Chromosomal Aberration of the 11Q23 Locus in Acute Leukemia and Frequency of MLL Gene Translocation Results in 378 Adult Patients

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 Downloaded from https://academic.oup.com/ajcp/article/122/2/298/1759501 by guest on 23 September 2021 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+) Structural abnormality of the 11q23 band (11q23+) bearing the MLL gene translocation (MLL+) is a recurrent chro- bearing the MLL gene translocation (MLL+) is a mosome change in leukemia described in acute myeloblastic recurrent chromosome change observed in 3% to 7% of leukemia (AML) and in acute lymphoblastic leukemia (ALL), acute lymphoblastic leukemias and in 3% to 4% of with a peak incidence in infant leukemia.1,2 A proposal by the acute myeloblastic leukemias. The resolution of World Health Organization specifies a separate category for conventional cytogenetics (CC) in detecting 11q23 AML with 11q23+/MLL+.3 This notion has been supported rearrangement is limited when the translocative partner recently by biologic studies: microarray analyses have shown has a telomeric location; furthermore, CC can barely that MLL+ acute leukemias (ALs) have a peculiar gene- discriminate between true 11q23+/MLL+ and profiling pattern that distinguishes them from all other ALs and rearrangements clustering within the 11q22~25 region that MLL+ leukemic blasts resemble very immature progenitor without MLL involvement (MLL–). We characterized a cells.4 Furthermore, these studies showed that MLL+ leukemias series of 378 consecutive patients with adult acute are a separate entity when compared with AML with MLL leukemia by using CC, fluorescence in situ partial tandem duplication (MLL-PTD), a recently identified hybridization (FISH), and multiplex karyotyping (M- genetic aberration observed in a sizable proportion of AMLs.5 FISH) analysis. Our aim was to define the frequency of Extensive cytogenetic and molecular studies have shown cryptic MLL+ cases and the frequency of MLL+ within that 11q23/MLL is a highly promiscuous locus: more than 50 11q22~25+ cases. As expected, FISH was more chromosomal loci have been described as 11q23 chromo- sensitive than CC in detecting MLL+ cases, but rather some partners, whereas more than 30 MLL partner genes unexpectedly, 9 (45%) of 20 patients with 11q22~25+ have been characterized.6 It also should be mentioned that were MLL–. A better characterization of t(11q23) might involve genes other than MLL7 and that 11q22~25+/MLL– leukemias is relevant for the conventional cytogenetics can barely discriminate between identification of new, recurrent translocations. true 11q23+/MLL+ and rearrangements clustering within the Moreover, these cases should be readily distinguishable 11q22~25 region without MLL involvement.8 from 11q23+/MLL+ cases. We recommend that Because t(9;11)(p21;q23) bearing the MLL/AF9 gene karyotypic analysis always be complemented by fusion in AML and t(4;11) with MLL/AF4 gene fusion in molecular or FISH methods to unravel MLL infant leukemia are the most common types,6 these transloca- rearrangements. tions 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.

298 Am J Clin Pathol 2004;122:298-306 © American Society for Clinical Pathology 298 DOI: 10.1309/RX27R8GJQM330C22 Hematopathology / ORIGINAL ARTICLE

