11q23 Abnormalities in Patients With Acute Myelogenous Leukemia ...

Hematopathology / 11q23 IN ACUTE MYELOGENOUS LEUKEMIA AND MYELODYSPLASTIC SYNDROME

11q23 Abnormalities in Patients With Acute Myelogenous Leukemia and Myelodysplastic Syndrome as Detected by Molecular and Cytogenetic Analyses Sherif Ibrahim, MD, PhD,1 Elihu H. Estey, MD,2 Sherry Pierce,2 Armand Glassman, MD,1 Michael Keating, MD,2 Susan O’Brien, MD,2 Hagop M. Kantarjian, MD,2 and Maher Albitar, MD1 Key Words: 11q23; Mixed lineage leukemia; MLL; Acute myelogenous leukemia; AML; Myelodysplastic syndrome; Molecular; Cytogenetic

Abstract 11q23 chromosomal abnormalities and rearrangement of the mixed lineage leukemia (MLL) gene are important prognostic factors in acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). However, the presence of 11q23 abnormalities does not always correlate with that of MLL gene rearrangement. We retrospectively compared the occurrence of 11q23 abnormalities (measured by karyotyping) and MLL gene rearrangement (measured by Southern blotting) in bone marrow from 311 consecutive adult patients with AML or MDS. 11q23 abnormalities were found in 18 patients (5.8%), of whom 7 (39%) did not have the MLL gene rearrangement. MLL gene rearrangement was detected in 35 patients (11.2%). Of these 35 patients, only 11 (31%) had cytogenetic evidence of 11q23 abnormalities. None of the 21 patients with chronic myelomonocytic leukemia had 11q23 abnormalities or MLL gene rearrangement. 11q23 abnormalities were associated with shorter survival than was a diploid karyotype. Both cytogenetic and molecular studies should be performed to detect 11q23 abnormalities in patients with AML or MDS.

© American Society of Clinical Pathologists

A variety of recurrent chromosomal abnormalities have been described in association with hematologic malignant neoplasms.1-4 One of the most common cytogenetic abnormalities, the 11q23 translocation, was first reported in 1970 by Van den Berghe et al.5 They described a case of acute lymphoblastic leukemia (ALL) with t(4;11)(q21;q23). This report was followed by a large number of others showing 11q23 translocations involving almost every other chromosome in various hematologic malignant neoplasms.6,7 All of these translocations involve the MLL gene (for mixed-lineage leukemia, also called HRX, ALL-1, and HTRX1) at 11q23 becoming fused with a new gene. The MLL gene seems to act as a transcription factor or regulator of other genes.8-12 Duplication within the MLL gene may be one mechanism by which this gene is expressed abnormally in leukemias.13 11q23 abnormalities are reported to be more common in leukemic cells that can differentiate into myelomonocytic cells.14 11q23 translocation is present in 7% to 10% of ALL, 5% to 8% of acute myelogenous leukemia (AML), and in more than 50% of infants with acute leukemia regardless of lineage.15,16 11q23 abnormalities also have been reported in other hematologic malignant neoplasms, such as therapy-related AML, biphenotypic acute leukemia, malignant lymphoma, and myelodysplastic syndrome (MDS).8 Several DNA probes that can be used to detect rearrangement in the MLL gene have been designed by different groups.17-19 Multiple studies using these probes reported that the rearrangement of the MLL gene predicts a poor prognosis for patients with AML or ALL.20 Thus, patients with the MLL gene rearrangement should be considered to have aggressive disease and should be treated aggressively.8 Correlation between cytogenetic and molecular evidence of abnormalities at 11q23 has been poor. Hence, we used Am J Clin Pathol 2000;114:793-797 793

Ibrahim et al / 11q23 IN ACUTE MYELOGENOUS LEUKEMIA AND MYELODYSPLASTIC SYNDROME

cytogenetic and molecular techniques to assess 11q23 abnormalities in 311 consecutive adult patients with AML or MDS. Of these patients, 6% showed 11q23 abnormalities while 11% showed MLL gene rearrangement. Poor correlation between cytogenetic and molecular studies was observed. Thus, performing cytogenetic and molecular studies simultaneously might be necessary to detect various 11q23 abnormalities.

