Incidence of Philadelphia-chromosome in acute myelogenous leukemia and biphenotypic acute leukemia patients: And its role in their outcome

Leukemia Research 35 (2011) 1339–1344

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Incidence of Philadelphia-chromosome in acute myelogenous leukemia and biphenotypic acute leukemia patients: And its role in their outcome Maha Atfy ∗ , Nashwa M.A. Al Azizi, Amina M. Elnaggar Flow Cytometry and Cytogenetic Units, Department of Clinical Pathology, Zagazig University, 20 Atfy Street, Kawmeya, Zagazig, Sharkia Governate 44155, Egypt

a r t i c l e

i n f o

Article history: Received 16 January 2011 Received in revised form 11 April 2011 Accepted 12 April 2011 Available online 25 May 2011 Keywords: Acute myeloid leukemia Biphenotypic leukemia Philadelphia (Ph) chromosome

a b s t r a c t Background: Philadelphia-chromosome positive acute myeloid leukemia (Ph+ AML) is a rare entity and patient prognosis is poor, with short median survival. Biphenotypic acute leukemia (BAL) is a rare disorder that is difficult to diagnose and it displays features of both myeloid and lymphoid lineage. The aim of this study was to highlight the incidence of Philadelphia chromosome and its presence in cases of acute myeloid and biphenotypic leukemia and determine its role in the outcome of these leukemias. Subjects and methods: This study examined 464 subjects with newly diagnosed acute myeloid leukemia: 312 were males and 152 were females. All individuals were subjected to immunophenotyping and conventional karyotyping. FISH was used in failed cases of conventional cytogenetics analysis to quantify disease and to prove positive BCR-ABL fusion gene. Results: the incidence of Ph+ chromosome was found to be higher in BAL (38.4%) than in AML (1.99%). There was statistically significant difference according to the age and the median survival time between the two groups. Conclusion: Detection of specific chimeric transcripts in AML and BAL at the time of diagnosis was crucial since it plays an important role for accurate risk stratification and treatment management. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells, characterized by rapid growth of abnormal white blood cells that accumulate in bone marrow and interfere with the production of normal blood cells. AML is the most common acute leukemia affecting adults, and its incidence increases with age [1]. Even with morphology, cytochemistry, and immunological phenotype it is still difficult to differentiate in some patients whether or not the leukemia cells are derived from myeloid or lymphoid lineage. These cases were classified as acute leukemia of ambiguous lineage according to World Health Organization (WHO) classification of hemopoietic malignancies [2]. The European Group for Immunological Classification of Leukemias (EGIL) proposed a set of diagnostic criteria for biphenotypic acute leukemia (BAL). This scoring system is based on the number and degree of the specificity of certain markers for myeloid or T/B lymphoid blasts [3]. The World Health Organization (WHO) recently released a revised version of the Classification of Hematopoietic and Lymphoid Malignancies, which includes significant modifications of the

∗ Corresponding author. Tel.: +20 55 232 1727, fax: +20 55 232 1510. E-mail address: mahaatfi@hotmail.com (M. Atfy). 0145-2126/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2011.04.011

diagnostic criteria for acute leukemias of mixed phenotype. These criteria are more stringent than those of the EGIL and rely heavily on positivity for myeloperoxidase [4]. Acute myeloid leukemia is frequently associated with chromosomal alterations. However, the presence of the Philadelphia chromosome (Ph) is an infrequent finding in 1–2% of newly diagnosed patients only. The t(9;22)(q34;q11) results in the formation of a Philadelphia chromosome (Ph) and generates an active chimeric BCR-ABL tyrosine kinase [5]. BCR-ABL positive acute myeloid leukemia (AML) is a rare disease, characterized by a poor prognosis, with resistance to induction chemotherapy and frequent relapses in responsive patients [6]. The fused BCR-ABl protein interacts with the interleukin-3 receptor beta(c) subunit. The BCR-ABl transcript is constitutively active, i.e. it does not require activation by other cellular messaging proteins. In turn, BCR-Abl activates a number of cell cycle-controlling proteins and enzymes, speeding up cell division. Moreover, it inhibits DNA repair, causing genomic instability [7]. Conventional cytogenetics are the recommended test for detecting t(9;22) in newly diagnosed leukemia patients, it should be initially performed on all patients. Chromosome banding analysis has the advantage of high specificity and an ability to detect alternate or additional cytogenetic defects that are valuable in diag-

