.9%) pts were affected by combined TP53 alterations (molecular mutation and cytogenetic deletion), 23/105 (21.9%) pts by a molecular mutation only and 15/105 (14.3%) pts by a cytogenetic deletion only. The median number of CA was 6 (range, 3-41) in the entire cohort. Median overall survival for the entire cohort was 17 months.The degree of genomic imbalances was higher in pts with any TP53 alteration (molecular mutation and/or cytogenetic deletion) as compared to those without: The median number of CA was 8 (range, 3-41) vs. 4 (3-20) (P<0.001), the median number of fusions was 5 (0-13) vs. 2 (0-9) (P<0.001), and the median TGA size was 327 (97-511) vs. 105 (64-226) Mb (P<0.001). The extent of genomic imbalances was higher in pts with a TP53 deletion only compared to pts with a TP53 mutation only: The median number of CA was 10 (range, 4-21) vs. 8 (3-25) (P=0.093; n.s.); the median number of fusions was 7 (2-13) vs. 3 (0-10) (P=0.031).By univariate analysis, presence of ≥5 CA as compared to 3-4 CA increased the hazard ratio (HR) to 3.34 (P=0.017). When we limited the analysis to the subgroup of pts without evidence of a molecular TP53 mutation, presence of a cytogenetic TP53 deletion resulted in a HR of 5.67 (P=0.029). When the analysis was restricted to pts without a TP53 deletion, presence of a molecular TP53 mutation did not significantly change the HR (1.58, P=0.448; n.s.).Multivariate analysis that considered molecular TP53 mutation status, cytogenetic TP53 deletion status, the number of CA and treatment with azacitidine identified presence of a cytogenetic TP53 deletion as the most significant prognostic marker for OS (HR 15.1, P=0.010). HR was increased for pts with ≥5 CA compared to pts with 3-4 CA (HR 5.9, P=0.012). The molecular TP53 mutation status showed no significant impact on HR in our cohort.Conclusion: Presence of a cytogenetic TP53 deletion and a higher number of cytogenetic aberrations (≥5) showed a negative prognostic impact even within the unfavorable cytogenetic subgroup of MDS with complex abnormalities. In contrast, we found no strong prognostic impact for a molecular TP53 mutation in our cohort of MDS with complex abnormalities. The lower impact of TP53 mutations compared to TP53 deletions might be due to the lower degree of cytogenetic imbalances in pts with TP53 mutations only compared to pts with TP53 deletions only. However, molecular as well as cytogenetic TP53 aberrations were associated with a greater extent of chromosomal imbalances and displayed clear interdependencies. Our data suggest that TP53 alterations (molecular mutations and/or cytogenetic deletions) may not have an independent prognostic impact in the MDS subgroup with complex abnormalities. [Display omitted] [Display omitted] [Display omitted] DisclosuresPlatzbecker:Amgen: Honoraria, Research Funding; TEVA Pharmaceutical Industries: Honoraria, Research Funding; Janssen-Cilag: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Kroeger:Novartis: Honoraria, Research Funding.

The Human Specific microRNA-941 Is a Key Member of a microRNA Signature of Functionally Validated Leukemic Stem Cells from Patients with Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is initiated and maintained by a subpopulation of leukemic stem cells (LSCs), which are defined by their property to serially engraft immunocompromised mice. Based on this a LSC-associated gene expression was identified which was shown to be prognostically relevant in a retrospective analysis (Eppert et al, Nature Medicine 2011). microRNAs (miRs) play important roles in leukemogenesis, serve as potential tumor markers and novel therapeutic targets. In the current analysis we wanted to establish a miRNA profile of functionally validated LSCs from AML patients of various molecular and cytogenetic subgroups by conducting a comprehensive functional genomics study.Method: In total, 17 de novo AML samples were sorted for CD34+ granulocyte-macrophage progenitor (GMP), CD34+ lymphoid-primed multi-potential progenitor (LMPP) and CD34- subpopulations. Sorted cells were transplanted into sublethally irradiated non-obese diabetic/severe combined immunodeficient Gamma (NSG) mice. Those subpopulations that gave rise to human leukemic cell engraftment > 1% were classified as LSCs and sub-divided into LMPP-LSCs and GMP-LSCs and compared to the CD34- cell compartment which did not give rise to human leukemic cell engraftment (non-LSC). In parallel corresponding counterparts were highly purified from healthy bone marrow (BM). miRNA libraries were prepared using the Illumina TruSeqTM Small RNA Sample Preparation Kit and sequenced as single end reads on a Illumina HiSeq 2000. The bioconductor package limma was used for identifying differentially expressed miRNAs, the made4 package for correspondence analysis and between group comparisons. Quantitative RT-PCR validation on bulk and sorted populations (LMPP-LSC, GMP-LSC, CD34-) tested the expression levels of the identified miRNAs.Results: We assessed the engraftment potential of 17 AML samples, sorted into the LMPP, GMP and CD34- fraction. The LMPP or GMP fractions of 8 patients showed a leukemic graft in the NSG mouse model, whereas the CD34- fractions were not able to show a human engraftment in these mice. To the end subpopulations of 8 validated patient samples and corresponding stem-, progenitor, and CD34- subpopulations of normal bone marrow samples (n=3) were analyzed. Global miRNA expression profiles of subpopulations from normal and patient samples were compared using correspondence analysis based between group analysis. First, LSC subpopulations were compared to the leukemic CD34- Non-LSCs: between the LMPP-LSCs and the CD34- non-LSCs 15 miRNAs were differentially expressed in the LMPP-LSCs; between the GMP-LSCs and the CD34- non-LSCs only six miRNAs were found to be differentially expressed. Using these miRNAs, an unsupervised hierarchical clustering showed that the LMPP-LSCs, the GMP-LSCs and leukemic CD34- samples formed non-overlapping clusters. In normal bone marrow, 12 miRs showed differential expression between normal BM LMPP and CD34- population and 36 miRNAs between BM GMP vs. BM CD34- cells.We then compared the miRs that were differentially expressed in the patients compared to the differentially expressed miRs between the normal BM populations: except for one miR (miR-182), the fifteen differentially expressed miRs in the LMPP-LSC versus leukemic CD34- samples did not show overlap with the twelve miR significantly differentially expressed between normal BM LMPP and CD34- cells. When the GMP-LSC to leukemic CD34- Non-LSC comparison was cross-compared to the BM GMP to normal BM CD34- comparison, only two miRs (miR-148a, miR-320a) were unique to the patient samples. When we finally compared the LMPP-LSC specific 14 miR to the 2 GMP-LSC specific miRs, interestingly, we observed that the GMP-LSC miRs were a subset of the LMPP-miRs. Thus, 14 miRs defined a set of miRs differentially expressed in functionally validated AML LSCs. Importantly, 4 of these 14 miRs represent miR-941 isoforms. This miR has not been linked to leukemogenesis yet. It is one of the very few human-specific miRs which developed de novo in the human lineage after separation of the human and the chimpanzee lineages between six and one million years ago and preferentially targets genes in hedgehog- and insulin-signalling pathways. Taken together, combining functional validation of AML LSCs with next generation sequencing allows to identify miRs so far not linked to AML biology and LSC properties. [Display omitted] DisclosuresMulaw:NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