Material and Methods Patients and Tissue Samples Subjects in the study were 217 patients with AML and 94 patients with MDS. These patients were examined at the University of Texas M.D. Anderson Cancer Center between January 1992 and January 1999. The mean age was 58 years for patients with AML (range, 18-84 years) and 65 years for patients with MDS (range, 19-82 years). The classification of the 217 patients with AML according to the French-American-British criteria was as follows: M0, 36; M1, 31; M2, 60; M4, 54; M5, 25; M6, 10; and M7, 1 ❚Table 1❚. Of the 94 patients with MDS, 4 were classified as having refractory anemia, 3 as having refractory anemia with ring sideroblasts, 30 as having refractory anemia with excess blasts, and 36 as having refractory anemia with excess blasts in transformation (RAEB-T), and 21 patients had chronic myelomonocytic leukemia (CMML). Cases with 20% to 30% blasts were classified as RAEB-T. Diagnosis was established on the basis of morphologic, immunophenotypic, cytochemical, and molecular studies of bone marrow biopsy samples.

Immunophenotyping included the use of antibodies for the following antigens: CD3, CD7, CD10, CD13, CD14, CD19, CD33, CD34, CD64, CD117, terminal deoxynucleotidyl transferase, and multidrug resistance. Cytochemical studies included the use of immunoperoxidase, nonspecific esterase, periodic acid–Schiff, Sudan black, and iron stains. Cytogenetic Analysis Cytogenetic analysis of bone marrow samples was done by the M.D. Anderson Cancer Center Hematopathology Laboratory using standard techniques as previously described.20 Bone marrow samples were incubated overnight at 37°C in Ham F-10 medium supplemented with L-glutamine, 10% fetal bovine serum, and penicillin and streptomycin. Cultures were harvested and slides prepared by established methods. At least 20 metaphases of Giemsa-banded bone marrow samples were analyzed using routine procedures. Karyotypes were characterized according to the International System of Human Cytogenetic Nomenclature 1991.1 Molecular Analysis Southern blot analysis was done as described by Cimino et al. 21 Briefly, bone marrow DNA was digested with BamHI and HindIII enzymes and analyzed with the approximately 0.5-kb DdeI probe that spans the breakpoint on 11q23. The probe was provided as a generous gift of E. Canaani, MD. Statistical Analysis Patient characteristics and survival data were obtained from the M.D. Anderson Cancer Center leukemia database.

❚Table 1❚ Patient Characteristics Acute Myelogenous Leukemia (n = 217)

Characteristic Mean age (y)* Peripheral blood* WBCs/µL [× 109/L] Blasts Platelets, × 103/µL [× 109/L] Hemoglobin, g/dL [g/L] Monocytes/µL [× 109/L] Bone marrow (%)* Cellularity Blasts Monocytes Poor cytogenetics†‡ Therapy-related disease† Performance status (0-2)†§

Myelodysplastic Syndrome (n = 94)

11q23 Abnormalities Found (n = 42)

58 (18-84)

65 (19-82)

55 (25-77)

14,500 [14.5] (700-262,500 [0.7-262.5]) 28 (0-97) 53 [53] (3-708 [3-708]) 7.9 [79] (2.9-15 [29-150]) 5,000 [5.0] (0-94,000 [0.0-94.0])

5,350 [5.35] (700-32,000 [0.7-32]) 1 (0-28) 42 [42] (2-492 [2-492]) 7.8 [78] (1.7-12 [17-120]) 9,000 [9.0] (0-92,000 [0.0-92.0])

14,100 [14.1] (700-32,000 [0.7-32]) 11 (0-95) 48 [48] (5-1,355 [5-1,355]) 7.9 [79] (5-14.4 [50-144]) 8,000 [8.0] (0-94,000 [0.0-94.0])

80 (10-100) 55 (2-96) 2 (0-45) 70 (32.2) 32 (14.7) 196 (90.3)

65 (5-100) 9 (0-28) 4 (0-48) 39 (41) 20 (21) 88 (94)

80 (10-100) 52 (5-96) 2 (0-38) 0 (0) 20 (48) 40 (95)

*

Data are given as mean (range). Data are given as number (percentage). Includes –5, –7, or +8. § The rest of the patients scored 3-4. † ‡

794

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Hematopathology / ORIGINAL ARTICLE

Possible associations between characteristics, survival, and cytogenetic and molecular abnormalities were assessed using the Wilcoxon signed rank test. Survival was assessed using Kaplan-Meier and log-rank tests. A P value of less than .05 was considered statistically significant.

