Complex Aberrant Karyotype Research Articles

Abstract B03: Very poor outcome and chemoresistance of acute myeloid leukemia patients with TP53 mutations: Correlation with complex karyotype and clinical outcome.

Abstract Background: AML is a heterogeneous disease. The karyotype provides important prognostic information that influences therapy and outcome. Identification of AML patients (pts) with poor prognosis such as those with complex karyotype (CK) has great interest and impact on therapeutic strategies. TP53 is the most frequently mutated gene in human tumours. TP53 mutation rate in AML was reported to be low (2.1%), but the incidence of TP53 mutations in AML with a complex aberrant karyotype is still debated. Aims: To investigate the frequency of TP53 mutations in adult AML pts, the types of mutations, the associations with recurrent cytogenetic abnormalities and their relationship with response to therapy, clinical outcome and finally their prognostic role. To this aim, we focused on a subgroup of 172/886 AML pts treated at the Seràgnoli Institute of Bologna between 2002 and 2013. Patients and Methods: 886 AML patients were analysed for morphology, immunophenotype, cytogenetic and for a panel of genetic alterations (FLT3, NPM1, DNMT3A, IDH1, IDH2 mutations, WT-1 expression, CBF fusion transcripts). Of these, 172 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, Next-Generation Deep-Sequencing (Roche) and HiSeq 2000 (Illumina) platform. 40 samples were genotyped with Genome-Wide Human SNP 6.0 arrays or with CytoScan HD Array (Affymetrix) and analysed by Nexus Copy Number™ v7.5 (BioDiscovery). Results: Of the 886 AML patients, 172 pts were screened for TP53 mutations. Sanger sequencing analysis detected TP53 mutations in 29/172 AML patients with 36 different types of mutations; seven pts (4%) had 2 mutations. At diagnosis, the median age of TP53 mutated and wild type patients was 68 years (range 42-86), and 65 years (range 22-97) respectively. Median WBC count was 8955/mmc (range 580-74360/mmc) and 1240/mmc (range 400-238000/mmc). Conventional cytogenetics showed that: a) 52 pts (30,2%) had 3 or more chromosome abnormalities, i.e. complex karyotype; b) 71 (41,3%) presented with one or two cytogenetic abnormalities (other-AML); c) 34 pts (19,8%) had normal karyotype. Most of the TP53 mutated pts (23/29, 79.3%) had complex karyiotype, whereas only 6/29 mutated pts had “no complex Karyotype” (21% and 3% of the entire screened population, respectively). Overall, TP53 frequency was 44.2% in the complex karyotype group, suggesting a pathogenetic role of TP53 mutations in this subgroup of leukemias. As far as the types of TP53 alterations regards, the majority of mutations (32) were deleterious. Copy Number Alterations (CNAs) analysis performed on 40 cases by Affymetrix SNP arrays showed the presence of several CNAs in all cases: they ranged from loss or gain of the full chromosome (chr) arm to focal deletions and gains targeting one or few genes involving macroscopic (>1.5 Mbps), submicroscopic genomic intervals (50 Kbps - 1.5 Mbps) and LOH (>5 Mbps) events. Of relevance, gains located on chr 8 were statistically associated with TP53 mutations (p = 0.001). In addition to the trisomy of the chr 8, others CNAs, located on chromosomes 5q, 3, 12, 17 are significantly associated (p = 0.05) with TP53 mutations. WES analysis was performed in 37 pts: 32 TP53 were wt while 5 pts were TP53 mutated. Interestingly, TP53 mutated patients had more incidence of complex karyotype, more aneuploidy state, more number of somatic mutations (median mutation rate 30/case vs 10/case, respectively). Regarding the clinical outcome, as previously reported (Grossmann V. et Al. Blood 2013), alterations of TP53 were significantly associated with poor outcome in terms of both overall survival (OS) (median survival: 4 and 31 months in TP53 mutated and wild type patients, respectively; p<0.0001) and relapse free-survival (RFS) (p < 0.0001). Conclusions: Our data demonstrated that TP53 mutations are more frequent at diagnosis in the subgroup of complex karyotype AML (16.86%) (p< 0.0001–Fisher's exact test). They are mostly deleterious mutations and are significantly correlated with worst prognosis, fail to respond to therapy and rapidly progress. We recommend TP53 mutation screening at least in AML pts carrying either complex karyotype or chr. 8 gain. Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Cristina Papayannidis, Anna Ferrari, Stefania Paolini, Carmen Baldazzi, Chiara Sartor, Maria Chiara Abbenante, Sarah Parisi, Francesca Volpato, Ilaria Iacobucci, Antonella Padella, Viviana Guadagnuolo, Margherita Perricone, Valentina Robustelli, Claudia Venturi, Giorgia Simonetti, Elisa Zuffa, Eugenia Franchini, Emanuela Ottaviani, Nicoletta Testoni, Giovanni Martinelli. Very poor outcome and chemoresistance of acute myeloid leukemia patients with TP53 mutations: Correlation with complex karyotype and clinical outcome. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B03.

Complex Aberrant Karyotype in Patients with Post-Transplant Relapses of Acute Myeloid and Lymphoid Leukemias Evaluated By Serial Cytogenetic Assays, Including mFISH

Background. Prognosis of acute myeloid leukemia (AML) with complex aberrant karyotype (CAK) is poor. These patients (pts) are often refractory to both chemotherapy and allogeneic HSCT (alloHSCT). At diagnosis the proportion of these pts is about 6-8 %, but their frequency essentially increases in relapses after chemotherapy and alloHSCT. Mechanism of CAK development and its effect on relapses have been studied insufficiently.Material and methods. Serial cytogenetic assays, including multicolor FISH, were performed on bone marrow cells from 99 patients with post-transplant relapses (PTR) AML (n=61) and ALL (n=38). Median ages for AML and ALL pts at HSCT were 23 and 17 years (range, 2-60 and 0.8-51 years), respectively.Results. Aberrant karyotypes were found in 90 % AML and 97 % ALL pts, respectively (Table 1). Of note, the proportion of CAK in general group of ALL patients, as well as in that with more 4 chromosomal abnormalities was significantly higher, compared to AML (66% vs. 36%, P=0.003 and 61% vs. 33%, P=0.006, respectively).Table 1Frequency and characteristics CAKs in AML and ALL patients with the PTRsLeukemia typeAMLALLPPatients, n6138Karyotype, n (%)Normal6 (10 %)1 (3 %)NSAbnormal55 (90 %)37 (97 %)NSComplex karyotype, n (%)22 (36 %)25 (66 %)0.0033 abnormalities2 (3 %)2 (5 %)NS³4 abnormalities20 (33 %)23 (61%)0.006NS - not significantThe above difference in frequency of CAK in ALL and AML might be explained by higher frequency of myeloablative conditioning regimes in the ALL group, compared to AML (55% vs. 36 %, respectively; P=0.09). Of notice also, that fraction of CAK with 4 and more chromosomal abnormality in the group of children from 1 to 18 years was significantly higher in patients with ALL, as compared with AML (60% vs. 30%, respectively; P=0.03). Furthermore, a similar tendency was revealed also in the group of patients aged 19-40 years (difference insignificant; P=0.08) (Table 2).Table 2The incidence of CAKs with ³4 chromosomal abnormalities in PTRs in different age groups.Patients, n (%)Age (years)AMLALLP1-187/23 (30 %)15/25 (60 %)0.0319-408/25 (32 %)7/11 (64 %)0.0841-605/13 (39 %)1/2 (50 %)0.5Finally, our data demonstrate, the proportion of CAK to be significantly higher in PTR of ALL pts compared with those of AML, when they were transplanted in active disease phase (70% vs. 32%; P=0.007; Table 3).Table 3The incidence of CAKs with ³4 chromosomal abnormalities in PTRs, depending on the clinical status at alloHSCT.Patients, n (%)PClinical status at HSCTAMLALLCR 13/13 (23 %)2/7 (29 %)0.6CR ³26/14 (43 %)7/11 (64 %)0.2Active disease11/34 (32 %)14/20 (70 %)0.007Discussion. Our study shows that number of karyotype abnormalities in acute leukemia with CAK+ is closely associated with previous chemotherapy and/or conditioning regimes. Despite it, only alloHSCT gives a hope for treatment of these pts. In order to improve overall results of treatment several modifications of alloHSCT and post-transplant treatment have been recently suggested. The main of them, called as early alloHSCT, includes modified FLAMSA-RIC conditioning regime, alloHSCT before obtaining results of standard courses chemotherapy followed by DLI in escalating doses at post-transplant period (Schmid et al., 2012). If such a possibility for early alloHSCT is lost, another variant of PTR prevention may be used. The latter can include the same DLI, hypomethylating agents, ATRA etc. As for PTR prevention in CAK+ ALL it may be different from AML. In our opinion, in these pts should be treated at the first with such target agents as tyrosine-kinase inhibitors, rituximab, ATRA etc.Conclusion. According to our data, the frequency of CAK in PTRs is high not only in AML, but ALL too. Mechanisms of CAK formation as well as treatment of CAK+ leukemia began to elucidate. In our opinion, the leading place in this treatment is to be given an early alloHSCT.Reference. Schmid C., et al. Early allo-SCT for AML with a complex aberrant karyotype – results from prospective pilot study. Bone Marrow Transplantation 2012; 47: 46-53. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometry-Based Maturity Score As a Novel Prognostic Parameter in AML

