MLL gene amplification in acute myeloid leukemia and myelodysplastic syndromes is associated with characteristic clinicopathological findings and TP53 gene mutation

Human Pathology (2015) 46, 65–73

www.elsevier.com/locate/humpath

Original contribution

MLL gene amplification in acute myeloid leukemia and myelodysplastic syndromes is associated with characteristic clinicopathological findings and TP53 gene mutation☆ Guilin Tang MD, PhD a,⁎, Courtney DiNardo MD b , Liping Zhang MD a , Farhad Ravandi MD b , Joseph D. Khoury MD a , Yang O. Huh MD a , Tariq Muzzafar MD a , L. Jeffrey Medeiros MD a , Sa A. Wang MD a , Carlos E. Bueso-Ramos MD a a

Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA

b

Received 12 August 2014; revised 15 September 2014; accepted 17 September 2014

Keywords: AML/MDS; MLL amplification; Pathologic features; Cytogenetics; TP53 mutation

Summary MLL gene rearrangements are well-recognized aberrations in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). In contrast, MLL gene amplification in AML/MDS remains poorly characterized. Here, we report a series of 21 patients with myeloid neoplasms associated with MLL gene amplification from 1 institution. This series included 13 men and 8 women, with a median age of 64 years. Eleven patients presented as AML with myelodysplasia-related changes, 6 as therapy-related AML, and 4 as therapy-related MDS. All patients had a highly complex karyotype, including frequent −5/del(5q), −18, and −17/del(17p) abnormalities; 16 patients were hypodiploid. TP53 mutations were detected in all 12 patients tested, and 3 patients showed TP53 mutation before MLL amplification. Morphologically, the leukemic cells frequently showed cytoplasmic vacuoles, bilobed nuclei, and were associated with background dyspoiesis. Immunophenotypically, 15 patients had a myeloid and 4 had myelomonocytic immunophenotype. Laboratory coagulopathies were common; 7 patients developed disseminated intravascular coagulopathy, and 3 died of intracranial bleeding. All patients were refractory to therapy; the median overall survival was 1 month, after MLL gene amplification was detected. We concluded that AML/MDS with MLL gene amplification is likely a subset of therapy-related AML/MDS or AML with myelodysplasia-related changes, associated with distinct clinicopathological features, frequent disseminated intravascular coagulopathy, a highly complex karyotype, TP53 deletion/mutation, and an aggressive clinical course. © 2014 Elsevier Inc. All rights reserved.

☆

All authors report no conflict of interest and funding disclosures. ⁎ Corresponding author. Department of Hematopathology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 72, Houston, Texas 77030-4009. E-mail address: [email protected] (G. Tang). http://dx.doi.org/10.1016/j.humpath.2014.09.008 0046-8177/© 2014 Elsevier Inc. All rights reserved.

1. Introduction Gene amplification occurs in a broad spectrum of malignancies, often leading to the inappropriate activation or amplification of oncogene expression. Activation of

66 oncogenes in this way is often associated with aggressive tumor growth and poor prognosis [1]. By using conventional cytogenetic analysis, amplification of oncogenes can be located extrachromosomally in double minutes; intrachromosomally in homogeneously staining regions; or as an unidentified marker/derivative chromosome, ring chromosome, or isochromosome [2-12]. The frequency of cytogenetically detectable gene amplification in AML is approximately 1% [13]. Although uncommon, MLL gene amplification has been recognized as a recurrent event that occurs in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and rarely therapy-related B-lymphoblastic leukemia (B-ALL) [212,14-16]. In the relatively few cases that have been reported to date, MLL amplification has been often associated with an older age, a complex karyotype, and a very poor prognosis [2-12,14]. Yet, because of the rarity of subjects described to date, it is not known if MLL-amplified AML or MDS is associated with any distinct morphologic or clinical features. It is also not known if AML/MDS associated with MLL amplification should be classified as a unique subset of AML/MDS. In this study, we conducted a detailed clinical chart review and pathology assessment of 21 patients with confirmed MLL gene amplification at our institution to answer the above questions. We also performed next-generation sequencing (NGS) molecular analysis in a subset of these patients to evaluate associated mutation patterns.