A Favorable BCL-2 Family Expression Profile May Explain the Increased Susceptibility of the t(11;14) Multiple Myeloma Subgroup to Single Agent Venetoclax

Background: In preclinical studies, multiple myeloma (MM) cells are sensitive to selective inhibition of the pro-survival protein BCL-2 by venetoclax (VEN). Resistance to VEN in these models was mediated through BCL-XL and MCL-1, and MCL-1 mediated resistance was neutralized by co-treatment with the proteasome inhibitor bortezomib (BTZ). MM cells harboring the t(11;14) translocation have a higher ratio of BCL-2 relative to MCL-1 mRNA and increased sensitivity to VEN alone, compared to cells with other cytogenetic abnormalities, suggesting that MM subgroups with a favorable BCL-2 family profile might be particularly sensitive to VEN.Clinically, VEN has shown activity in CLL, a disease typically expressing high BCL-2 and low BCL-XL and MCL-1 expression. In MM, VEN is being evaluated as a single agent (NCT01794520) and in combination with BTZ and dexamethasone (Dex) (NCT01794507, NCT02755597) in relapsed/refractory (R/R) patients. As a single agent, improved objective response rates were observed in patients with R/R t(11;14) MM (40% in t(11;14) vs 6% in non-t(11;14)-). In the BTZ/Dex combination study, patients with high BCL-2 expression had an increased objective response rate. Thus, BCL-2 family profiling may explain increased susceptibility of certain MM subgroups to antitumor activity of VEN both as a single agent and in combination. We analyzed MM subgroups for expression of BCL-2 family members to better understand and identify the patient population that can benefit with VEN.Methods: We used publically available data (GSE4581; GSE9782) to analyze BCL-2 family mRNA expression within molecular/cytogenetic defined subgroups among newly diagnosed (NDMM n=414), and R/R (n=264) MM patients, and in comparison to other hematological malignancies (genealogic database).Results: BCL-2, BCL-XL and MCL-1 expression was assessed in MM and compared with 12 major hematologic malignancies. Based on median expression, BCL-2 in MM ranked 6th of 12 tested, with highest BCL-2 expression observed in small lymphocytic leukemia and mantle cell lymphoma. In MM, BCL-2 expression varied significantly across molecular and cytogenetic subgroups. Highest expression was among patients with t(11;14) molecular subtypes (CD1, CD2), in addition to subtypes that are not enriched with t(11;14) like the hyperdiploid and low bone disease subtypes, and lowest in the high-risk subtypes (proliferation, MMSET, MAF/MAFB) (p=0.0001), both in NDMM and R/R MM.BCL-XL and MCL-1 were highly expressed in MM relative to other hematologic malignancies. Expression of BCL-XL and MCL-1 were lowest in the t(11;14) MM population (both p<0.001). Correspondingly, t(11;14) MM was enriched for the highest ratios of BCL-2/MCL-1 (p<0.0001) and BCL-2/BCL-XL (p<0.0001), further supporting the strong single agent VEN activity observed in these patients in clinical study. The t(11;14) positive CD2 subtype can be further characterized by high expression of CD20, (p<0.0001, CD2 vs other), and by other B-cell phenotypic markers (high for PAX5, CD79A and VPREB, and low for CD56). Thus, the CD2 molecular subtype may identify a CLL-like subset of t(11;14) MM that is uniquely susceptible to VEN single agent activity. Finally, when we assessed BCL-2 family expression in R/R MM across lines of treatment, we found patients with 1-3 prior lines of therapy had a higher BCL-2/MCL-1 ratio (p<0.001) compared to patients with greater lines of therapy, due primarily to lower MCL-1 expression (p<0.001), but similar BCL-2 expression.Conclusions: Our analysis suggests that MM subgroups are associated with distinct BCL-2 family expression profiles, and the t(11;14) subgroup amongst MM subgroups may be particularly suited for single agent VEN activity due to a high BCL-2/MCL-1 and BCL-2/BCL-XL ratio. In the non-t(11;14) MM subgroups, VEN treatment in earlier lines of therapy, as well as combination strategies with agents that will down-modulate BCL-XL and/or MCL-1, such as BTZ, may enable maximum VEN activity, and will be further assessed in clinical studies. [Display omitted] DisclosuresWu:Genentech: Employment. Ross:AbbVie: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Shaughnessy:Signal Genetics: Consultancy, Equity Ownership. Van Laar:Signal Genetics, Inc.: Employment. Morgan:Takeda: Consultancy, Honoraria; Janssen: Research Funding; Bristol Meyers: Consultancy, Honoraria; Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding. Venstrom:Genentech: Employment. Punnoose:Genetech, Inc.: Employment.