Background: Based on cytogenetic and molecular phenotypes, the European LeukemiaNet (ELN) classification is widely accepted for risk stratification of patients with acute myeloid leukemia (AML). Single leukemic blast cell antigens were also shown to have prognostic value, but the influence of AML blast maturity within the different ELN risk groups is largely unknown.Methods: Our study includes 300 AML patients, who were diagnosed at our institution between 01/2003 and 04/2012 and in whom morphologic, immunophenotypic, cytogenetic and molecular data from primary diagnosis were available. A flow cytometric maturity score was developed in order to distinguish “mature” AML (AML-ma) from “immature” AML (AML-im) by means of a quantitative analysis of the expression of early progenitor cell antigens (CD34, CD117, and TdT). Clinical outcome analysis was performed both in the whole cohort and in the different ELN risk groups using Chi Square and Fisher’s Exact Test (CR/CRi) and Logrank Test (RFS, OS).Results: A mature AML blast phenotype (AML-ma) was associated with a significantly longer RFS (7.5 vs. 4.3 years) and OS (5.3 vs. 2.7 years) than an immature phenotype (AML-im) (p < 0.001). Low-risk phenotypes according to ELN (NPM1, PML-RARA) significantly accumulated in the AML-ma group, whereas high-risk aberrations (monosomal and complex aberrant karyotypes, -5 or del(5q), -7) had a higher frequency in AML-im.Interestingly, the differences in RFS and OS were maintained in a subgroup analysis within the different ELN risk groups. Statistical significance was obtained in the “intermediate risk” group with AML-ma being superior to AML-im (RFS: 7.0 years (AML-ma) vs. 3.3 years (AML-im); p = 0.002; OS: 5.1 years (AML-ma) vs. 3.0 years (AML-im); p = 0.022). In contrast, there was no correlation between AML blast maturity and CR/CRi rates.Conclusions: Our novel flow cytometric score easily determines AML blast maturity and can predict clinical outcome, even within the different ELN risk groups. Particularly for “intermediate risk” patients according to ELN, the score may be useful in post remission therapy decision-making. It remains to be clarified whether these results reflect differences in genetic instability and/or a different frequency of leukemia stem cells in AML-ma and AML-im. [Display omitted] [Display omitted] RFS (A) and OS (B) of AML patients with mature (AML-ma) and immature (AML-im) blast phenotype within the ELN “intermediate risk” group. AML-ma show a significantly longer RFS and OS compared to AML-im. DisclosuresNo relevant conflicts of interest to declare.

In-Vitro Actvity of Pixantrone (PIX) in Plasma Cell Leukaemia (PCL) and Early Clinical Activity in a Pixantrone–Pomalidomide–Dexamethasone (PiPoD) Combination

IntroductionPlasma cell leukemia (PCL) is a leukemic variant of Myeloma arising either de novo or from clinically pre-existent Multiple Myeloma (MM). Treatment options are limited and no drug has ever been explicitly registered in this indication. Furthermore, there is no generally accepted standard of care, although most haematologists would, depending on previous treatments, use combinations of MM drugs and chemotherapy as induction therapy.Pixantrone (PIX) is an aza-anthracenedione which has both alkylating and intercalating activity as well as a unique method of action the induction of cell death through mitotic perturbations and subsequent aberrant cell divisions. PIX is registered in both the US and EC for the treatment of relapsed or refractory aggressive NHL that has failed to respond to at least two previous regimens. Our group has previously shown significant in-vitro activity of PIX in both MM cell lines and primary patient materials*.MethodsPeripheral blood mononuclear cells from patient samples containing > 80% plasma cells at high leucocyte counts were isolated by density gradient using Ficoll and incubated for 24 and 48h with various pharmacokinetically realistic concentrations of PIX ranging from 0.01 – 5.00 µM at 37°C in a humidified air atmosphere. Apoptosis was determined by annexin/propidium iodide staining of the whole population (Pt. 1) and 7-AAD staining of the CD38 high/CD45 low cells on a FACSCanto using Diva software (BD).CasesPt 1.was a 63 year old female diagnosed MAR 2012 with an IgG lambda ISS stage I, Durie & Salmon stage III A MM. Cytogenetics showed a t(11;14). 1st line treatment consisted of VTD -> ASCT with a resulting PR. Post ASCT the pt. was stable on Rd – maintenance until JAN 13 when a leukemic transformation and a light chain escape phenomenon occurred. Cytogenetics showed clonal evolution with acquisition of additional aberrations (+18, amp 1p36, amp 11q22.3). From APR 2013 – JUL 2013 the pt. could be stabilized with a Bortezomib-liposomal Anthracyclin-Dexamethason combination. On progression she opted for palliative care and succumbed to PCL in AUG 2013. Her PCL cells were evaluated in-vitro.Pt. 2was a 51 years old female diagnosed with FLC kappa ISS stage III, Durie & Salmon stage III A MM in NOV 2011. In JUN 2014 the pt. presented with a 2nd relapse having been pre-treated with VTD-ASCT in 1st line, and Rd in 2nd line. Out of PR under continuous Rd for 9 months she developed a rapidly progressive PCL with a complex aberrant karyotype and massively elevated sFLC kappa. The patient was severely anaemic and thrombopenic from presentation onwards. Her PCL cells were evaluated in-vitro while treatment was initiated with the Pi-Po-D regime:Table 1Pi-Po-D regimeDRUGAPPLICATIONDOSAGESCHEDULEREPETITIONPixantronei.v.50mg/m²d1-8-15d28Pomalidomidep.o.4mgd1-21d28Dexamethasonp.o.40mgd1-4, 8,15d28In-Vitro-ResultsPt. 1 showed a significant induction of apoptosis concomitantly with a reduction of living cells after an incubation period of 24h starting with a PIX concentration of 2.50 µM and with 1.0 µM at 48h (Table 2). Similar results were obtained with the cells of Pt. 2, despite methodological problems however, plasma cells proofed not to be as sensitive as the whole cell population.Abstract 5732. Table 2In-Vitro activity of Pixantrone in pt. 1Pixantrone Concentration (µM)Living cells after 24 h (%)Apoptotic cells after 24h (%)Living cells after 48h (%)Apoptotic cells after 48h (%)0.0095.8504.1597.3502.650.0196.1503.8596.4503.550.0595.0504.9596.7503.250.1093.3506.6595.7004.300.2590.7509.2593.7006.300.5085.4014.6084.2515.751.0079.9520.0561.1038.902.5031.3068.7006.6593.355.0000.1599.8505.0099.70Clinical course with Pi-Po-D in PCLPt. 2 received the Pi-Po-D regime as no suitable clinical alternative was available and in-vitro data were promising. We noticed a sharp decline of sFLC (> 30%/3weeks) and peripheral plasma cell counts within cycle I as early sign of clinical activity. Unfortunately the pt. had a severe fall while being grade IV CTC thrombopenic and died from unstoppable intracranial haemorrhage.DiscussionPixantrone shows promising in-vitro activity in PCL and MM* at pharmacologically realistic concentrations and first signs of clinical activity in combination therapy. In our opinion PIX deserves further pre-clinical and clinical evaluation in MM and PCL.*J Clin Oncol 32, 2014 (suppl; abstr e19569) [Display omitted] DisclosuresOff Label Use: Use of Pixantrone in Plasma Cell Leukaemia.