G. Tang et al. white blood cell (WBC) count, hemoglobin level and platelet count, prothrombin time, partial thromboplastin time, fibrinogen, and D-dimer results.

2.3. Flow cytometric immunophenotyping Flow cytometry immunophenotypic analysis was performed using a panel of antibodies designed for acute leukemia and further analyzed by extended panels designed to characterize AML or precursor B-ALL or T-lymphoblastic leukemia as indicated according to the methods described previously [17].

2.4. Conventional karyotyping and FISH Conventional chromosomal analysis was performed on G-banded metaphase cells prepared from unstimulated BM aspirate cultures (24 and 48 hours) using standard techniques. Twenty metaphases were analyzed, and the results were reported using the International System for Human Cytogenetic Nomenclature (2013) [18]. FISH for MLL (dual-color break-apart probe; Abbott Molecular/Vysis, Des Plaines, IL) was performed on freshly harvested BM cells (metaphase or interphase) or BM aspirate smear (interphase). Cases with greater than or equal to 5 MLL copies were considered as gene amplification and included in this study.

2.5. Molecular studies

2. Materials and methods 2.1. Case selection We searched the database of the Clinical Cytogenetics Laboratory, Department of Hematopathology, The University of Texas MD Anderson Cancer Center, for cases with MLL gene amplification (fluorescence in situ hybridization [FISH] study) between the years 2007 and 2014. Cases with 5 or more MLL copies were included in the study. Cases with MLL gene rearrangement were not included. Clinical and laboratory data and overall patient outcome were obtained by retrospective review of medical records. All samples were collected following institutional guidelines with informed consent in accordance with the Declaration of Helsinki.

2.2. Laboratory data and morphologic examination Peripheral blood smears, bone marrow (BM) aspirate smears, and trephine biopsy specimens were reviewed in all cases. Blast percentage, BM cellularity, and background dysplasia were assessed. Myeloperoxidase (MPO) and nonspecific esterase by cytochemistry stain were performed in all acute leukemia cases. Laboratory data collected included

2.5.1. NGS The TruSeq Amplicon Cancer Panel (Illumina, San Diego, CA), customized to include probe pairs for 5 additional genes according to the manufacturer's protocol was used to screen for mutations in 53 cancer-related genes (Supplementary Table) as described previously [19]. The mutation results were confirmed by other assays including Sanger sequencing, pyrosequencing, and fragment analysis by capillary electrophoresis as indicated. Sanger sequencing for TP53 mutation: genomic DNA was extracted from BM aspirates or formalin-fixed, paraffinembedded BM clot section without decalcification. DNA was amplified and subjected to mutational analysis for TP53 (exons 2-11) by direct Sanger sequencing on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Carlsbad, CA) as described previously [20]. FLT3 mutation analysis for both internal tandem duplication (ITD) and tyrosine kinase domain mutation and CEBPA mutation were tested using separate polymerase chain reaction assays and capillary electrophoresis as described previously [21]. For other genes, including c-KIT, N-RAS, IDH1, IDH2, NPM1, the mutation analysis was either included in the NGS panel or performed separately as a part of institutional standard clinical workup for acute leukemia and MDS patients.

Demographic, clinical, and pathologic features of 21 patients with MLL amplification

Case Age/ no. sex

Diagnosis (WHO)

FAB

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

AML-MRC AML-MRC AML-MRC AML-MRC AML-MRC AML-MRC AML-MRC AML-MRC AML-MRC AML-MRC AML-MRC t-AML t-AML t-AML t-AML t-AML t-AML t-MDS t-MDS t-MDS t-MDS

AML-M2 AML-M4 AML-M4 AML-M2 AML-M4 AML-M1 AML-M2 AML-M2 AML-M1 AML-M0 AML-M4 AML-M2 AML-M1 AML-M1 RAEB-T RAEB-T AML-M2 RA RA RA RA