In older patients with AML (60 years or older), it is observed in newly diagnosed patients with AML and ALL (n = 170). rarely.9 The majority of 11q23+/MLL+ AML cases have mono- Furthermore, all residual archival AL samples (n = 208) also cytoid differentiation features and are classified in the M4 and were analyzed by FISH for MLL rearrangement. Overall, M5 leukemia French-American-British (FAB) subtypes.10 In 378 samples from consecutive patients with newly diagnosed adult ALL, the overall incidence of 11q23+/MLL+ is reported AL were the basis of this study without further selection. to be around 3% to 7%,11 but in pro-B-cell ALL, it accounts for Leukemia was classified according to FAB criteria21,22 and more than 30% of chromosomal aberrations.12,13 immunophenotyping of leukemic cells.23 While t(4;11) ALL has an established dismal prognosis, Of the 378 cases, 327 (86.5%) were classified as AML, the clinical outcome of 11q23+/MLL+ AML is more hetero- 47 (12.4%) as ALL, and 4 (1.1%) as biphenotypic leukemia. geneous.14-16 The Medical Research Council14 and the Leukemia subtypes are summarized in ❚Table 1❚. Southwest Oncology Group15 classify the risk for patients In 56 (17.1%) of 327 patients, AML had developed after Downloaded from https://academic.oup.com/ajcp/article/122/2/298/1759501 by guest on 23 September 2021 with AML with t(9;11) as intermediate and poor, respec- a primary malignancy; all ALL and B-cell AL cases were de tively. Even more disagreement surrounds the prognostic novo. The mean age was 58 years in patients with AML relevance of classic t(9;11) vs v11q23: some clinical trials (range, 14-81 years), 33 years in ALL (range, 14-74 years), reported that patients with t(9;11) fared better than patients and 26 years in B-cell AL (range, 19-32 years). with v11q23,16,17 whereas other studies failed to identify All patients with AML who were eligible for intensive differences.9,14 These discrepancies probably reflect the chemotherapy were enrolled in consecutive trials of the marked biologic heterogeneity of 11q23 aberrations. Further- GIMEMA (Gruppo Italiano Malattie ematologiche more, because many v11q23 translocations are rare translo- dell’adulto)–European Organization for Research and Treat- cations, the clinical impact of specific single variants is diffi- ment of Cancer cooperative group (AML8, AML10, AML12, cult to extrapolate, even from large studies on 11q23 AL.14-16 AML11, AML13, AML15). Patients younger than 60 years Recently, the combined use of conventional cytoge- who had an HLA-identical sibling donor underwent allo- netics, reverse transcriptase–polymerase chain reaction (RT- geneic bone marrow or peripheral blood transplantation. ALL PCR), Southern blot analysis, and fluorescence in situ patients eligible for intensive treatment were enrolled in the hybridization (FISH) in limited AL series has revealed that conventional induction regimen ALL 0288 (GIMEMA)24; discrepant results with MLL involvement might become some patients with standard-risk and most with high-risk evident,18 and a high incidence of patients with cryptic disease were given intensive induction chemotherapy based MLL+ leukemia were observed in 2 pediatric series.19,20 on high-dose cytarabine and mitoxantrone plus prednisone, Because the majority of clinical trials include only karyotype which, after a similar consolidation cycle, was followed by data,14-16 it is reasonable to speculate that beyond biologic autologous or allogeneic transplantation.25 heterogeneity, these discrepancies also are due partly to the low accuracy of conventional cytogenetics. ❚Table 1❚ We describe our findings in a series of 378 consecutive Distribution of Patients With AL in Subcategories and cases of adult AL, studied with conventional cytogenetics, Incidence of MLL+ and 11q22~25+/MLL– Cases Within * FISH, and multiplex karyotyping (M-FISH) analysis. The Different ALs aim of the study was to define the incidence of cryptic MLL Subcategory No. of Patients MLL+ 11q22~25/MLL– gene translocation and the incidence of MLL gene rearrange- Acute myeloblastic leukemia ment within 11q22~25+ cases. M0 27 2 (7) 2 (7) M1 69 1 (1) 1 (1) M2 82 1 (1) 0 (0) M3 25 0 (0) 0 (0) M4 47 2 (4) 1 (2) Materials and Methods M5a 30 6 (20) 1 (3) M5b 30 3 (10) 0 (0) M6 13 0 (0) 2 (15) Cases M7 4 0 (0) 0 (0) Acute lymphoblastic leukemia Between November 1992 and September 2003, 478 AL Pro-B cell 4 0 (0) 0 (0) samples from newly diagnosed adult patients (older than 14 Pre-B cell, common 25 0 (0) 1 (4) Burkitt 7 0 (0) 0 (0) years) were sent to our laboratory for conventional cytoge- Pre-T cell 4 0 (0) 0 (0) netic analysis. Conventional cytogenetics was done T cell 7 0 (0) 0 (0) B-cell AL 4 0 (0) 1 (25) following standard methods, and residual pellets were stored Total 378 15 (4.0) 9 (2.4) at –20°C in Carnoy solution. From January 2000, FISH analysis with the MLL gene probe (Vysis, Downers Grove, AL, acute leukemia. * Data are given as number (percentage). AMLs are listed according to the French- IL) was combined routinely with conventional cytogenetics American-British Classification.