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nosis and prognosis. However, it is also vulnerable to false negative results and requires viable marrow cells or more than 10% blasts in the peripheral blood to reliably culture the cells and visualize metaphases [8]. Fluorescence in situ hybridization (FISH) allows detection of the BCR-ABL translocation and detects cryptic and complex BCR-ABL [9]. The aim of this study was to illuminate on the incidence of existence of Philadelphia chromosome positive, and the presence of BCR-ABL translocation, in cases of acute myeloid and biphenotyping leukemia. As the awareness of the genetic status in leukemia cases help to determine its role in prognosis and in choosing the best approach to treatment. 2. Subjects and methods 2.1. Subjects The study included 464 subjects with newly diagnosed acute myeloid leukemia ranging in age from 20 to 72 years; 312 were males and 145 were females. The present study was conducted in Internal Medicine (Medical Oncology & Hematology Unit) and Clinical Pathology Departments, Zagazig University Hospitals from March 2006 to December 2009. Medical histories were taken from patients with emphasis on symptoms of anemia, fever, bleeding tendency, bone aches and abdominal enlargement, or any other previous ailments and treatment. Patients with a history of chronic myeloid leukemia were excluded from this study. Prior to receiving any medication, 1 ml of venous blood was withdrawn from each subject under aseptic precaution in a vacutainer tube containing EDTA for a complete blood count. Two ml of bone marrow samples were taken aseptically upon presentation, one ml on lithium heparin to prevent clotting, and the other one ml was added to a sterile tube containing EDTA for flow cytometric analysis and was stored in sterile conditions. 2.2. Methods Complete blood count (CBC) and bone marrow aspirates were examined for the presence of blast cells and the diagnosis of each leukemias subtype was established according to morphological, cytochemical and immunological criteria set forth by the French-American-British (FAB) and World Health Organization (WHO) classifications [10]. For immunophenotyping, bone marrow cells were stained with various combinations of fluorescein isothiocyanate (FITC), phycoerythrin (PE) and peridinchlorophyll protein (PerCP)-labeled monoclonal antibodies against the following antigens: CD3, CD5, CD13, CD14, CD19, CD20, CD34 and CD45 (BD, Biosciences, San Jose, CA), CD7, CD24, CD33, CD64 and CD10 (Dako). Other antibodies were used to identify cytoplasmic antigens as a triple color MPO, CD3, CD79a (BD), and nuclear terminal deoxynucleotidyl transferase (TdT) (Dako). The cutoff of positive marker was defined above 20% for lymphatic markers and 30% for myeloid markers. 2.3. Conventional karyotyping Culture: the BM cells are cultured in a medium {RPMI 1640} supplemented with fetal calf serum (Gibco laboratories, USA), l-glutamine (PAA laboratories, USA), penicillin and streptomycin 10000 u/ml and 10 mg/ml, respectively (Biochrom KG A2213) incubated at 37 ◦ C in a strict sterile condition. All cultures were set up for 24–72 h. Harvesting was done using 10 ␮g/ml colcemid (Gibco BRL KaryoMAX) and potassium chloride (5.59 g/L), chromosome spreading was done using 3–4 drops of cells suspended in fixative (freshly prepared by mixing 3 parts of absolute methanol with 1 part glacial acetic acid) were allowed to fall on a pre-cleaned glass slide from a height of about 40–60 cm (wet, cold, ethanol cleaned slides were used). G Banding was done, using trypsin Sigma solution (ready for use) stored at 4 ◦ C, and phosphate buffered saline (PBS). 2.4. Microscopy and automated karyotyping At least 20 metaphases were examined unless a clone was detected with lesser number; an automated karyotyping system was used for analysis. Karyotyping was done according to the International System for Human Cytogenetic Nomenclature (ISCN, 1995) [11]. There were 35 cases showing no metaphases; FISH was performed for these failed cases, only three cases were proved positive for BCR-ABL fusion gene. 2.5. Fluorescence in situ hybridization (FISH) is a highly sensitive molecular genetics technique, which enables detection of (BCR-ABL) complex not only in metaphase, but also in interphase cells. It was

Table 1 The incidence of Ph+ chromosome in all patients.