66/M 78/F 35/M 74/M 85/M 65/M 64/M 52/M 77/M 46/M 76/M 48/F 56/M 53/M 75/F 72/F 60/F 63/F 64/F 60/M 69/F

BM morphology and pathologic diagnosis a Interval b (mo) Diagnosis

Blasts (%) Dysplasia Cytoplasmic vacuoles Bilobular nuclei

Immunophenotype Response to Survival therapy

5 8 1 0 0 0 2 4 9 0 0 3 16 0 0 6 9 7 1 1 0

48 33 20 40 59 91 56 67 38 96 48 62 12 85 22 67 33 10 19 12 10

Myeloid Myelomonocytic Myelomonocytic Myeloid Myelomonocytic Myeloid Myeloid Myeloid Myeloid Myeloid Myelomonocytic Myeloid Myeloid Myeloid Myeloid Myeloid Myeloid NA Myeloid Myeloid NA

Persistent AML Relapsed AML Persistent AML AML-MRC AML-MRC AML-MRC Persistent AML Persistent AML Relapsed AML AML-MRC AML-MRC Persistent AML Persistent AML t-AML t-AML Persistent AML Persistent AML Persistent MDS Persistent MDS Persistent MDS t-MDS

Yes Yes Yes Yes Yes DE Yes Yes Yes DE Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Yes Yes Yes No Yes Yes No Yes Yes No Yes Yes Yes Yes Yes No No No Yes Yes No

Yes No Yes No Yes Yes No No No No No Yes Yes No No Yes Yes Yes Yes No Yes

No No No No No No No No No No No No No No No No No No No No No

D/26 d D/18 d D/2 mo D/12 d D/14 d D/5 d D/22 d D/6 mo A/1 mo D/4 mo D/34 d D/2 mo A/1 mo D/41 d A/3 mo D/2 mo D/3 mo D/2 mo D/4 mo D/9 mo D/4 mo

MLL gene amplification in AML/MDS

Table 1

Abbreviations: A, alive; AML, acute myeloid leukemia; BM, bone marrow; D, dead; DE, difficult to evaluate; F, female; FAB, French-American-British classification; M, male; MDS, myelodysplastic syndromes; MRC, myelodysplasis-related changes; RA, refractory anemia; RAEB-T, refractory anemia with excess blasts in transformation; t-, therapy-related; WHO, World Health Organization classification. a At the time when MLL amplification was detected. b From the onset of disease to the detection of MLL amplification.

67

68

3. Results 3.1. Clinical and laboratory findings MLL amplification was identified in 13 men and 8 women patients, with a median age of 64 years (range 35-85) (Table 1). Ten patients had prior cytotoxic therapies for breast cancer (cases 12 and 15), large B-cell lymphoma (cases 16 and 17), chronic lymphocytic leukemia (cases 18 and 21), Hodgkin lymphoma (case 14), rheumatoid arthritis (case 19), prostate cancer (case 13), and lung adenocarcinoma (case 20). Of note, cases 13 and 15 received radiation therapy only, case 18 received alkylating agents only, case 19 received antimetabolites only, and the other 6 patients received both alkylating agents and topoisomerase II inhibitors. All patients had anemia (hemoglobin level, median 9.5 g/dL; range, 6.6-11 g/dL) and thrombocytopenia (platelet, median 26 × 109/L; range 5-110 × 109/L). WBC was variable: 10 patients showed leukopenia, 7 patients showed leukocytosis, and 4 patients had normal WBC counts. Eighteen patients had circulating blasts (approximately 2%-90%). Seventeen patients had laboratory evidence of coagulopathy, including elevated D-dimer (n = 17), decreased fibrinogen (n = 7), prolonged partial thromboplastin time (n = 10), and prolonged prothrombin time (n = 14). Seven patients developed clinically significant disseminated intravascular coagulopathy (cases 1, 3, 6, 7, 9, 11, and 20); 3 patients (cases 6, 7, and 9) died of massive intracranial hemorrhage.