A Transcriptional- and Posttranscriptional-Based Prognostic Model in Pediatric Acute Myeloid Leukemia

Background. We have recently investigated transcriptional and posttranscriptional changes in AML cells (Oncotarget, 2016, 7, 2889-909) and identified ABCA3 expression, TET2 expression and TET2 exon 2 skipping (TET2E2S) as prognostic factors in pediatric AML (See companion abstracts from Ceraulo et al.). The present study was conducted in order to test whether the combination of these prognostic factors might help stratifying patients according to their relapse risk.Methods. Samples derived from 120 patients with available high-quality RNA and enrolled in the French ELAM2 protocol. Patients were classified according to their standardized cytogenetic and molecular (NPM1 mutations, FLT3-ITD, CEBPA double mutations) risk subgroups. Treatment consisted of 1 induction course (AraC and mitoxantrone) and 3 consolidation courses (course 1 and 3 with high dose AraC); all children with either intermediate or high-risk disease were candidates for hematopoietic stem cell transplant in complete remission (CR) after 1 to 2 consolidation courses. qRTPCR amplification of 2 conserved ABCA3 (exons 6-7 and exon 19-20) and TET2 (exon 4 and exon 2-3) mRNA sequences were performed with GUS and ABL as reference genes. TET2E2S was quantified as already described (Oncotarget, 2016, 7, 2889-909). The concordance index (c-index) was used to compare the relative statistical power of the new model compared with the standardized cytogenetic and molecular-based scoring system.Results. ABCA3 expression [HR 2.6 (95% CI 1.3-5), p=0.006], TET2 expression (HR 0.5 (0.2-0.9) p=0.04, and TET2E2S (HR 2.5 (1.2-5.1), p=0.01) represented independent prognostic factors for EFS. To design a convenient prognostic score, continuous variables were dichotomized using cutoff points that were based using the Maximally Selected Rank Statistics (maxstat R-statistics package, from the R-Project). One point was attributed for lower TET2 expression (n=85), higher ABCA3 expression (n=44), and TET2E2S (n=61). Three groups were formed: lower-risk patients (0 point, n=33), intermediate-risk patients (1 point, n=41), and higher-risk patients (either 2 or 3 points, n=46). The present score identified subgroups of patients with significantly different outcome (p<10-4) (Figure 1). Patients with a low TPS (Transcriptional-Posttranscriptional Score) had significantly better EFS than either patients with a high (p<10-4) or an intermediate TPS (p=0.03) while patients with intermediate TPS had significantly better EFS than patients with high TPS (p=0.02). For OS (p=0.0004), patients with a low TPS had significantly better OS than either patients with a high (p=0.0004) or an intermediate TPS (p=0.047) while patients with intermediate TPS had significantly better OS than patients with high TPS (p=0.024). For DFS (p=0.003), patients with a low TPS had significantly better DFS than either patients with a high (p=0.0012) or an intermediate TPS (p=0.036) whereas no significant difference was noticed between patients with intermediate versus high TPS (p=0.2). Figure 1 shows that FLT3-ITD was significantly associated with intermediate TPS, EVI1 expression with high TPS and NPM1 mutations and CBFB-MyH11 leukemias with low TPS. The distribution of CR rate and relapse rate was significantly distinct among the 3 TPS categories (Figure 1). The added value of the TPS within AML genetic risk groups was evaluated through univariate analysis. The TPS permitted to identify patients with different outcome in lower and intermediate-risk AML (EFS: Log-rank, p=0.011 and 0.005, respectively). Ten of the 20 lower-risk AML corresponded to intermediate (n=7) and high (n=3) TPS. Their CR rate was 100%, none of these patients was allografted and 4 patients relapsed as compared to 0/10 in the remaining lower-risk AML cases with low TPS (p=0.02). In intermediate risk AML, the CR and relapse rates of patients with low, intermediate and high TPS were 94 and 17%, 100 and 45%, and 73 and 50 %, (CR, p=0.001; relapse, p=0.036). Overall, the present TPS model had a better c-index (0.73) compared with the standardized cytogenetic and molecular-based scoring system (0.61).Conclusion. We propose a new prognostic model that includes transcriptional and posttranscriptional data and can predict survival in pediatric AML. A validation in a larger, independent, multicenter, data set of patients is important to facilitate the translation of this model into clinical practice. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RANK-RANKL Mediated Bone Destruction in B-Cell Acute Lymphoblastic Leukemia