AML BAL

Total

Ph+

Incidence

Fisher exact test

451 13

9 5

1.99% 38.4%

0.00089

S: significant.

performed in the failed cases (no metaphases) of conventional cytogenetics analysis. It was based on the precise annealing of a single stranded DNA or RNA probe labeled with a fluorophore to complementary target sequences. This hybridization was visualized by fluorescence microscopy. FISH assays were performed according to the probe manufacturer’s instructions. The LSI bcr-abl Dual color, Single fusion translocation probe (Vysis, Stuttgart, Germany) was used. Slides were analyzed using an epifluorescence microscope (Olympus, BX40) and computerized image analysis software (Vysis Quips XL Genetics workstation). A minimum of 200 cells per specimen/probe were scored by two independent investigators. 2.6. Treatment For all patients, treatment with 7 days of cytarabine (Ara-C) in conventional or high-dose schedules alone, or in combination with amsacrine, or daunorubicin for 3 days. No imatinib or any tyrosine kinase inhibitors are included in the protocol of treatment. 2.7. Follow-up All patients were monitored from diagnosis of the disease until the end of 2009. The median monitoring period was 4 months for BAL and 12 months for AML. 2.8. Statistical analysis Results were entered, checked, and analyzed using SPSS version 10.0 (Statistical package for social science, SPSS Inc., Chicago, USA). The student “t” test was applied for comparison of means of two independent groups. For categorical data, the differences between controls and patient groups were analyzed by Mann–Whitney “U” test. Kaplan–Meier method was used to estimate survival, and differences between groups were analyzed using Log rank test. p value of less than 0.05 was accepted as significant.

3. Results From a large group of 464 acute myeloid leukemia patients, 451 patients were established as acute myeloid leukemia and 13 patients were diagnosed as BAL using the 2008 WHO Classification of Leukemias. The overall incidence of BAL was 2.8% of all acute leukemia. Subsequent to WHO classification, it was found that 10(76.9%) patients had a myeloid/B-lymphoid (M/B) biphenotype; 2 (15.3%) had a myeloid and T-lymphoid (M/T) biphenotype; and one (7.7%) had a T-lymphoid/B-lymphoid/myeloid (M/B/T) triphenotype. Primarily, twenty-one patients, categorized as acute promeylocytic leukemia (M3), were not included at the outset of the study because of its particular biological features. In the current study, more restrictive criteria (proposed by the 2008 WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues) were applied to define BAL [10], rather than EGIL. 3.1. The incidence of Ph+ chromosome Detection of Ph+ chromosome by karyotyping and/or FISH was done for all AML and BAL patients. Nine cases only (1.99%) of AML patients could be diagnosed Ph+ AML, and 5 cases (38.4%) of BAL patients could be diagnosed as Ph+ BAL (Table 1). All patients with Ph+ chromosome had no other chromosome aberrations. 3.2. Immunophenotyping of the leukemic blast cells of Ph+ AML patients The most frequently observed positive markers were CD33 and CD13, which were found in all nine cases (100%), and CD64 in 8/9

M. Atfy et al. / Leukemia Research 35 (2011) 1339–1344 Table 2 Details of the immunophenotype of the leukemic blasts in AML Ph+ cases.

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Table 3 The immunophenotyping of the BAL Ph+ cases.

