Complex Aberrant Karyotype Research Articles

Prediction of Complete Remission and Survival in Acute Myeloid Leukemia Using Supervised Machine Learning

Prediction of Complete Remission and Survival in Acute Myeloid Leukemia Using Supervised Machine Learning

Whole Genome Sequencing Identifies Non-KIT Mutations and Cytogenetic Aberrations in Systemic Mastocytosis but Has Limited Sensitivity for Detection of KIT D816V

▪Background: Systemic mastocytosis (SM) is a hematologic neoplasm characterized by the infiltration of clonal mast cells in the bone marrow or other extra-cutaneous organs. The clinical course varies between advanced and non-advanced (indolent and smoldering SM) forms of SM. The vast majority of patients harbor the activating D816V mutation in the KIT tyrosine kinase. Additional somatic mutations in other genes have been recognized as risk factors in SM. Cytogenetic aberrations are rarely found in SM but have been associated with advanced disease. Whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) have been described as an alternative to cytogenetics and targeted molecular genetic analysis in myeloid cancers.Aim: To assess the ability of WGS/WTS to detect cytogenetic aberrations and recurrent somatic mutations in SM.Methods: 120 patients (51 female, 69 male) diagnosed with SM were analyzed with WGS/WTS and results were compared with orthogonal data of KIT D816V PCR, targeted sequencing, and cytogenetics. 47 patients (39%) were diagnosed with advanced SM (1 mast cell leukemia, 3 aggressive SM, 43 SM with associated hematologic neoplasm). For WGS, 2x151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). BaseSpace's Tumor/Normal app v3 was used to call variants with Strelka Somatic Variant Caller v2.4.7 and structural variants (aberrations with >50bp in size) with Manta (v0.28.0). Genomic DNA from a mixture of multiple anonymous donors (Promega, Fitchburg, WI, USA) was used as normal. For WTS, 2x101 bp paired-end reads were produced with a median of 50 mio. reads per sample, aligned with STAR v2.5.0, and variants were called using Isaac Variant Caller v2.3.13.Results: WGS/WTS detected cytogenetic aberrations in 21% of patients: 2 patients displayed a complex aberrant karyotype, 3 balanced structural aberrations, 16 copy number alterations, and 6 copy number neutral losses of heterozygosity. Aberrations detected by chromosome banding analysis were also found by WGS in all but three patients (small clones with aberrations present in ≤20% of metaphases and <10% of interphase nuclei as determined by FISH). In contrast, WGS/WTS detected additional aberrations in 16 patients. The frequency of chromosomal aberrations detected by WGS/WTS was higher in advanced compared to non-advanced SM (34% vs. 12%, p<0.05). KIT D816V was detected by PCR in 98%, by WGS in 21% and by WTS in 46% of patients. The detection rate by WGS was significantly higher in advanced (36%) compared to non-advanced SM (12%, p<0.05) while no difference was observed for WTS (45% vs. 47%). Somatic mutations outside of KIT were analyzed within a subset of 121 genes recurrently mutated in hematologic neoplasms. 46% of patients showed non-KIT mutations with a median of 2 mutations per patient. Both frequency of non-KIT mutations as well as the median number of mutations per patient was higher in advanced (83%; n=3) compared to non-advanced SM (22%, n=1, p<0.05). Finally, we analyzed the impact of genetic aberrations on survival in our SM cohort. Patients were grouped according to the presence of chromosomal aberrations and gene mutations (non-KIT) as assessed by WGS/WTS. SM patients with both types of aberrations (n=16), one type of aberration (n=47; gene mutations only n=38; chromosomal aberrations only n=8), or no aberration but KIT D816V (n=57) showed significant differences in overall survival (p<0.05, Figure 1).Conclusions: WGS/WTS has limited sensitivity for detection of KIT D816V in SM. This finding can be explained by the low KIT D816V mutation burden typically found in bone marrow aspirates of SM patients. In line, we observed a slightly higher detection rate in advanced SM and in RNA-based WTS analysis. As WGS/WTS will be applied for the diagnostic workup of myeloid malignancies in the future and SM associated with other hematologic neoplasms may be overlooked if not specifically investigated, additional PCR-based techniques are still mandatory to rule out KIT D816V as a diagnostic criterion for SM. In contrast, WGS/WTS detects both chromosomal aberrations and additional gene mutations in patients with SM and can be used as an alternative to cytogenetics and targeted sequencing for risk assessment. In particular, the absence of genetic aberrations in WGS/WTS identifies SM patients with indolent course of the disease and favorable prognosis. [Display omitted] DisclosuresHoermann: Novartis: Honoraria. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Impact of 9q Deletions on the Classification in AML

