Leukemia & Lymphoma, 2015; Early Online: 1–3 © 2015 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2014.1003053

Letter to the Editor­

Clinical usefulness of fluorescence in situ hybridization for detection of MLL rearrangements in acute myeloid leukemia Neus Ruiz-Xivillé1, Isabel Granada1, Diana Campos1, Adela Cisneros1, Javier Grau1, Marisol Xandri1, Montserrat Arnan2, Natalia Lloveras3, Lourdes Escoda4, Llorenç Font5, Fuensanta Millá1 & Josep-Maria Ribera1

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1Hematology Department, ICO-Hospital Germans Trias i Pujol, José Carreras Leukemia Research Institute, Badalona, UAB,

Spain, 2Hematology Department, ICO-Hospital Duran i Reynals, Hospitalet de Llobregat, Spain, 3Hematology Department, ICO-Hospital Josep Trueta, Girona, Spain, 4Hematology Department, Hospital Joan XXIII, Tarragona, Spain and 5Hematology Department, Hospital Verge de la Cinta, Tortosa, Spain

Acute myeloid leukemia (AML) is a heterogeneous group of hematological neoplasms with distinct clinical and genetic features. Chromosomal abnormalities are detectable by conventional cytogenetic analysis (CCA) in approximately 55% of de novo AML in adults [1]. The karyotype is one of the most important independent prognostic factors in AML in all age groups, and is used to stratify patients into prognostic categories. Large multicenter studies have determined that acute promyelocytic leukemia (APL) with t(15;17)(q22;q21) and core binding factor (CBF) leukemias with t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22) are associated with a favorable outcome, whereas AML with 3q26 abnormalities,  5/del(5q),  7/del(7q), 11q23 translocations [excluding t(9;11)] and complex karyotypes (CKs), among others, are associated with a very poor prognosis [2–5]. Nevertheless, 40–50% of adults with de novo AML show a normal karyotype (NK) and are classified in the intermediate prognostic category [3–5]. Although molecular techniques have allowed the detection of genetic markers with a significant prognostic value (e.g. FLT3 internal tandem duplication or NPM1 and CCAAT/enhancer-binding protein a [CEBPA] mutations) [2], an important percentage of AML still shows a NK without prognostically significant molecular abnormalities. Some apparently NK leukemias by CCA may have hidden chromosome aberrations. These abnormalities may be overlooked because of poor morphology of chromosomes, low or missing proliferation of leukemic cells in vitro or their cryptic nature. Additional techniques such as interphase fluorescence in situ hybridization (FISH) can be useful in detecting cryptic rearrangements, including cytogenetically invisible chromosomal rearrangements involving the MLL gene located at 11q23. For this reason, the guidelines for cytogenetic quality assessment [6] highly recommend FISH analysis to screen for MLL (and partner chromosome) rearrangements in all

diagnostic AML samples with NK or non-obtainable metaphases, as this abnormality has an important prognostic impact and may be cryptic or overlooked by CCA. However, there is currently no information about the diagnostic value of such systematic FISH testing for MLL rearrangements. The aim of this study was to evaluate the clinical usefulness of MLL rearrangements detected by FISH in patients with AML with NK or non-assessable metaphases. During the period 2005–2013, 778 samples of patients with de novo or secondary AML [excluding APL with t(15;17) and CBF leukemias] from five different hospitals were referred to a single cytogenetic laboratory for CCA. Two parallel unstimulated 24 h cultures of fresh bone marrow (BM) aspirates or peripheral blood (PB) samples were performed in each patient. Chromosome G-banding was carried out using standard techniques, and karyotypes were described according to the current version of the International System for Human Cytogenetic Nomenclature (ISCN) at the time of diagnosis. A minimum of 20 normal metaphases were required to define a NK, and in these cases at least 10 additional metaphases from the second culture were analyzed by a different cytogeneticist in order to minimize the possibility of an overlooked abnormality. Interphase FISH analysis was performed on fixed cell suspensions resulting from cultured and processed samples. FISH was carried out with a commercially available probe, LSI 11q23 (MLL) Dual Color Break Apart (Abbott, Des Plaines, IL) and performed according to the protocol of the manufacturer. Two hundred interphase cells were scored in each sample and the cut-off for MLL rearrangements was 2%. A total of 778 cases, 752 BM aspirates and 26 PB samples, were analyzed by CCA. Clonal abnormalities were found in 405 (52%) cases, whereas 337 cases (43.3%) had NK and 36 cases (4.6%) showed no mitosis. Rearrangements of

Correspondence: Neus Ruiz-Xivillé, Crta. Canyet s/n, 08916, Badalona, Catalonia, Spain. Tel:  34-934978868. Fax:  34-934978794. E-mail: nruiz@ iconcologia.net Received 17 July 2014; revised 18 December 2014; accepted 21 December 2014

1

2  N. Ruiz-Xivillé et al. Table I. Summary of conventional cytogenetic analysis and fluorescence in situ hybridization results. Total   Abnormal karyotype   Normal karyotype   Culture failure 11q23 rearrangements by CCA   t(9;11)   t(6;11)   t(11;19)   Other 11q23 Total tested MLL by FISH (NK  culture failure)   Total positive MLL by FISH   Positive MLL in NK   Positive MLL in culture failure

No. of cases

%

778/778 405 337 36 36/405 11 12 3 10 360/373 4 2 2

52 43.3 4.6 8.9 30.5 33.3 8.3 27.7 96.5 1.1 0.6 5.9

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­ CA, conventional cytogenetic analysis; FISH, fluorescence in situ hybridization; C NK, normal karyotype.