Survival rates for pediatric B-Cell Acute Lymphoblastic Leukemia (B-ALL) have improved dramatically over the past 40 years approaching a current long-term survival rate of 85%. However childhood B-ALL patients continue to confront co-morbidities and their long-term consequences. For example, osteopenia and osteoporosis associated fractures are a common complication of pediatric leukemia at diagnosis, during treatment and in long-term B-ALL survivors. The STeroid-associated Osteoporosis in the Pediatric Population (STOPP) study reported that at ALL diagnosis, 16% of children and adolescents present with bone pain, vertebral compression and low vertebral Bone Mineral Density (BMD) scores, with the greatest incidence of vertebral fractures (VF) seen in the first year following diagnosis (J Clin Endocrinol Metab. 2015, 100:3408-17). Glucocorticoid treatment further elevated fracture risk in this population. These data underscore the need to identify molecular mechanism by which leukemic cells contribute to bone loss, and provide targeted therapies to limit these effects.Our laboratory previously showed that Rag2-/- p53-/- Prkdcscid/scid triple mutant (TM) and p53-/- Prkdcscid/scid double mutant (DM) mice develop spontaneous B-ALL, but only TM animals exhibit dissemination of leukemic blasts to the leptomeninges of the CNS, a poor prognosis feature observed in pediatric and adult ALL patients. We observed that TM leukemic mice also displayed fragile vertebral bones. Using comparative transcriptome analysis, we found that RANKL (Receptor Activator of the Nuclear factor-kB Ligand), a Tumor Necrosis Factor (TNF) superfamily member ligand and a key regulator of B cell and osteoclast differentiation, was expressed at greater levels in TM compared to the DM leukemia cells. RANKL binds to its receptor RANK, which is expressed in osteoclast precursor cells. RANK-RANKL interaction induces signaling in the osteoclast precursors and drives their differentiation into mature bone resorbing osteoclasts (Proc. Natl. Acad. Sci. 1999, 96:3540-3545). Upon adoptive leukemia cell transfer into immune deficient mice, RANKL+ TM but not DM cells caused decreased vertebral trabecular bone density in the recipients. Treatment with the recombinant RANKL antagonist protein Osteoprotegerin (OPG-Fc) inhibited the growth and dissemination of RANKL+TM leukemic cells and attenuated bone destruction in the recipient mice. These data suggested that TM mouse leukemia cells cause bone loss in the absence of glucocorticoid or other chemotherapy agents.We then examined the potential role of RANKL in osteoporosis associated with human B-ALL. RANKL mRNA was expressed by a majority of primary human adult and pediatric B-ALL. To determine whether primary patient B-ALL can cause bone loss, we transplanted RANKL+ human B-ALL samples of multiple cytogenetic high-risk subgroups (Complex, hypo-diploid and Mixed Lineage Leukemia (MLL) rearranged) into NOD.SCID.gC-/-(NSG) recipient mice. Micro-CT imaging and bone density measures in the xenotransplant recipients revealed extensive vertebral trabecular bone destruction. Immuno-histological analysis of the human B-ALL recipient mice demonstrated extensive osteoporotic damage of the long bones and marked RANKL protein expression in the long bones of mice harboring extensive human B-ALL cell burden compared to NSG control mice. To determine whether RANKL-RANK interaction was required for the B-ALL mediated bone destruction, cohorts of NSG mice engrafted with human B-ALL were treated with recombinant OPG-Fc compared to a matched Fc control protein. OPG-Fc treatment did not attenuate leukemia cell expansion and bone marrow burden, but despite bulky disease, the treatment conferred robust protection from bone destruction suggesting that RANKL was a critical mediator of this clinical complication.Our data demonstrate a central role of the RANK-RANKL axis in B-ALL-mediated bone disease and identify an actionable therapeutic target to reduce acute and long-term morbidity. Denosumab, an anti-RANKL antibody has been approved for the treatment of bone metastasis by solid tumors and for post-menopausal osteoporosis. Our pre-clinical studies suggest that Denosumab and other agents that inhibit the RANK-RANKL pathway may be efficacious in patients with B-ALL associated bone degeneration. DisclosuresNo relevant conflicts of interest to declare.

Increased Idarubicin Dosage during Consolidation Therapy for Adult Acute Myeloid Leukemia Improves Leukemia-Free Survival

Background: Anthracylines are one of the major classes of drugs active against acute myeloid leukemia (AML). Increased doses of daunorubicin during induction therapy for AML have been shown to improve remission rates and survival. The ALLG used idarubicin in induction therapy at a dose of 9 mg/m2 x 3 days (total dose 27 mg/m2) in combination with high-dose cytarabine and etoposide (Blood 2005, 105:481), but showed that a total idarubicin dose of 36 mg/m2 was too toxic in this context (Leukemia 2001, 15:1331). In order to further improve outcomes in adult AML by anthracycline dose escalation, we conducted a phase 3 trial comparing standard to an increased idarubicin dose during consolidation therapy. Methods: Patients achieving complete remission after 1 or 2 cycles of intensive induction therapy (idarubicin 9 mg/m2 daily x3, cytarabine 3 g/m2 twice daily on days 1,3,5 and 7, and etoposide 75 mg/m2 daily x7; ICE protocol) were randomized to receive 2 cycles of consolidation therapy with cytarabine 100 mg/m2 per day for 5 days, etoposide 75 mg/m2 for 5 days, and idarubicin 9mg/m2 daily for either 2 or 3 days (standard and intensive arms respectively). No further protocol therapy was given. The primary endpoint was leukemia-free survival from randomization to consolidation therapy (LFS) with overall survival (OS) as secondary endpoint. Results: A total of 422 patients with AML (excluding cases with CBF rearrangements or APL) aged 16 to 60 years were enrolled between 2003-10, with 345 (82%) achieving complete remission, and 293 being randomized to standard (n=146) or intensive (n=147) consolidation arms. The median age was 45 years in both arms (range 16- 60), and both groups were balanced for intermediate versus unfavorable karyotypes and for frequency of mutations involving FLT3-ITD and NPM1 genes. Of the randomized patients, 120 in the standard arm (82%) and 95 in the intensive arm (65%) received the second consolidation cycle (p Conclusion: We conclude that in adult patients in complete remission after intensive induction chemotherapy an increased dose of idarubicin delivered during consolidation therapy results in improved LFS, without increased non-hematologic toxicity. Disclosures Szer:Ra Pharma: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Alexion Pharmaceuticals, Inc.: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Alnylam: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Marlton:Amgen: Honoraria, Membership on an entity9s Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Wei:Novartis: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; CTI: Consultancy, Honoraria; Abbvie: Honoraria, Research Funding; Servier: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Cartwright:ROCHE: Consultancy, Membership on an entity9s Board of Directors or advisory committees. Roberts:Servier: Research Funding; Janssen: Research Funding; Genentech: Research Funding; AbbVie: Research Funding. Mills:Novartis: Membership on an entity9s Board of Directors or advisory committees, Other: Meeting attendance sponsorship. Gill:Janssen: Membership on an entity9s Board of Directors or advisory committees. Seymour:Roche: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Genentech: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Other: Travel support, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees.