Background: In the 2016 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia the deletion of the long arm of chromosome 9 (del(9q)) was excluded as a defining cytogenetic abnormality for AML with myelodysplasia-related changes due to its frequent association with NPM1 and biallelic CEBPA mutations.Aim: Investigation of the frequency of del(9q) in AML and its accompanying molecular and cytogenetic abnormalities to assess whether or not del(9q) is associated with a myelodysplasia-related mutation profile.Patients and Methods: We evaluated 9762 AML patients (pts) diagnosed between 2005 and 2017 for del(9q) using chromosome banding analysis (CBA). Pts with del(9q) were further analyzed for mutations in NPM1 , CEBPA and RUNX1 with amplicon sequencing to categorize them according to the WHO classification. Pts without a class defining aberration or a complex aberrant karyotype were screened for AML- or MDS-related mutations (ASXL1, BCOR, EZH2, FLT3- ITD , FLT3- TKD , SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, WT1 and ZRSR2 ). Variants of unknown significance were excluded from statistical analysis. The size of the deleted region on chromosome 9 was evaluated by array CGH (Agilent Technologies, Santa Clara, CA) and NUP98 -rearrangements were investigated by FISH(MetaSystems, Altlussheim, Germany).Results: In 114/9762 pts (1 %) a del(9q) was detected by CBA. This cohort with del(9q) comprised 60 male and 54 female pts, median age was 63 years (range: 16 - 90 years). Del(9q) was accompanied by the following rearrangements: RUNX1-RUNX1T1 rearrangement in 20/114 pts (18 %), PML-RARA in 5/114 pts (4 %), KMT2A in 2/114 pts (2 %) and MECOM in 1/114 pts (1 %). In 20/114 pts (18 %) a NPM1 mutation and in 7/114 pts (6 %) a biallelic CEBPA mutation was observed. Furthermore, RUNX1 mutations were found in 3/114 pts (3 %). The remaining 56 pts (49 %) were divided into the following three groups: pts with del(9q) as a sole cytogenetic abnormality (del(9q) sole, 8/114 pts, 7 %), pts with one or two additional cytogenetic aberrations (del(9q) other, 19/114 pts, 17 %) and pts with complex aberrant karyotype (complex, 29/114 pts, 20 %) harboring 4 to 29 cytogenetic aberrations (median: 8). Cases with del(9q) sole and other (n = 27) were further characterized. NUP98 -FISH analysis revealed a NUP98 -rearrangement in 5/27 pts (19 %) in the del(9q) sole and other group. Moreover, two pts with del(9q) showed AML specific rearrangements (t(8;16)(p11;p13) and t(3;5)(q25;q35)). The most frequently mutated genes in del(9q) sole and other were: FLT3 -ITD (8/23 pts, 35 %), TET2 (7/27 pts, 26 %), FLT3 -TKD (4/21 pts, 19 %) and WT1 (3/26 pts, 11 %). A very high frequency of FLT3 -ITD and FLT3 -TKD were found in pts with del(9q) and balanced rearrangements (5/7 pts, 71 %).To identify whether pts with del(9q) sole and other rather resemble a molecular mutation profile typical for secondary AML arising from MDS (s-AML), the presence of gene mutations related to s-AML (according to Lindsley, et al., 2015, Blood) was evaluated. 9 pts (33 %) had a mutation in at least one of these genes, 14 pts (52 %) showed no such mutation and 4 pts (15 %) could not be fully analyzed.Array CGH showed that in pts with del(9q) sole the deletion size ranged from 14 - 42 Mb (median: 28 Mb), in pts with del(9q) other from 19 - 70 Mb (median: 32 Mb). A minimal deleted region for pts with del(9q) sole and other could not be determined for all pts due to the strong variation between the respective deleted regions.Follow up data was available for 75 pts with a median follow-up of 27 months. The median overall survival (OS) for pts with RUNX1-RUNX1T1 , NPM1 and bialleic CEBPA mutation was not reached. It was 73 month for pts with del(9q) sole and other and 16 month for pts with complex karyotype, RUNX1 mutations or MECOM rearrangements. The OS of first two groups did not differ significantly, but was significantly distinct from the last group (p = 0.03 and 0.03, respectively).Conclusion: In 1 % of AML pts a del(9q) was present. Del(9) frequently co-occured with RUNX1-RUNX1T1 , biallelic CEBPA and NPM1 mutations, NUP98 -rearrangements and other AML-typical translocations. A mutation signature typical for s-AML was infrequent. Thus, it seems reasonable that the del(9q) is no longer regarded as a defining cytogenetic abnormality for AML with myelodysplasia-related changes, in particular as prognosis in del(9q) cases with non-complex karyotype is favorable. DisclosuresBalk:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Genomic Profiling of Blast Phase in Philadelphia-Negative Myeloproliferative Neoplasms

