IgH and TCRγ gene rearrangements, cyclin A1 and HOXA9 gene expression in biphenotypic acute leukemias

IgH and TCRγ gene rearrangements, cyclin A1 and HOXA9 gene expression in biphenotypic acute leukemias

Leukemia Research 30 (2006) 211–221 IgH and TCR␥ gene rearrangements, cyclin A1 and HOXA9 gene expression in biphenotypic acute leukemias M. Golemovi...

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Leukemia Research 30 (2006) 211–221

IgH and TCR␥ gene rearrangements, cyclin A1 and HOXA9 gene expression in biphenotypic acute leukemias M. Golemovi´c a,1 , M. Suˇci´c a , R. Zadro a , S. Mrsi´c a , M. Mikuli´c b , B. Labar b , L.j. Raji´c c , D. Batini´c a,∗ a

c

Division of Immunology, Clinical Institute of Laboratory Diagnosis, Zagreb University School of Medicine, Zagreb Clinical Hospital Center, 10000 Zagreb, Croatia b Department of Hematology, Zagreb University School of Medicine and Clinical Hospital Center, 10000 Zagreb, Croatia Department of Pediatrics–Salata, Zagreb University School of Medicine and Clinical Hospital Center, 10000 Zagreb, Croatia Received 5 April 2005; received in revised form 6 July 2005; accepted 6 July 2005 Available online 15 August 2005

Abstract In this study we investigated IgH and TCR␥ gene rearrangements, cyclin A1 and HOXA9 gene expression as well as the in vitro growth of biphenotypic acute leukemia (BAL) blasts in relation to acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The aim of the study was to correlate BAL morphology and its biological parameters in order to get information that might be used for additional stratification of BAL. This rare form of AL was identified in a total of 10 patients, comprising 4.3% of adult and 3.0% of pediatric patients with de novo AL referred to our institution during the 1999–2003 period. Our results indicate that IgH and TCR␥ gene rearrangements correlated well with lymphoid BAL morphology, whereas the expression of cyclin A1 correlated with myeloid and undifferentiated BAL morphology. Surprisingly, HOXA9 expression, a marker associated with myeloid cell lineage, showed no strong correlation with BAL morphology. Finally, in vitro growth of blasts during a 7-day culture showed autonomous cell growth in 3/10 AML and 3/8 myeloid BAL samples tested, but not in any of the AL with lymphoid features. Further studies are needed to confirm these findings and to extend research to a broader spectrum of cell markers. © 2005 Elsevier Ltd. All rights reserved. Keywords: Biphenotypic acute leukemia; Morphology; IgH; TCR; Gene rearrangement; Cyclin A1; HOXA9

1. Introduction Acute leukemia (AL) expressing multiple cross-lineage antigens can either be leukemia with two separate populations of blasts, each from a different lineage, or leukemia with a single blast population expressing multiple lineage markers. The new World Health Organization classification system refers to these two groups of AL as bilineal acute leukemia and biphenotypic acute leukemia (BAL), respectively. Bilineal and biphenotypic AL should be recognized as different forms ∗

Corresponding author. E-mail addresses: [email protected] (M. Golemovi´c), [email protected] (D. Batini´c). 1 Tel.: +385 1 2388 336; fax: +385 1 2312 079. 0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.07.001

of AL and clearly distinguished from those with aberrant expression of a marker from another lineage (e.g. the expression of CD19 in AML-M2). Based on a previously described scoring system [1], the European Group for the Immunological Classification of Leukemias (EGIL) proposed diagnostic criteria for BAL [2]. The scoring system is based on the number and specificity of the lymphoid and myeloid markers expressed by blasts, distinguishing true BAL from ALL and AML with aberrant marker co-expression. According to the studies using EGIL diagnostic criteria, most of BAL cases have combined myeloid and Blymphoid or myeloid and T-lymphoid immunophenotype, whereas combinations of B- and T-lymphoid markers and those with trilineage differentiation are rare [3–5]. The cases of AL showing cross-lineage co-expression of leukocyte dif-

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ferentiation antigens represent more than 20% of all AL cases [6,7], whereas the reported incidence of BAL is in the range of 4–8.5% [5,8]. So far, only a few biological features of this most probably heterogeneous leukemia subtype have been reported, including expression of CD34 and the multidrug resistancerelated P-glycoprotein [8]. Regarding other cell surface markers with potential diagnostic and/or prognostic importance, the expression of CD133 and CD90 does not further distinguish BAL from other types of acute leukemia [9]. Although there is no single chromosome specific abnormality, BAL often reveals t(9;22) and rearrangements involving 11q23 [5,7,8,10]. Chromosomal translocations involving 11q23 (mixed lineage leukemia gene, MLL) result in leukemia with aberrant expression of some native MLL target genes, including HOX genes. BAL is generally associated with poor response to treatment and poor prognosis. The reported causes of poor prognosis in BAL have been related to intrinsic cell characteristics such as unfavorable karyotypes, over-expression of Pgp (MDR-gene over-expression) and immature cell marker (CD34) [5]. In addition, there has been a lack of uniform treatment of BAL patients. The use of combined lymphoid and myeloid drugs for induction is a highly toxic therapy resulting in a high incidence of early deaths [3,7]. Better knowledge concerning biological features of this disease could contribute to improved diagnosis and classification of AL as well as result in new potential therapeutic strategies. The aim of this study was to analyze biological properties of BAL blasts from 10 patients referred to our institution during the 1999–2003 period. Clinical and biological data were compared to 15 AML and 15 ALL patients treated during the same period of observation. Apart from routine multiparametric diagnostic parameters (i.e. the immunophenotype, morphology and cytogenetics), we investigated the rearrangements of immunoglobulin heavy chain (IgH) and T-cell receptor ␥ (TCR␥) genes, expression of cyclin A1 (CycA1) and HOXA9 genes as well as in vitro parameters of leukemia cells growth.