Results

❚Table 3❚ Karyotypes of Cases With 11q23 Cytogenetic Abnormalities

t(9;11)(q22;q23) del(11)(11q23) t(11;19)(q23;p13) t(1;11)(q24;q23) t(X;11)(q22;q23) *

The characteristics of the 311 patients are shown in Table 1. Patients with 11q23 abnormalities measured by cytogenetic or molecular analysis were younger (P = .06), but had overall shorter survival (P = .03) than those without 11q23 abnormalities. More patients with AML had 11q23 abnormalities (17.1%) than did patients with MDS (5%). Peripheral blood cell counts and bone marrow characteristics in patients with 11q23 abnormalities were close to those in patients with AML. On the other hand, more patients with 11q23 abnormalities had therapy-related disease than those who did not have 11q23 abnormalities (P = .0012). Discrepancy in Detecting 11q23 Abnormalities Nine of the 311 patients studied in this series had insufficient metaphases, and 135 patients showed diploid karyotypes. The remaining 167 patients (53.7%) had clonal cytogenetic aberrations. All patients studied were previously untreated. Conventional cytogenetic analysis revealed abnormalities in 11q23 in 17 patients with AML (7.8%) and in 1 patient with MDS (1%) ❚Table 2❚. Of these 18 patients, 12 had monocytic lineage involvement. The details of the

No. of Patients*

Cytogenetic Characteristics

8 (2) 5 (3) 3 (1) 1 (1) 1 (0)

Data in parentheses are the number of patients who lack the MLL (mixed lineage leukemia) gene rearrangement as shown by Southern blot analysis.

karyotyping analyses are shown in ❚Table 3❚. Seven (39%) of the 18 patients with 11q23 abnormalities did not have MLL gene rearrangement as shown by Southern blot analysis (see the next paragraph). Results from Southern blot analyses ❚Figure 1❚ are summarized in Table 2. MLL gene rearrangements were detected in 31 (14.3%) of 217 patients with AML and in 4 (4%) of 94 patients with MDS. All 4 patients with MDS had the RAEB-T subtype. Of the 35 cases with MLL gene rearrangements by Southern blot, 24 (68%) did not show 11q23 abnormality by conventional cytogenetics at the time of diagnosis, although 5 of these patients developed 11q23 abnormalities later during treatment. The karyotypic analyses of these patients are summarized in ❚Table 4❚. The incidence of 11q23 abnormalities was highest in patients with AML-M5 (11 of 25 cases), followed by those with AML-M4 (11 of 54 cases). None of the patients with CMML had 11q23 abnormalities by either evaluation method.

❚Table 2❚ Cytogenetic and Molecular Abnormalities in 11q23 in Patients With AML or MDS Disease Subtype AML* M0 M1 M2 M3 M4 M5 M6 M7 Subtotal MDS RA RARS RAEB RAEB-T CMML Subtotal Total

No. of Cases

11q23 Abnormality Found MLL Gene Rearrangement Found by Cytogenetics by Southern Blot Analysis

11q23 Abnormalities Found by Molecular and Cytogenetic Studies

36 31 60 0 54 25 10 1 217

0 2 3 — 6 6 0 0 17

2 3 7 — 8 10 1 0 31

2 4 8 — 11 11 1 0 37

4 3 30 36 21 94 311

0 0 1 0 0 1 18

0 0 0 4 0 4 35

0 0 1 4 0 5 42

AML, acute myelogenous leukemia; CMML, chronic myelomonocytic leukemia; MDS, myelodysplastic syndrome; MLL, mixed lineage leukemia; RA, refractory anemia; RAEB, refractory anemia with excess blasts; RAEB-T, refractory anemia with excess blasts in transformation; RARS, refractory anemia with ring sideroblasts. * AML subtypes are according to the French-American-British classification.


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Ibrahim et al / 11q23 IN ACUTE MYELOGENOUS LEUKEMIA AND MYELODYSPLASTIC SYNDROME

HindIII M

N

P1

P2

BamHI P3

N

P1

P2

P3

C

❚Figure 1❚ Southern blot analysis of MLL (mixed lineage leukemia) gene rearrangement study in control patient’s (C) and 3 patients’ (P1, P2, P3) samples digested with the restriction enzymes. Rearrangement is seen for patient P1. Lane M shows the size marker.