Age/sex

55/M

45/F

52/M

44/M

29/F

38/M

25/F

49/M

32/M

cCD79a CD19 CD20 CD24 CD10 CD34 cCD3 CD5 CD7 TDT CD13 CD33 CD64 CD14 MPO

− − − + − + − − + − + + + − +

− + − − − − − − − − + + + − +

− − − − − + − − − − + + + − +

− − − − − + − − + − + + + − +

+ − − − − − − − − − + + + − +

− − − − − + − − + + + + − − +

− − − − − + − − + − + + + − +

− − − − − + − − − − + + + + +

− + − − − − − − − − + + + − +

(88.9%) of patients. MPO activity was assessed by flow cytometry and/or cytochemistry in all cases. MPO was positive in 9/9 (100%) of AML patients by flow cytometry and positive in 7/9(60%) by cytochemistry. For the B-lymphoid phenotype, CD79a was tested in patients; it was negative in all AML cases, except one 1/9 (11.1%). The most frequently found positive T-lymphoid marker was CD7, found in (4/9; 44.4%) of AML cases. So, this marker was considered to be apparently expressed. CD3, CD5, CD20 and CD10 were negative in all AML patients (9/9; 100%). The other less-encountered lymphatic marker was CD24 found in one case only. Unlike, CD19 was found in two AML cases that could be considered as M2. So, 7/9 (77.8%) of AML patients had aberrant expression of lymphoid (B or/and T) markers. Stem cell markers CD34 were positive in 6/9 (66%) of AML patients, and TdT was positive in 1/9 (11.1%) cases (Table 2). According to the FAB, one case was diagnosed as M0, 3 cases as M1, 4 cases as M2, and one case as M4. 3.3. AML group with Ph+ chromosome The median age of this group was 38 years (range 25–55 years). Six patients were males and 3 patients were females. The mean ± SD of WBC count was 49.9 ± 27.4 (range 22–110 × 109 /L); the mean ± SD of hemoglobin levels in this group of patients was 7.5 ± 1.9 (range 4–9.8 mg/dl); and the platelet counts ranged from 30 to 110 × 109 /L with mean ± SD of 67.1 ± 29.7. A comparison between the two groups showed that there was statistically significant difference according to the age and the TDT expression (p < 0.05). No statistical differences were found as regard the WBCs, Hb, platelets count, CD34 expression, or the rate of CR after the 1st induction therapy (p > 0.05) (Table 3). 3.4. Immunophenotyping of the leukemic blast cells of Ph+ BAL patients The most frequently observed positive markers were CD33 and CD13 in 5/5 (100%) and 4/5 (80%), respectively, and CD64 in 5/5 (100%) patients. For the B-lymphoid phenotype, CD79a was tested in patients and was positive in 4/5 (80%) of them. The other frequently encountered markers were CD19 (4/5; 80%), CD24 (3/5; 60%) and CD10 (3/5; 60%). T-lymphoid markers cyCD3 and CD5 were positive in patients who had the T-phenotypic association. The most frequently found positive T-lymphoid marker was CD7. It was found in 3 patients, including one patient who did not have T-cell-specific markers and therefore, it was apparently expressed. The stem cell markers CD34 and TdT were positive in 5/5 (100%),

Age/sex

M/B

M/T

M/T/B

61/M

38/M

53/F

60/M

48/M

B-Lineage cCD79a CD19 CD20 CD24 CD10 CD34

+ + + − + +

+ + − + − +

+ + − + + +

− − − − − +

+ + − + + +

T-lineage cCD3 CD5 CD7 TDT

− − − −

− − + +

− − − +

+ + + +

+ + + +

Myeloid lineage CD13 CD33 CD64 MPO

+ + + +

+ + + +

+ + + +

+ + + +

− + + +

Fig. 1. Kaplan–Meier estimates the overall survival probability of BAL and AML groups patients (p = 0.0312). Group 1: BAL (Ph+) group; Group 2: AML (Ph+) group.