2. Patients and methods 2.1. Patients and cells A total of 40 cases of AL included in this study were reviewed for their biological properties at the Laboratory of Immunology, Zagreb Clinical Hospital Center (Croatia), during the 1999–2003 period. There was a total of 10 patients (5 children and 5 adults) diagnosed as having BAL based on morphology, immunocytochemistry and immunophenotyping by flow cytometry. For a better comparison of observed biological properties, the study also included bone marrow cells from 15 patients with de novo ALL (10 children and 5 adults) and 15 patients with AML (1 child and 14 adults). The patients were treated at the Department of Hematology and

Oncology, Department of Pediatrics and at the Department of Hematology, Zagreb Clinical Hospital Center. Normal bone mononuclear cells (BMMNC) were obtained from healthy bone marrow donors. All subjects in the study gave informed consent to the use of their biological samples. Bone marrow mononuclear cells (BMMNC) from healthy and leukemic patients were separated by ficoll density gradient separation, followed by freezing of the cells in the freezing medium containing 10% DMSO and storage in liquid nitrogen. Control cells (for RNA analysis) included the following acute leukemia cell lines: NALM-6 (human pre-B ALL), SUDHL4 (human follicular B-lymphoma), HL60 (human acute promyelocytic leukemia) and K562 (human chronic myelogenous leukemia). 2.2. Diagnosis of AL All leukemia cases included in this study were classified according to the French–American–British Cooperative Group (FAB) criteria. The immunophenotype was assessed in all but one case with a standard procedure according to EGIL [2]. Cytogenetic analysis (G-banding and FISH) was successfully made in 21 of 40 cases (Table 1). 2.3. Flow cytometry Following density gradient separation, bone marrow cells were immunophenotyped by using a standard panel of monoclonal antibodies and flow cytometry (FACScan, Becton Dickinson, USA). The following antibodies were used in double and triple staining procedures: CD1a, CD2, CD3, CD5, CD7, CD13, CD15, CD16, CD19, CD23, CD33, CD38, CD41, CD64, CD65w, CD71, CD79a, c-kit/CD117, antiglycophorin A, anti-MPO, anti-Tdt (all from DAKO, Denmark); CD4, CD8, CD10, CD11c, CD14, CD20, CD22, CD34, CD45, CD56, anti-HLA DR (all from Becton Dickinson, USA); CD3, CD4, CD8, CD13, CD14, CD15, CD19, CD33, CD64, anti-lysozyme (all from Caltag, USA). Criteria used for the classification of BAL were based on the previously described scoring system by the European Group of Immunological Classification of Leukemias (EGIL) [2,11]. To determine cell surface antigens, cells were washed in PBS and 1 × 106 viable cells in 100 ␮l volume were incubated with specific monoclonal antibody for 15 min in the dark at room temperature. Following the incubation, cells were washed, resuspended in 0.5 ml PBS and analyzed on flow cytometer. Forward and side scatters and fluorescence were measured for each sample on a FACScan flow cytometer (Becton Dickinson). Data were acquired and analyzed using CellQuest software (Becton Dickinson). A region was drawn around the abnormal cell population (blast gate) which could be clearly separated from the remaining cells due to a high number of blasts in the majority of cases (Table 1). In some cases the blast gate was further modified using standard diagnostic panel of antibody-combination in order to contain a minimum of normal cell contamination (mostly T

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213

Table 1 Clinical and laboratory data of acute leukemia patients Pt. #

Age (years)

FAB

Blasts (%)

IF

CD-markers

ALL 1

15

L2

50

T

cCD3+CD5+ HLA−DR+

2 3 4 5 6 7 8 9 10 11 12 13 14 15

4 8 3 7 2 9 10 3 4 23 24 38 56 28

L2 L1 L2 L2 L2 L2 L3 L3 L1 L3 L1 L2 L1 L2

90 56 95 92 90 90 90 87 90 65 99 98 96 51

T T T Common Common Common Common Common Common B-ALL Pro-B Common Common T

cCD3+CD2+CD5+ cCD3+CD5+CD10+ cCD3+CD5+CD10+CD13+ cCD79a+CD19+CD10+ cCD79a+CD19+CD10+CD13+ cCD79a+CD19+CD10+CD13+ cCD79a+CD19+CD10+ cCD79a+CD19+CD10+ cCD79a+CD19+CD10+CD13+CD33+ cCD79a+CD19+CD10+mIgM+ cCD79a+CD19+HLA−DR+ CD10+CD19+HLA−DR+MPO+ cCD79a+CD19+CD10+ ND (at diagnosis)

AML 16 46 17 59

M1 M1

97 97

AML AML

CD13+CD33+MPO+CD14−HLA−DR− MPO+CD33+CD13−CD14−CD15− CD65−HLA−DR− CD13+CD33+HLA−DR+CD14−CD15− MPO+HLA−DR+CD13−CD14−CD33−/ MPO+CD13+CD33+ CD13+HLA−DR+ MPO+CD65+CD15+HLA−DR+CD13−CD33− CD13+CD33+MPO+HLA−DR+CD14− CD13+CD14+CD33−HLA−DR− CD33+MPO+CD13−CD14−CD117−

18 19

44 44

M1 M2

53 85

AML AML

20 21 22 23 24

59 24 49 21 22

M2 M2 M2 M3 M3

83 45 80 97 65

AML AML AML AML AML

25 26 27 28

37 26 32 12

M4 M4 M4 M5

84 85 75 93

AML AML AML AML

29 30

36 32

M5a M5a

87 86

AML AML

95

BAL

80

BAL

BAL 31

0.4

32

4

L1+ M5b L2

33 34

9 11

L2 M5a

95 97

BAL BAL

35 36

15 30

L2 M1

90 95

BAL BAL

37 38

65 45

M5 M5a

88 90

BAL BAL

39 40

45 29

AUL AUL

80 95

BAL BAL

CD34 (%)