Conventional Cytogenetics at room temperature in 2× SSC for 1 minute. Slides then Samples obtained before February 1997 were cultured were counterstained with 4'-6'-diamidino-8-phenylindole at 24 and 48 hours without synchronization. Starting from (DAPI), 0.1 µg/mL (Vysis), and analyzed using an Olympus 1997, 3 short-term cell cultures were set up from each BX2 microscope (Olympus, Tokyo, Japan) equipped with a harvest: 2 synchronized cultures at 24 and 48 hours and 1 100-W lamp and a complete set of filters. overnight Colcemid-treated culture (0.0025 µg/mL) to obtain In the first 80 samples, 200 nuclei were analyzed per a high rate of mitotic cells. The synchronization procedure patient, per probe; in the following 311 samples, only 100 cells was carried out by incubating cells with methotrexate for 17 per patient were recorded for each probe tested. The slides hours (final concentration, 10–7 mol/L), and then thymidine were analyzed blindly by 2 experienced operators (M.C.C. and solution (final concentration, 10–5 mol/L) was added for 5 P.P.) who were unaware of each other’s results and of the additional hours. Cells were exposed to Colcemid (0.05 conventional cytogenetics results. The cutoff value of the MLL Downloaded from https://academic.oup.com/ajcp/article/122/2/298/1759501 by guest on 23 September 2021 µg/mL) for the last 15 minutes before centrifugation and probe (Vysis) was predetermined as 4.3% (mean plus 3 SD in standard processing.26 In the ALL samples, direct prepara- 20,000 nuclei from 20 control bone marrow samples). When- tions were carried out after 1 hour of Colcemid exposure. ever abnormalities were found, a second test and incidental Karyotypes were set up on GTG-banded chromosomes cohybridization with a control probe were carried out. In the following the 1995 International System for Human Cytoge- presence of mitotic cells, metaphase FISH analysis also was netic Nomenclature.27 To define a structural clonal aberra- done. Additional FISH with centromeric, telomeric, or other tion, at least 2 cells with the same chromosomal change were locus-specific probes was done when necessary to refine chro- to be found and at least 3 abnormal metaphases had to be mosomal breakpoints or identify aneuploidies. identified to define chromosomal aneuploidy. In 89% of cases with a normal karyotype, 20 or more metaphases were Multiplex Karyotyping analyzed, while in the remaining cases, at least 10 We analyzed 11 AL samples by M-FISH. In 7 AML metaphases were scored. cases, M-FISH was done to characterize a complex karyotype or ill-defined aberrations. In the other 4 AML cases, Fluorescence In Situ Hybridization with normal conventional cytogenetic results, M-FISH was FISH was done using commercial double-colored probe performed to check for cryptic translocations. None of these sets (Vysis). The SpectrumGreen-labeled probe covers a cases had apparent 11q aberrations. 350-kilobase portion centromeric to the MLL gene break- The slides, after aging overnight at room temperature, point, and the SpectrumOrange-labeled probe, a 190-kilo- were treated with pepsin (0.005%) for 2.5 minutes at 37°C base portion largely telomeric of the breakpoint cluster and then were fixed in a 10% formaldehyde solution for 2 region. minutes at room temperature. The slides were washed for 5 Cytogenetic pellets from direct or short-term cultures minutes in phosphate-buffered saline, dehydrated in an were used for hybridization: 20 µL of cytogenetic pellet alcohol series (70%, 85%, 100%) for 1 minute, and dried at fixed in Carnoy solution was dropped with a micropipette on room temperature. The slides were codenatured with the a cleaned slide. The slides then were aged for 20 minutes at SpectraVysion assay probe (Vysis) at 68°C for 5 minutes 80°C on a hot plate and dehydrated at room temperature in and hybridized overnight at 37°C in the PCR In Situ 1000. 70%, 80%, and 100% ethanol (2 minutes each). Gene- After 16 to 18 hours, slides were washed at 71°C in 0.4× frames (Abgene, Epsom, England) were applied to dried SSC/0.3% NP40 (Sigma-Aldrich, Milan, Italy) for 2 slides to mark and separate the hybridization areas of single minutes, at room temperature in 2× SSC for 2 minutes, and probes. Slides were placed on a hot plate at 37°C, and 2.5 µL then dried and counterstained with DAPI III (Vysis). Fluo- of each probe-buffer solution was applied inside the area of rescent images were captured using an Olympus (Japan) the slide delineated by the frame (probes were prepared microscope equipped with a CCD camera. The images were following the manufacturer’s instructions). The slide was processed and analyzed using the QUIPS M-FISH program covered with a plastic coverslip (Abgene) and placed in the (Applied Biosystems, Foster City, CA). A minimum of 5 PCR In Situ 1000 (Perkin-Elmer, Fremont, CA) device or metaphases were analyzed in each case and chromosomal the Hybrite machine (Vysis). Codenaturation was carried out aberrations confirmed with additional hybridization using at 72°C for 5 minutes and hybridization at 42°C for 90 specific whole-chromosome painting telomeric probes (Q- minutes. If overnight hybridization was preferred, the co- Biogene, Vysis) or region-specific probes (Q-Biogene). denaturation was carried out at 68°C for 4 minutes and hybridization at 37°C. Slides then were removed and the Statistical Analysis coverslip discarded. Posthybridization washing was done at The χ2 test was used to determine differences between 71°C in 0.4× saline sodium citrate (SSC) for 2 minutes and variables in 2 × 2 tables. The Kaplan-Meier method was