Background Classic Ph-negative myeloproliferative neoplasms (MPN) include essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Clonal evolution can lead MPN patients in chronic phase (CP) to developacute myeloid leukemia (AML), called blast phase (BP), a rare and late event in the natural history of MPN.Aim To evaluate differences in clinical, cytogenetic and molecular features and outcome in 92 patients with BP MPN, according to MPN diagnosis and mutational profile.Methods We identified in our database all patients affected with ET, PV and PMF who developed AML according to 2016 WHO criteria and for whom at least one DNA sample was available to define the mutational status of the 3 MPN driver genes (JAK2, CALR, MPL). Target sequencing was performed using the Illumina TruSight Myeloid Sequencing Panel kit on the HiSeq 2000 platform. For each patient circulating granulocytes or CD34+ cells were analyzed as neoplastic tissue and CD3+ T cells as control tissue; data were analyzed with the pipeline for somatic variant calling implemented at CeMM. Treatment used in BP was classified as palliation (supportive care or low intensity chemotherapy) or induction chemotherapy (de novo AML-like therapy).Results We retrospectively identified 92 patients who progressed to BP, with a known molecular profile: 29 ET (18 JAK2 V617F , 9 CALR, 1 MPL mutated and 1 triple-negative), 39 PV (all JAK2 V617F mutated) and 24 PMF patients (18 JAK2 V617F, 1 CALR, 2 MPL mutated and 3 triple-negative).Median age at BP was 70.8 years (range 46.3-86); age at evolution was not significantly different (P 0.182) in the 3 diagnostic categories (ET, PV, PMF).The complete blood count at leukemic evolution was not influenced by the initial diagnosis.Median time to leukemic evolution was 110 months (range 3.6-367.6) and it was shorter in PMF compared to ET (29.8 vs 153.4 months, P < 0.001) and PV (29.8 vs 129.7 months, P < 0.001). Moreover, time to leukemic evolution was shorter in JAK2 V617F positive ET compared to CALR mutated ET (117.2 vs 210.9 months, P 0.027), but not statistically different in JAK2 mutated compared to CALR mutated PMF (P 0.312).At the time of BP, 35 out of 49 patients (71%) for whom cytogenetic analysis was available showed an abnormal karyotype (22 patients with complex karyotype or high risk aberrations).JAK2 mutated MPN can evolve into JAK2 wild type AML (8 of 32 patients with blast DNA available), whereas CALR and MPL mutations were identified also in AML blasts in all patients for which DNA was available (3 for CALR and 3 for MPL mutated AML).We performed targeted re-sequencing in 53 patients, in 23 studying both the CP and the BP sample. Overall, we identified 176 variants in 39 different genes (68% were missense mutations); the most commonly involved pathways were DNA methylation (22%), transcriptional regulation (20%) and chromatine regulation (18%). The most frequently involved genes were TET2, TP53, RUNX1 and ASXL1 . In the 23 paired cases, we observed a statistically significant increase in the median number of mutations in the BP, compared to CP [4 (range 1-10) vs 2 (range 0-9); P 0.002]. This was observed in JAK2 mutated patients (P 0.006) and in those with PV (P 0.008), but not in subjects with TE or PMF. In our paired-sample cohort, BP evolution was associated with the acquisition of mutations (e.g. IDH1) and/or an increase in mutation occurrence or variant allele frequency (VAF) of genes implicated in leukemogenesis including TET2 (P 0.026), RUNX1 (P 0.003), ETV6 (P 0.068) and TP53 (P 0.003), the latter known to be involved also in JAK2 mutated MPN evolving into JAK2 wild type AML. In addition, VAFs suggested an heterogeneous clonal architecture and evolution of somatic mutations in these patients.Outcome was dismal, with a median OS of 3.8 months (range 3.1-5.3), independently from MPN diagnosis (P 0.603) and treatment during BP (5.6 months with induction chemotherapy vs 3.5 months with palliation; P 0.171).Conclusion Clinical phenotype and outcome of BP is not influenced neither by the diagnosis in CP nor by the type of driver mutation; likewise the outcome is poor irrespective of treatment. PMF patients have a shorter time to BP than ET and PV patients; in ET JAK2 V617F mutation is associated with a shorter time to BP compared to CALR mutation. TP53, RUNX1, TET2 and ETV6 are more frequently involved in leukemic evolution and the number of mutations increases from CP to BP. DisclosuresNo relevant conflicts of interest to declare.

High Frequency of P53 Mutations Detected by a Microarray Resequencing Assay, the AmpliChip P53 Test, in AML with a Complex Aberrant Karyotype.

Acute myeloid leukemia (AML) is heterogeneous in morphology, cytogenetics, and prognosis. On the basis of cytogenetics it can be subdivided into three groups with favorable, intermediate, or unfavorable prognosis. Accounting for 10%–20% of all AML cases, AML with complex aberrant karyotypes is more frequent at higher ages and is the subgroup with the worst prognosis. AML with complex aberrant karyotype is generally defined by the presence of three or more clonal chromosome abnormalities. Genetically, it has a characteristic pattern of chromosomal gains and losses, resulting also in a unique gene expression pattern including upregulation of genes involved in DNA repair. One of the important characteristics of AML with complex aberrant karyotype is the high incidence of deletions and/or mutations in the tumor suppressor p53 gene (60%–80%) which occur very unfrequently in other AML subgroups. Here we present data on 119 AML cases with complex aberrant karyotypes that were analyzed with the new AmpliChip p53 Test, a microarray-based resequencing test that is designed to detect single nucleotide changes in the entire coding region of the p53 gene. The AmpliChip p53 Test queries for the presence of sequence alterations through comparative analysis of the microarray hybridization pattern of a series of probes from sample DNA to wild type (wt) reference DNA. Starting from genomic DNA, the 10-hour assay includes PCR amplification, fragmentation and labeling, hybridization to the microarray, washing and staining, and mutation data analysis. By use of this technique we have detected p53 mutations located in exons 4–10 in 89/119 (74.8%) samples. These mutations included missense mutations, frameshift mutations, splice site mutations, silent mutations, and nonsense mutations. All 119 samples had also been analyzed in parallel for p53 mutations by a combination of several alternative methods including the predecessor Affymetrix p53 GeneChip microarray, denaturing high performance liquid chromatography (DHPLC), as well as conventional sequencing. The specificity and sensitivity of the AmpliChip p53 Test were compared to the results obtained with these alternative assays. Discordant results were observed in 27 samples when comparing the AmpliChip p53 Test to the alternative assays. Among these 27 samples the AmpliChip p53 Test detected mutations in Exon 10 in 2 samples which had not been tested by DHPLC and further exclusively detected the R72P polymorphism in 9 samples. The remaining group of discordant samples contained:5 samples with a I03 dup 16bp +40 (acctggagggctgggg);3 samples containing a > 2 bp deletion;2 samples with insertion mutations; andone with a mutation in intron 3.Due to technical reasons, all of these aberrations noted above are out of the specifications of the AmpliChip p53 Test. In 5 samples the AmpliChip p53 Test detected additional mutations that were not identified by conventional methods. In conclusion, the AmpliChip p53 Test is a robust and rapid diagnostic tool that allows to identify a poor prognostic subset of AML. Unraveling the biology of this disease may provide new pathophysiologic insights applicable to therapeutic intervention, especially for the targeted therapy of AML with complex aberrant karyotype.