and 4/5 (80%), respectively in BAL patients. CD20 was positive in 1/5 cases only (20%). MPO activity was evaluated by flow cytometry and/or cytochemistry in all cases. MPO was positive in 5/5 (100%) patients by flow cytometry and positive in 3/5 (60%) by cytochemistry (Table 4). 3.5. BAL group with Ph+ chromosome The median age of Ph + BAL group was 52 years (range 38–61 years). Four patients were males and one patient was female. The mean ± SD of WBC count was 58.6 ± 41.9 (range 18–109 × 109 /L); the mean ± SD of hemoglobin level in this group of patients was 9.02 ± 1.2 (range 7.2–10.5 mg/dl); and the platelet counts ranged from 60 to 110 × 109 /L with mean ± SD of 86.2 ± 19.4. Patients were monitored for a median of 4 months (range 1–7 months) for BAL patients, and for a median of 12 months (range 4–22 months) for AML patients Ph+ chromosome. There was significant difference in overall survival (OS) between BAL patients and AML Ph+ chromosome patients (p = 0.0312), Fig. 1. 4. Discussion Cytogenetic characteristics played an important role in acute leukemia and have appeared as one of the most important prognostic factors [12]. The Philadelphia chromosome (Ph), resulting

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Table 4 The patients’ characteristics in BAL and AML with Ph+ chromosome.

Age (years)a WBC (×109 /L) Hemoglobin (g/dl) Platelets count (×109 /L) CD34+ b TDTb CR after the 1st induction therapyb

BAL (n = 5)

AML (n = 9)

p

50 (38–61) 58.6 ± 41.9 (18–109) 9.02 ± 1.2 (7.2–10.5) 86.2 ± 19.4 (60–110) 5 (100%) 4 (80%) 2 (40%)

38 (25–55) 49.9 ± 27.4 (22–110) 7.5 ± 1.9 (4–9.8) 67.1 ± 29.7 (30–110) 6 (66.7%) 1 (11.1%) 5 (55.6%)

0.035 (S) 0.644 0.134 0.224 0.298 0.022 (S) 0.499

Data presented as mean ± SD (range). CR: complete remission; S: significant. a Data are presented as median (range). b Data presented as number (%).

from t(9;22)(q34;q11.2), occurs in 2–5% of children with ALL, and in approximately 25% of adults with ALL. The outcome for both children and adults with Ph ALL is poor [13] (Fig. 2). Unlike more commonly seen acute leukemias classified as B or T lymphoid or myeloid lineage, BAL is a type of acute leukemia with uncommon biological and clinical features [14]. In this study, an evaluation was done to shed light on the incidence of existence of Philadelphia chromosome positive, and the presence of BCR-ABL translocation, in cases of acute myeloid and biphenotyping leukemia. The incidence of BAL in this series was 2.8% of all acute myeloid leukemia. Xu et al. [14] reported that 4.6% of their AML were diagnosed as BAL. Earlier reports on BAL showed variability in the incidence; however, due to this objectivity in definition, the incidence of BAL has recently shown some consistency (2–5%). Lastly, in pediatric studies, the incidence of BAL was 3.8% [15]. Previous studies have reported incidences of the Ph+ chromosome among AML cases ranging from 1% to 3%, whereas latest

studies have reported incidences between 0.6% and 0.9% [16,17]. After exclusion of biphenotypic leukemias and cases in which Ph+ AML developed in patients with a preexisting myeloid neoplasm, the incidence of de novo Ph+ AML was only 0.35% of all AML cases [18]. These results do not exactly conform to this study and that may be due to the limitation of this current study of relatively small numbers, and imperative conclusions can still be drawn. In several earlier studies, incidence of t(9;22) in BAL was high, ranging from 28% to 35% [14]. These findings are in harmony with the findings in this study, as it was found that the Ph+ chromosome was established in 38.4% of cases. It was found that age, as a biological parameter, was significantly increased in Ph+ chromosome BAL patients. Philadelphia chromosome positive leukemias are more common in older age groups [15]. The CR rate after the first time induction therapy was 40% only in Ph+ chromosome BAL. There was no statistical difference when comparing with Ph+ chromosome AML. The median survival time in

Fig. 2. Flow data analysis of three cases. The first case is AML with apparent expression of CD7 (positive: MPO, CD33, HLA-DR and CD7 and negative: cCD79a and cCD3). The second case is BAL (M/T) (positive: MPO, cCD3, CD33, CD7 and HLA-DR and negative: cCD79a). The third case is BAL (M/B) (positive: MPO cCD79a, CD33, HLA-DR, CD19, CD10 and CD7 and negative: cCD3).

