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Hematopathology / 11Q23 ABERRATIONS AND MLL GENE STATUS IN ACUTE LEUKEMIA Chromosomal Aberration of the 11q23 Locus in Acute Leukemia and Frequency of MLL Gene Translocation Results in 378 Adult Patients M. Christina Cox, MD, PhD,1 Paola Panetta,1 Francesco Lo-Coco, MD,1 Giovanni Del Poeta, MD,1 Adriano Venditti, MD, PhD,1 Luca Maurillo, MD, PhD,1 M. Ilaria Del Principe, MD, PhD,1 Alessandro Mauriello, MD,2 Lucia Anemona, MD, PhD,2 Antonio Bruno,1 Carla Mazzone, MD,1 Paolo Palombo, MD,3 and Sergio Amadori, MD, PhD1 Key Words: MLL; 11q23; AML; Acute myeloblastic leukemia; ALL; Acute lymphoblastic leukemia; AL; Acute leukemia; Chromosomal aberrations; FISH; Fluorescence in situ hybridization; 11q22~25 DOI: 10.1309/RX27R8GJQM330C22 Abstract Structural abnormality of the 11q23 band (11q23+) bearing the MLL gene translocation (MLL+) is a recurrent chromosome change observed in 3% to 7% of acute lymphoblastic leukemias and in 3% to 4% of acute myeloblastic leukemias. The resolution of conventional cytogenetics (CC) in detecting 11q23 rearrangement is limited when the translocative partner has a telomeric location; furthermore, CC can barely discriminate between true 11q23+/MLL+ and rearrangements clustering within the 11q22~25 region without MLL involvement (MLL–). We characterized a series of 378 consecutive patients with adult acute leukemia by using CC, fluorescence in situ hybridization (FISH), and multiplex karyotyping (MFISH) analysis. Our aim was to define the frequency of cryptic MLL+ cases and the frequency of MLL+ within 11q22~25+ cases. As expected, FISH was more sensitive than CC in detecting MLL+ cases, but rather unexpectedly, 9 (45%) of 20 patients with 11q22~25+ were MLL–. A better characterization of 11q22~25+/MLL– leukemias is relevant for the identification of new, recurrent translocations. Moreover, these cases should be readily distinguishable from 11q23+/MLL+ cases. We recommend that karyotypic analysis always be complemented by molecular or FISH methods to unravel MLL rearrangements. 298 298 Am J Clin Pathol 2004;122:298-306 DOI: 10.1309/RX27R8GJQM330C22 Structural abnormality of the 11q23 band (11q23+) bearing the MLL gene translocation (MLL+) is a recurrent chromosome change in leukemia described in acute myeloblastic leukemia (AML) and in acute lymphoblastic leukemia (ALL), with a peak incidence in infant leukemia.1,2 A proposal by the World Health Organization specifies a separate category for AML with 11q23+/MLL+.3 This notion has been supported recently by biologic studies: microarray analyses have shown that MLL+ acute leukemias (ALs) have a peculiar geneprofiling pattern that distinguishes them from all other ALs and that MLL+ leukemic blasts resemble very immature progenitor cells.4 Furthermore, these studies showed that MLL+ leukemias are a separate entity when compared with AML with MLL partial tandem duplication (MLL-PTD), a recently identified genetic aberration observed in a sizable proportion of AMLs.5 Extensive cytogenetic and molecular studies have shown that 11q23/MLL is a highly promiscuous locus: more than 50 chromosomal loci have been described as 11q23 chromosome partners, whereas more than 30 MLL partner genes have been characterized.6 It also should be mentioned that t(11q23) might involve genes other than MLL7 and that conventional cytogenetics can barely discriminate between true 11q23+/MLL+ and rearrangements clustering within the 11q22~25 region without MLL involvement.8 Because t(9;11)(p21;q23) bearing the MLL/AF9 gene fusion in AML and t(4;11) with MLL/AF4 gene fusion in infant leukemia are the most common types,6 these translocations often are referred to as classic translocation, whereas all other variants are reported as v11q23. 11q23+/MLL+ is described in 3% to 4% of AML cases and is more frequent in younger subjects with de novo (5%-7%) AML or with t-AML (10%-15%) evolving after chemotherapy. © American Society for Clinical Pathology Hematopathology / ORIGINAL ARTICLE In older patients with AML (60 years or older), it is observed rarely.9 The majority of 11q23+/MLL+ AML cases have monocytoid differentiation features and are classified in the M4 and M5 leukemia French-American-British (FAB) subtypes.10 In adult ALL, the overall incidence of 11q23+/MLL+ is reported to be around 3% to 7%,11 but in pro-B-cell ALL, it accounts for more than 30% of chromosomal aberrations.12,13 While t(4;11) ALL has an established dismal prognosis, the clinical outcome of 11q23+/MLL+ AML is more heterogeneous. 