1.18

0.0 3.0 97.7 78.7 19.0 ND 54.6 4.5 98.0 0.0 57.4 98.7 11.0 1.0

Cytogenetics

42,xy, del 1p (p34), −4,−9,−9, −10,−15,−21,−22/49,xy,del 1p,−6,+8,+15,+20,−22,+2mar ND ND 46, xy ND ND 46, xx, t(1;11), t(5;12) ND ND ND ND 46,xy ND FISH = MLL− 46,xy/,49,xy,del(1p), 6,+8,+15, +20,+2mar/, 42,xy,del(1p) (p34−tel),−4,−9,−9, −10,−15,−21,−22

4.2 0.1

46, xx 46, xy

40.0 44.6

ND 46, xy

0.0 82.0 ND 8.0 0.0 24.4 64.6 2.0 79.0

ND 46, xy 46, xx ND 46,xx,t(15;17) FISH 75% t(15;17) 46, xx, inv(16) 46, xx ND 46, xy, t(7;14), inv(9)

2.0 88.0

ND ND

cCD79a+CD19+CD11b//MPO+CD11b+CD13+CD14+ CD15+CD33+CD117+CD7+CD19+CD20 CD19+CD22+MPO+CD13+ HLA−DR+

22.5

46, xx, t(11;19); 11q23

75.4

CD19+cCD79+CD10+cIgM+CD13+MPO+HLA−DR+ CD19+ cCD79+cCD3+ CD117+CD13+CD33+ HLA−DR+a cCD79+CD10+ TdT+MPO+CD13+CD33+ cCD3+CD7+ TdT+CD13+MPO+HLA−DR+

99.0 96.7

50, xx, der(1)ad(1)(p36) dup(1)(q21),+5+8+16+21/2 ND 47,xy,+11

cCD79a+TdT+CD13+CD14+CD15+CD33+HLA−DR+ cCD3+cCD79a+CD7+TdT+MPO+Lisozyme+CD117 +CD13+ cCD79+CD19+ CD10+CD13+CD33+MPO+HLA−DR+ CD2+cCD3+CD7+ MPO+CD13+CD11b+HLA−DR+

0.0 93.0

ND 47,xx,+mar/FISHmar = cen8(D8Z1+) 11q23 ND

62.0 69.0

ND 46,xy/FISH = MLL−

CD13+CD33+CD14+HLA−DR+ CD13+CD33+CD14+HLA−DR+CD15−CD2+ CD13+CD14+MPO+HLA−DR+ HLA−DR+CD33+MPO−CD13−CD14−CD117− CD7+CD19+ CD13+CD33+MPO+CD117+HLA−DR+CD7+CD19+ CD13+CD33+CD117+MPO+HLA−DR+CD7+CD19+

ND 76.3

FAB: French–American–British classification; blasts: percentage of blasts in processed bone marrow specimens; IF: immunophenotype; CD markers of other cell lineage indicating co-expression are marked in bold; ND: no determination. a ANAE+ (inhibited with NaF).

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cells). Samples were considered positive for antigen binding when more than 20% of the cells in blast gate were stained with a particular antibody in excess of the isotypic negative control. Intracytoplasmic antigens (CD3, CD79a, ␮-chain, MPO and lysozime) and nuclear Tdt were determined as previously reported [12,13]. Briefly, cells were permeabilized and fixed in IntraStain Reagents (DAKO, Denmark). Following the pre-treatment, cells were incubated with specific monoclonal antibody and then processed as described above. The number of blast cells in each sample was assessed by May–Gr¨unwald–Giemsa staining of ficoll-separated cells and/or by flow cytometry as described above. 2.4. Clonal rearrangements of IgH and TCR-γ genes 2.4.1. IgH primers Primers used for amplifying the IgH gene involving CDRIII region were adopted from Yamada et al. [14]. The finding of the single 100–120 bp size PCR product identified with vertical polyacrylamid gel electrophoresis was considered a proof of clonality. Primers for analysis of IgH rearrangements involving CDRI region were adopted from Yamada et al. [14] (Jhspecific primer) and Salo et al. [15] (Vh-specific primers). In the analysis including CDRI region, three different combinations of primers were used: one Jh primer and three different Vh-specific primers (Vh1, Vh2, Vh3). The finding of the single 300–350 bp size PCR product identified with vertical polyacrylamid gel electrophoresis was considered a proof of clonality. 2.4.2. TCRγ primers To assess the rearrangements of TCR␥ gene, multiplex PCR method was used. The primers used to detect variable genes of all four families of V␥ genes (V␥I-2, 3, 4, 5, 8; V␥II-9; V␥III-10 and V␥IV-11) were adopted from F¨odinger et al. [16], whereas J␥-specific primers were adopted from Trainor et al. [17]. The expected product size of clonal TCR␥ rearrangement was 170–230 bp. 2.5. Expression of cyclin A1 and HOXA9 genes 2.5.1. Total RNA isolation Total RNA was isolated according to the manufacturer’s recommendations. Promega’s RNAgents® Total RNA Isolation System kit was used. Briefly, totRNA was isolated from 2.5 × 106 cells. According to the cell number, appropriate volumes of the kit reagents were added. Firstly, cells were pelleted by centrifugation at 300 × g for 5 min at 4 ◦ C. The pellet was washed once more in cold PBS. Pre-chilled denaturing solution (Promega, USA) was added and the material was mixed. Then 2 M sodium acetate (pH 4.0) was added and mixed thoroughly by inverting the tube four to five times. Recommended amount of phenol:chloroform:isoamyl alcohol (Promega, USA) was added to the tube (only lower