Cytogenetic and Molecular Genetic Abnormalities of CIITA Gene in Patients with Primary Mediastinal (Thymic) Large B-Cell Lymphoma

Background. Primary mediastinal (thymic) large B-cell lymphoma (PMBCL) is an aggressive malignant lymphoproliférative disease which accounts for 2-3 % of all non-Hodgkin's lymphomas. In 40 % of PMBCL cases rearrangements of the MHC class II activator, i.e. CIITA gene, are observed. CIITA abnormalities lead to decreasing protein expression and surface expression of MHC class II, which results in lack of adaptive cell immunity targeted at tumor cells. Aim. To assess the rate and spectrum of cytogenetic and molecular genetic abnormalities of CIITA gene in PMBCL patients. Materials &amp; Methods. The study enrolled 37 patients with diagnosed PMBCL: 10 men and 27 women aged 21-61 years (median of 31 years). Sanger sequencing was performed in 36 patients. In 20 patients CIITA/16p13.13 FISH and in 15 patients standard cytogenetic analysis were carried out. Results. In 3 (8.3 %) out of 36 patients the sequencing method detected mutations impairing CIITA gene function, as well as microdeletion in exon 1, deletion and nucleotide substitution in a splice donor site. Multiple somatic variations in intron 1 were identified in 21 (58.3 %) patients: in 11 (52.4 %) cases there were deletions and single nucleotide variants (SNV); the other 10 (47.6 %) patients showed only SNVs. In 13 (61.9 %) out of 21 cases the abnormalities of promoter IV and/or alternative exon 1 were observed. In 5 (25 %) out of 20 patients the FISH assay identified CIITA gene translocation. Standard cytogenetic analysis detected complex karyotype in 7 (46.6 %) out of 15 patients. The comparison of data showed hypermutagenesis in 8 out of 10 patients with FISH-detected chromosome aberrations, and in 3 (37.5 %) of them complex karyotype aberrations were found as well. Conclusion. Molecular genetic methods identified different somatic variations in CIITA gene affecting its functionally important regions, which can be of special interest for further studying the biology of tumors, including PMBCL.

Prevalence of Inherited Predisposition Syndromes in Young Patients with Acute Myeloid Leukemia and Aberrant Karyotype

Prevalence of Inherited Predisposition Syndromes in Young Patients with Acute Myeloid Leukemia and Aberrant Karyotype

TP53 Status As Well As Cytogenetic Complexity Significantly Impact on Prognosis in Myelodysplastic Syndromes with Complex (≥3 anomalies) Aberrant Karyotypes

TP53 Status As Well As Cytogenetic Complexity Significantly Impact on Prognosis in Myelodysplastic Syndromes with Complex (≥3 anomalies) Aberrant Karyotypes

TP53 deficiency permits chromosome abnormalities and karyotype heterogeneity in acute myeloid leukemia.

Abnormal karyotypes are common in cancer cells and frequently observed in acute myeloid leukemia (AML), in which complex karyotype aberrations are associated with poor prognosis. How exactly abnormal karyotypes arise and are propagated in AML is unclear. TP53 mutations and deletions are frequent in complex karyotype AML, suggesting a role of TP53 alterations in the development of chromosome abnormalities. Here, we generated isogenic TP53-knockout versions of the euploid AML cell line EEB to investigate the impact of TP53 on karyotype stability. We show that chromosome abnormalities spontaneously arise in TP53-deficient cells. Numerical aneuploidy could, to some extent, be propagated in a TP53-proficient setting, indicating that it does not necessarily trigger TP53 activation. In contrast, tolerance to structural chromosome aberrations was almost entirely restricted to TP53-knockout clones, all of which were able to continue proliferation in the presence of damaged DNA. Mechanistically, as a source of chromosome aberrations, limited numerical but not structural chromosomal instability was tolerated by TP53-wildtype cells. In contrast, structural instability was found only in TP53-knockout cells. Together, in myeloid cells TP53 loss allows for the development of complex karyotype aberrations and karyotype heterogeneity by perpetuation of chromosome segregation errors.

PF553 CLONES WITH COMPLEX ABERRATIONS AND TP53 MUTATIONS RESPOND TO AZACITIDINE IN MYELODYSPLASTIC SYNDROMES AND ACUTE MYELOID LEUKEMIA