14-16 The Medical Research Council 14 and the Southwest Oncology Group15 classify the risk for patients with AML with t(9;11) as intermediate and poor, respectively. Even more disagreement surrounds the prognostic relevance of classic t(9;11) vs v11q23: some clinical trials reported that patients with t(9;11) fared better than patients with v11q23,16,17 whereas other studies failed to identify differences.9,14 These discrepancies probably reflect the marked biologic heterogeneity of 11q23 aberrations. Furthermore, because many v11q23 translocations are rare translocations, the clinical impact of specific single variants is difficult to extrapolate, even from large studies on 11q23 AL.14-16 Recently, the combined use of conventional cytogenetics, reverse transcriptase–polymerase chain reaction (RTPCR), Southern blot analysis, and fluorescence in situ hybridization (FISH) in limited AL series has revealed that discrepant results with MLL involvement might become evident,18 and a high incidence of patients with cryptic MLL+ leukemia were observed in 2 pediatric series.19,20 Because the majority of clinical trials include only karyotype data,14-16 it is reasonable to speculate that beyond biologic heterogeneity, these discrepancies also are due partly to the low accuracy of conventional cytogenetics. We describe our findings in a series of 378 consecutive cases of adult AL, studied with conventional cytogenetics, FISH, and multiplex karyotyping (M-FISH) analysis. The aim of the study was to define the incidence of cryptic MLL gene translocation and the incidence of MLL gene rearrangement within 11q22~25+ cases. Materials and Methods Cases Between November 1992 and September 2003, 478 AL samples from newly diagnosed adult patients (older than 14 years) were sent to our laboratory for conventional cytogenetic analysis. Conventional cytogenetics was done following standard methods, and residual pellets were stored at –20°C in Carnoy solution. From January 2000, FISH analysis with the MLL gene probe (Vysis, Downers Grove, IL) was combined routinely with conventional cytogenetics in newly diagnosed patients with AML and ALL (n = 170). Furthermore, all residual archival AL samples (n = 208) also were analyzed by FISH for MLL rearrangement. Overall, 378 samples from consecutive patients with newly diagnosed AL were the basis of this study without further selection. Leukemia was classified according to FAB criteria21,22 and immunophenotyping of leukemic cells.23 Of the 378 cases, 327 (86.5%) were classified as AML, 47 (12.4%) as ALL, and 4 (1.1%) as biphenotypic leukemia. Leukemia subtypes are summarized in ❚Table 1❚. In 56 (17.1%) of 327 patients, AML had developed after a primary malignancy; all ALL and B-cell AL cases were de novo. The mean age was 58 years in patients with AML (range, 14-81 years), 33 years in ALL (range, 14-74 years), and 26 years in B-cell AL (range, 19-32 years). All patients with AML who were eligible for intensive chemotherapy were enrolled in consecutive trials of the GIMEMA (Gruppo Italiano Malattie ematologiche dell’adulto)–European Organization for Research and Treatment of Cancer cooperative group (AML8, AML10, AML12, AML11, AML13, AML15). Patients younger than 60 years who had an HLA-identical sibling donor underwent allogeneic bone marrow or peripheral blood transplantation. ALL patients eligible for intensive treatment were enrolled in the conventional induction regimen ALL 0288 (GIMEMA)24; some patients with standard-risk and most with high-risk disease were given intensive induction chemotherapy based on high-dose cytarabine and mitoxantrone plus prednisone, which, after a similar consolidation cycle, was followed by autologous or allogeneic transplantation.25 ❚Table 1❚ Distribution of Patients With AL in Subcategories and Incidence of MLL+ and 11q22~25+/MLL– Cases Within Different ALs* Subcategory No. of Patients Acute myeloblastic leukemia M0 27 M1 69 M2 82 M3 25 M4 47 M5a 30 M5b 30 M6 13 M7 4 Acute lymphoblastic leukemia Pro-B cell 4 