organic phase in the bottle was used). The tube was mixed by inversion three to five times, shaken vigorously for 10 s and then chilled on ice for 15 min. The mixture was then transferred to the fresh tube and centrifuged at 10,000 × g for 20 min at 4 ◦ C. The top aqueous phase that contained RNA was carefully removed to a fresh DEPC-treated tube, an equal volume of the provided isopropanol (Promega, USA) was added and the sample was incubated at −20 ◦ C for additional 30 min. The RNA was pelleted by centrifugation at 10,000 × g for 10 min at 4 ◦ C. The pellet was washed once more with 1 ml of 75% ethanol by centrifugation, air-dried and then dissolved in 20 ␮l nuclease-free water (Promega, USA). Prepared samples were stored at −20 ◦ C. The purity and concentration of isolated RNA was determined spectrophotometrically by measuring relative absorbance at 260 and 280 nm. 2.5.2. RT-PCR The reverse transcription system kit (Promega, USA) was used for cDNA generation. For the reaction, 1 ␮g RNA was used in total reaction volume of 20 ␮l containing 5 mM MgCl2 (25 mM), 1× RT-buffer, 1 mM dNTPs (10 mM), 1 U/␮l recombinant RNasin ribonuclease inhibitor, 15 U AMV reverse transcriptase (20 U/␮l) and 0.5 ␮g random primers (0.5 mg/ml). The RNA sample was preincubated at 70 ◦ C for 10 min and then the reaction mixture was added. The reverse transcription was performed on a thermal cycler (Applied Biosystems, USA) by using the following parameters: 25 ◦ C for 10 min; 42 ◦ C for 30 min; 99 ◦ C for 5 min; 4 ◦ C for 5 min; hold 4 ◦ C. The amplified cDNA was used to assess the CycA1 and HOXA9 gene expression in tested cells. PCR reaction was performed with an Applied Biosystems thermal cycler (Applied Biosystems, USA) using the following conditions: 92 ◦ C for 2 min followed by 40 cycles at 94 ◦ C for 45 s; 60 ◦ C for 45 s; 72 ◦ C for 45 s followed by 72 ◦ C for 7 min and 4 ◦ C hold. The reaction mixture (total of 50 ␮l) contained 4 ␮l cDNA, 1.5 mM MgCl2 (25 mM; Promega, USA), 1× thermophilic DNA polymerase reaction buffer (10×; Promega, USA), 200 ␮M dNTPs (40 mM; Promega, USA), 1 U Taq DNA polymerase enzyme (5 U/␮l; Promega, USA) and 1 ␮M of each primer (MWG, Germany). The primers for U1A housekeeping gene were adopted from Oostveen et al. [18], for CycA1 gene from Kr¨amer et al. [19], and for HOXA9 gene from Kawagoe et al. [20]. To detect amplified PCR products, horizontal electrophoresis in 1.5% agarose gel was performed. 2.6. Short term liquid cell culture After thawing, BMMNCs were counted and viability was determined. The BMMNC were seeded in BIT9500 serum-free medium (Stem Cell Technologies, Vancouver, BC, Canada) in 24-well plates (1 × 106 ml−1 ) under three different in vitro growth conditions. Cell culture designated with letter “S” indicated the condition without addition of exoge-

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nous growth factors (GFs). Growth conditions designated with letters “L” and “M” indicated addition of FL + IL-7, and FL + GM-CSF, respectively. The cytokines IL-7 and FL (Flt3-ligand) were purchased from Serotec (UK), whereas GM-CSF was purchased from Sandoz (Switzerland). On days 0, 3, 5 and 7, the cell count and viability were determined. On day 0 of experiment, RNA and DNA were extracted from BMMNC and used for analysis of IgH and TCR␥ gene rearrangements and analysis of expression of CycA1 and HOXA9 genes. 2.7. Cell count and viability To assess the viability of cells cultured in different growth conditions on days 0, 3, 5 and 7, the cells were collected and then washed in phosphate-buffered saline (PBS). To assess cell viability, a small volume of cell suspension was mixed with an equal volume of saline-containing trypan blue stain (Flow Laboratories, UK). To assess the cell number, a small volume of cell suspension was mixed with an equal volume of T¨urk solution (Kemika, Croatia) that induced the lysis of red blood cells. Cell counting was performed by using B¨urker–T¨urk hemocytometer (Fein-Optic, Germany).

3. Results 3.1. Patients In a total of 40 de novo AL patients included in this study, there were 15 ALL patients, 15 AML patients and 10 BAL patients (Table 1). In the group consisting of ALL patients, there were 10 children and 5 adults. Median age in the group of childhood ALL was 5.5 years (range 2–15 years), with a male to female ratio 4:1. Median age in adult ALL was 28 years (range 23–56), with a male to female ratio 4:1. In the group of AML patients, there was only one child (a boy aged 12) and 14 adult patients. The median age of these patients was 36.5 years (range 21–59), with a male to female ratio of 1:1. In the group of BAL patients, five patients were under 15 years of age and five were over 15 years. Median age for children was 9 years (range 0.4–16 years), with a male to female ratio 1:1.5. Median age in adult BAL group was 45 years (range 29–65), with a male to female ratio 4:1. 3.2. Morphology, immunophenotype and cytogenetics of AL blasts In the case of childhood AL, ALL was the most frequent type of AL (>90% of childhood ALs) [12], while the most frequent type of adult AL was AML. According to the records of the Zagreb Clinical Hospital Center, the incidence of adult acute leukemia subtypes during the 1999–2003 period was