Background:Mutations in TP53 are associated with poor outcome in pts with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). This holds also true for allogeneic stem cell transplantation (Lindsley et al, NEJM, 2017). Regarding hypomethylating agents, it was suggested that pts with TP53 mutations preferentially respond to decitabine (Welch et al, NEJM, 2016), although more recent studies also suggest response to azacitidine (AZA) (Döhner et al, Leukemia, 2018).Aims:Our aim was to retrospectively study real life data in terms of response to AZA in pts with TP53 mutations.Methods:We included 34 pts diagnosed with MDS or AML that were analyzed by banding analysis, fluorescence in situ hybridization (FISH‐panel, 10 probes), and sequencing (54 genes). The cohort comprised pts with TP53 mutations (N = 16, all with complex aberrant karyotypes, 9/16 with additional mutations) and without TP53 mutations (N = 18, 6x normal karyotype, 10x 1–2 cytogenetic aberrations, 2x complex aberrations, 17/18 with mutations other than TP53). For 6 pts with TP53 mutations treated with AZA at least two sequential genetic analyses (before and during treatment) were available. For the other 10 pts with TP53 mutations clinical but not genetic follow up was available or they received other treatment than AZA. All 18 pts without TP53 mutations received at least four cycles of AZA and genetic analyses were available from at least two time points. The IWG criteria (Cheson et al, Blood, 2006) for cytogenetic response were adapted and genetic response was defined as 50% reduction in FISH clone size or in variant allele frequency (VAF).Results:Clones with a TP53 mutation responded to AZA (50% VAF reduction) in 6/6 pts with TP53 mutations and genetic follow up (Figure 1A). Two of these pts were subjected to allogeneic transplantation, best clinical response for the other four pts was 3x hematologic improvement and 1x stable disease.From the other 10 pts with TP53 mutations, two responded to induction chemotherapy and were subjected to allogeneic transplantation; six started or intended to start treatment with AZA (5x) or decitabine (1x); two received best supportive care.At least one clone responded to AZA (50% reduction in FISH or VAF) in 10/18 pts (56%) without TP53 mutations. Aberrations that responded were: ASXL1, IDH2, RUNX1, SF3B1, SRSF2, TET2, U2AF1, +1, t(3;3), 5q‐, −7, i(12q), 17p‐, 17q‐, 20q‐. Although med. overall survival (OS) was shorter in pts with responding TP53 mutations than in pts without TP53 mutations but other responding clones (432 vs 682 days, P = 0.02, Figure 1B), it was longer than in pts with TP53 mutations where no genetic follow up analysis was available that could prove genetic response (266 days, P = 0.08).Summary/Conclusion:While pts without proven reduction of the TP53 clone size showed the poor outcome (med. OS 266 days) expected due to the presence of this high‐risk genetic abnormality, pts with documented reduction of the TP53 clone size also clinically profited with a med. OS of 432 days. We want to emphasize that a TP53‐mutated clone responded in 6/6 pts with TP53 mutations and genetic follow up analysis. This response rate of 100% is remarkably, as only 56% of pts with other mutations than TP53 had genetic aberrations responding to AZA. Although a retrospective analysis on a small patient number was performed, our observation supports the suggestion that pts with TP53 mutations not only respond to decitabine, but also to AZA. Even though response is not persistent, treatment with AZA could gain survival time, postpone progression and bridge interval until transplantation.image

Cohesin Subunit SMC3 Levels Impact on Karyotype and Outcome in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disease with highest incidence in the elderly. Advancing age and complex karyotype aberrations, two strong adverse prognostic factors in AML, are closely interconnected and responsible for poor treatment outcome. Chromosomal instability (CIN) is a major contributor to genetic heterogeneity and clonal diversification in AML and its level increases with advancing age both in normal and malignant cells. In elderly healthy individuals clonal mosaicism for chromosomal aberrations in blood cells is associated with an increased risk of a subsequent hematological malignancy. Despite major advances in understanding the genetic landscape of AML and its impact on disease pathophysiology, why genomic integrity declines with age is barely uncovered. For accurate chromosome segregation, sister chromatids are tightly associated until the spindle assembly checkpoint is satisfied, allowing for anaphase‐promoting complex (APC/C)‐mediated anaphase onset. Sister chromatid cohesion is established by the ring‐shaped cohesin complex consisting of four subunits (SMC1A, SMC3, Rad21, and either SA‐1 or SA‐2). Age‐associated CIN in human oocytes seems to be the consequence of cohesin decay with increasing maternal age.Aims and methods: Based on frequent loss‐of‐function mutations in cohesin complex components in human malignancies including AML, we sought to investigate whether decreased cohesin subunit protein levels are associated with aging and leukemogenesis in human individuals by analyzing mononuclear cells (MNCs) from 95 healthy blood donors and 48 AML patients.Results: Western Blot analysis of peripheral blood MNCs from 95 healthy individuals with a median age of 48.5 years (range 19-90) revealed that SMC3 and SMC1A cohesin subunit levels were significantly reduced with advancing age (Fig. 1A). Interestingly, SMC3 protein levels were most prominently reduced in individuals older than 60 years. For SA-1, a borderline significant age-associated decline in protein expression was observed. Surprisingly, compared to healthy donors, Western blot analysis of MNCs from peripheral blood (n=) or bone marrow (n=) from 48 AML patients (median age: 59 years, range 17-91) showed significantly elevated protein expression levels of all cohesin complex components (Fig. 1B). Nevertheless, SMC3 protein levels significantly declined with advancing age in AML patients, similar to the situation in healthy individuals. High SMC3 protein levels were significantly associated with ELN favorable risk group classification (low versus high protein levels, p=0.0010) (Fig. 1C). Moreover, the occurrence of numerical chromosome abnormalities and complex karyotypes increased with decreasing SMC3 protein levels (low versus high protein levels, p=0.0185). The association between age and SMC3 protein levels remained significant after adjusting for karyotype and risk group in multivariate linear regression analysis with age (p=0.03), risk group and numerical karyotype aberrations being independently associated with SMC3 expression (adverse versus favorable risk, p=0.027; numerical aberrations versus normal karyotype, p=0.048). No correlation of SMC3 levels with the mutational status of NPM1 and FLT3 or the CBFB‐MYH11 fusion nor an association of other cohesin subunit expression levels with any of the parameters analyzed was found. Compared to AML patients with lower SMC3 expression, patients with higher levels had a significantly longer overall survival. No survival differences between patients with low versus high expression levels of other cohesin subunits were found. Interestingly, patients with elevated levels of both SMC3 and SA-2 experienced the longest overall survival.Conclusions: In summary, SMC3 cohesin subunit protein levels decline with advancing age in both healthy individuals and AML patients. Reduced SMC3 levels are associated with numerical chromosome abnormalities, complex karyotypes and outcome in AML. These findings are remarkable as overall cohesin mutations are neither associated with karyotype aberrations nor with an impact on survival and possibly explained by the fact that with the exception of SMC3 for all of the other cohesin subunits at least two versions exist, which ‐ at least partially ‐ complement each other. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cutaneous Leukemic Infiltrates Successfully Treated With Biomodulatory Therapy in a Rare Case of Therapy-Related High Risk MDS/AML.