Very Poor Outcome and Chemoresistance of Acute Myeloid Leukemia Patients with TP53 Mutations: Correlation with Complex Karyotype and Clinical Outcome

Background: AML is a heterogeneous disease. The karyotype provides important prognostic information that influences therapy and outcome. Identification of AML patients (pts) with poor prognosis such as those with complex karyotype (CK) has great interest and impact on therapeutic strategies. TP53 is the most frequently mutated gene in human tumours. TP53 mutation rate in AML was reported to be low (2.1%), but the incidence of TP53 mutations in AML with a complex aberrant karyotype is still debated.Aims: To investigate the frequency of TP53 mutations in adult AML pts, the types of mutations, the associations with recurrent cytogenetic abnormalities and their relationship with response to therapy, clinical outcome and finally their prognostic role. To this aim, we focused on a subgroup of TOT/886 AML pts treated at the Serˆgnoli Institute of Bologna between 2002 and 2013.Patients and Methods: 886 AML patients were analysed for morphology, immunophenotype, cytogenetic and for a panel of genetic alterations (FLT3, NPM1, DNMT3A, IDH1, IDH2 mutations, WT-1 expression, CBF fusion transcripts). Of these, 172 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, Next-Generation Deep-Sequencing (Roche) and HiSeq 2000 (Illumina) platform. 40 samples were genotyped with Genome-Wide Human SNP 6.0 arrays or with CytoScan HD Array (Affymetrix) and analysed by Nexus Copy Numberª v7.5 (BioDiscovery).Results: Of the 886 AML patients, 172 pts were screened for TP53 mutations. Sanger sequencing analysis detected TP53 mutations in 29/172 AML patients with 36 different types of mutations; seven pts (4%) had 2 mutations. At diagnosis, the median age of TP53 mutated and wild type patients was 68 years (range 42-86), and 65 years (range 22-97) respectively. Median WBC count was 8955/mmc (range 580-74360/mmc) and 1240/mmc (range 400-238000/mmc).Conventional cytogenetics showed that: a) 52 pts (30,2%) had 3 or more chromosome abnormalities, i.e. complex karyotype; b) 71 (41,3%) presented with one or two cytogenetic abnormalities (other-AML); c) 34 pts (19,8%) had normal karyotype.Most of the TP53 mutated pts (23/29, 79.3%) had complex karyiotype, whereas only 6/29 mutated pts had “no complex Karyotype” (21% and 3% of the entire screened population, respectively). Overall, TP53 frequency was 44.2% in the complex karyotype group, suggesting a pathogenetic role of TP53 mutations in this subgroup of leukemias. As far as the types of TP53 alterations regards, the majority of mutations (32) were deleterious..Copy Number Alterations (CNAs) analysis performed on 40 cases by Affymetrix SNP arrays showed the presence of several CNAs in all cases: they ranged from loss or gain of the full chromosome (chr) arm to focal deletions and gains targeting one or few genes involving macroscopic (>1.5 Mbps), submicroscopic genomic intervals (50 Kbps - 1.5 Mbps) and LOH (>5 Mbps) events. Of relevance, gains located on chr 8 were statistically associated with TP53 mutations (p = 0.001). In addition to the trisomy of the chr 8, others CNAs, located on chromosomes 5q, 3, 12, 17 are significantly associated (p = 0.05) with TP53 mutations. WES analysis was performed in 37 pts: 32 TP53 were wt while 5 pts were TP53 mutated. Interestingly, TP53 mutated patients had more incidence of complex karyotype, more aneuploidy state, more number of somatic mutations (median mutation rate 30/case vs 10/case, respectively).Regarding the clinical outcome, as previously reported (Grossmann V. et Al. Blood 2013), alterations of TP53 were significantly associated with poor outcome in terms of both overall survival (median survival: 4 and 31 months in TP53 mutated and wild type patients, respectively; p<0.0001) and relapse free-survival (RFS) (p < 0.0001). (Figure 1)Figure 1: Overall Survival curve of 172 AML patients with (red) or without (blue) TP53 mutations (p< 0.0001).Conclusions: Our data demonstrated that TP53 mutations are more frequent at diagnosis in the subgroup of complex karyotype AML (16.86%) (p< 0.0001–Fisher’s exact test). They are mostly deleterious mutations and are significantly correlated with worst prognosis, fail to respond to therapy and rapidly progress. We recommend TP53 mutation screening at least in AML pts carrying either complex karyotype or chr. 8 gain.Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Universitˆ 2010-12 (L. Bolondi), FP7 NGS-PTL project. DisclosuresNo relevant conflicts of interest to declare.

Leukemia Associated TP53 Mutations in AML Patients ARE Strongly Associated with Complex Karyotype and Poor Outcome

Background: AML is a heterogeneous disease with various chromosomal aberrations. The karyotype at diagnosis provides important prognostic information that influences therapy and outcome, and patients (pts) with complex karyotype (CK) have generally a poor outcome. TP53 is the most frequently mutated gene in human tumors. The reported TP53 mutation rate in AML is low (2.1%). In contrast, the incidence of TP53 mutations in AML with a complex aberrant karyotype is higher (69-78%).Aims: To investigate the frequency, the types of mutations, the associated cytogenetic abnormalities and the prognostic role of TP53 mutations in adult AML pts, we focused the screening on subgroups of AML with chromosome abnormalities.Patients and Methods: 886 AML patients were analysed at the Seràgnoli Institute of Bologna between 2002 and 2013 for morphology, immunophenotype, cytogenetic and for a panel of genetic alterations (FLT3, NPM, WT1, CBF fusion transcripts, DNMT3A, IDH1, IDH2, etc). Of these, 172 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, Next-Generation deep-Sequencing (Roche) and HiSeq 2000 (Illumina) platform (35/172 pts). 40 samples were genotyped with Genome-Wide Human SNP 6.0 arrays or with CytoScan HD Array (Affymetrix) and analysed by Nexus Copy Number™ v7.5 (BioDiscovery).Results: Of the 886 AML patients beforehand analysed, 172 pts were screened for TP53 mutations and were correlated with cytogenetic analysis (excluding 15 pts where the karyotype was not available). 1. Fifty-two pts (30,2%) have 3 or more chromosome abnormalities, i.e. complex karyotype; 2. 71 (41,3%) presented one or two cytogenetic abnormalities (other-AML) and 3. 34 pts (19,8%) have normal karyotype. Sanger sequencing analysis detected TP53 mutations on 29 patients with 36 different types of mutations; seven pts (4%) have 2 mutations. Mostly (23/29) of the TP53 mutated pts (79.3%) had complex karyotype while only 6/29 mutated pts have “no CK” (21% and 3% of the entire screened population). Overall, between pts with complex karyotype, TP53 frequency is 44.2%. Regarding the types of the TP53 alterations, 32 were deleterious point mutations (http://p53.iarc.fr/TP53GeneVariations.aspx) and 4 deletions.Forty pts were also analysed for Copy Number Alterations (CNAs) by Affymetrix SNP arrays: several CNAs were found ranged from loss or gain of complete chromosome (chr) arms to focal deletions and gains targeting one or few genes involving macroscopic (>1.5 Mbps), submicroscopic genomic intervals (50 Kbps - 1.5 Mbps) and LOH (>5 Mbps) events. Of relevance, gains located on chr 8 were statistically associated with TP53 mutations (p = 0.001). Seven genes are included in these regions (RGS20, TCEA1, LINC01299, ARMC1, MTFR1, RAD54B, KIAA1429). In addition to the trisomy of the chr 8, others CNAs, located on other chromosomes are significantly associated (p = 0.05) with TP53 mutations: loss of chr 5q, chr 3 (p22.3), chr 12 (p12.3) and the gain of chr 17 (p11.2), chr 16 (p11.2-11.3) and chr 14 (q32.33). The zinc finger gene ZNF705B, implicated in the regulation of transcription was the most differentially associated gene (gain).WES analysis was done in 37 pts, 32 TP53 were wt while 5 pts were TP53 mutated: of importance, CDC27, PLIN4 and MUC4 were found also mutated in 3 out of 5 TP53 mutated (60%).Clinical outcome: as previously reported, alterations of TP53 were significantly associated with poor outcome in terms of both overall survival and disease free-survival (P < 0.0001).Conclusions: Our data demonstrated that TP53 mutations occur in 16.86% of AML with a higher frequency in the subgroup of complex karyotype AML (p< 0.0001–Fischer’s exact test). Since TP53 mutations have predicted to be deleterious and significantly correlated with prognosis, TP53 mutation screening should be recommended at least in complex karyotype AML pts.Furthermore, although further studies in larger numbers of patients are needed, the gain of chromosome 8 was observed to be significantly associated to TP53 mutations pts.Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. DisclosuresMartinelli:Novartis: Speakers Bureau; Bristol Mayers Squibb: Speakers Bureau; Pfizer: Speakers Bureau.