215

69.8% for AML, 25.4% for ALL, 4.3% for BAL and 0.2% for AUL. The incidence of childhood BAL was even lower, amounting to only 3%. In the ALL group of patients, all FAB types were included (L1–L3) (Table 1) and the frequencies of different ALL immunophenotypes were in accordance with their distribution in vivo (common > T > pro > B-ALL). In the case of AML patients, only FAB subclasses M0, M6 and M7 were not collected for this study. Some ALL (5/15) and AML (5/15) patients had leukemia blasts co-expressing markers of different blood lines: however, they did not fulfill the criteria to be classified as BAL according to EGIL recommendations [2,11]. Of 10 BALs studied, lymphoid BAL (L2) was identified in three children and none of the adult patients, myeloid BAL (M1 and M5 types exclusively) was identified in three adults and one child, BAL of undifferentiated cytomorphology (AUL) in two adult patients, whereas one 5-month-old child presented with two separate blast cell populations (L1 + M5b, bilineal BAL) (Table 1). With regard to immunophenotype, the group of BAL patients was also heterogeneous: 8/10 expressed combination of myeloid and B-lymphoid antigens and 2/10 expressed myeloid and T-lymphoid antigens (Table 1). The chromosomal analysis of BAL patients was available in 6 of 10 patients (Table 1). Normal karyotype was found in one patient with undifferentiated morphology, three patients had chromosome 11 abnormalities, whereas aneuploidy was found in three patients. The expression of CD34 was seen in 50% of AML, 50% of ALL and 89% of BAL cases (Table 1). This distribution of CD34-positive acute leukemias among three major AL groups reflects the exact distribution as in ALs recorded during the last 6 years in our institution. 3.3. IgH and TCRγ rearrangements The clonal rearrangements of IgH and TCR␥ genes were tested in 37 AL samples, including several control cell samples (Table 2). PCR with IgH primers detecting rearrangements involving CDRIII region resulted in one or two discrete bands of amplified material, 100–120 bp in size, while the primers detecting rearrangements involving CDRI region resulted in products of 300–350 bp in size (Fig. 1A and B). The multiplex PCR with any combination of used TCR␥ primers resulted in one or two discrete bands of amplified material, 170–230 bp in size (Fig. 1C). Monoclonal amplified material was mostly observed in lymphoid leukemias. Monoclonal amplification with the IgH primers was observed in six of nine (67%) cases of B-lineage ALL, but also in one of five (20%) cases of T-lineage ALL and 1 of 11 (9%) AML cases. Similarly, the monoclonal amplification with the TCR␥ primers was not completely lineage-specific: TCR␥ amplification was observed in two of three (67%) cases of T-lineage ALL, but also in two of

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216

Table 2 IgH and TCR␥-gene rearrangements in different types of acute leukemia Disease

Total no. of patients studied

IgH (positive/studied)

TCR␥ (positive/studied)

AML

11

1/11

0/11

5 7 1 1

1/5 4/7 1/1 1/1

2/3 2/7 0/1 0/1

2 3 1 3 1

1/2 0/3 0/1 3/3 0/1

1/2 0/3 0/1 1/3 0/1

3

3/3 Positive

0/3 ND

ALL T-ALL Common ALL B-ALL Pro-B ALL BAL BAL-AUL BAL-M5 BAL-M1 BAL-L2 BAL-bilineal Control B-CLL SU-DHL4 ND: no determination.

nine (22.2%) cases of B-lineage ALL (Table 2). As shown in Table 3, some of the cases showed more than one IgH gene rearrangement or combination of IgH and TCR␥ gene rearrangements, indicating the coexistence of multiple leukemia clones at the presentation of the disease. As summarized in Tables 2 and 3, monoclonal or oligoclonal amplification in the group of BAL patients was mostly specific for the cases with lymphoid morphology, with the exception of one case with undifferentiated morphology. None of the BAL cases with myeloid FAB-morphology showed IgH and/or TCR␥ monoclonal amplifications. All three cases of lymphoid morphology (BAL-L2) showed IgH rearrangements, with one of them showing an additional rearrangement of TCR␥ gene (Tables 2 and 3). One case of BAL with undifferentiated morphology also showed a combined pattern of gene rearrangements (Table 3).

Fig. 1. (A) PCR-detection of leukemia-specific rearrangements of immunoglobuline heavy chain gene involving CDRIII sequence. Lanes 1–4 and 6, single PCR-CDRIII product (Pts. # 31, 33, 39, 8 and 1). Patient samples in lanes 5, 7 and 8 showed no CDRIII sequence specific rearrangement. Lane 9, positive control. (B) PCR-detection of IgH-CDRI specific leukemia rearrangement (Jh/Vh4 primers). Lane 2, single PCR-CDRI product (Pt. # 1). Lanes 1 and 3, negative control and blank. (C) Lanes 1–4, monoclonal rearrangements of TCR␥ detected by multiplex PCR (Pts. # 31, 39, 34 and 9). Lanes 5 and 6, positive controls. Lane 7, blank. A 1 kb ladder was used as a molecular weight marker.

10 BAL and 11 control samples (Table 4). The majority of samples tested (33/40 or 82.5%) contained >80% of blasts (median 90%, range 80–99%). Only 7 out of 40 samples (17.5%) had <80% blasts at the time of the analysis (median 53%, range 45–75%). As presented in Fig. 2 and Table 4, CycA1 expression was detected in 3/3 samples of AML-M1, 2/4 AML-M2, 2/2 AML-M3, 1/3 AML-M4 and 3/3 AMLM5, amounting to a total of 73% positive AML cases. There was only 1 of 15 ALL cases that expressed CycA1 (Pt. #

3.4. Cyclin A1 Human cyclin A1 (CycA1) is highly expressed in AML blasts [21]. To determine whether its expression in BAL cases correlates with FAB-classification, the expression of CycA1 was tested by RT-PCR. Expected size of amplified product was 197 bp (Fig. 2). Analysis included 15 AML, 15 ALL,

Table 3 Patients with more than one antigen receptor gene rearrangements at the presentation of the disease Patient’s #

IF/FAB

CDRIII

CDRI-Vh1

CDRI-Vh3

CDRI-Vh4

TCR␥

12 7 8 13 11

ALL-pro B ALL-common ALL-common ALL-common B-ALL

+ + + + −

− − − − +

– + + + +

+ − − − −

− − + + −

33 35 39

BAL-L2 BAL-L2 BAL-AUL

+ – +

+ + −

+ − −

− − −

− + +

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Fig. 2. (A) Representative examples showing results of RT-PCR of CycA1. Lanes 1–5, cDNA from patients with AML-M1, M2, M3, M4 and M5 at diagnosis. Lanes 6–8, cDNA from patients with common, T, and B-ALL. Lanes 9–11, cDNA from patients with BAL: L2, M5a and bilineal BAL, respectively. (B) CycA1 in control leukemia cell lines. Lane 1, cDNA from K562 cells was used as negative control; lane 2, cDNA from HL60 cells was used as positive control; lane 3, blank; lane M, 100 bp marker.