Age and Gemtuzumab Ozogamicin Influence the Prognostic Impact of TET2 Expression and Splicing in Intensively Treated Patients with Acute Myeloid Leukemia

Background. In AML, the frequency of TET2 mutations increases with age ranging from 1-5% in children to 6-27% in adults. These mutations have both been reported to exert unfavorable or neutral effects on patient outcomes. Besides mutations, fluctuation in TET2 expression has been evidenced in AML while we recently found that TET2 exon 2 skipping (TET2E2S) is associated with favorable outcome in adult AML (Mint-Mohamed et al., ASH 2014 and submitted). Here we investigated the effect TET2 expression and TET2E2S in homogeneously treated adult and pediatric AML patients with intensive chemotherapy (IC).Methods. Samples derived from 341 patients with available high-quality RNA and enrolled in the ELAM2 protocol (n=120) and the ALFA-0701 protocol (n=221).qRTPCR amplification of 2 conserved TET2 mRNA sequences was performed with GUS and ABL as reference genes. TET2 exon 2 skipping was quantified throughexon(E)-specific qRTPCR (qESPCR) amplification of E1E3 (spliced) and E2E3 (unspliced) TET2 isoforms. TET2E2S was quantified by calculating the ratio E1E3/E2E3. Patients were classified according to their standardized cytogenetic and molecular (NPM1 mutations, FLT3-ITD, CEBPA double mutations) risk subgroups. In children, treatment consisted in 1 induction course (AraC and mitoxantrone) and 3 consolidation courses (course 1 and 3 with high dose AraC); children with either intermediate or high-risk disease were candidates for hematopoietic stem cell transplant (HSCT) in complete remission (CR) after 1 to 2 consolidation courses. Adult patients were given a 3+7 induction course without (control group, n=110) or with fractionated intravenousgemetuzumab ozogamicin (GO) (n=111) as already described (Castaigne S, Lancet 2012; 379:1508-1516).Results. In pediatric AML, median age, median WBC, CR rate, relapse rate, median follow-up, 3-years EFS, DFS, and OS were 9.4 years, 19 G/L, 95%, 29%, 48 months, 60±5%, 63±6%, and 72±5%, respectively. Lower TET2 expression was associated with inferior EFS (3-years: 44±10%vs 69±6%, log-rank p = 0.01), DFS (51±8%vs 64±5% p=0.02), and OS (60±8%vs 83±3%, p=0,0003) while TET2E2S was associated with inferior EFS (44±8%vs 70±7%, p = 0.006) and OS (61±7%vs 81±4%, p=0.01). For TET2 expression, multivariate analysis identified WBC, age, risk group, and TET2 expression as independent prognostic factors for EFS and OS, and age, WBC, and TET2 expression for DFS (Table 1). For TET2 splicing, WBC and TET2E2S independently predicted EFS while TET2E2S alone independently predicted DFS (Table 1). In the low- (n=29) and intermediate-risk (n=62) groups, lower TET2 expression remained associated with shorter 3-years EFS. There was no significant correlation between TET2 expression and TET2E2S and TET2E2S retained its negative prognostic impact in the 85 patients with higher TET2 expression (3-years EFS 48±8vs 84±6%, p=0.004). In adult AML, median age, median WBC, CR rate, relapse rate, median follow-up, 3-years EFS, DFS, and OS were 62 years, 8 G/L, 76%, 59%, 47 months, 26±3%, 30±4%, and 44±4%, respectively. In the entire population, TET2 expression and splicing had no significant effect on outcome. In the 110 patients treated with IC alone, TET2 expression was associated with inferior OS (25±9vs 29±9%, p=0.02) while TET2E2S was associated with better EFS (17±5vs 7±6%, p=0.008), DFS (28±8vs 8±6%, p=0.02), and OS (46±8vs 29±8%, p=0.05). TET2E2S, age, and disease risk represented independent prognostic factors for EFS (Table 1). In the GO arm, TET2 expression had no significant effect on outcome whereas TET2E2S was associated with inferior EFS (27±5vs 60±12%, p=0.01), DFS (31±6vs 63±12%, p=0.05), and OS (39vs 72±11%, p=0.04).Conclusion. Inchildren, higher TET2 expression confers a favorable outcome whereas TET2E2S represents an independent unfavorable prognostic factor. In adult treated with IC alone, present results confirm the favorable prognostic impact of TET2E2S and the weak prognostic effect of TET2 expression. In contrast, in patients fulfilling the same inclusion criteria, TET2E2S loses its favorable prognostic effect and is even associated with unfavorable outcome when GO is associated to IC. While TET2 expression and splicing might represent useful biomarkers, the data highlight the strong difference in tumor biology between adult and pediatric AML and suggest distinct relationships between TET2E2S and the cytotoxic effect of GO plus IC versus IC alone. [Display omitted] DisclosuresThomas:Pfizer: Consultancy.