Pre-B cell, common 25 Burkitt 7 Pre-T cell 4 T cell 7 B-cell AL 4 Total 378 MLL+ 11q22~25/MLL– 2 (7) 1 (1) 1 (1) 0 (0) 2 (4) 6 (20) 3 (10) 0 (0) 0 (0) 2 (7) 1 (1) 0 (0) 0 (0) 1 (2) 1 (3) 0 (0) 2 (15) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 15 (4.0) 0 (0) 1 (4) 0 (0) 0 (0) 0 (0) 1 (25) 9 (2.4) AL, acute leukemia. * Data are given as number (percentage). AMLs are listed according to the FrenchAmerican-British Classification. Am J Clin Pathol 2004;122:298-306 © American Society for Clinical Pathology 299 DOI: 10.1309/RX27R8GJQM330C22 299 299 Cox et al / 11Q23 ABERRATIONS AND MLL GENE STATUS IN ACUTE LEUKEMIA Conventional Cytogenetics Samples obtained before February 1997 were cultured at 24 and 48 hours without synchronization. Starting from 1997, 3 short-term cell cultures were set up from each harvest: 2 synchronized cultures at 24 and 48 hours and 1 overnight Colcemid-treated culture (0.0025 µg/mL) to obtain a high rate of mitotic cells. The synchronization procedure was carried out by incubating cells with methotrexate for 17 hours (final concentration, 10–7 mol/L), and then thymidine solution (final concentration, 10–5 mol/L) was added for 5 additional hours. Cells were exposed to Colcemid (0.05 µg/mL) for the last 15 minutes before centrifugation and standard processing.26 In the ALL samples, direct preparations were carried out after 1 hour of Colcemid exposure. Karyotypes were set up on GTG-banded chromosomes following the 1995 International System for Human Cytogenetic Nomenclature.27 To define a structural clonal aberration, at least 2 cells with the same chromosomal change were to be found and at least 3 abnormal metaphases had to be identified to define chromosomal aneuploidy. In 89% of cases with a normal karyotype, 20 or more metaphases were analyzed, while in the remaining cases, at least 10 metaphases were scored. Fluorescence In Situ Hybridization FISH was done using commercial double-colored probe sets (Vysis). The SpectrumGreen-labeled probe covers a 350-kilobase portion centromeric to the MLL gene breakpoint, and the SpectrumOrange-labeled probe, a 190-kilobase portion largely telomeric of the breakpoint cluster region. Cytogenetic pellets from direct or short-term cultures were used for hybridization: 20 µL of cytogenetic pellet fixed in Carnoy solution was dropped with a micropipette on a cleaned slide. The slides then were aged for 20 minutes at 80°C on a hot plate and dehydrated at room temperature in 70%, 80%, and 100% ethanol (2 minutes each). Geneframes (Abgene, Epsom, England) were applied to dried slides to mark and separate the hybridization areas of single probes. Slides were placed on a hot plate at 37°C, and 2.5 µL of each probe-buffer solution was applied inside the area of the slide delineated by the frame (probes were prepared following the manufacturer’s instructions). The slide was covered with a plastic coverslip (Abgene) and placed in the PCR In Situ 1000 (Perkin-Elmer, Fremont, CA) device or the Hybrite machine (Vysis). Codenaturation was carried out at 72°C for 5 minutes and hybridization at 42°C for 90 minutes. If overnight hybridization was preferred, the codenaturation was carried out at 68°C for 4 minutes and hybridization at 37°C. Slides then were removed and the coverslip discarded. Posthybridization washing was done at 71°C in 0.4× saline sodium citrate (SSC) for 2 minutes and 300 300 Am J Clin Pathol 2004;122:298-306 DOI: 10.1309/RX27R8GJQM330C22 at room temperature in 2× SSC for 1 minute. Slides then were counterstained with 4'-6'-diamidino-8-phenylindole (DAPI), 0.1 µg/mL (Vysis), and analyzed using an Olympus BX2 microscope (Olympus, Tokyo, Japan) equipped with a 100-W lamp and a complete set of filters. In the first 80 samples, 200 nuclei were analyzed per patient, per probe; in the following 311 samples, only 100 cells per patient were recorded for each probe tested. The slides were analyzed blindly by 2 experienced operators (M.C.C. and P.P.) who were unaware of each other’s results and of the conventional cytogenetics results. The cutoff value of the MLL probe (Vysis) was predetermined as 4.3% (mean plus 3 SD in 20,000 nuclei from 20 control bone marrow samples). Whenever abnormalities were found, a second test and incidental cohybridization with a control probe