3.2. Response to therapy and follow-up For patients younger than 60 years old (cases 3, 8, 10, 12, 13, and 17), they received standard chemotherapy for AML (cytarabine or plus fludarabine and idarubicin); for patients older than 60 years old or with comorbidities (cases 1, 4, 6, 7, 9, 11, 14, 15, and 16), they were treated with hypomethylating agents, such as vidaza, decitabine, and SGI-110 (under clinical trial). Cases 2 and 5 only received hydrea and supporting treatments. Four MDS patients received hypomethylating agents. All 21 patients in this study cohort failed to achieve remission. Two patients with MDS transformed into AML after 2 to 4 months (cases 19 and 20). Eighteen patients died, 3 patients (cases 9, 13, and 15) were alive at last follow-up (after 1-3 months), and all had persistent disease. The median survival after detection of MLL gene amplification was 1 month (range, 3 days to 9 months).

3.3. Morphologic findings Based on the 2008 World Health Organization classification [22], 11 cases (cases 1-11) were classified as AML with myelodysplasia-related changes (AML-MRC), 6 cases as therapy-related AML (t-AML), and 4 cases as therapyrelated MDS (t-MDS). Using the French-American-British

G. Tang et al. (FAB) classification system [23], these cases were classified as follows: 1 AML-M0, 4 AML-M1, 6 AML-M2, 4 AMLM4, 4 refractory anemia, and 2 refractory anemia with excess blasts (Table 1). The median percentage of BM blasts was 40% (range, 10%-96%). Except for 2 cases (cases 6 and 10) with an overwhelming infiltrate of blasts (≥90%), dysplasia was present in all other cases. Dyserythropoiesis included cytoplasmic vacuolization, ring sideroblasts, nuclear budding, and internuclear bridging; dysgranulopoiesis includes bilobed nuclei or nuclear hypolobation in neutrophils (eg, pseudo–Pelger-Huet anomaly), cytoplasmic vacuoles, and hypogranulation; dysmegakaryocytosis included micromegakaryocytes and nuclear hypolobation. Myeloblasts, maturing myeloid cells, and monoblasts containing cytoplasmic vacuoles (Fig. 1A and C) were observed in 14 cases (64%) and bilobed nuclei in maturing myeloid lineage (Fig. 1C and D) in 11 (50%) cases (Table 1). Eight cases showed prominent nucleoli (Fig. 1A and B). In 2 cases (cases 8 and 9), which had combined morphologic-FISH analysis, MLL amplification seemed to be restricted to the cells with cytoplasmic vacuoles (Fig. 2). Mild myelofibrosis (MF-1) was noted in 2 cases (case 3 and 21). MPO cytochemistry was performed on 11 cases (cases 1, 2, 4, 5, 6, 8, 9, 12, 15, 16, and 19), and all 11 cases were positive for MPO.

3.4. Immunophenotypic findings Flow cytometric immunophenotypic analysis was performed on all AML cases and 2 MDS cases with AML transformation (cases 19 and 20). Most cases showed a typical myeloblast immunophenotype with CD34 and CD117 expression (n = 15); 4 cases showed both myeloid and monocytic differentiation (cases 2, 3, 5, and 11) (Table 1).

3.5. Karyotype and MLL FISH The karyotype is summarized in Table 2. All 21 cases showed a complex karyotype, and 16 (76%) cases showed hypodiploidy. Abnormalities involving chromosome 11 were detected in all cases. Other commonly detected abnormalities included −5/del(5q) (n = 17), −17/del(17p) (n = 8), −18 (n = 8), and −7/del(7q) (n = 7). Eight cases had chromosomal analysis before (cases 7, 9, 12, 13, 17, and 18) or after (case 8, 16, and 18) detection of MLL amplification; all 8 cases showed rapidly increased karyotypic complexity (numerically and/or structurally) at the later time points compared with the earlier time point. For example, case 7 had a complex karyotype at the time when AML was diagnosed: 44 to 46, XY,add(2)(q23),−5,add(7)(q22),−13,−16,add(19)(p13.2),+1 ~ 3mar[cp17]/46,XY[3]; 2 months later (MLL amplification was detected), the karyotype became more complex: 38 ~ 44, X,−Y,add(2)(q23),−5,del(6)(q13),i(6)(p10),add(7)(q22),−11, −13,−16,−17,add(19)(p13.3),+1 ~ 3mar[cp20].