Abstract 570: Tp53 mutation screening in adult acute myeloid leukemia (AML) patients shows a strong association with complex karyotype and poor outcome

Abstract Introduction: AML is a heterogeneous disease with various chromosomal aberrations. The karyotype at diagnosis provides important prognostic information that influences therapy and outcome. The TP53 gene is the most frequently mutated gene in human tumors. The reported TP53 mutation rate in AML is low (2.1%). In contrast, the incidence of TP53 mutations in AML with a complex aberrant karyotype (CK-AML) is higher (69-78%). Aims: to investigate the frequency and the prognostic role of TP53 mutations in adult AML patients (pts) focusing the screening on subgroups of pts with chromosome abnormalities. Patients and Methods: 106 adult AML pts with FAB-M0, M1, M2, M3, M4, M5, miscellaneous cytogenetic abnormalities and normal karyotype (nK-AML, 14/106 pts) were examined. Forty-four pts (41.1%) showed 3 or more chromosome abnormalities (CK-AML), 42 (39.6%) presented one or two cytogenetic abnormalities (other-AML) and in 6 cases the karyotype was not available. Genomic DNA and/or cDNA were isolated from mononuclear AML blast cells. TP53 mutation screening was performed on all 106 AML pts, in particular in 42 from exon (ex) 2 to 11, in 48 from ex 4 to 11 and in 16 pts from ex 2 to 8. Analysis was focused on coding sequences (RefSeq GRCh37/hg19 NG_017013.2). Results: By PCR and subsequent Sanger sequencing, mutations of TP53 were detected in 23 pts (21.1%). Seven pts revealed 2 mutations. 83% of all mutated pts had CK (19/23) by contrast the frequency of mutations was very low in “no CK-AML” pts (6.5%). Overall, mutated patients included 19/44 with CK-AML (43.2%); 1/6 (16.7%) with nK and 3/42 (7.1%) with other-AML. 26 TP53 point mutations [19 missense, 1 silent, 5 intron and 1 3’ untranslated region (UTR)] and 4 TP53 deletions were found. Twenty-six out of 30 mutations/deletions (86.6%) were located in the DNA binding domain, 2 in the carboxyl-terminal tetramerization and regulatory domains and 2 in the transcriptional activation domain. All mutations in coding regions were classified by the IARC database (http://p53.iarc.fr/TP53GeneVariations.aspx) as deleterious. Of note, alterations of TP53 were significantly associated with poor outcomes in terms of overall survival and disease free-survival (P &amp;lt; 0.0001). Conclusions: Our data demonstrated that mutations of TP53 occur in 21.7% of AML with a higher frequency in the subgroup of CK-AML (p&amp;lt; 0.0001-Fischer's exact test). Since TP53 mutations have predicted to be deleterious and significantly correlated with prognosis, TP53 mutation screening should be recommended in CK-AML pts. Supported by: EuropeanLeukemiaNet, AIL, AIRC, PRIN 2010-2011, Fondazione del Monte di Bologna e Ravenna, European Union Seventh Framework Programme [FP7/2007-2013]. Citation Format: Anna Ferrari, Ilaria Iacobucci, Cristina Papayannidis, Carmen Baldazzi, Chiara Sartor, Emanuela Ottaviani, Nicoletta Testoni, Valentina Robustelli, Margherita Perricone, Claudia Venturi, Maria Chiara Abbenante, Viviana Guadagnuolo, Antonella Padella, Federica Cattina, Giorgia Simonetti, Domenico Russo, Giovanni Martinelli. Tp53 mutation screening in adult acute myeloid leukemia (AML) patients shows a strong association with complex karyotype and poor outcome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 570. doi:10.1158/1538-7445.AM2014-570

Efficacy and feasibility of cyclophosphamide combined with intermediate- dose or high-dose cytarabine for relapsed and refractory acute myeloid leukemia (AML).

Approximately, 70 % of adult patients with de novo acute myeloid leukemia (AML) achieve a complete remission (CR) while 10-20 % of AML are refractory to induction chemotherapy. Furthermore, a significant proportion of AML patients in CR will relapse during or after consolidation treatment. There is no evidence for a standard salvage regimen and most centers use a combination of an anthracycline and cytarabine (AraC). The aim of this study was to investigate the impact of two age-adjusted regimens containing AraC and cyclophosphamide applied for the treatment of relapsed or refractory AML. We retrospectively analyzed 60 patients (24 male, 36 female; median age 56 years) with relapsed or refractory AML who were treated with a combination of AraC and cyclophosphamide monocentrically between October 2000 and January 2013. Two different protocols containing either high-dose (hAC) or intermediate-dose cytarabin (iAC) have been applied dependent on age and performance status. We demonstrate an overall response rate (CR + PR) induced by hAC and iAC of 56.7 %. Importantly, a complete remission rate (CR + CRp) of 52.2 % was found in patients who received the hAC regimen while only 8.8 % of patients achieved a CR following the iAC protocol (p < 0.001). The rate of refractory disease was 26.1 and 47.1 %, respectively. High-risk cytogenetics, i.e., a complex aberrant or monosomal karyotype had no effect on achievement of CR after hAC. In addition, there was no impact of activating FLT3 mutations on response to treatment according to the hAC regimen. In the cohort of patients treated with the iAC protocol, treatment-related mortality of 11.8 % within 60 days was observed but none of the patients who received the hAC regimen died within the first 2 months following chemotherapy. The toxicity profile was acceptable at both cytarabine dose levels. Importantly, 19 patients (82.6 %) of the hAC cohort underwent allogeneic hematopoietic stem cell transplantation (HSCT) as consecutive treatment. The hAC regimen represents a promising therapeutic approach to induce a second CR in younger patients with relapsed or refractory AML prior to HSCT without using anthracyclines.

Numerical Aberrations Involving The X Chromosome As a New Recurrent Aberration In Patients With Chronic Lymphocytic Leukemia

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.