were carried out. In the presence of mitotic cells, metaphase FISH analysis also was done. Additional FISH with centromeric, telomeric, or other locus-specific probes was done when necessary to refine chromosomal breakpoints or identify aneuploidies. Multiplex Karyotyping We analyzed 11 AL samples by M-FISH. In 7 AML cases, M-FISH was done to characterize a complex karyotype or ill-defined aberrations. In the other 4 AML cases, with normal conventional cytogenetic results, M-FISH was performed to check for cryptic translocations. None of these cases had apparent 11q aberrations. The slides, after aging overnight at room temperature, were treated with pepsin (0.005%) for 2.5 minutes at 37°C and then were fixed in a 10% formaldehyde solution for 2 minutes at room temperature. The slides were washed for 5 minutes in phosphate-buffered saline, dehydrated in an alcohol series (70%, 85%, 100%) for 1 minute, and dried at room temperature. The slides were codenatured with the SpectraVysion assay probe (Vysis) at 68°C for 5 minutes and hybridized overnight at 37°C in the PCR In Situ 1000. After 16 to 18 hours, slides were washed at 71°C in 0.4× SSC/0.3% NP40 (Sigma-Aldrich, Milan, Italy) for 2 minutes, at room temperature in 2× SSC for 2 minutes, and then dried and counterstained with DAPI III (Vysis). Fluorescent images were captured using an Olympus (Japan) microscope equipped with a CCD camera. The images were processed and analyzed using the QUIPS M-FISH program (Applied Biosystems, Foster City, CA). A minimum of 5 metaphases were analyzed in each case and chromosomal aberrations confirmed with additional hybridization using specific whole-chromosome painting telomeric probes (QBiogene, Vysis) or region-specific probes (Q-Biogene). Statistical Analysis The χ2 test was used to determine differences between variables in 2 × 2 tables. The Kaplan-Meier method was © American Society for Clinical Pathology Hematopathology / ORIGINAL ARTICLE Results Conventional Cytogenetics Conventional cytogenetics showed clonal abnormalities in 50.0% (189/378), a normal karyotype in 36.0% (136/378), and failed in 14.0% (53/378) of the analyzed cases. 11q22~25 rearrangements were observed in 18 of 324 cases (5.6%) with assessable conventional cytogenetics: 12 were balanced translocations, and in 6 cases, there was no evidence of a translocative partner chromosome ❚Table 2❚ and ❚Table 3❚. Fluorescence In Situ Hybridization FISH was carried out successfully in all samples analyzed and revealed MLL+ in 15 (4.0%) of 378 cases. Of the 18 cases showing rearrangement within the 11q22~25 bands by conventional cytogenetic analysis, only 11 had MLL gene splitting (61%). Of 15 MLL+ samples, 4 (27%) were missed by conventional cytogenetics. Two of these were truly cryptic 11q23 rearrangements, add(16)(p13), revised to t(16;11)(p13;q23) and t(10;11)(p13;q13). The latter case had an unusual FISH pattern showing 2 yellow spots and 1 green signal in interphase cells that was interpreted as MLL insertion.28 The 2 remaining MLL+ cases had no metaphases suitable for evaluation. In 4 additional cases, FISH detected abnormalities of the MLL gene pattern as follows: 1 with MLL gene amplification; 1 with an extra copy of the MLL gene owing to a cryptic unbalanced translocation (Table 3) that was detected with M-FISH; 2 with an extra copy of the MLL gene in which trisomy 11 was disclosed by additional hybridization with a chromosome 11 centromeric probe (no assessable conventional cytogenetics analysis). Multiplex Karyotyping M-FISH was carried out successfully in 11 cases and identified 2 cases with an undetected 11q translocation, respectively, t(9;11)(p21~22;q23~24), and an unbalanced der(8)t(8;11)(p2?;q21~22). The latter case was mentioned in the preceding section because FISH analysis showed an extra copy of the MLL gene ❚Image 1❚. Characteristics of Patients in MLL+ Cases We did not find MLL+ cases in ALL (0/47) or B-cell AL (0/4); all MLL+ cases were classified as AML. The overall incidence of MLL+ in patients with AML was 4.6% (15/327). All MLL+ cases involved younger patients (younger than 60 years) with a mean age of 44 years (range, 24-57 years; mean age for all 327 patients with AML, 58 years; range, 14-81 years). Considering only the younger patients, the incidence