MLL gene amplification in AML/MDS

69

Fig. 1 Characteristic BM morphologic features. A, Large blasts with prominent nucleoli, moderate amount of basophilic cytoplasm with cytoplasmic vacuoles (case 6, 91% blasts). B, Large blasts with prominent nucleoli and fine cytoplasmic vacuoles, dyserythropoiesis, and dysgranulopoiesis (case 11, 48% blasts). C, Blasts with cytoplasmic vacuoles, dyserythropoiesis, and dysgranulopoiesis (prominent bilobed nuclei, hypogranularity, and agranularity) (case 19, 19% blasts). D, Most cells with bilobed nuclei, dyserythropoiesis, and dysgranulopoiesis (case 15, 22% blasts) (Wright-Giemsa stain, original magnification ×100).

Certain chromosomal aberrations were highly correlated with the presence of MLL amplification, including amplification of chromosome 11(q21q23) (n = 2) (Fig. 3A), homogeneously staining regions (n = 5) (Fig. 3B), marker chromosome (n = 13), ring chromosome (n = 2), triplication (n = 2), and double minutes (n = 1). Ten cases showed greater than or equal to 10 MLL copies, and 11 cases showed approximately 5 to 9 copies (Table 2). Interestingly, all MDS cases had less than 10 MLL copies. Higher MLL copy number was associated with shorter survival: patients with greater than or equal to 10 copies had a median overall survival of 22 days (5 days to 2 months); patients with approximately 5 to 9 copies had a median overall survival of 3 months (34 days to 9 months) (P = .001)

showed TP53 mutation, including missense (n = 9), nonsense (n = 1, case 15), and frame shift (n = 2, case 19 and 20). Three cases (cases 9, 13, and 18) were confirmed to have TP53 mutation before MLL gene amplification. Mutation analysis, either by NGS or Sanger sequencing, was performed for FLT3 (n = 21), CEBPA (n = 16), KIT (n = 20), neuroblastoma (n = 20), IDH1 (n = 13), IDH2 (n = 13), and NPM1 (n = 18). One patient (case 14) showed FLT3 ITD mutation; all other patients were negative for mutations for all genes analyzed.

3.6. Gene mutation

MLL gene amplification in AML and MDS is an uncommon event. In this study, we show that patients with AML/MDS associated with MLL gene amplification have distinctive clinicopathological as well as molecular genetic features. Importantly, we wish to emphasize that cases of AML/MDS associated with MLL gene amplification are not synonymous with hematologic neoplasms associated with MLL gene rearrangements.

NGS was performed on BM aspirate of 9 patients (cases 5, 6, 8, 9, 13, 14, 15, 18, and 19), 8 patients showed TP53 gene as the sole gene mutated, and 1 patient showed TP53 mutation combined with DNMT3A and NOTCH1 mutation (Table 2). Three patients (cases 4, 10, and 20) were tested for TP53 mutation by Sanger sequencing. All 12 patients tested

4. Discussion

70

Fig. 2 Combined morphology and FISH analysis (case 9). Cells with cytoplasmic vacuoles show MLL gene amplification. A, Wright-Giemsa stain of BM smear. B, FISH analysis with MLL 2-color break-apart probe (Wright-Giemsa stain, original magnification ×100 [A and B]).

The MLL gene, located on chromosome 11q23, encodes a histone methyltransferase that has a critical role in epigenetic regulation of transcription. The functions of MLL are also important for normal embryonic development and hematopoiesis [24]. The most common abnormality involving MLL is reciprocal translocation, with more than 70 partner genes forming chimeric fusion proteins reported in AML, MDS, and acute lymphoblastic leukemia patients to date [25]. The major difference between MLL amplification and MLL translocation is that the former results in increasing MLL copy number or transcriptional activity, whereas the latter results in a production of novel chimeric proteins with partner genes. Because of the promiscuity of partner genes, the leukemogenesis mechanism of MLL gene fusions is mostly studied through targeted genes. Gene expression profiling and animal studies showed that leukemia cells with MLL gene fusions have a distinct gene expression profile associated with overexpression of HOXA5, HOX7, HOXA9, MEIS1, et al, genes [26]. In MLL gene amplification, MLL has been amplified as part of a large amplicon encompassing up to 10 MB of genomic sequence, not only leading to overexpression of a wild-type MLL transcript but also several target genes including HOXA9 and MEIS1 [12,27]. Although there is similarity in gene expression profile of cases with MLL rearrangement versus MLL amplification,