13 with common ALL), but it should be noted that leukemic blast in this patient co-expressed MPO, but not pan-myeloid surface markers. These findings correlated well with tested control samples: out of four acute leukemia cell lines tested, only AML cell line (HL60) gave positive result. The MNC isolated from bone marrow and peripheral blood of healthy donor did not express CycA1, whereas selected population

Table 4 Expression of CycA1 gene by RT-PCR in different types of acute leukemia Disease

AL subtype

No. positive/ tested

Positive cases (Pt. #)

AML

AML-M1 AML-M2 AML-M3 AML-M4 AML-M5

3/3 2/4 2/2 1/3 3/3

17, 18, 16 20, 22 23, 24 27 28, 29, 30

ALL

T-ALL Pro-B ALL Common-ALL B-ALL

0/5 0/1 1/8 0/1

– – 13 –

BAL

BAL-M1 BAL-M5 BAL-L2 BAL-AUL BAL-bilineal

1/1 2/3 1/3 1/2 1/1

36 34, 38 32 39 31

Control cell samples

CLL

0/4

PBMMNCa BMMNCa CD34+ cellsb SU-DHL4 NALM-6 K562

– – Weak positive – –

HL60

+

CLL: B-chronic lymphocytic leukemia; PBMC: peripheral blood mononuclear cells; BMMNC: bone marrow mononuclear cells. a Healthy donors. b Positively selected peripheral blood CD34+ cells.

of peripheral blood CD34+ cells gave a weak signal. This is consistent with a hypothesized role of CycA1 in proliferation and differentiation of hematopoietic cells [21]. In the group of BAL patients, CycA1 expression was found in 1/1 of BAL-M1, 2/3 BAL-M5, 1/3 BAL-L2, 1/2 BALAUL and 1/1 bilineal (L1 + M5b) BAL. Since most of BAL patients with myeloid morphology showed CycA1 expression, it seems that CycA1 expression is an additional marker of myeloid cell lineage determination. 3.5. HOXA9 In the studies involving classification of hematopoietic malignancies and outcome prediction based on gene expression analysis [22], several parameters, including expression of HOXA9 gene, showed promising results. In this report, HOXA9 expression was investigated in 14 AML, 15 ALL, 9 BAL patients and several control samples. The majority of samples tested (30/38 or 79%) contained >80% of blasts (median 90%, range 80–99%). Out of 38 samples, only 8 (21%) had <80% blasts at the time of the analysis (median 54.5%, range 45–75%). As summarized in Fig. 3 and Table 5, the HOXA9 expression was detected in only 3/15 ALL (20%) cases, all with B-lineage immunophenotype. These findings are in consistency with previous studies [22,23] showing the involvement of HOXA9 expression not only in myeloid, but also in early Bcell development. In AML, HOXA9 expression was detected in 11/14 cases (2/3 AML-M1, 2/4 AML-M2, 2/2 AML-M3, 3/3 AML-M4, and 2/2 AML-M5), amounting to a total of 78.6% positive AML. The expression of HOXA9 in control samples showed the same pattern (Table 5): cell lines HL60 (APL), K562 (CML-BC) and NALM-6 (pre-B ALL) expressed HOXA9, whereas SU-DHL4 cells (more mature form of lymphoblasts) did not show HOXA9 expression. The MNC isolated from BM were positive for HOXA9, whereas MNC isolated from PB of a healthy donor did not express HOXA9. This finding could be easily explained by the higher

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218

Fig. 3. (A) Representative examples HOXA9 RT-PCR. Lanes 1–5, cDNA from patients with AML-M1, M2, M3, M4 and M5 at diagnosis. Lanes 6–8, cDNA from patients with common, T and B-ALL. Lanes 9–11, cDNA from patients with BAL-M1, L2 and AUL. (B) RT-PCR of HOXA9 in control cell samples. Lane 1, cDNA from NALM-6 cell line; lane 2, SU-DHL4 cell line; lane 3, HL60 cell line; lane 4, K562 cell line; lane 5, cDNA from BMMNC of healthy donor; lane 6, cDNA isolated from human tonsils; lane 7, cDNA from patient with B-CLL; lane 8, blank; lane M, 100 bp marker.

incidence of immature/progenitor cells in bone marrow specimen. In the group of BAL patients, HOXA9 expression was heterogeneous: 1/1 of BAL-M1, 0/2, BAL-M5, 1/3 BAL-L2, 2/2 BAL-AUL and 1/1 of bilineal BAL expressed HOXA9.

Since only 5/9 (56%) of tested BAL showed HOXA9 expression, and among them only 1/3 of myeloid BAL, it seems that HOXA9 expression cannot be used as a criteria for BAL sub-stratification. 3.6. Leukemia growth in vitro

Table 5 Expression of HOXA9 gene by RT-PCR in different types of acute leukemia Disease

AL subtype

No. positive/ tested

Positive cases (Pt. #)

AML

AML-M1 AML-M2 AML-M3 AML-M4 AML-M5

2/3 2/4 2/2 3/3 2/2

16, 18 20, 22 23, 24 25, 26, 27 29, 30

ALL

T-ALL Pro-B ALL Common ALL B-ALL

0/5 1/1 1/8 1/1

– 12 13 11

BAL

BAL-M1 BAL-M5 BAL-L2 BAL-AUL BAL-bilineal

1/1 0/2 1/3 2/2 1/1

36 – 35 39, 40 31

Control cell samples

CLL

0/4

PBMMNCa BMMNCa CD34+ cellsb SU-DHL4 NALM-6 K562 HL60

– + – – + + +

Tonsilar lymphocytes

+

CLL: B-chronic lymphocytic leukemia; PBMC: peripheral blood mononuclear cells; BMMNC: bone marrow mononuclear cells. a Healthy donors. b Positively selected peripheral blood CD34+ cells.