Postremission Therapy with Repeated Courses of HD-Arac, Idarubicin and Limited Autologous Stem Cell Support Achieves a Very Good Long-Term Outcome in ELN Favourable and Intermediate-Risk AML Except in NPM-Mutated Patients Where Relapse May Occur Even after Five Years

Introduction: Postremission treatment in adult acute myeloid leukemia (AML) is mandatory to improve long-term remission duration. Allogeneic stem-cell transplantation (allo-SCT) is considered gold standard in younger patients (pts) with higher risk of relapse, whereas in pts categorized as standard-risk (SR) AML the optimal treatment remains an open issue. High-dose cytarabine (HD-ARAC) is considered the backbone of postremission treatment in SR AML. The optimal dose and schedule, and the clinical benefit of additional chemotherapeutic agents remain controversial (Schaich, 2011- Lowenberg, 2011- Lowenberg, 2013) as well as the relative efficacy of HD-ARAC in the different cytogenetic and molecular subgroups of SR AML. We analyzed the very long term outcome of consecutive unselected AML pts treated according to AML 00 NILG protocol (Bassan, Blood, 2003: 102; 11) with three repetitive HD-ARAC-based cycles, associated with idarubicin, followed by limited peripheral blood stem cell (PBSC) support, in order to reduce toxicities.Methods: We evaluated 263 consecutive AML pts, diagnosed at our Institution, from January 2001 to March 2016 (median age 52 years - range 15-65). The treatment plan included induction with ICE (Idarubicin-Cytarabine-Etoposide) followed by IC consolidation or HD-ARAC/HD-idarubicin (SPLIT therapy) in refractory cases. ARAC 1g/mq bd for 4 days (A8) was given to CR pts, to collect 3-6x10^6 CD34/kg in 3 aliquots. Patients with ELN favorable or intermediate risk AML, considered SR AML, received 3 cycles of HD-ARAC (2g/mq bd for 5 days) plus idarubicin (8mg/mq for 2 days) with PBSC reinfusion (1-2x10^6 CD34/Kg) (A20). The pts with insufficient CD34 cells yield, received 2 courses HD-ARAC (1g/mq bd for 5 days) plus idarubicin (10mg/mq day 1) (A10). The cumulative dose of ARAC and idarubicin given according to A20 protocol were 69,4 g/mq and 162 mg/mq, respectively.Results: Among 263 pts, 142 (54%) pts were considered SR and 116 (44.1%) HR. Only 5 pts were lacking molecular or cytogenetic information (1.9%) to allow ELN risk classification. Accordingly, 65 pts (25.2%) were favorable [35.4% core binding factor (CBF) positive, 9.2% CEPBA mutated and 55.4% NPM mutated], 87 (33.7%) Intermediate-I, 45 (17.4%) Intermediate II and 61 (23.7%) adverse risk. CR rate after ICE treatment was 77.6% (204/263), 95.8% in SR and 56% in HR pts and after ICE+SPLIT it was 88.2% (232/263); early or aplastic death was 6%. A8 course was administered in 218 pts, of whom 179 were successfully mobilized (82.1%). Among 136 SR pts, 127 were received HD-ARAC (A20 or A10), 4 allo-SCT for early relapse, 3 pts did not proceed to A20 because of prolonged cytopenia and 2 are ongoing. Hematological toxicity was acceptable. ANC recovered at a median of 10 days after CD34+ cells reinfusion. Two death in CR occurred within 100 days after A20 therapy (1.6%). After a median follow-up of 53.3 months (range 1-172), the median survival of SR AML patients was 118.9 months, the DFS and OS at 5 y were 51+/-4.8% and 56.5+/-4.7%, and at 10 y 43.3+/-5.9% and 49.6+/-5.7%, respectively. According to ELN risk, the OS at 5 y and 10 y was 78.3+/-6% and 68+/-8.7% in favorable (median OS not reached), 52.3+/-8% and 46+/-9.4% in Intermediate-I (median OS 118.8 months), and 42+10% and 33.6+/-10.9% in Intermediate-II (median OS 33.1 months) (p0.0034) (Figure1). According to molecular characteristics, the OS at 5-y was 100% in CEBPA-mutated, 77+/-9% (+/-SE) in CBF-mutated and 71+/-11% in NPM-mutated subgroups, respectively (Figure 2). In NPM-mutated pts it was 56+/-14% if FLT3 was mutated and 75+/-10% if not. At 8-y OS was not evaluable in CEBPA-mutated, it remained 77+/-9% in CBF mutated but dropped to 47+/-15% in NPM-mutated pts due to late relapses (28%), which occurred in pts without FLT3. The median time to relapse in NPM pts was 17 m, the latest relapse occurred after 97 months.Conclusion: A postremission program with repeated courses of HD-ARAC associated to idarubicin and limited autologous CD34+ cells support was feasible, well tolerated and very effective, also long-term, in pts with ELN favourable or intermediate risk AML. The late relapse tendency of NPM-mutated AML is puzzling and should be considered by innovative programs aimed at improving these results. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

SWOG S1203: A Randomized Phase III Study of Standard Cytarabine Plus Daunorubicin (7+3) Therapy Versus Idarubicin with High Dose Cytarabine (IA) with or without Vorinostat (IA+V) in Younger Patients with Previously Untreated Acute Myeloid Leukemia (AML)

SWOG S1203: A Randomized Phase III Study of Standard Cytarabine Plus Daunorubicin (7+3) Therapy Versus Idarubicin with High Dose Cytarabine (IA) with or without Vorinostat (IA+V) in Younger Patients with Previously Untreated Acute Myeloid Leukemia (AML)