Cutaneous manifestations in hematologic malignancies, especially in leukemia, are not common and may be very variable. Here we report a very unusual case of a patient (female, 70 years old) who was admitted to the hospital in 2016 because of skin lesions on the face, the trunk of the body and the extremities. She had a history of breast cancer in the year 2004 (pT1b, pN0, cM0, L0, V0, R0) which had been resected and treated with adjuvant radiation and chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracile) as well as psoriasis treated with methotrexate and cyclosporine. Because of mild cytopenia a bone marrow aspirate/biopsy was performed showing myelodysplastic syndrome (MDS) with multilineage dysplasia. Cytogenetic review revealed a complex aberrant karyotype denoting adverse outcome. Simultaneously, a skin biopsy could confirm leukemic skin infiltration. Consequently, a therapy with azacitidine was started. After the first cycle the patient developed severe pancytopenia with a percentage of 13% peripheral blasts (previously 0–2%) as well as fever without evidence for infection which was interpreted as progressive disease. Therefore, the therapeutic regimen was changed to a biomodulatory therapy consisting of low-dose azacitidine 75 mg/day (given sc d1-7 of 28), pioglitazone 45 mg/day per os, and all-trans-retinoic acid (ATRA) 45 mg/m2/day per os. After cycle 1 of this combined biomodulatory therapy the patient showed hematologic recovery; besides a mild anemia (hemoglobin 11.1 g/dl) she developed a normal blood count. Moreover, the cutaneous leukemic infiltrates which had been unaffected by the azacitidine ameliorated tremendously after 2 cycles resulting in a complete remission of the skin lesions after cycle 6. In conclusion, we report a very unusual case with cutaneous infiltrates being the first clinical manifestation of hematologic disease, preceding the development of acute myeloid leukemia. While azacitidine alone was ineffective, a combined biomodulatory approach resulted in a complete remission of the cutaneous manifestation.

Prognostic factors in newly diagnosed multiple myeloma patients with 1q21 amplification/gain treated with bortezomib-based regimens followed by autologous hematopoietic stem cell transplantation

目的探讨硼替佐米为基础的化疗序贯自体造血干细胞移植（ASCT）治疗伴有1q21扩增的初治多发性骨髓瘤（MM）患者的疗效和预后影响因素。方法回顾性分析2008年1月至2015年8月以硼替佐米为基础的化疗序贯ASCT以及移植后维持和（或）巩固治疗伴有1q21扩增的35例初治MM患者资料。结果①35例患者中，男22例，女13例，中位发病年龄49（33~63）岁。单纯检出1q21扩增者仅为3例（8.6%），其余32例均合并其他细胞遗传学异常，包括13q14缺失、t（11;14）、t（4;14）、t（14;16）、17p缺失和复杂核型。②ASCT后获得完全缓解者20例（57.0%），获得非常好的部分缓解者13例（37.1%），获得部分缓解者2例（5.7%）。中位随访24（8~85）个月，预期3年无进展生存（PFS）率和总生存（OS）率分别为（66.5±9.7）%和（69.6±9.9）%。③按合并其他高危或非高危细胞遗传学异常对患者进行分组，高危组（13例）中位PFS和OS时间分别为26个月和28个月，非高危组（22例）中位PFS和OS时间分别为54个月和未达到，高危组与低危组3年预期PFS率[（28.0±15.9）%对（71.5±12.7）%，χ2=5.404，P=0.020]和OS率[（36.5±16.4）%对（92.3±7.4）%，χ2=7.596，P=0.006]差异均有统计学意义。结论1q21扩增很少单独发生，常伴发其他细胞遗传学异常。硼替佐米为基础的化疗序贯ASCT治疗初治MM患者疗效良好，合并其他高危细胞遗传学异常是影响该亚组患者PFS和OS的不良预后因素。

Marker chromosomes can arise from chromothripsis and predict adverse prognosis in acute myeloid leukemia

AbstractMetaphase karyotyping is an established diagnostic standard in acute myeloid leukemia (AML) for risk stratification. One of the cytogenetic findings in AML is structurally highly abnormal marker chromosomes. In this study, we have assessed frequency, cytogenetic characteristics, prognostic impact, and underlying biological origin of marker chromosomes. Given their inherent gross structural chromosomal damage, we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event. In 2 large consecutive prospective, randomized, multicenter, intensive chemotherapy trials (AML96, AML2003) from the Study Alliance Leukemia, marker chromosomes were detectable in 165/1026 (16.1%) of aberrant non–core-binding-factor (CBF) karyotype patients. Adverse-risk karyotypes displayed a higher frequency of marker chromosomes (26.5% in adverse-risk, 40.3% in complex aberrant, and 41.2% in abnormality(17p) karyotypes, P < .0001 each). Marker chromosomes were associated with a poorer prognosis compared with other non-CBF aberrant karyotypes and led to lower remission rates (complete remission + complete remission with incomplete recovery), inferior event-free survival as well as overall survival in both trials. In multivariate analysis, marker chromosomes independently predicted poor prognosis in the AML96 trial ≤60 years. As detected by array comparative genomic hybridization, about one-third of marker chromosomes (18/49) had arisen from chromothripsis, whereas this phenomenon was virtually undetectable in a control group of marker chromosome-negative complex aberrant karyotypes (1/34). The chromothripsis-positive cases were characterized by a particularly high degree of karyotype complexity, TP53 mutations, and dismal prognosis. In conclusion, marker chromosomes are indicative of chromothripsis and associated with poor prognosis per se and not merely by association with other adverse cytogenetic features.