M. Atfy et al. / Leukemia Research 35 (2011) 1339–1344

Ph+ chromosome BAL was only four months. This was much shorter than those of Ph+ chromosome AML. Ph+ AML have an aggressive clinical course, and responses to therapy were of limited duration [18]. The poor prognosis of these cases suggests that the t(9;22) cytogenetic abnormality be added to the group of poor cytogenetic risk factors in AML. The prognosis of biphenotypic acute leukemia patients is poor when compared with de novo acute myeloid leukemia or acute lymphoblastic leukemia [3]. This suggests that the Ph+ chromosome might be a poor prognosis indicator for BAL patients. Contrarily, the prognosis of children with BAL was good in another study [15], as their survival being comparable to that of patients with high-risk ALL and better than that of patients with AML. This good prognosis was retained even when the diagnostic criteria for BAL were those of the more stringent WHO classification. The phenotype distribution in our BAL patients was no different from previously published distributions, with the majority of patients having a B-lymphoid/myeloid phenotype (3/5), and the BAL patients with T-lymphoid/myeloid phenotype were considered the next most common phenotype after the B-lymphoid/myeloid phenotype [15]. Our cases of Ph chromosome-positive AML and BAL demonstrated a high degree of immaturity. Most AML cases showed high prevalence of immature FAB subtypes (M0, M1 and M2). TDT expression was detected in more than half of AML Ph+ chromosome and in most of BAL Ph+ chromosome. Immunophenotyping of Ph-positive AML discloses CD34 coexpression in 6/9 patients. CD34 positive was predominant in virtually all BAL patients compared to AML (even it is not significant p > 0.05). Other researchers have found that the percentage of CD34 and TdT positive was more than 80%. These results support the supposition that BAL may originate from the stem/progenitor cells [14]. Conventional karyotyping is the routine method for chromosomal diagnosis from well spread metaphases. It is not always possible to achieve good quality metaphases for chromosomal diagnosis and treatment monitoring. Fluorescence in situ hybridization is a very sensitive technique, which can detect Ph chromosome (BCR-ABL fusion gene) even in interphase cells or poor quality metaphases. FISH should be used in the failed cases of conventional cytogenetic analysis. In the current study, the overall incidence of de novo Ph+ AML among all cases of AML was 1.99%, whereas 38.4% of patients with acute biphenotypic leukemia were diagnosed Ph+. For the failed cases, only three cases were proven to be positive for BCR-ABL fusion gene [19]. Patients with Ph+ chromosomes treated only with conventional chemotherapy have a poor long-term survival [2]. Finally, most reported cases of Ph + AML predate imatinib and were treated with such conventional AML therapy. In one series, 4 (36%) of 11 patients treated with conventional chemotherapy regimens achieved complete responses, with duration of 3–14 months, and a median survival of 7 months [13]. However, none of six patients with Ph+ AML in another study treated with conventional chemotherapy achieved remission [13]. STI571 (imatinib) is a selective tyrosine kinase inhibitor of the ABL1 tyrosine kinase, and induces high rates of complete cytogenetic remission. No patient in this study treated with imatinib or others tyrosine kinase inhibitors. So, generally there is little information on the response of patients diagnosed with Ph+ AML to imatinib, with only rare case reports available. Ongoing and future clinical trials will establish whether front-line therapy with second-generation ABL kinase inhibitors, i.e., dasatinib are superior to imatinib. Results may differ depending on their use as single-agents or combination ther-