of MLL+ was 8.3% (13/157) in de novo AML and 11% (2/19) in t-AML. Of the 2 patients with t-AML, AML-M5a developed in one 18 months after chemotherapy containing topoisomerase-II inhibitors for testicular seminoma (unique patient number [UPN], 97111), while the disease in the other evolved into AML-M0 after a myelodysplastic phase diagnosed 8 months before (UPN, 03589). The incidence of MLL+ cases was 7% in AML-M0 (n = 2), 6% in AML-M4 (n = 2), 20% in AML-M5a (n = 6), 10% in AML-M5b (n = 3), 1% in AML-M1 and AML-M2 (n = 1 each), and 0% in other FAB subgroups (Table 1). In total, 73% of MLL+ cases (11/15) showed involvement of the monocytic lineage. The partner chromosomes of the MLL gene were as follows: 9p22 (2 [13%]); 10p12 (3 [20%]); 17q21-25 (2 [13%]); 19p13 (2 [13%]); 4q21, 6q22, 16p13, 22q13 (1 case each [7%]); and unknown (2 [13%]). Clinical follow-up data were available for all 15 MLL+ cases. Complete remission (CR) was achieved in 9 patients (60%); 5 (33%) had resistant disease, and 1 patient died during induction chemotherapy. After a mean follow-up of 14.6 months, 9 patients died (60%). Of the remaining 6 patients, follow-up was very short for 2 (Table 2), and the other 4 were in prolonged, continuous CR after having undergone autotransplantation (n = 2) or allotransplantation (n = 2) during the first (n = 3) or second (n = 1) CR. Of these 4 patients (Table 3), 1 had a classic t(9;11), and the 100 MLL+ Unfavorable Intermediate Favorable 90 80 70 Percent used to calculate the survival curves and the log-rank test to determine significance ❚Figure 1❚. 60 50 40 30 20 10 0 0 20 40 60 80 100 Time (mo) 120 140 160 ❚Figure 1❚ Cumulative proportion of surviving patients (Kaplan-Meier survival curves). Patients were grouped according to cytogenetic or fluorescence in situ hybridization data in 4 classes (favorable, intermediate, unfavorable, and MLL+). The log-rank test was used to determine significance. Patients older than 60 years (MLL+, 0) or diagnosed with t-AML (MLL+, 2) were excluded from the computation. circles, complete data; +, censored. Am J Clin Pathol 2004;122:298-306 © American Society for Clinical Pathology 301 DOI: 10.1309/RX27R8GJQM330C22 301 301 Cox et al / 11Q23 ABERRATIONS AND MLL GENE STATUS IN ACUTE LEUKEMIA ❚Table 2❚ Clinical and Cytogenetic Features of 15 Patients With MLL+ Acute Leukemia UPN/Sex/Age (y) AML Type* Secondary 98257/F/41 97111/M/32 M0 M5a No Yes (CHT) 97210/M/50 98079/F/44 97198/F/24 01424/M/44 96259/F/57 97212/M/24 M5a M4 M5a M1 M5b M2 No No No No No No 03535/M/51 03589/F/51 96334/F/38 97222/F/41 95078/M/28 98124/M/51 97025/F/56 M5b M0 M5a M5a M5a M5b M4 No Yes (MDS) No No No No No Karyotype MLL Localization CR OS (mo) 4q21;11q23 11q23; 9p2? Yes Yes 14 10 9p22; 11q23 10p12;11q23 NE 11q23; 17q2? 11q23; 17q25 11q23; 19p13 Yes No No Yes Yes Yes +57 8 20 +24 +77 +56 11q23; 22q13 11q23; 16p13 6q22; 11q23 10p11-12; 11q23 11q23;19p13 NE NE Yes Yes No Yes No NE No +1 +4 7 12 1 1 1 47,XX,t(4;11)(q21;q23),+m 47,XY,t(9;11)(p21;q23),+der(8)t(8;22) (q24;q12)c 46,XY,t(9;11)(p22;q23) 46,XX,t(10;11)(p12;q23) 46,XX,t(10;11)(p13;q13)/46,XX 46,XY,t(11;17)(q23;q2?)/ 46,XX,t(11;17)(q23;q24)/46,XX 46,XY,t(11;19)(q23;p13)/47,XY,t(11;19) (q23;p13),+19/46,XY 46,XYt(11;22)(q23;q13) 46,XX,add(16)(p13) 46,XX,add(11)(q23)/46,XX 46,XX,add(11)(q23) 46,XY,del(11)(q23) — — AML, acute myeloblastic leukemia; CHT, chemotherapy; CR, complete remission; MDS, myelodysplastic syndrome; NE, not evaluable; OS, overall survival; UPN, unique patient number. * Diagnoses are listed according to the French-American-British Classification. ❚Table 3❚ Clinical and Cytogenetic Features of 9 Patients With 11q22~25+/MLL– Acute Leukemia UPN/Sex/Age(y) Diagnosis Secondary 95056/M/45 98230/F/24 99042/F/62 98207/F/14 00383/M/33 02058/F/58 AML-M6 B-cell AL AML-M0 AML-M1 AML-M0 ALL, pre-B cell Yes (MDS) No No Yes (CHT) No No 95035/M/59 AML-M6 No 02418/M/40 AML-M4 Yes (MDS) 02061/F/46 AML-M5a No Karyotype Breakpoint 46,XY,t(2;11)(p21;q23) 46,XX,t(11;16)(q23;p13)/46,XX 46,XX,t(11;12)(q23;q24)/46,XX 46,XX,t(11;15)(q23;q12) 46,XY,add(1)(q42),del(11)(q13q23),del(12)(p13) 46,XX,t(2;3)(q2?;q3?),add(11)(q23)ish.dup(11) (q23q24)(MLLx1,Tel11q+)/46,XX 46-48,XY,–11,–17,–18,del(4)(q21),inv(6) (p21.3q13),der(12)t(11;12)(q13;p13), +(3-5)markers.ish