G. Tang et al. we believe that the pathogenesis of these MLL abnormalities is different. This is manifested by the following aspects: (1) MLL amplification is more restricted to AML and MDS patients, whereas MLL gene rearrangements have also been seen in B-ALL and less T-lymphoblastic leukemia; (2) the most common AML subtypes with typical MLL rearrangements are acute myelomonocytic leukemia (FAB AML-M4) and acute monoblastic and monocytic leukemia (AML-M5) [28]; in contrast, AML with MLL amplification does not appear to associate with a specific FAB subtype and has also been described in AML-M0, M1, M2, and M6 [3,4,6,8,10,12], comprising approximately 40% of the AML cases with MLL amplification; (3) both MLL rearrangement and MLL amplification are common in t-AML/MDS; however, t-AML with MLL rearrangement is most commonly associated with topoisomerase II inhibitors therapy [22], whereas t-AML/ MDS with MLL amplification can be observed in patients treated by radiation, alkylating agents, or antimetabolites [3,14]; (4) AML/MDS with MLL gene amplification is associated with a very complex karyotype and high frequency of TP53 mutations, 100% in this study; and (5) some MLL rearrangements are associated with intermediate—t(9;11)) and favorable (t(1;11)—prognosis, whereas MLL amplifications are associated with universally poor prognosis. Coagulopathy was a common phenomenon found in our patient cohort. The mechanism of developing coagulopathy is not very clear but could be multifactorial [29]: (a) leukemic cells contain enzymes, which are capable of degrading capillary basement membrane; (b) increased procoagulant activity and thromboplastic material in leukemic cells; and (c) the increased blood viscosity, penetration of vascular walls by leukemic cells, and hypoxia in the microcirculation could further compromise the integrity of vascular walls. Complex karyotypes are observed in approximately 15% of de novo AML and MDS cases and up to 50% of t-AML/ t-MDS cases [30,31]. Loss of one allele of TP53 is uncommon in AML overall [31,32] but can be as high as 40% to 60% in cases with complex karyotype [31,33]. The TP53 gene has important functions in maintaining genomic stability and integrity, and mutational inactivation of TP53 has been shown experimentally to result in gene amplification as well as in aneuploidy [34,35]. Two studies have demonstrated a close relationship between TP53 deletions/ mutations and MLL amplification [14,15]. In a study by Andersen et al [14], 7 of 8 patients with t-MDS and AML showed TP53 mutation; and in another study by Zatkova et al [15], 15 of 16 AML/MDS cases had TP53 deletion and/or mutation. In our study cohort, 8 (38%) of 21 cases showed monosomy 17/del(17p) resulting in loss of TP53; and all 12 cases tested showed TP53 mutation. Interestingly, 3 patients showed TP53 mutation before MLL amplification. In these 3 cases, the emerging MLL amplification was associated with markedly increased karyotypical complexity of leukemic cells. This evolution of the patients' karyotype, which occurred over a short time interval, was also observed by Maitta et al [7]. Taking all these findings together, it seems

Summary of karyotype, MLL gene copies, and gene mutations of 21 patients

Case Karyotype no.