Unbalanced Translocation t(5;17) Resulting In a TP53 Loss As Recurrent Aberration In Myelodysplastic Syndrome With Complex Karyotype

A complex karyotype, detected in approximately 10%-15% of patients with myelodysplastic syndrome (MDS), is associated with a very short median survival and a high risk of transformation into AML. The most frequent chromosome aberrations in complex karyotypes are deletion of 5q (del(5q)) and deletion of 17p (del(17p)) harboring the tumor suppressor gene TP53. It is still unclear, how complex karyotypes develop.We have identified an unbalanced translocation der(5)t(5;17)(q11∼q13;q11∼q13) in 199 patients with MDS or secondary AML after MDS. The cohort consists of 111 male (56 %) and 88 (44 %) female patients between 4 and 91 years of age (median age 68 years).In order to better understand the underlying pathomechanism of this aberration, we have investigated these cases in greater detail. For all patients, we performed cytogenetic banding analysis, fluorescence in-situ hybridization (FISH) and, in about one third, multicolor-FISH.In all patients, a complex aberrant karyotype with a median of 7 aberrations was observed, indicating high chromosomal instability. In 30 patients, clonal evolution was identified. To identify the breakpoints in 5q and 17q more precisely, array-CGH was performed in 7 patients. The breakpoints on 5q and 17p were located between the centromere of chromosome 5 and 5p15 and between the centromere of chromosome 17 and 17q22, respectively. The breakpoints were in gene-poor regions, suggesting that no fusion genes would result from these rearrangements. Notably, the breakpoints were all very close to the centromeric region and heterochromatin. It is known that an altered methylation of heterochromatic regions plays an important role in tumor development. Therefore, alterations of DNA methylation or histone modifications may be involved in the generation of the unbalanced translocation t(5;17).Using whole exome sequencing, we sought to define the mutational spectrum of complex karyotypes with t(5;17). In one patient we were able to analyse bone marrow cells from different time points: complex karyotype at diagnosis, complete remission and relapse with complex karyotype again. As possible candidate genes for driver mutations we identified mutations in the genes NF1, ETV6 (TEL) and KMT2C (MLL3). Of note, in this patient the allele frequencies of mutations affecting NF1 and KMT2C (MLL3) increased during the course of the disease, whereas the ETV6 (TEL) mutation found at diagnosis was lost at relapse indicating clonal evolution. Especially the identification of a mutation in NF1, a negative regulator of the RAS pathway, is of great significance. NF1 is encoded on 17q11.2. A possible underlying mechanism could be a downregulation of NF1 by a mutation of one allele and by a deletion evolved from the unbalanced translocation t(5;17) of the second allele. These data provide further evidence that the inactivation of NF1 seems to play an important role in clonal evolution and leukemic progression. Disclosures:No relevant conflicts of interest to declare.

Karyotypic Complexity In Acute Myeloid Leukemia In The Context Of Adverse Prognosis

IntroductionCytogenetic analysis is a mandatory component in the diagnostic evaluation of acute myeloid leukemia (AML) providing information regarding the biology of the disease including response or resistance to therapy. One of the cytogenetic markers which reflect an adverse outcome in conventional chemotherapy regimens is the complex aberrant karyotype consisting of multiple unrelated cytogenetic abnormalities. In AML two definitions have been established which differ in the perception of unbalanced aberrations as well as the number of single aberrations. The ELN classification scheme adopts three unrelated abnormalities while the UK MRC recently recommended four abnormalities as the most informative cut-off of complexity in the context of an adverse prognosis. The aim of this work was to study the best cut-off defining complexity (3 vs. 4) in AML with other cytogenetic high-risk markers. MethodsThe databases of three clinical multicentric, randomized, and prospective SAL trials (NCT 00180115, 00180102, and 00180167) were analyzed for AML patients with multiple cytogenetic aberrations as well as normal karyotypes (control group). Unbalanced abnormalities were counted as two aberrations according to the recommendations of the MRC (i.e. a single unbalanced translocation leading to gain and loss of chromosomal material as two unique abnormalities). The following single aberrations associated with an adverse prognosis according to ELN as well as UK MRC recommendations were included: inv(3), t(3;3), abn(3q), -5, del(5q), t(5q), t(6;9), -7, add(7q)/del(7q), t(11;v)(q23;v) (except t(9;11)), and abnl(17p). ResultsComplete data were analyzed from 2056 patients: normal karyotype (NK) n=1590, three aberrations (K3) n=65, ≥ four aberrations (K4) n=355, t(8;21)/inv(16)/t(16;16) and at least two additional aberrations n=46. All four groups differed significantly in 5–year overall survival (OS): 35% [95% CI 32–37], 19% [95% CI 9–29], 7% [95% CI 4–10], 67% [95% CI 53–81], respectively, p≤0.001. The K4 group had a significant inferior 5–year OS as compared to the K3 group, 19% [95% CI 9–29] and 7% [95% CI 4–10], p≤0.001. HSCT was performed in first remission in 25% of patients with K3 (n=16) and 17% of patients with K4 (n=59) (p=n.s.). As demonstrated earlier, multiple aberrations additional to the good risk anomalies (t(8;21), inv(16), or t(16;16)) did not impact on the favourable prognosis of the respective group. In the K3 and K4 groups single adverse risk abnormalities were found in 55% (abnl(17p) 12%) and 83% (abnl(17p) 37%) in these patients, respectively. A hyperdiploid karyotype (HDK) with gains of whole chromosomes without any structural aberration or monosomy was present in 14% of K3 and 3% of K4-patients. Interestingly, HDK with three trisomies as well as ≥ four trisomies led to a survival similar to K4 patients without HDK. Therefore, the K3 group lost its inferior survival as compared to NK when patients with adverse risk, which induce a worse prognosis per se, as well as HDK were excluded (5y–OS: 29% [9–44] vs. 35%, [95% CI 32–37], p=n.s.). HDK patients or patients with additional single adverse risk abnormalities had a worse survival compared to NK (5y–OS: 11%, [95% CI 0–32], p=0.012; and 15%, [95% CI 3–28], p=0.004 vs. 35%, [95% CI 32–37], respectively). In contrast, when comparing the K4 group after exclusion of adverse risk and HDK patients to NK, the K4 group remained its inferior OS as compared to NK, p<0.001. ConclusionsHence, our investigation confirms and therefore favors the ≥4 cut-off of complexity in the context of an adverse prognosis as proposed by the MRC with the exception of HDK patients. HDK patients should be considered as high-risk independent of the level of complexity. Whether K3 patients without single adverse risk abnormalities and HDK should be treated as intermediate risk, as suggested by our results, needs to be investigated prospectively in clinical trials. Disclosures:Platzbecker:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Clonal Heterogeneity As Detected by Metaphase Karyotyping Is an Indicator of Poor Prognosis in Acute Myeloid Leukemia

In acute myeloid leukemia (AML), studies based on whole-genome sequencing have shown genomic diversity within leukemic clones. The aim of this study was to address clonal heterogeneity in AML based on metaphase cytogenetics. This analysis included all patients enrolled onto two consecutive, prospective, randomized multicenter trials of the Study Alliance Leukemia. Patients were newly diagnosed with non-M3 AML and were fit for intensive chemotherapy. Cytogenetic subclones were detected in 418 (15.8%) of 2,639 patients from the whole study population and in 418 (32.8%) of 1,274 patients with aberrant karyotypes. Among those, 252 karyotypes (60.3%) displayed a defined number of distinct subclones, and 166 (39.7%) were classified as composite karyotypes. Subclone formation was particularly frequent in the cytogenetically adverse group, with subclone formation in 69.0%, 67.1%, and 64.8% of patients with complex aberrant, monosomal, and abnl(17p) karyotypes (P < .001 each). Two-subclone patterns typically followed a mother-daughter evolution, whereas for ≥ three subclones, a branched pattern prevailed. In non-core binding factor AML, subclone formation was associated with inferior event-free and overall survival and was confirmed as an independent predictor of poor prognosis in multivariate analysis. Subgroup analysis showed that subclone formation adds prognostic information particularly in the cytogenetic adverse-risk group. Allogeneic stem-cell transplantation improved the prognosis of patients with subclone karyotypes as shown in landmark analyses. Cytogenetic subclones are frequent in AML and permit tracing of clonal evolution and architecture. They bear prognostic significance with clonal heterogeneity as an independent adverse prognostic marker in cytogenetically adverse-risk AML.