der(11)amp(11)(q23) (WCP11+,MLL>30 )/idem,–Y,–5 45,XY,–7,dic(12;22)(p13;q10),der(13;13) (q10;q10),+13 M-FISH: der(8)t(8;11)(p22;q21-22) 46,XX,der(7)add(7)(p10) M-FISH: 46,XX,ish.der(7)t(7;19)(p10;p10) (WCP7+,WCP19+),t(9;11)(p21-22;q23-24) (WCP9+,WCP11+) CR OS (mo) Telomeric to MLL NE Telomeric to MLL Telomeric to MLL Centromeric to MLL NE Yes Yes Yes Yes 1 23 12 24 20 Telomeric to MLL MLL amplification (>30 spots) Yes No 1 1 Centromeric to MLL NE NE NE No 1 AL, acute leukemia; ALL, acute lymphoblastic leukemia; AML, acute myeloblastic leukemia; CHT, chemotherapy; CR, complete remission; MDS, myelodysplastic syndrome; NE, not evaluable; OS, overall survival; UPN, unique patient number; WCP, whole chromosome painting. other 3 had t(11;17)(q23;q21), t(11;17)(q23;q25), and t(11;19)(q23;p13), respectively. We compared overall survival in patients with MLL+ AML and 122 patients with MLL– AML. The latter group of patients was divided by karyotype into 3 categories: (1) favorable (n = 41), ie, t15;17; t8;21, inv16, or t16;16; (2) intermediate (n = 61), ie, AML with a normal karyotype or with abnormalities not defined as favorable or unfavorable; and (3) unfavorable (n = 20), ie, 5q–/–5, 7q–/–7, inv3/t3;3; t(6;9); t(9;22), 12p–; 9q–; 17p and 21q abnormalities; or 20q–, complex aberrant karyotype with 3 or more aberrations. The median overall survival was 8.2 months in the MLL+ group compared with 35 months, 11.5 months, and 302 302 Am J Clin Pathol 2004;122:298-306 DOI: 10.1309/RX27R8GJQM330C22 2.4 months in the favorable, intermediate, and unfavorable groups, respectively (P < .001; Figure 1). Patients older than 60 years (MLL+, 0) or diagnosed with t-AML (MLL+, 2) were excluded from the computation. Characteristics of Patients With 11q22~25+/MLL– Leukemia Of 327 cases analyzed by cytogenetics, 9 showed rearrangements clustering within the 11q22~25 region without MLL gene splitting (2.8%). In 8 of these cases, the involved region was within the 11q23~25 bands, and in 1 case, it was within the 11q13~22 bands. Seven rearrangements were detected by conventional cytogenetics and 2 only © American Society for Clinical Pathology Hematopathology / ORIGINAL ARTICLE B A MLL probe MLL probe t (2;11)(p21;q23) t (11;15)(q23;q12) der(11) 11 11 der(11) D C E WCP11 probe; WCP19 probe MLL probe 11 der(7) 11 der(8) der(11) t (11;12)(q23~24;q24) 19 19 der(9) 11 der(7)t(7;19)(p10;p10); t(9;11)(p21;q23) ❚Image 1❚ A, Partial karyotype showing t(2;11)(p21;q23) (left); fluorescence in situ hybridization (FISH) analysis with the MLL doublecolor break-apart probe (Vysis, Downers Grove, IL) (right). The probe localized on normal chromosome 11 and on der(11) (unique patient number [UPN], 95056). The red and the green spots colocalized in a fusion red-green signal; the MLL gene is not rearranged. B, Partial karyotype showing t(11;15)(q23;q12) (left); FISH with the MLL probe, which localized on chromosome 11 and on der(11) (right). The MLL probe appears as a fusion red-green signal; MLL is not rearranged (UPN, 98207). C, FISH analysis with the MLL probe that localized on normal chromosome 11 (2×) and on der(8). The probe appears as a fusion red-green signal (MLL not rearranged) (UPN, 02418). D, Partial karyotype showing t(11;12)(q23~24;q24). E, FISH analysis with whole chromosome painting 11(red) and whole chromosome painting 19 (green) probes, disclosing der(7)t(7;19)(p10;p10); t(9;11)(p21;q23) (UPN, 02061). by M-FISH. In 4 cases, the rearranged locus was telomeric to MLL, in 2 it was centromeric to MLL, in 1 case the MLL gene was amplified, and in the remaining 2 cases, no metaphase was available to assess MLL status. In 3 cases, the 11q rearrangement was associated with a complex karyotype (3 or more aberrations), in 4 it was the sole chromosomal change, and in 2 it was associated with another structural abnormality (Table 3). Of 9 patients, 7 had AML and were a median age of 42 years (range, 14-62 years). The FAB subtypes represented were as follows: AML-M0, 2; AML-M1, 1; AML-M4, 1; AML-M5a, 1; and AML-M6, 2 (Table 3). Three patients (33%) had secondary AML: 2 were diagnosed with myelodysplasia 12 (UPN, 02418) and 20 (UPN, 95056) months before the leukemia outbreak; in 1 patient, AML recurred after chemotherapy and allotransplantation for previous AML that at disease onset