To correlate in vitro growth of AL blasts with their biological properties, the blasts isolated from BM of AL patients were cultured for 7 days in different in vitro conditions. As described in Section 2, blasts were cultured in a serum free medium in three culture conditions: without addition of GFs, and in the presence of FL + IL7 or FL + GM-CSF (growth conditions designated with letters S, L and M, respectively). The follow up of cell count during a 7-day experiment showed a very heterogeneous growth pattern, even among patients with the same AL subtype. In other words, in the group of AML, ALL or BAL patients, there were cases of AL blasts that showed in vitro growth, while, on the other hand, there were cases of AL blasts that showed rapid in vitro cell death. Nonparametric Friedman test showed significant difference in viable cell number upon culture of AML or ALL blasts in different culture conditions, while in the BAL group of patients there was no clear difference in cell number among various growth conditions (Table 6). The blasts of BAL patients showed almost equal in vitro growth in all culture conditions, indicating that their growth was less dependent on the addition of exogenous GFs. Furthermore, the analysis of samples that had in vitro growth showed that none of ALL blast samples had autonomous growth in a serum free medium without addition of GFs, whereas 3/10 AML and 3/8 BAL samples showed spontaneous increase in viable cell number. Three cases showing an autonomous serum-free and cytokine-free growth in the culture were of AML-M1 and M4 morphology

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219

Table 6 In vitro growth of blasts of different types of AL expressed as the number of viable cells on day 7 relative to the number of viable cells on day 0 Disease

ALL (n = 8) median (range) AML (n = 10) median (range) BAL (n = 8) median (range)

Friedman testa , p

Viable cells (% input) w/o GFs (S)

FL + IL-7 (L)

FL + GM-CSF (M)

34 (14–90) 45 (9–270) 75 (33–140)

70 (19–112) 61 (8–150) 80 (30–230)

56 (15–110) 82 (13–190) 69 (21–203)

<0.05 <0.05 NS

FL: Flt3-ligand. a Nonparametric Friedman test.

(for classical AML cases), and BAL-M1 and BAL-M5 in the group of BAL patients (Pts. # 34, 36 and 37).

4. Discussion BAL is a rare type of AL with the reported incidence of 4.0–8.5% [3,6–8], which is also shown in this study. However, the recognition of BAL is important for several reasons. First of all, BAL needs more precise biological definition from the diagnostic as well as from therapeutic point of view. Indeed, this form of AL is usually associated with poor prognosis [3,5,8], although there is an urge for large prospective studies using the uniform diagnostic and therapeutic approach to answer this question [8]. According to literature data, BAL has poor prognosis when compared to AML and ALL in terms of complete remission and a 4-year overall survival [8]. Finally, BAL is an attractive model for in vitro studies of biological behavior of leukemic cells, especially in the context of associated chromosomal abnormalities. The aim of this study was to analyze some of the biological properties of BAL cells in vitro in relation to control samples of more conventional AML and ALL. According to the AL records taken during the last 6-year period in our institution, the incidence of BAL was 4.3%; therefore, the number of BAL samples in this study was relatively low. The most common type of BAL was that in which blasts co-expressed myeloid and B-lymphoid antigens (80%), whereas myeloid and T-lineage antigens were found less commonly (20%). As reported before [5,8], the majority of BAL expressed stem-cell marker CD34 (89%) and also HLADR. When it comes to cytogenetics, the chromosomal analysis was available only in 6 of 10 BAL patients due to a relatively high number of diagnostic failures. Interestingly, Ph+ chromosome cases were not found among BAL group of patients. In contrast, abnormalities of chromosome 11 were detected at the frequencies similar to that reported previously [5,7,10]. We can only speculate that some of our diagnostic failures, actually, belonged to Ph+ category. However, it should be mentioned that we have observed several Ph+ CML patients in blast crisis expressing BAL properties, but those patients were subsequently excluded from this study. Studies of IgH and TCR␥ gene rearrangements in lymphoproliferative diseases show distinction of monoclonality

from polyclonality at the level of blast population, thus providing information of their biological properties as well as important diagnostic data. B-cell clonality in ALL has been demonstrated in many reports by detecting rearrangement of IgH gene. According to the results obtained in the control group of ALL and AML patients, IgH rearrangements were shown to be predominantly a B-lineage leukemia feature, as previously described [15]. However, IgH rearrangements were also found in 1/15 AML and 1/5 T-ALL, which is not surprising since more than 10% of acute non-lymphocytic leukemias and more than 10% of T-cell leukemias and lymphomas have been shown to contain IgH gene rearrangements [14,24]. In the group of BAL patients, all cases with lymphoid and one with undifferentiated morphology showed IgH gene rearrangement. In contrast, none of BAL with myeloid morphology showed rearranged IgH, suggesting a rather good correlation of this biological parameter with BAL morphology. TCR␥ gene rearrangements were found in 2/10 BAL cases, one with lymphoid (L2) and one with undifferentiated (AUL) morphology. Similar to IgH, TCR␥ gene rearrangement was not detected in any of BAL with myeloid morphology. In the control group of patients, TCR␥ showed predominant, but not absolute T-ALL specificity, as previously observed [17,24]. In three cases of BAL with positive IgH and/or TCR␥ gene rearrangements, we also found oligoclonal amplification of antigen-receptor genes. This finding indicates the coexistence of several malignant clones at the time of the presentation of the disease. Oligoclonal malignancies have been described in >20% of ALL, thus providing an insight into clonal evolution in AL lymphoid precursors [14]. In an attempt to locate “informative genes” whose expression would correlate with AML–ALL distinction, Golub et al. [22] found that the single most highly correlated gene out of 6817 genes was HOXA9, which was over-expressed in AML patients with treatment failure. A general role for HOXA9 expression in predicting AML outcome has not been suggested prior to that study. HOXA9 is Class I homeobox gene coding for a homeodomain containing transcriptional factor potentially involved in myeloid differentiation. In an experimental setting, Thorsteindottir et al. [25] showed that a high proportion of mice transplanted with bone marrow cells over-expressing