Prognostic Impact of ABCA3 Expression in Pediatric Acute Myeloid Leukemia

Abstract Background. Despite progress in the molecular and genetic classification of pediatric acute myeloid leukemia (AML), the prognosis remains heterogeneous. The ATP-binding cassette transporter A3 (ABCA3) seems specifically involved in the resistance of pediatric AML to intensive chemotherapy. However, studies having investigated the prognostic impact of ABCA3 expression have yielded conflicting results with respect to patient outcomes while the small sample size of these studies precluded the use of multivariate analysis. Here we investigated the prognostic impact of ABCA3 expression in a representative series of homogeneously treated pediatric AML. Methods. Samples derived from 233 patients with available high-quality RNA and enrolled in the ELAM2 protocol (NCT00149162). qRTPCR amplification of 2 conserved ABCA3 mRNA sequences was performed with GUS and ABL as reference genes. Primer sets were complementary to exons 6-7 and exons 19-20 junctions. Patients were classified according to their standardized cytogenetic and molecular (NPM1 mutations, FLT3-ITD, CEBPA double mutations) risk subgroups (Rubnitz JE, Blood 2012;119:5980-5988, Creutzig U, Blood 2012;120:3187-3205). Treatment consisted of 1 induction course (AraC and mitoxantrone) and 3 consolidation courses (course 1 and 3 with high dose AraC); all children with either intermediate or high-risk disease were candidates for hematopoietic stem cell transplant (HSCT) in complete remission (CR) after 1 to 2 consolidation courses. Results. The discovery cohort included 120 patients. Median age, median WBC, CR rate, relapse rate, median follow-up, 5-years EFS, DFS, and OS were 9.4 years, 19.3 G/L, 95%, 29%, 60 months, 58±6%, 61±6%, and 71±5 months, respectively. The two primer sets yielded consistent results (R=0.9, p&lt;10-4, Spearman Rank Correlation). Lower ABCA3 expression was positively associated with CBFB-MYH11 AML (p=0.002) and thereby with favorable cytogenetics (p=0.036) and low-risk AML (p=0.027). Higher ABCA3 expression was associated with higher relapse rate (p=0.006), shorter EFS (5-years, 34±9 vs 61±6 % p=0.0005), DFS (36±9 vs 62±6% p=0.0028), and OS (49±12 vs 79.5±5% p=0.0007). Multivariate analyses identified age, WBC, risk group, and ABCA3 expression as independent prognostic factors for EFS, DFS, and OS (Table 1). The validation cohort included 113 patients in whom the proportions of AML1-ETO- and MLL-positive AML were significantly higher than in the discovery cohort: 26,5% vs 6,7% (p&lt;10-4) and 24.8 vs 14.2% (p=0.03). There was no significant difference in patients' outcome between the 2 cohorts. Using the same cutoff value in the validation cohort, higher ABCA3 expression remained significantly associated with shorter 5-years EFS: 63±7% vs 43±9% (p=0.025) with a trend for shorter DFS: 45±9 vs 53±11% (p=0.065). Multivariate analyses identified ABCA3 expression as an independent negative prognostic factor for EFS and DFS (Table 1). In the entire patients population, ABCA3 expression independently predicted EFS, DFS, and OS (not shown). In the low- (n=74) and adverse-risk (n=59) groups, higher ABCA3 expression remained associated with shorter 5-years EFS (low: 46±12 vs 75±7%, p=0.006; adverse: 12±10 vs 44±16%, p=0.018), DFS (low: 49±13 vs 75±7%; high: 12±11 vs 45±16%, p=0.016), and OS (low: 76±10 vs 94±4%; adverse: 32±14 vs 57±18%, p=0.046). Conclusion. ABCA3 expression represents an independent prognostic factor in pediatric AML. As they indicate that the level of ABCA3 expression is significantly associated with survival for currently accepted cytogenetic and molecular prognostic categories, our findings suggest that assessing ABCA3 expression will permit a better assessment of disease risk. Finally our results suggest that inhibiting ABCA3 expression, such as with indomethacin, could be beneficial in order to overcome drug resistance in pediatric AML. Disclosures No relevant conflicts of interest to declare.

The Clinical and Biological Effects of Expression of Dedicator of Cytokinesis 1 (DOCK1) in Myelodysplastic Syndrome