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apy with specific attention to sequence and dosing of these agents [20]. 5. Conclusion The Ph+ chromosome is a rare molecular abnormality in AML patients but it is more common in BAL patients, and usually implies a poor prognosis. Treatment with the tyrosine kinase inhibitor STI571 may improve outcomes. Further trials with a larger patient number are worth exploring along with the role of imatinib in Phpositive AML. Conflicts of interests The authors declare that they have no competing interests. Acknowledgements Funding: No funding to declare. Authors’ contributions: MA carried out the flow cytometry analysis and wrote most of the manuscript. NA and AE carried out karyotyping and Fish. MA, NA and AE contributed to concept and design and provided data. MA performed statistical analysis. All authors read and approved the final manuscript. References [1] Burnett AK. Acute myeloid leukemia: treatment of adults under 60 years. Rev Clin Exp Hematol 2002;6:26–45. [2] Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009;114(5):937–51. [3] Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao A, et al. Proposals for the immunological classification of acute leukemias European Group for the Immunological Characterization of Leukemias (EGIL). Leukemia 1995;9:1783–6. [4] Borowitz MJ, Bene MC, Harris NL, Porwit A, Matutes E. Acute leukemias of ambiguous lineage. In: Swerdlow SH, Campo E, Harris NL, Pileri ES, Stein SA, Thiele H, Vardiman J, editors. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyons: IARC Press; 2008. p. 150–5. [5] Quartarone E, Allegra A, Alonci A, Toscano G, Bellomo G, Corigliano E, et al. Imatinib myselate treatment in a patient with chemoresistant ph+ acute myeloid leukemia: possible role of Wilms’ tumor gene 1. Tumori 2007;93:230–1. [6] Kurzrock R, Kantarjian HM, Druker BJ, Talpaz M. Philadelphia chromosomepositive leukemias: from basic mechanisms to molecular therapeutics. Ann Intern Med 2003;138:819–30. [7] Cimino G, Pane F, Elia L, Finolezzi E, Fazi P, Annino L, et al. The role of BCR/ABL isoforms in the presentation and outcome of patients with Philadelphiapositive acute lymphoblastic leukemia: a seven-year update of the GIMEMA 0496 trial. Haematologica 2006;91:377–80. [8] Jha CB, Kucheria K, Choudhary P. Diagnostic role of conventional cytogenetics and fluorescence in situ hybridization (FISH) in chronic myeloid leukemia patients. Kathmandu Univ Med J 2006;4(2):171–5. [9] Heim S, Mitelman F. Chromosomal and molecular genetics aberrations of tumor cells. In: Cancer cytogenetics. 3rd ed. Hoboken, New Jersey: Wiley-Blackwell; 2009. [10] Zhao XF, Gojo I, York T, Ning Y, Baer MR. Diagnosis of biphenotypic acute leukemia: a paradigmatic approach. Int J Clin Exp Pathol 2010;3(1):75–86. [11] Mitelman F. ISCN 1995: an international system for human cytogenetics nomenclature. Basel, Karger; 1995. Recommendations of the International Standing Committee on Human Cytogenetics Nomenclature. Memphic, Tennessee, USA; 1994. [12] Hassanzadeh-Nazarabadi M, Modarressi A, Miranpour H. The role of chromosomal aberration in childhood leukemia. J Sci, Islamic Republic of Iran 2004;15(3):219–25. [13] Heerema NA, Harbott J, Galimberti S, Camitta BM, Gaynon PS, Janka-Schaub G, et al. Secondary cytogenetic aberrations in childhood Philadelphia chromosome positive acute lymphoblastic leukemia are nonrandom and may be associated with outcome. Leukemia 2004;18:693–702. [14] Xu XQ, Wang JM, Lüsq, Chen L, Yang JM, Zhang WP, et al. Clinical and biological characteristics of adult biphenotypic acute leukemia in comparison with that of acute myeloid leukemia and acute lymphoblastic leukemia: a case series of a Chinese population. Haematologica 2009;94(7):919–27. [15] Al-Seraihy AS, Owaidah TM, Ayas M, El-Solh H, Al-Mahr M, Ali AA, et al. Clinical characteristics and outcome of children with biphenotypic acute leukemia. Haematologica 2009;94(12):1682–90.

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