MLL Positive gene group mutation

1 2

A A

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

43,X,-Y,del(7)(q22),der(11)dup(11)(q13q23)dup(11)(q23q23),der(12)t(5;12)(p12;p 11.2),add(15)(p13),-16,add(21)(p13)[20] 45,XX,der(4)t(4;11)(p16;q21)t(4;11)(q21;q21)amp(11)(q21q23),-5,der(7)t(4;7)(q25;q22),der(11)t(4;11)(p16;q21), add(12)(p11.1)[6]/46,idem,+mar[5]/ 45~48,idem,del(3)(q27q29),-17,+1~4mar[cp9] 44,XY,-5,der(11)add(11)(p11.2)hrs(11)(q23),der(11)add(11)(p15)hrs(11)(q23),-18,add(22)(p11.2),-22,+mar1[7]/44,idem,-12, add(21)(p11.2),+mar2 [3]/40~45,idem,der(11)add(11)(p11.2)hrs(11)(q23),-15,add(21)(p11.2),+1~3mar[cp9]/46,XY[1] 46,XY,add(3)(p11.2),add(5)(q11.2)[3]/76,XXY,+Y,+1,-4,add(4)(q25),-5,del(5)(q13q33)x2,add(7)(p15),+9,+9,+10,+11,+11, psudic(11;15)(q25; p11.2)hsr(11)(q23)x2,+13,+14,-18,+22[16]/46,XY,add(3)(p11.2), add(5)(q11.2),t(10;19)(p11.2;q13.1)[1] 45~47,XY,der(2)ins(2;11)(p13;q22q23),r(7),add(11)(q24),r(11)x2,-15,-17,-18,+der(?)ins(?;11)(?;q22q23), +2~5mar[cp13]/46,XY[7] 46,XY,del(5)(q22q35),add(11)(q13),-13,-16,-21,+r1,+r2,+mar[14]/46~47,idem,+1~2mar[cp6] 38~44,X,-Y,add(2)(q23),-5,del(6)(q13),i(6)(p10),add(7)(q22),-11,-13,-16,-17,add(19)(p13.3),+1~3mar[cp20] 45,XY,-2,-5,del(7)(q32q34),psu dic(11;14)(q23;q11.2),del(12)(p11.2),-14,+r,+mar[7]/45,idem,del(17)(p11.2)[5]/46,idem,+8[3]/45~46,XY,-2,del(3) (p13),-4,-5,del(7)(q32q34),+8,+2~4mar[cp5] 60~63,XY,-Y,-2,del(5)(q22q35),del(6)(q21),der(7)add(7)(p13)del(7)(q22q32),+8,del(9)(q22),del(11)(q22q23),+der(11)trp(11)(q13q23)hsr(11)(q23), -12,-13,-15,add(15)(p11.2),-16,-17,del(18)(q21)x2,-19,-22,+0~3mar[cp16]/46,XY[4] 44,XY,del(5)(q13q33),-11,add(11)(q25),-13,-17,-18,add(20)(q13.3),+2mar[18]/46,XY[2] 50-51,XY,+1,+5,der(5;17)(p10;q10)x2,-7,+8,+9,+10,+11,psu dic(11;22)(q25;p13)dup(11)(q25q13),+14,add(16)(p13.1),+17,+17, del(20) (q11.2q13.1),psu idic(22)(p11.2)ins(22;11)(p11.2;q25q13)[cp19]/46,XY[1] 44,XX,-2,der(5)t(2;5)(q11.2;q22),-5,add(7)(q11.2),der(7)hsr(7p22)hsr(7q32),del(11)(q13q23),add(17)(p11.2),-18[4]/42~45,XX,-2, der(5)t(2;5) (q11.2;q2),del(6)(q13q21),add(7)(q11.2),der(7)hsr(7p22)hsr(7q32),+8, del(11)(q13q23),add(17)(p11.2),-17,-18,+1~3mar[cp16] 46,XY,del(5)(q13q33)[9]/46,X,-Y,del(5)(q13q33),der(11)amp(11)(q21q23),+der(11)del(11)(p11.1)amp(11)(q21q23)[6]/46,XY[5] 45,XY,der(5)hsr(5)(p15.3)del(5)(q13q33),del(9)(q13q14),-11[11]/46,idem,+r[9] 45,XX,psu dic(5;12)(q15;p11.2),-11,add(11)(q24),+r[1]/44,idem,-18[4]/45~46,X,-X,+1,del(1)(p32p36.3),der(1;5)(p10;q10), del(3)(p13p25), del(5) (q22q35) psu dic(5;12)(q15;p11.2),del(7)(q22q32),+8, der(8)t(5;8)(p14;p21), der(8)t(5;8)(p13;p21)add(8)(q24.3),-13,+0~3mar[cp15] 44,XX,del(7)(q22q34),add(11)(q23),-16,-18[2]/44,idem,del(4)(q25q31)[17]/46,XX[1] 44,X,-X,del(5)(q31q35),-7,der(11)ins(11;?)dup(11)(q21q24),der(19)t(1;19)(q31;p13),+2mar[7]/43,idem,-19[5]/44,XX,del(5)(q31q33),-7,-11, −14, der(19)t(1;19)(q31;p13.3),+2mar[8] 43,XX,der(4)add(4)(p16)trp ins(?;11)(?;q23q23),-5,der(7)add(7)(q32)ins(?;11)(?;q23q23), −18,add(22)(p11.2)[20] 43~44,XX,der(3)t(3;5)(p21;p15.2),-5,add(5)(p12),+6,del(6)(q13q23),der(11)ins(11)(p11.2q13q23)t(11;?)(p11.2;?) invtrp(11)(q23q13)t(5;11)(p14; q23),der(14)t(11;14)(q13;p11.2)invtrp(11)(q23q13) -17,-18[cp12]/49,idem, +3,+14,+21[2] 44,XY,del(5)(q22q32),del(7)(q22q31),+8,i(11)(q10),-16,-17,-18,add(21)(q22),-22,+mar[2]/43-46,idem,+3,+0-2mar[cp8]/46,XY[10] 45,XX,add(6)(p23),add(7)(q22),der(11)inv(11)(q21q25)dup(11)(q23q23),-18,4~5dmin[cp20]