chromosome 11q23 were detected in 36 cases (8.9% of abnormal karyotypes), with t(6;11) and t(9;11) being the most frequent translocations, observed in 12 and 11 cases, respectively. Three hundred and sixty cases (96.5%) with NK or with culture failure were tested by FISH for MLL rearrangements. Positive MLL rearrangements were detected in four cases (1.1%), two showing a NK and two with unsuccessful cultures, representing rates of 0.6% and 5.9% of additional MLL rearrangements revealed by FISH, respectively (Table I). Metaphase FISH screening for the MLL partner was possible in one NK case, showing a cryptic translocation between chromosomes 10p11 and 11q23 (Figure 1). In the present study, CCA showed clonal abnormalities in 52% of cases of AML analyzed. The frequency of the chromosome 11q23 rearrangements was 8.9%, which is consistent with previously published studies [7]. There are no recent published studies evaluating the usefulness of FISH for the detection of MLL rearrangements in large series of patients. In a Korean study, no additional MLL rearrangements were detected in 48 cases of AML analyzed [8]. De Braekeleer et al. gathered 77 published chromosomal abnormalities involving 11q23 rearrangements, and 32 cases were cryptic by CCA but detected by FISH [9]. They found a higher proportion of MLL rearrangements due to the selection of cases. In the present study, FISH analysis with the MLL break-apart probe detected MLL rearrangements in only four cases (1.1%) of the 360 cases negative for MLL translocations by CCA. These results confirm the feasibility of detecting cryptic MLL rearrangements by FISH analysis, but they also demonstrate that

the diagnostic yield of FISH testing for the detection of MLL rearrangements not found by CCA is poor. Taking our data into account, a systematic MLL analysis is probably not justified for all cases of AML with NK. Some morphologic and immunophenotypic features have been associated with AML positive for MLL rearrangements, including myelomonocytic or monoblastic morphology and immunophenotypic features (monocytic differentiation, variable expression of immature markers and neuron-glial antigen 2 [NG2] positivity). MLL rearrangements have also been reported with increased frequency in infants, in mixed phenotype acute leukemias and therapy-related leukemias after topoisomerase II inhibitor therapy. Until the results of this study are validated in additional large patient series, a sensible approach could be to perform FISH analysis for MLL rearrangements in those cases of AML with NK which fulfill the criteria described above. Chromosome 11q23 translocations result in the creation of fusion transcripts with various partner genes, depending on the other chromosomes involved. Recent studies have demonstrated the distinct prognostic impact of 11q23 translocations, depending on the partner chromosome. In the most recent AML classification from the Medical Research Council (MRC), cases with 11q23 rearrangements had an adverse prognosis, excluding translocations t(9;11) (p21∼22;q23) and t(11;19)(q23;p13), which were moved to the intermediate-risk group [3]. In order to find the partner chromosomes in our cases, metaphase FISH was performed in both patients with NK and a MLL positive rearrangement, and showed a cryptic t(10;11)(p11∼13;q23) in one. In current treatment trials, this patient would be classified in the adverse-risk group. Therefore, an attempt to determine MLL partner genes should always be made in cases in which a MLL rearrangement is confirmed in order to detect those that may lead to reclassification of patients with AML in prognostic subgroups.­­ Potential conflict of interest:  Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.

References [1]  Mrózek K, Heerema NA, Bloomfield CD. Cytogenetics in acute leukemia. Blood Rev 2004;18:115–136.

Figure 1. Metaphase FISH for the MLL gene and G-banding analysis showing a cryptic translocation between chromosomes 10p11 and 11q23.

Letter to the Editor  3

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[2]  Smith ML, Hills RK, Grimwade D. Independent prognostic variables in acute myeloid leukaemia. Blood Rev 2011;25:39–51. [3]  Grimwade D, Hills RK, Moorman AV, et  al. Refinement of cytogenetic classification in acute myeloid leukemia:determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood 2010;116:354–365. [4]  Slovak ML, Kopecky KJ, Cassileth PA, et  al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern Cooperative Oncology Group Study. Blood 2000;96:4075–4083. [5]  Byrd JC, Mrózek K, Dodge RK, et  al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002;100:4325–4336.

[6]  Hastings R, Howell R, Betts D, editors. Guidelines and quality assurance for acquired cytogenetics. A common European framework for quality assessment for banded chromosome studies and molecular cytogenetic investigations of acquired abnormalities. European Cytogeneticists Association Newsletter No. 31, January 2013. Neuilly, France: ECA; 2013. [7]  De Braekeleer M, Morel F, Le Bris MJ, et  al. The MLL gene and translocations involving chromosomal band 11q23 in acute leukemia. Anticancer Res 2005;25:1931–1944. [8]  Kwon WK, Lee JY, Mun YC, et  al. Clinical utility of FISH analysis in addition to G-banded karyotype in hematologic malignancies and proposal of a practical approach. Korean J Hematol 2010;45:171–176. [9]  De Braekeleer E, Meyer C, Douet-Guilbert N, et al. Complex and cryptic chromosomal rearrangements involving the MLL gene in acute leukemia: a study of 7 patients and review of the literature. Blood Cells Mol Dis 2010;44:268–274.

Clinical usefulness of fluorescence in situ hybridization for detection of MLL rearrangements in acute myeloid leukemia.

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