Chromosome Banding Analysis of Peripheral Blood Lymphocytes Stimulated with IL-2 and CpG Oligonucleotide DSP30 in Patients with Chronic Lymphocytic Leukemia

Chromosomal aberrations play an important role as prognostic factors in chronic lymphocytic leukemia (CLL). These aberrations are mostly detected by fluorescent in situ hybridization (FISH), as chromosomal banding analysis has been scarce due to low proliferative activity of malignant B-lymphocytes in vitro. In 2006, a new method using stimulation with IL-2 and CpG oligonucleotide DSP30 for metaphase generation in CLL was published [1]. The objective of our study was to verify the efficacy of stimulation and to evaluate if the method is suitable for routine diagnostics. In total, peripheral blood samples of 369 CLL patients were analyzed in parallel by chromosomal banding analysis and by FISH probes for 13q14, 11q22-23, CEP12 and 17p13. Out of 369 patients, 307 (83%) were successfully stimulated for metaphase generation. Chromosomal aberrations were detected in 243 (79%) out of 307 patients evaluated by chromosomal banding analysis. Other aberrations that are not included into standard FISH panel were detected in patients karyotypes, e.g. del(6q), del(14q), t(14;18)(q32;q21), t(11;14)(q13;q32) and t(18;22)(q21;q11). One hundred and three (42%) patients showed complex aberrant karyotype not detected by FISH analysis. Stimulation with IL-2 and oligonucleotide DSP30 is an efficient method how to induce proliferation of malignant B-lymphocytes and allows detection of a substantial number of chromosomal aberrations in addition to those detected by standard FISH panel. Using this method in routine diagnostics is helpful particularly in identification of patients with complex aberrant karyotype.

Acute Erythroleukemia Morphology Does Not Confer an Unfavourable Prognosis in Itself. Results of a Subgroup Analysis of the Prospective Randomized AML96-, AML2003 and AML60+ Studies of the Study Alliance Leukemia (SAL)

AbstractAbstract 1481▪▪This icon denotes a clinically relevant abstract IntroductionAcute erythroleukemia (AEL) represents a rare type of acute myeloid leukemia accounting for less than 5% of all cases. So far, according to WHO classification this AML entity is thought to have a poor prognosis in itself. Design3267 patients with newly diagnosed AML were treated according to the protocols of the AML96, AML2003 or AML60+ studies of the Study Alliance Leukemia (SAL). 116 of these patients had acute erythroleukemia (AEL). The median age both for patients with AEL and non-AEL was 57 years.We assessed the influence of relevant clinical and demographic parameters, FLT3-ITD, NPM1 status and cytogenetics on complete remission rates (CR), overall survival (OS) and event free survival (EFS) separately in AEL and non-AEL patients. ResultsCompared to non-AEL AML, significantly more AEL were due to secondary causes than non-AEL AML (31% versus 20.4%; p=0.024). NPM1 mutations were found in 11.1% (out of 99) of the patients with AEL and in 32.8% (out of 2693) of the patients with non-AEL AML (p<0.001). Surprisingly, both the FLT3-ITD mutation and high FLT3-ITD ratios > 0.8 were found less frequently in AEL (FLT3-ITD mutation in 4.9% vs 23.3%; p=0.001; FLT3-ITD ratio >0.8 in 0% vs. 33.4%; p<0.001).The cytogenetic aberrations +8, −7 and complex aberrant karyotype (>/= 3 independent aberrations) were found more often in the AEL cohort (14.8%, 10.3% and 26.7%) than in the non-AEL AML cohort (8.9%, 5% and 13.2%) (p=0.036, p= 0.01 and p <0.001, respectively).Despite these differences, no significant differences in CR rates, OS and EFS were found between both groups. This finding was confirmed in a multivariate analysis including cytogenetics, molecular markers and clinical parameters (LDH, WBC, blast count, platelet count and ECOG). According to the analysis, ALE morphology was not an independent prognostic factor for OS and EFS.With the AEL group, patients with monosomy 7 (n=12) had a higher median blast count (NEC) of 62% and shorter median OS with 5.7 months compared to patients with AEL and no monosomy 7 (n=104) with a median blast count (NEC) of 43% (p=0.013) and an median OS with 15.7 months (p=0.016).A complex aberrant karyotype was found more often in patients with secondary AEL than in patients with de novo AEL (p=0.037). Significant differences were also seen in patients with AEL and complex aberrant karyotype compared to AEL without complex aberrant karyotype with respect to CR rates (54.8% versus 77.6%; p=0.016), OS (6.2 versus 17.4 months; p<0.000) and EFS (2.9 versus 6.2 months; p=0.007).In patients with AEL and abn17p/-17 (n=15), lower median platelet counts (31 versus 48 ×106/μl; p=0.017), a worse OS (6.4 vs 15.7 months; p=0.011) and EFS were observed (1.7 vs 5.7 months; p=0.046). ConclusionsAccording to our data, the characteristic morphological features of acute erythroleukemia do not confer an unfavorable prognosis in itself. Furthermore, we can confirm the relevant influence of established prognostic factors such as cytogenetics and disease status within the AEL subgroup. Disclosures:Off Label Use: sorafenib for the treatment of acute myeloid leukemia.

Sequential Evolution Versus a Single Catastrophic Event (Chromothripsis) in the Pathogenesis of AML with Complex Aberrant Karyotype

Abstract 517 Background:In AML, the concept of gradual evolution through a sequence of genetic alterations and clonal expansion was favored thus far, but has been recently challenged by a hypothesis that one catastrophic event generates multiple lesions across the genome in a single step. The term “chromothripsis” was introduced for a single catastophic event leading to the shattering of a single chromosome followed by rejoining and thereby resulting in a highly recombined chromosome (Stephens PJ et al., Cell 2011). Aim:We addressed the question whether AML with complex karyotype - defined as 4 or more abnormalities - evolves by sequential gradual acquisition of chromosome abnormalities or by a single catastrophic event. Patients and Methods:We selected 889 AML cases (de novo: n=634, secondary AML: n=164, therapy-related AML: n=91) presenting a complex karyotype at diagnosis. These were analyzed by chromosome banding analysis, 24-color-FISH, interphase-FISH, array CGH (n=78) and TP53 mutation analysis (n=195). Results:In 518/889 (58.3%) cases at least one subclone was observed that showed extra chromosome aberrations, thus demonstrating clonal evolution already at the timepoint of AML diagnosis. Within these, 77/518 (14.9%) cases showed a primary clone with only one cytogenetic abnormality. Two of these were recurrent single abnormalities: del(5q) (n=62), +8 (n=4). Clonal evolution was more frequent in cases with del(5q) as compared to those without (404/666 (60.7%) vs 117/223 (52.5%); p=0.034) while no association with loss of 7q, loss of 17p, TP53 mutation or type of AML (de novo vs secondary vs therapy-related AML) was observed. In 46 cases which evolved from MDS (n=43) or MPN (n=3) chromosome banding analysis had been performed prior to the diagnosis of AML. In 21/46 (45.7%) cases karyotype had not changed while in 25/46 (54.3%) cases clonal evolution had occurred. 57 cases were analyzed at relapse of AML; in 28 (49.1%) cases clonal evolution was detected.Additionally, 78/889 cases were evaluated by array CGH. The occurrence of chromothripsis was analyzed following the definition by Rausch et al. (Cell 2012) with at least 10 segmental copy-number changes involving two or three distinct copy-number states on a single chromosome. Evidence of at least one “shattered” chromosome was found in 24/78 (30.8%) cases. In 21 cases only one chromosome fulfilled these criteria, while in 3 cases chromothripsis affected two or more chromosomes. The chromosome most frequently affected by “shattering” was chromosome 11, observed in 18 (85.7%) cases, followed by chromosomes 2 and 21, which were affected in 2 cases each. Chromosomes 1, 5, 7, 13, 15, 16 and 20 showed signs of chromothripsis in single cases only. In 19/24 (79.2%) cases showing evidence of chromothripsis a high level amplification was observed for the MLL gene (11q23) in 17 cases and for the ERG gene (21q22) in 2 cases. Thus, amplifications were more frequent than in cases without chromothripsis (21/54, 38.89%; p=0.001), while no association was observed between chromothripsis and deletions of 5q, 7q or 17p or TP53 mut, presence of clonal evolution or type of AML (de novo vs secondary vs therapy-related AML). With respect to outcome within the subgroup of AML with complex karyotype only TP53 mutations and the presence of 5q deletions were significantly associated with overall survival (relative risk (RR) for shorter OS in TP53 mut cases: 3.19, p<0.0001, and in del(5q) cases: 1.61, p=0.006; median OS in TP53mut vs TP53wt cases: 4.6 vs 22.0 months, p<0.0001; median OS in del(5q) vs non-del(5q) cases: 5.7 vs 14.4 months, p=0.006), while presence of deletions of 7q, or 17p, chromothripsis and clonal evolution showed no impact on outcome. In multivariable Cox regression analysis only TP53mut had an independent association with shorter OS (RR: 3.12, p=0.001). Conclusions:1. In AML with complex karyotype harboring a 5q deletion the acquisition of additional abnormalities is more frequent than in cases without del(5q). 2. Chromothripsis does occur in AML with complex karyotype. However, the “shattering” of one chromosome was never observed as the sole abnormality, indicating that chromothripsis and sequential genetic evolution are not alternative but more likely combined mechanisms in AML. 3. Our data demonstrates that stepwise clonal evolution is more frequent than chromothripsis and thus likely to be more important in the pathogenesis of AML with complex karyotype. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