220

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HOXA9 developed AML [25,26]. Although aberrant expression of HOXA9 also severely perturbed B-lymphopoiesis, none of the transgenic mice developed lymphoid malignancies [25]. As quantitative HOXA9 over-expression was not observed in ALL, this gene appeared as a genetic marker for AML [27]. In this study, HOXA9 expression was investigated in acute leukemia patients and several control samples. The majority (79%) of leukemic samples contained >80% of blasts, whereas the minority of samples (21%) had <80% of blast cells (range 45–75%). However, even in this later group of patients there was an equal number of HOXA9 positive and negative patients, indicating that the positive result did not come from a normal myeloid precursor but rather from leukemic cells. This is further supported by the finding indicating that the patients with the lowest number of blasts (i.e. 45%, 50% and 56%) showed no expression of HOXA9 at all. We detected HOXA9 expression in 79% of AML cases tested, among which the FAB-M2 type showed somewhat lower incidence of HOXA9 expression. An association between FAB-M2 morphology and a lower expression of HOXA9 was previously described by Casas et al. [27]. Despite that fact, predominant expression of HOXA9 in myeloid leukemia was confirmed by its positive findings in myeloid leukemic cell lines. Our RT-PCR analysis also detected HOXA9 in 3/15 ALL cases, all of them showing features of B-lineage ALL. The HOXA9 expression was also confirmed in NALM-6 cell line (pre-B ALL), suggesting previously hypothesized HOXA9 involvement in early B-lymphopoiesis [28,29]. We investigated HOXA9 expression pattern in nine cases of BAL in order to get additional information on the possible lineage path in BAL with different morphology. Despite expectations, HOXA9 expression was not limited to myeloid BAL and was also missing in two BAL-M5 cases. Furthermore, HOXA9 expression was also found in 1/3 BAL-L2, 2/2 BAL-AUL and 1/1 bilineal BAL, suggesting that it did not correlate with a BAL morphology and also did not give better stratification of BAL. Finally, when investigating HOXA9 involvement in leukemogenesis, additional parameters should be taken into consideration, such as MLL status of cells, especially in the cases of lymphoid neoplasias [28,29]. Many studies showed that elevated levels of CycA1 are predominantly expressed in myeloid hematological malignancies [19,21,30,31]. Despite demonstrating a clear correlation with the development of the disease, CycA1 overexpression is not self sufficient for leukemogenesis [31]. Since CycA1 expression is also evident (but much weaker) in normal hematopoietic progenitor cells [19,21,30], it is believed that this gene has a role in the proliferation and differentiation of myeloid cells [21,32]. Furthermore, its over-expression in AML originates from the leukemic cells arrested at the stage of differentiation when CycA1 is normally expressed [19,21]. In this study, CycA1 expression turned out to be myeloid-specific in case of the control group of patients with ALL or AML disease. Since CycA1 expres-

sion was not detected in normal BMMNC, it is unlikely that its expression in the cases of samples with the lower content of blast cells could have originated from normal residual cells. We also confirmed the findings of Yang et al. [21] who showed lower incidence of CycA1 expression in AML-M4 subtype. There was only 1 of 15 ALL patients (Pt. # 13, My+ common-ALL) whose blasts co-expressed MPO and CycA1 in the absence of cell surface myeloid markers (CD13, CD14, CD33 and CD117). In the group of BAL patients, CycA1 expression correlated better with myeloid morphology than HOXA9 expression. Although not quantitative, our study showed that CycA1 was present in the majority of leukemias with myeloid and undifferentiated morphology. The assays concerning in vitro growth of leukemia blasts in different culture conditions revealed that BAL showed similar in vitro growth in all three culture conditions, indicating lesser GF-dependent growth. Interesting was the finding of three AML and three BAL cases showing autonomous proliferation in a serum free medium during 7 days of culture in vitro. The autonomous cell proliferation was previously described in AML associated with unfavorable prognosis [33–35]. In our study, three BAL patients showing autonomous growth in vitro had AML-M1 and AML-M5 morphology. Since the number of investigated patients was too small, it was impossible to connect this biological parameter with any parameter other than myeloid morphology of the disease. The importance of the autonomous proliferation in vitro may indicate a more likely regeneration of the leukemic clone following therapy at the expense of normal hematopoiesis [33], thus contributing to the poorer prognosis [33–35]. To sum up, we present a group of de novo BAL patients whose blasts were investigated for additional biological parameters in vitro aiming to give a better definition of this rare group of acute leukemias associated with poor prognosis. We observed that IgH and/or TCR␥ gene rearrangements, CycA1 expression and autonomous proliferation in vitro correlated with morphology of BAL, whereas the expression of HOXA9 could not be related to leukemic cell morphology.

Acknowledgements This work was supported by a grant from Ministry of Science and Technology of Croatia (no. 214006). The authors express their gratitude to Drs Mirjana Markovic-Glamocak, Suncica Ries and Koraljka Gjadrov for cytomorphological analyses and to Maja Rupcic, Zaklina Cavar, Jadranka Jerkovic and Marijana Jurak for their technical expertise. Contributions. M. Golemovi´c performed the experiments, collected the data and prepared the manuscript. M. Suˇci´c was responsible for cytology and immunocytochemistry. R. Zadro contributed to set up the molecular biology techniques. S. Mrsi´c was responsible for cytogenetics including FISH. M. Mikuli´c, B. Labar and L.j. Raji´c contributed to the conception of the study and interpretation of data. D. Batini´c

M. Golemovi´c et al. / Leukemia Research 30 (2006) 211–221

is the author taking primary responsibility for the paper. The authors declare that they have no potential conflict of interest.

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