showed a normal karyotype (UPN, 98207). Of 7 patients with AML, clinical outcome could be evaluated for 6, and 1 was lost to follow-up. Two died during the chemotherapy induction phase, 3 achieved CR, and the remaining 1 had resistant disease (Table 3). All 6 patients died (median survival, 10 months; range, 1-24 months). The remaining 2 patients with 11q22~25+/MLL– were characterized as having pre-B-cell ALL (UPN, 02058) and B-cell AL with T-cell and myeloid markers (UPN, 98230); both cases were de novo leukemias. Pre-B-cell ALL was Am J Clin Pathol 2004;122:298-306 © American Society for Clinical Pathology 303 DOI: 10.1309/RX27R8GJQM330C22 303 303 Cox et al / 11Q23 ABERRATIONS AND MLL GENE STATUS IN ACUTE LEUKEMIA diagnosed in a 58-year-old woman who died of disease recurrence after achieving CR that lasted 420 days. B-cell AL was diagnosed in a 24-year-old woman who died of infection during the course of aploidentical stem cell transplantation. In 5 patients with 11q22~25+/MLL– leukemia, 5 different balanced translocations cytogenetically indistinguishable (Table 3) from typical 11q23+/MLL+ were observed: (1) The t(2;11)(p21;q23) was found in a case of AML-M6 evolved after myelodysplastic syndrome. t(2;11)(p21;q23) has been described in t-AML and in myelodysplastic syndrome.29 The MLL gene rearrangement was shown at the molecular level in 2 cases and excluded in 1 patient. (2) The t(11;16)(q23;p13) was found in a young patient with a B-cell AL with T and myeloid markers. To our knowledge this is the only reported case of t(11;16)(q23;p13) without MLL+ leukemia.30,31 (3) The t(11;12)(q23;q24) was found. By studying printed research, we found only 1 other case with a similar translocation, but it showed an MLL splitting pattern by FISH analysis.9 (4) The t(11;15)(q23;q12) was observed in a patient with AML at the time of leukemia recurrence. t(11;15)(q23;q12~15) is a rare 11q23v. In a few cases studied at the molecular level, MLL rearrangement has been ascertained and a partner gene named AF15 has been cloned.32 (5) The t(9;11)(p21-22;q23-24) was identified incidentally by M-FISH and confirmed by whole chromosome painting. This case was previously reported,30 and to our knowledge, no other such case has been described. The remaining 4 patients had various unbalanced 11q22~25 translocations always associated with other chromosomal changes: (1) The del(11)(q13q23) was associated with 2 additional aberrations. (2) An unbalanced t(8;11)(p21~22; q13~22) was identified through M-FISH in a complex karyotype. (3) The add(11)(q23) was identified by conventional cytogenetic analysis; it was revised after FISH (using MLL and 11q telomeric probes) to dup(11)(q23~25). (4) FISH analysis showed MLL amplification33 on the derivative markers of chromosome 11 within a complex karyotype also bearing a t(11;12)(q13;p13) without MLL rearrangement. The 9 11q22~25+/MLL– cases were clinically and cytogenetically heterogeneous, and statistical consideration regarding survival is not feasible. Nevertheless, the overall outcome was rather poor; no patients survived more than 24 months. Discussion In the forthcoming era of tailored, targeted therapy, the identification of genetic aberration in leukemia will become more and more important for assigning patients to more specific or more intensive treatment. The combining of conventional cytogenetics and molecular and FISH methods greatly increases the accuracy of information; nevertheless, 304 304 Am J Clin Pathol 2004;122:298-306 DOI: 10.1309/RX27R8GJQM330C22 the majority of multicenter clinical trials still base their data only on karyotype results.14-17 In the present study, conventional cytogenetics was combined with FISH analysis19,28 using a commercial probe that should permit the identification of all MLL rearrangements (not MLL-PTD) and of the translocated partner chromosome.34 The overall incidence of MLL+ cases in patients with AML (4.6%) is comparable to that recently reported by Schoch et al.9 MLL+ cases were not detected in the 47 cases of ALL and 4 cases of B-cell AL analyzed by FISH. We attribute this finding to the low number of cases studied. Notably 2 (4%) of these 51 patients had 11q23+ without MLL rearrangement. Compared with FISH, the sensitivity of conventional cytogenetics was 73%: FISH permitted the identification of 4 additional MLL+ samples (15/378 vs 11/378; Table 1). Of the 15 cases, 2 (13%) were truly cryptic MLL+ cases, which is similar to the rate reported in adult AML.9,10 In infant and childhood AL,19,20 the incidence of cryptic MLL+ cases might be higher. A recently published series showed that 25% of MLL+ cases were missed by conventional cytogenetic analysis,19 and several of these cases had insertion of the MLL gene. We found only 1 case of cryptic MLL insertion in a patient with the t(10;11)(p12;q13) (UPN, 97198). Extensive FISH studies have shown that t(10;11) is a complex translocation that implies inversion of translocated chromosomes with multiple breaks. 35 Furthermore, in patients with the t(10;11), the possibility of AF10/CALM gene fusion without MLL gene involvement should be ruled out. AF10/CALM is a nonrandom translocation described in AML, ALL, and non-Hodgkin lymphoma.36,37 Not infrequently, reciprocal MLL translocation appears at the chromosomal level as an unbalanced rearrangement and is referred to as add(11)(q23) or del(11)(q23).38 In the present series, FISH permitted the revision of 3 (20%) of 15 cases reported as unbalanced 11q23+ aberrations. A del(11)(q23) was revised to t(11;19)(q23;p13), and 2 cases with add(11)(q23) were reclassified as t(6;11)(q22;q23)39 and t(10;11)(p12;q23), respectively. Although the analysis of clinical outcome is beyond the scope of this work, we briefly report that the overall survival of patients with de novo MLL+ AML was comparable to that of the intermediate-risk group (Figure 1). Notably 3 (75%) of 4 patients in prolonged, continuous CR had 11q23v (Table 3). Several studies comparing FISH and conventional cytogenetics for the diagnosis of MLL+ have shown greater sensitivity of FISH. Conversely, except for sporadic articles,7,19 little emphasis has been given to the incidence of cases displaying 11q22~25 aberrations without MLL rearrangement in AL. Only recently, Tanaka et al8 reported a series of 11q+/MLL– cases in Japanese patients affected by various hematologic malignant neoplasms. They identified several restricted breakpoint sites involved in translocations, deletions, © American Society for Clinical Pathology Hematopathology / ORIGINAL ARTICLE or both. The candidate targets of these rearrangements might be a few genes known to map at the 11q23 locus that are implicated in hematopoietic malignant neoplasms.40-46 Overall in our series, 9 (45%) of 20 11q22~25+ cases showed no translocation of the MLL gene. Seven of these were identified by conventional cytogenetic analysis, and 2 were observed incidentally in a group of 11 AML cases analyzed by M-FISH.47,48 Cryptic rearrangement of 11q in the form of balanced or unbalanced translocation has been reported in published series of M-FISH and spectral karyotyping.47,48 Our findings further strengthen the notion that wider use of these technologies could give a relevant hint about the chromosomal changes in AL. A better characterization of 11q22~25+/MLL– leukemias is relevant for the identification of new recurrent translocations, cloning of genes, and elucidation of the pathogenic mechanism involved in AL. Moreover, these cases should be readily distinguished from 11q23+/MLL+ cases. Beyond FISH, MLL rearrangement also can be detected by Southern blot and RT-PCR. Southern blot is sensitive and capable of identifying all MLL translocations and MLL-PTD but is not used routinely because it is laborious and unable to discriminate different MLL rearrangements. RT-PCR is the most sensitive approach for detecting specific subtypes of MLL rearrangements. The main drawback of this method is that the partner gene needs to be known. To overcome this limitation, a multiplex RT-PCR approach has been devised.49 This method is useful because it identifies, in a single reaction, the most common MLL translocations: (4;11)(q21;q23) (MLL/AF4); t(6;11)(q27;q23) (MLL/AF6); t(9;11)(p21-22;q23) (MLL/AF9); t(10;11)(p11-13;q23) (MLL/AF10); t(11;19)(q23;p13.1) (MLL/ELL); and t(11;19)(q23;p13.3) (MLL/ENL). Whatever the method for determining MLL gene status, the diagnosis of 11q23+/MLL+ leukemia should not be done without confirmation by molecular or FISH methods. 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