The Landscape of RUNX1 Mutations in Acute Myeloid Leukemia: Investigations On Stability of Mutations At Relapse and Utility As a Marker for Minimal Residual Disease

AbstractAbstract 657 Introduction:RUNX1 mutations constitute disease-defining aberrations in acute myeloid leukemia (AML) and were demonstrated to be particularly frequent in secondary and de novo AML with normal karyotype or non-complex alterations and to confer an unfavorable prognosis. Monitoring minimal residual disease (MRD) in AML has been shown to provide prognostic information and is increasingly used for treatment decisions. A variety of molecular markers has been identified suitable for MRD assessment, yet there still is a lack of such markers in a significant number of patients. The use of RUNX1 mutations may bridge a gap. Aims:1.Evaluate the stability of RUNX1 mutations between diagnosis and relapse.2.Test the utility and clinical relevance of RUNX1 mutations as MRD marker. Patients and Methods:RUNX1 mutation screening was prospectively performed in 814 patients with AML at diagnosis (645 de novo, 109 s-AML, and 60 t-AML). The median age of the patients was 69.6 years (range: 1 – 93 years), including 375 female and 439 male patients, respectively. 50.5% (411/814) of cases presented with a normal karyotype, 38.8% (316/814) with non-complex cytogenetic alterations, 9.6% (78/814) with a complex aberrant karyotype, and 1.1% (9/814) with prognostically favorable cytogenetics. Mutation analysis was performed using a sensitive next-generation amplicon deep-sequencing assay (454 Life Sciences, Branford, CT). Moreover, in a subset of 44 AML patients and additional 59 retrospectively analyzed cases the prognostic impact of MRD levels of RUNX1 mutations was studied at a second time point after completion of intensive induction therapy (median sampling interval: 128 days after diagnosis; range 60 – 180 days). In these follow-up samples the RUNX1 mutations already detected at diagnosis were investigated with a higher coverage (835-fold median coverage) as compared to the diagnostic assessment (759-fold median coverage) resulting in a sensitivity level of 1%. Furthermore, in 57 patients paired samples from diagnosis and relapse were analyzed to assess the stability of RUNX1 mutations. Results:211/814 patients (25.9%) were detected to carry RUNX1 mutations. The median clone size was 39% and revealed a significant heterogeneity ranging from 2% to 96%. 73.9% (156/211) of mutated patients carried one mutation only, whereas 26.1% (55/211) harbored two (n=46) or more (n=9) mutations. In detail, the 211 patients harbored a total number of 275 alterations in RUNX1: 42.5% (117/275) frame-shift mutations, 34.9% (96/275) missense, 14.2% (39/275) nonsense, 4.4% (12/275) exon-skipping/splicing, and 4.0% (11/275) in-frame insertion/deletion alterations, respectively. Regarding MRD assessment, patients were separated according to the median MRD level (3.92%; range 0.03% - 48.00%) into “good responders” (n=78) with MRD levels below 3.92% and “poor responders” (n=25) with MRD levels above 3.92%. This resulted in significant differences in both event-free survival (median 21.4 vs 5.7 months, p<0.001) and overall survival (73.3% vs 66.1% at 2 years, p=0.016). Moreover, in 57 cases the stability of individual RUNX1 mutations was studied at the time of relapse. In 46/57 (80.7%) cases the same alterations detected at diagnosis were present at relapse, whilst in 2/57 (3.5%) cases the RUNX1 mutation from the diagnostic sample was no longer detectable at relapse. Importantly, in 7/57 (12.3%) patients novel RUNX1 mutations were detected in regions different from those affected at diagnosis. Conclusion:Next-generation deep-sequencing accurately detects and quantifies RUNX1 mutations in AML with high sensitivity. RUNX1 mutations qualify as patient-specific markers for individualized disease monitoring. Thus, the measurement of mutation load by next-generation sequencing may contribute to refine the assignment into distinct risk categories in AML. Analysis of RUNX1 mutations should be considered for the complete coding region at relapse to detect new RUNX1 mutations. Disclosures:Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

Prognostic value of monosomal karyotype in comparison to complex aberrant karyotype in acute myeloid leukemia: a study on 824 cases with aberrant karyotype

AbstractIn acute myeloid leukemia (AML) the subset with complex karyotype (CK) is traditionally regarded as the worst prognostic group. However, ≥ 3, ≥ 4, or ≥ 5 abnormalities have been variably used for its definition. Recently, monosomal karyotype (MSK) was suggested to indicate an even inferior outcome. We tested which definition fits best to identify the most unfavorable subgroup. After excluding patients with t(15;17)/PML-RARA, t(8;21)/RUNX1-RUNX1T1, inv (16)/t(16;16)/CBFB-MYH11, and normal karyotype, 824 patients with AML with cytogenetic abnormalities were analyzed. Patients with MSK or CK defined as ≥ 3, ≥ 4, or ≥ 5 abnormalities showed an inferior overall survival compared with the respective remaining patients not fulfilling these criteria (for all, P < .001). Hazard ratios were 1.93, 1.68, 1.94, and 1.92. CK ≥ 4 as a single parameter identified the largest proportion of patients with very poor risk. However, combining CK ≥ 4 and MSK detected an even larger number of patients with very unfavorable outcome (261 of 824; 31.7%).

Molecular cytogenetics : an indispensable tool for cancer diagnosis

Cytogenetic aberrations may escape detection or recognition in traditional karyotyping. The past decade has seen an explosion of methodological advances in molecular cytogenetics technology. These cytogenetics techniques add color to the black and white world of conventional banding. Fluorescence in-situ hybridization (FISH) study has emerged as an indispensable tool for both basic and clinical research, as well as diagnostics, in leukemia and cancers. FISH can be used to identify chromosomal abnormalities through fluorescent labeled DNA probes that target specific DNA sequences. Subsequently, FISH-based tests such as multicolor karyotyping, comparative genomic hybridization (CGH) and array CGH have been used in emerging clinical applications as they enable resolution of complex karyotypic aberrations and whole global scanning of genomic imbalances. More recently, crossspecies array CGH analysis has also been employed in cancer gene identification. The clinical impact of FISH is pivotal, especially in the diagnosis, prognosis and treatment decisions for hematological diseases, all of which facilitate the practice of personalized medicine. This review summarizes the methodology and current utilization of these FISH techniques in unraveling chromosomal changes and highlights how the field is moving away from conventional methods towards molecular cytogenetics approaches. In addition, the potential of the more recently developed FISH tests in contributing information to genetic abnormalities is illustrated.