Background: DOCK1 is an archetype member of the CDM (CED-5, Dock180, Myoblast city) superfamily of guanine nucleotide exchange factors and functions as an RAC activator.DOCK1 is involved in diverse biological processes including cytoskeleton rearrangement, cell motility and phagocytosis. Expression of DOCK1 is ubiquitous except in hematopoietic cells. Several studies highlighted the functional implications of DOCK1 in migratory and invasive properties of cancer cells, yet the roles of DOCK1 in hematological malignancies are not known.Materials and Methods: In this study, we focused on myelodysplastic syndrome (MDS) according to the diagnostic criteria of 2008 WHO classification, a clinically heterogeneous myeloid cancer whose pathogenesis remains enigmatic. We employed Affymetrix Human Transcriptome Array 2.0 to quantify the global expression patterns of the marrow mononuclear cells (MNC) in 243 newly diagnosed MDS patient recruited in the National Taiwan University Hospital (NTUH). We specifically analyzed the clinical, genetic, and prognostic significance of DOCK1 expression in various stages of MDS.Results: Our data showed that higher expression of DOCK1 conferred an unfavorable prognosis in MDS patients. Increased expression levels of DOCK1 was associated with higher-risk MDS defined by either revised international scoring system (IPSS-R) or WHO classification. Notably, DOCK1 expression levels in lower-risk MDS such as RA, RARS, RCMD, RCMD-RS, in which the marrow blast percentages were not increased, were still significantly higher than normal marrow (Figure 1), implying that the higher DOCK1 expression was independent of blast numbers in the samples, at least in these subgroups of patients. Among the 51 patients documented with leukemic transformation, 36 patients (70.6%) had higher DOCK1 expression compared with 70 out of 168 patients (41.7%) without transformation (P < 0.001). Regarding the genetic alterations, mutations in RUNX1, EZH2, SETBP1, ZRSR2, ASXL1 and TP53 were more frequently seen in the patients with higher DOCK1 expression, whilst SF3B1 mutation showed the contrary pattern. Remarkably, we demonstrated step-wise decreasing DOCK1 expression in the marrow MNC from higher-risk MDS, lower-risk MDS, to normal marrow, suggesting an impact of DOCK1 in pathogenesis in MDS. With a median follow up of 37.4 months, higher expression of DOCK1 was associated with shorter overall survival (OS) (median 13.7 months vs. 69.9 months) and shorter interval to leukemic transformation (median 23.6 months vs. not reached) after a follow up of 20.0 months (both P < 0.001) (Figure 3A and 3B). Independent analysis of DOCK1 expression showed higher level in the very poor cytogenetic subgroup in MDS (P < 0.001). In the multivariate analysis, higher DOCK1 expression proved to be a strong unfavorable prognostic factor for OS and leukemic transformation (P = 0.005 and P = 0.001, respectively) independent of other unfavorable factors including age, karyotype, and mutations of ASXL1, RUNX1, EZH2 and TP53.Conclusion: To our knowledge, our study is the first to show the clinical, genetic, prognostic, and biological association of DOCK1 expression in MDS patients. The step-wise increasing expression levels of DOCK1 in MNC from normal marrow, lower-risk MDS to higher-risk MDS patients highlight the hitherto unappreciated role of DOCK1 in the pathogenesis of MDS. Further studies on large and prospective cohorts are necessary to confirm our observations. Animal studies are needed to elucidate the in vivo roles of DOCK1 in MDS. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Improved Diagnosis of Intrachromosomal Amplification of Chromosome 21 (iAMP21) By Copy Number Profiling

Abstract Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of 2% childhood acute lymphoblastic leukaemia (ALL). iAMP21-ALL patients have precursor B-cell ALL, are older (median age 9 years), generally present with low white cell counts and have an inferior outcome when treated with standard therapy. Stratification to high risk treatment arms has significantly reduced their relapse risk, thus accurate diagnosis is essential. We have identified iAMP21 as a complex structure of one copy of chromosome 21, comprising multiple regions of gain, amplification, inversion and deletion, initiated through breakage-fusion-bridge cycles and chromothripsis. Currently, fluorescence in situ hybridisation (FISH), using probes directed to RUNX1, is the most reliable and convenient detection method, in which four or more copies of RUNX1 on a single abnormal chromosome 21 defines iAMP21. From our examination of several hundred cases of iAMP21-ALL, we have noted that, in the absence of metaphase FISH and/or in cases with an unusual cytogenetic presentation, reliance on FISH alone for accurate detection may be problematic. Among a collection of 210 patients with iAMP21-ALL, we performed SNP6.0 and Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA kit P327 iAMP21-ERG (MRC Holland) on 57 and 45 patients, respectively. Although chromosome 21 structure is highly variable between patients, a characteristic copy number profile emerged from the SNP6.0 data (Figure 1). In common, all cases were amplified across a 5.1Mb region, between 32,813,553 and 37,941,425bp that includes RUNX1 and 46 other known protein-coding genes. Asub-telomeric deletion occurred in 88% patients. We propose that this distinctive chromosome 21 copy number profile is used in addition to FISH for the definitive diagnosis of iAMP21-ALL in problematic cases. Support for this approach is provided by results from 5 patients from the Children's Oncology Group (North America, Australia and New Zealand) and ALL2003 (UK) treatment trials who met the iAMP21 FISH criteria but had atypical karyotypes or an unusual distribution of the additional RUNX1 signals that made confident diagnosis challenging. Clinical and cytogenetic data were collected and SNP6.0 and MLPA were performed to clarify the genomic alterations present in these cases. In patient #1, although five RUNX1 signals were observed per cell, two normal copies of chromosome 21 (each with one RUNX1 signal) were present with only three signals located to the abnormal chromosome 21. In patient #2, the additional signals were distributed between 2 different abnormal copies of chromosome 21. Metaphase FISH of patient #3 indicated that the RUNX1 signals were distributed between one normal copy of chromosome 21 and four small chromosomes, which were identified to originate from chromosome 21 by chromosome painting with a chromosome 21 specific probe (wcp21). In a further 2 cases (patients #4 and #5) the signals were too tightly clustered for the number to be discerned. In these 5 cases the characteristic SNP6.0 profile definitively confirmed the suspected diagnosis of iAMP21-ALL (Figure 2). Our previous SNP6.0 data have shown that the copy number profile of the iAMP21 chromosome remains stable between diagnosis and relapse, as well as in serial xenografts successfully transplanted with iAMP21-ALL cells over several generations of mice. However, while FISH analysis of patient #6 at diagnosis and relapse showed the same signal pattern at both time points, there was a change in the karyotype, involving translocation of part of the iAMP21 chromosome onto chromosome 11 at relapse. These observations indicate that the iAMP21 chromosome may become fragmented and distributed throughout the genome without changing its genomic profile. Collectively these observations indicate that the amplified segments resulting from the formation of iAMP21 chromosomes, with typical copy number profiles, can be distributed throughout the genome, either by translocation or fragmentation. Thus FISH together with chromosome 21 copy number profiling provide more accurate diagnosis of iAMP21-ALL than cytogenetics, which may be misleading. For laboratories with no access to SNP6.0 or other copy number arrays, we have shown that MLPA, with a kit specifically designed to detect chromosome 21 copy number changes, provides a reliable alternative (Figure 3). Disclosures No relevant conflicts of interest to declare.