Symptomatic central nervous system involvement in adult patients with acute myeloid leukemia.

IntroductionAcute myeloid leukemia (AML) rarely involves the central nervous system (CNS). Little is known about the clinical course in adult AML patients since most studies examined pediatric patients. Therefore, this study analyzed the data of patients treated in three prospective trials of the “Study Alliance Leukemia” (SAL) study group for CNS involvement.MethodsIn all, 3,261 AML patients included in the prospective AML96, AML2003, and AML60+ trials of the SAL study group were analyzed. Symptomatic patients underwent cerebrospinal fluid (CSF) puncture and CNS involvement was diagnosed depending on morphology and/or flow cytometry of the CSF. Cytogenetic, molecular, clinical, and laboratory parameters were analyzed in order to identify risk factors.ResultsA total of 55 patients had proven symptomatic CNS involvement. Significantly more patients revealed CNS involvement at relapse (34 patients, 2.9%) compared with first diagnosis (21 patients, 0.6%), p<0.001. CNS involvement at initial diagnosis had a significantly higher frequency in patients with complex aberrant karyotypes, high serum lactate dehydrogenase activity, French–American–British M5 subtype, FLT3–internal tandem duplication (ITD) mutations alone, and co-occurrence of a FLT3–ITD and NPM1 mutation. Furthermore, AML patients with CNS involvement at diagnosis had an inferior outcome compared with patients without CNS involvement even if treated with intrathecal chemotherapy with an overall survival of 11% versus 30% at 5 years, p=0.004.ConclusionThis study analyzed the largest data set of adult AML patients with proven CNS involvement reported so far. The data demonstrated very low prevalence of CNS involvement at initial diagnosis in adult patients with AML, and described new risk factors. In patients with risk factors, intense diagnostic and treatment strategies should be employed in the future.

Marker Chromosomes Can Arise from Chromothripsis and Predict Adverse Prognosis in Acute Myeloid Leukemia

Introduction: Cytogenetic testing is routinely performed in newly diagnosed acute myeloid leukemia (AML) for risk stratification. Elaborate risk classifications based on karyotyping are provided by both the European Leukemia Net (ELN) and the Medical Research Council (MRC). Complex aberrant, monosomal and abnl(17p) karyotypes confer a poor prognosis. In cytogenetic studies, chromosome aberrations that cannot be identified due to gross rearrangement, thereby preventing the allocation to a specific chromosome, are designated as "Marker Chromosomes" (MC). The significance of MC as prognostic factor for AML has remained elusive so far. In this study we have assessed frequency, cytogenetic characteristics and prognostic impact of MC as well as their underlying biological origin. Given the gross structural chromosomal damage inherent to MC we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event.Patients and Methods: Patients recruited intwo large consecutive, prospective, randomized, multicenter clinical trials for newly diagnosed non-M3 AML patients from the German Study Alliance Leukemia (SAL) were analyzed (AML96, NCT00180115; AML2003, NCT00180102). All karyotypes were retrospectively screened for MC. For the detection of chromothripsis array-CGH was used. For each sample 50 ng of DNA were hybridized to an Affymetrix® CytoScan HD Oligo/SNP-array and scanned with the Affymetrix GeneChip® Scanner 3000 7G. Chromothripsis was defined according to the criteria of Rausch et al., which require at least 10 switches in segmental copy number involving two or three distinct copy number states on a single chromosome.Results: MC were detectable in 165/1026 (16.1%) of aberrant non-CBF karyotype cases. Adverse-risk karyotypes displayed a higher frequency of MC (40.3% in complex aberrant, 26.5% in adverse-risk as defined by MRC criteria and 41.2% in abnl(17p) karyotypes, p<.001 each). MC were associated with a poorer prognosis compared to other non-CBF aberrant karyotypes as well as with lower remission rates (CR+CRi; 36.0% vs. 55.8% in AML96 ≤60 years, p=0.01; 14.3% vs. 44.1% in AML2003, p<0.001), inferior event-free survival (2.24 vs. 6.54 months, p<0.001; 3.45 vs. 8.03 months, p<0.001) and overall survival (5.72 vs. 11.87 months, p<0.001; 8.68 vs. 20.78, p=0.01). In multivariate analysis with co-variables age, prior MDS, therapy-related AML and adverse-risk cytogenetics according to MRC criteria, MC independently predicted poor prognosis in AML96 ≤60 years but not in AML2003 with its higher allogeneic transplantation rate.As detected by array-CGH, in about one third of MC karyotypes (18/49, 36.7%, including 3 cases with 8 or 9 copy number switches) MC had arisen from chromothripsis, whereas this phenomenon was virtually undetectable in a control group of complex aberrant karyotypes without MC (1/34) (p<0.001). Chromothripsis in MC karyotypes typically involved one single chromosome (n=11), with two or three chromosomes affected in 5 and 2 patients, respectively. There was no predilection for a particular chromosome. MC karyotypes positive for chromothripsis were characterized by a particularly high degree of karyotype complexity as compared to those that were negative for chromothripsis (complex aberrant 100% vs. 64.5% p<0.01; abnl(17p) 50.0% vs. 16.1%, p=0.01). In 12/18 (66.7%) cases, at least one of the chromothriptic chromosomes was reported as loss in the karyotype formula, suggesting that the grouping of a chromothriptic chromosome as a marker is paralleled by a putative loss of the affected chromosome. The chromothripsis positive MC karyotype subgroup had a particularly dismal prognosis with a combined CR+CRi rate of 2/16 vs. 10/31 (p=0.14). It also displayed inferior event-free and overall survival, though statistical significance was not reached for either endpoint, likely due to the already poor prognosis of the entire MC positive group.Conclusion: This is the first study showing that MC are a frequent finding predominantly in adverse-risk AML and associated with particularly poor prognosis. Our data provide evidence that a substantial portion of MC arise from chromothripsis. DisclosuresThiede:AgenDix: Employment, Other: Ownership.