Gene Amplifications Are Rare Events in AML and MDS and Are Associated with Complex Karyotype, TP53 Deletions and Very Poor Survival

Abstract 2524 Background:Gene amplifications are usually defined as the presence of more than 6 copies of a gene per cell. These supernumerary copies are located either extrachromosomally in double minutes (small acentric chromosome structures) or intrachromosomally in homogeneously staining regions. Such gene amplifications are rare but recurrent phenomenons in AML and MDS. So far, only small case studies have been reported. Aims: 1) to determine the frequency of gene amplifications in a large AML and MDS cohort, 2) to characterize the amplified regions and accompanying abnormalities, 3) to analyze the impact of specific amplifications on outcome. Patients and Methods: Out of 4,248 AML and 3,689 MDS studied by chromosome banding analysis (CBA) we identified 105 AML patients (2.5%) with gene amplifications (80/3,478 (2.3%) de novo AML, 7/478 (1.5%) s-AML, 18/292 (6.2%) t-AML) and 46 (1.2%) MDS. All cases with gene amplification were studied by 24-color FISH in addition to CBA in order to characterize the amplified regions and the accompanying abnormalities in detail. Further, interphase (IP)-FISH was performed with probes for TP73, HOXD cluster, EVI1, CMYC, JAK2, NUP214, MLL, ZNF4, GLTSCR1, ERG, RUNX1, BCR and CLRF, if 24-color FISH suggested amplification of these genes. In a subcohort of 12 patients genomic arrays (Human CGH Whole-Genome Array, NimbleGen, Madison, WI; Genome-Wide Human SNP Array 6.0, Affymetrix, Santa Clara, CA) were performed to characterize the amplified region in more detail. Results: In 28/151 pts (18.5%) the amplification was located in double minutes and in the remaining 123 cases intrachromosomally (81.5%). The following regions were found to be amplified: 1p (n=1, containing TP73), 2q (n=1, containing HOXD cluster), 3q (n=1, containing EVI1), 7p (n=1), 8q (n=29, containing CMYC in 28/29 pts), 9p (n=2, containing JAK2), 9q (n=1, containing NUP214), 11q (n=81, containing MLL in 80/81 cases), 13q (n=2), chromosome 19 (n=10, containing ZNF4 in 5 cases and GLTSCR1 in 2 cases), 21q (n=19, containing ERG in 16 and RUNX1 in 6 cases), 22q (n=2, containing BCR) and Xp (n=1, containing CLRF). In median, 8 accompanying chromosomal aberrations per cases were observed (range 0–21). 124/151 (82.1%) cases had a complex aberrant karyotype, defined as 4 or more abnormalities. However, in 2 cases the double minutes were the sole abnormalities. Gene amplifications were not observed in patients with disease defining aberration like t(8;21), inv(16), t(15;17) or those carrying NPM1 or CEPBA mutations (mutation status available in 89 and 37 patients, respectively). However, 2 cases with t(6;11)(q27;q23)/MLL-AF6 harbored an amplification of CMYC. In 88 cases the copy number status of TP53 was determined by IP-FISH. A TP53 deletion was detected in 49 (55.7%) pts. Interestingly, 14/16 (87.5%) cases with double minutes compared to 35/72 (48.6%) patients with intrachromosomal gene amplifications showed a TP53 deletion (p=0.004). Only 3 chromosomal regions were amplified in double minutes: 8q24/CMYC (n=14), 11q23/MLL (n=12) and 13q (n=2). In 6 cases with 8q amplification, 2 cases with 11q amplification and 4 cases with 21q amplification genomic arrays were performed. While the amplified region was quite homogeneous in cases with 8q amplification and contained in all cases the CMYC gene, amplified regions on 11q and 21q were heterogeneous and amplified regions were interspersed with regions of deletions. Interestingly, MLL and CBL were amplified in all analyzed cases with 11q23 amplification. In all analyzed cases with 21q22 amplification ERG was located within the amplified region while RUNX1 was amplified in 3/4 cases and deleted in the remaining case. In AML, overall survival was short in cases with gene amplification (median OS 11.3 months) and was particularly short in cases accompanied by complex karyotype (6.3 mo vs 18.6 mo in cases with non-complex karyotype, p=0.049). Conclusions: 1) MLL is the most frequently amplified gene in AML and MDS. 2) Gene amplifications occur predominantly in complex aberrant karyotypes. 3) Prognosis is poor in this subset of cases, and even more dismal if these amplifications are accompanied by complex karyotype. 4) The association of gene amplifications, complex karyotypes and TP53 deletions suggests that the unfavorable prognosis is due to chromosome instability facilitating the occurrence of additional genetic aberrations triggering resistance to chemotherapy. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Incidence of Central Nervous System Involvement in AML: A SAL-Tor Vergata Analysis

Abstract 4236 Background:Acute myeloid leukemia (AML) involves infrequently the central nervous system (CNS). However, most studies examined paediatric patients. Thus little is known regarding CNS-involvement in adult AML. Therefore, we analyzed the data of patients with AML treated in SAL study group and Tor Vergata study group for CNS involvement. Methods:In this retrospective analysis in adult AML patients included in the prospective AML96- (NCT00180115), AML2003- (NCT00180102), AML60+ trials (NCT00180167) of the SAL study group and patients that were admitted to the University Hospital of Rome Tor Vergata from 2005 to 2011, 3863 patients were evaluated for CNS involvement of AML. The median age of the patients was 49 years (range, 18 –74 years). A total of 30 patients had CNS involvement at time of initial presentation and 30 patients had CNS involvement at first, second or third relapse. CNS involvement was proven depending on morphology and/ or flow cytometry of the Cerebrospinal fluid (CSF). Clinical variables of statistical significance were compared using the χ2-test for dichotomized variables and the Mann-Whitney U-test for continuous variables. The method of Kaplan Meier was used to estimate OS and event-free survival (EFS). Survival distributions were compared using the log-rank test. Results:21 of 1862 patients (1%) in the AML96-trial, 26 patients of 1182 (2.2%) in the AML2003 trial, 3 of 486 patients (0.6%) in the AML60+ trial, and 10 of 270 patients (3.7%) in the University of Rome Tor Vergata studies had CNS involvement resulting to a total of 60 patients with a combined overall incidence of 1.6%. 30 patients had CNS involvement at the initial presentation and 30 patients at relapse of AML. The majority of patients (n=53, 88%) had de novo AML. Data from the Tor Vergata study group need further analysis therefore in this abstract patients from SAL study group were included. Extramedullary AML other than CNS involvement was observed in 21 patients (42 %) as compared to 246 (7%) in patients without CNS involvement, p < 0.001. Complex aberrant karyotype was not significantly higher in patients with CNS involvement as compared to those without, whereas patients with CNS involvement had a higher tendency for trisomy of chromosome 8 (n=8, 16%) in comparison with patients without CNS involvement (n= 277, 8%), p=0.03. AML FAB M5 occurred more frequently in patients with CNS involvement (n= 15, 30%) as compared to those without (n=459, 13%), p=0.01. Patients with CNS involvement at diagnosis had significantly higher lactate dehydrogenase (LDH) levels (1318 vs. 665 IE/l, p< 0.001) and higher white blood cell count (WBC) (64 vs. 35 Gpt/l, p<0.001). Only 7 of 30 patients (23%) with CNS involvement at initial diagnosis developed relapse and none of them had CNS involvement at the time of relapse, whereas almost all patients with CNS involvement at relapse developed again systemic and CNS-relapse. On the other hand, there was no significant difference in the number of patients who achieved complete remission between these two groups. Comparing patients with CNS involvement with patients without CNS involvement at the initial diagnosis no significant difference in overall survival at 2 years (40% vs. 41%) and event free survival at 2 years (20% vs. 26%) was observed. Conclusion:CNS involvement in AML is a rare entity and it is accompanied with higher incidence of other extramedullary AML. This represents the largest analysis of AML patients with CNS involvement studied in the literature. Factors associated with an increased risk of CNS involvment include: trisomy of chromosome 8, FAB M5, higher LDH levels and higher WBC count at diagnosis. Patients with CNS disease at initial diagnosis of AML have similar survival outcomes as compared to those without CNS disease if treated by intrathecal therapy, whereas extramedullary relapse within the CNS is associated with poor outcome. Disclosures:No relevant conflicts of interest to declare.