❚Table 4❚ Karyotype of Cases With MLL Gene Rearrangements Cytogenetic Characteristic Diploid 11q23 t(3;21)(q26;q22) inv(9)(p11;q12) del(7)(q21) t(16;17)(q13;p13)

No. of Patients 19* 11 2 1 1 1

MLL, mixed lineage leukemia. * Five of these patients developed 11q23 abnormalities at various times after diagnosis.

Discussion A recent proposal by the World Health Organization for classifying hematologic malignant neoplasms emphasized the importance of cytogenetic changes as diagnostic and prognostic markers.4 This proposal specifies a separate category for AML with a recurrent cytogenetic abnormality that includes 11q23. Our finding that cytogenetic and molecular evidence of 11q23 abnormalities is not always consistent underscores the difficulty classifying leukemias in the absence of morphologic criteria. The discrepancy between results obtained by conventional cytogenetic analyses and molecular analyses is important and may have clinical implications.17 796

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Unlike previous reports that have involved small numbers of patients in multicenter analyses, our study included a series of 311 adult patients diagnosed and treated at a single institution, with long follow-up and complete clinical data.8,16 Our findings of a 6% incidence of 11q23 abnormalities on cytogenetic analysis (7.8% for patients with AML) agree with previously published values. However, 39% of these patients with proven 11q23 abnormalities did not show the MLL gene rearrangement on Southern blot analysis. This discrepancy may have resulted from failure of the probe to detect all of the abnormalities in the MLL gene22 or, perhaps, involvement of genes other than MLL in the 11q23 abnormality in patients with AML or MDS. Other genes, such as LARG, IGSF4, and Pafah1a2 have been described at the 11q23 region, any of which may have a role in the leukemogenic process associated with the 11q23 abnormality.23-25 Fluorescence in situ hybridization (FISH) and reverse transcriptase–polymerase chain reaction (RT-PCR) are additional methods that are suggested by several studies to have significant values in detecting 11q23 abnormalities.26-28 Mathew and colleagues27 examined 67 children with known 11q23 abnormalities using FISH and Southern blot techniques and concluded that FISH is a relatively accurate and rapid screening test for detecting 11q23 abnormalities.27 While RT-PCR is faster and less labor intensive compared with cytogenetic and Southern blotting techniques, it requires knowing the partner gene and does not detect abnormalities at 11q23 that do not involve the MLL gene. In addition, using RT-PCR, MLL gene rearrangement was detectable in healthy people, which makes it difficult to interpret positive results when detected by RT-PCR alone in patients with AML or MDS.28 Overall, 42 (13.5%) of 311 patients showed abnormalities in 11q23 by either cytogenetic and or molecular studies. With regard to disease subtype, 44% of patients with M5 disease (11/25) and 20% of patients with M4 disease (11/54) showed 11q23 abnormalities. This strong correlation between MLL gene rearrangement and monocyticmyelomonocytic (M4 and M5) disease has been reported previously.14,29 Most of these patients develop leukemia secondary to treatment with topoisomerase II inhibitor therapy.30 In our study, the 21 patients with CMML showed no 11q23 abnormalities. Mauritzson and others31 reported similar findings in their series of 65 CMML cases. These observations suggest that although CMML and M4 and M5 diseases are all of monocytic origin, their biology is different. Although the percentage of MLL-positive RAEB-T cases (4/36 [11%]) is similar to that of MLL-positive AML cases (31/217 [14.3%]), the number of patients studied precludes drawing any conclusions. Interestingly, all 4 patients with MLL-positive RAEB-T showed Auer rods. © American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE

We conclude that significant number of cases with abnormalities at 11q23 can be missed if conventional cytogenetic analysis alone is used. Furthermore, not all cases with the 11q23 abnormality can be detected by Southern blot analysis alone. Additional evaluation is necessary to establish whether there is biologic and clinical relevance in detecting 11q23 abnormalities by cytogenetics alone or by Southern blot analysis alone in patients with AML or MDS. From the Departments of 1Hematopathology and 2Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, TX. Address reprint requests to Dr Albitar: Dept of Hematopathology, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 72, Houston, TX 77030-4095.

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