Complex karyotypes and KRAS and POT1 mutations impact outcome in CLL after chlorambucil-based chemotherapy or chemoimmunotherapy

AbstractGenetic instability is a feature of chronic lymphocytic leukemia (CLL) with adverse prognosis. We hypothesized that chromosomal translocations or complex karyotypes and distinct somatic mutations may impact outcome after first-line chemoimmunotherapy of CLL patients. We performed metaphase karyotyping and next-generation sequencing (NGS) of 85 genes in pretreatment blood samples obtained from 161 patients registered for CLL11, a 3-arm phase 3 trial comparing frontline chlorambucil (Clb) vs Clb plus rituximab (Clb-R) or Clb plus obinutuzumab in CLL patients with significant comorbidity. Chromosomal aberrations as assessed by karyotyping were observed in 68.8% of 154 patients, 31.2% carried translocations, and 19.5% showed complex karyotypes. NGS revealed 198 missense/nonsense mutations and 76 small indels in 76.4% of patients. The most frequently mutated genes were NOTCH1, SF3B1, ATM, TP53, BIRC3, POT1, XPO1, and KRAS. Sole chemotherapy, treatment with Clb-R, or genetic lesions in TP53 (9.9% of patients) and KRAS (6.2% of patients) were significantly associated with nonresponse to study therapy. In multivariate models, complex karyotypes and POT1 mutations (8.1% of patients) represented significant prognostic factors for an unfavorable survival, independently of IGHV mutation status, Binet stage, and serum β-2-microglobuline. Patients with the copresence of complex karyotypes and deletions/mutations involving TP53 demonstrated a particularly short survival. In summary, this is the first prospective, controlled study in CLL patients that shows a role of complex karyotype aberrations as an independent prognostic factor for survival after front-line therapy. Moreover, the study identifies mutations in KRAS and POT1 as novel determinants of outcome after chemoimmunotherapy using chlorambucil and anti-CD20 treatment.

Cytogentic and prognostic characteristic of acute myeloid leukemia with monosomal karyotype

目的探讨成人单体核型急性髓系白血病（AML）的细胞遗传学和预后特点。方法2002年9月至2014年11月在中国医学科学院血液病医院诊断、治疗的96例细胞遗传学预后不良AML患者和1例预后中等单纯单体核型AML患者纳入研究，对比分析单体核型患者与其他不良遗传学预后患者的临床特征。结果97例患者中，单体核型者31例（占同期收治的AML患者的2.5%），按照美国西南肿瘤研究组预后分层标准，其中30例染色体异常≥3种的复杂核型者归入预后不良组，另1例单纯单体核型者归入预后中等组；非单体核型者66例。单体核型常见的染色体单体为−17、−5、−7、−21、−8、−22。96例细胞遗传学预后不良患者中，单体核型患者中位总生存（OS）时间为6.1个月，非单体核型患者中位OS时间未达到，两组中位无复发生存（RFS）时间分别为3.1和18.6个月，差异均有统计学意义（P值分别为0.001和<0.001）。49例复杂核型患者中，单体核型（30例）和非单体核型（19例）组中位OS时间分别为6.1和10.8个月（P=0.088），中位RFS时间分别为3.1和8.6个月（P=0.009）。结论单体核型主要见于预后不良组中的复杂核型患者，在预后不良及复杂核型群体中单体核型患者具有比非单体核型患者更差的预后。

Karyotype complexity and prognosis in acute myeloid leukemia.

A complex aberrant karyotype consisting of multiple unrelated cytogenetic abnormalities is associated with poor prognosis in patients with acute myeloid leukemia (AML). The European Leukemia Net classification and the UK Medical Research Council recommendation provide prognostic categories that differ in the definition of unbalanced aberrations as well as the number of single aberrations. The aim of this study on 3526 AML patients was to redefine and validate a cutoff for karyotype complexity in AML with regard to adverse prognosis. Our study demonstrated that (1) patients with a pure hyperdiploid karyotype have an adverse risk irrespective of the number of chromosomal gains, (2) patients with translocation t(9;11)(p21∼22;q23) have an intermediate risk independent of the number of additional aberrations, (3) patients with ⩾4 abnormalities have an adverse risk per se and (4) patients with three aberrations in the absence of abnormalities of strong influence (hyperdiploid karyotype, t(9;11)(p21∼22;q23), CBF-AML, unique adverse-risk aberrations) have borderline intermediate/adverse risk with a reduced overall survival compared with patients with a normal karyotype.

Helpful Tool or Oversimplification? Concept of the Monosomal Karyotype from the Clinical and Cytogenetic Point of View.

Helpful Tool or Oversimplification? Concept of the Monosomal Karyotype from the Clinical and Cytogenetic Point of View.