A A

TP53

A A A B

TP53 TP53 TP53

B

TP53

B B

TP53

MLL gene amplification in AML/MDS

Table 2

A A A B

TP53 TP53, FLT3-ITD TP53, DNMT3A, NOTCH1

B B B B

TP53 TP53

B B

TP53

NOTE. MLL group A, MLL copy number greater than or equal to 10; MLL group B, MLL copy number approximately 5 to 9. Cases 5, 6, 8, 9, 13, 14, 15, 18, and 19 had NGS on a 53-gene panel. Cases 4, 10, and 20 were tested for TP53 mutation by Sanger sequencing. All cases tested are positive for TP53 mutation.

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Fig. 3 FISH analysis with MLL 2-color break-apart probe. A, MLL gene amplification from derivative chromosome 4 with t(4;11) and amplification of 11(q21q23) (case 2). B, MLL gene amplification on homogeneously staining regions (case 4).

very likely that TP53 deletions/mutations occur before MLL amplification, leading to genome instability and predisposing to MLL amplification. Pathologic features of AML/MDS cases associated with MLL amplification have rarely been described, with only generalized “dysplasia” mentioned previously [3,10]. In our study cohort, dysplasia is noted in all cases with adequate cells to evaluate (19 cases). Among the dysplastic features, cytoplasmic vacuoles and bilobed nuclei are the 2 most prominent and distinct features, which were observed in mature and maturing granulocytes and blasts. Loss of 17p has been associated with small vacuolated neutrophils and pseudo–Pelger-Huet forms in MDS/AML patients [32]; however, large and prominent vacuolated blasts and bilobed maturing granulocytes have not been reported before. We strongly suggest performing FISH for MLL gene amplification in cases with cytoplasmic vacuoles or bilobed nuclei and/or with hints of presence of oncogene amplification by conventional chromosomal analysis (such as double minutes, homogeneously staining region, etc). The nature and pathogenic role of these cytoplasmic vacuoles and the relationship with MLL amplification are an important and unanswered question.

In summary, AML/MDS with MLL amplification is likely a subset of t-AML/MDS or AML-MRC. MLL amplification typically follows or accompanies TP53 deletion and/or mutation. MLL amplification in AML/MDS is associated with characteristic morphologic findings such as cytoplasmic vacuoles and bilobed nuclei, a highly complex karyotype, frequent disseminated intravascular coagulopathy, an aggressive clinical course, and poor response to chemotherapy.

Supplementary materials Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.humpath.2014.09.008.

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