300 Am J Clin Pathol 2004;122:298-306 © American Society for Clinical Pathology 300 DOI: 10.1309/RX27R8GJQM330C22 Hematopathology / ORIGINAL ARTICLE used to calculate the survival curves and the log-rank test to 24-57 years; mean age for all 327 patients with AML, 58 determine significance ❚Figure 1❚. 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 Results chemotherapy containing topoisomerase-II inhibitors for testicular seminoma (unique patient number [UPN], 97111), Conventional Cytogenetics while the disease in the other evolved into AML-M0 after a Conventional cytogenetics showed clonal abnormalities myelodysplastic phase diagnosed 8 months before (UPN, in 50.0% (189/378), a normal karyotype in 36.0% (136/378), 03589). and failed in 14.0% (53/378) of the analyzed cases. The incidence of MLL+ cases was 7% in AML-M0 (n = Downloaded from https://academic.oup.com/ajcp/article/122/2/298/1759501 by guest on 23 September 2021 11q22~25 rearrangements were observed in 18 of 324 cases 2), 6% in AML-M4 (n = 2), 20% in AML-M5a (n = 6), 10% (5.6%) with assessable conventional cytogenetics: 12 were in AML-M5b (n = 3), 1% in AML-M1 and AML-M2 (n = 1 balanced translocations, and in 6 cases, there was no each), and 0% in other FAB subgroups (Table 1). In total, evidence of a translocative partner chromosome ❚Table 2❚ 73% of MLL+ cases (11/15) showed involvement of the and ❚Table 3❚. monocytic lineage. The partner chromosomes of the MLL gene were as follows: 9p22 (2 [13%]); 10p12 (3 [20%]); Fluorescence In Situ Hybridization 17q21-25 (2 [13%]); 19p13 (2 [13%]); 4q21, 6q22, 16p13, FISH was carried out successfully in all samples 22q13 (1 case each [7%]); and unknown (2 [13%]). analyzed and revealed MLL+ in 15 (4.0%) of 378 cases. Of Clinical follow-up data were available for all 15 MLL+ the 18 cases showing rearrangement within the 11q22~25 cases. Complete remission (CR) was achieved in 9 patients bands by conventional cytogenetic analysis, only 11 had (60%); 5 (33%) had resistant disease, and 1 patient died MLL gene splitting (61%). Of 15 MLL+ samples, 4 (27%) during induction chemotherapy. After a mean follow-up of were missed by conventional cytogenetics. Two of these 14.6 months, 9 patients died (60%). Of the remaining 6 were truly cryptic 11q23 rearrangements, add(16)(p13), patients, follow-up was very short for 2 (Table 2), and the revised to t(16;11)(p13;q23) and t(10;11)(p13;q13). The other 4 were in prolonged, continuous CR after having latter case had an unusual FISH pattern showing 2 yellow undergone autotransplantation (n = 2) or allotransplantation spots and 1 green signal in interphase cells that was inter- (n = 2) during the first (n = 3) or second (n = 1) CR. Of preted as MLL insertion.28 The 2 remaining MLL+ cases had these 4 patients (Table 3), 1 had a classic t(9;11), and the 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 100 of the MLL gene owing to a cryptic unbalanced translocation MLL+ 90 Unfavorable (Table 3) that was detected with M-FISH; 2 with an extra 80 Intermediate Favorable copy of the MLL gene in which trisomy 11 was disclosed by 70 additional hybridization with a chromosome 11 centromeric 60 probe (no assessable conventional cytogenetics analysis). 50 Percent Multiplex Karyotyping 40 30 M-FISH was carried out successfully in 11 cases and 20 identified 2 cases with an undetected 11q translocation, 10 respectively, t(9;11)(p21~22;q23~24), and an unbalanced 0 der(8)t(8;11)(p2?;q21~22). The latter case was mentioned in 0 20 40 60 80 100 120 140 160 the preceding section because FISH analysis showed an extra Time (mo) copy of the MLL gene ❚Image 1❚. ❚Figure 1❚ Cumulative proportion of surviving patients (Kaplan-Meier survival curves). Patients were grouped Characteristics of Patients in MLL+ Cases according to cytogenetic or fluorescence in situ hybridization We did not find MLL+ cases in ALL (0/47) or B-cell AL data in 4 classes (favorable, intermediate, unfavorable, and (0/4); all MLL+ cases were classified as AML. The overall MLL+). The log-rank test was used to determine incidence of MLL+ in patients with AML was 4.6% significance. Patients older than 60 years (MLL+, 0) or (15/327). All MLL+ cases involved younger patients diagnosed with t-AML (MLL+, 2) were excluded from the (younger than 60 years) with a mean age of 44 years (range, computation. circles, complete data; +, censored.

❚Table 2❚ Clinical and Cytogenetic Features of 15 Patients With MLL+ Acute Leukemia

UPN/Sex/Age (y) AML Type* Secondary Karyotype MLL Localization CR OS (mo)

98257/F/41 M0 No 47,XX,t(4;11)(q21;q23),+m[6] 4q21;11q23 Yes 14 97111/M/32 M5a Yes (CHT) 47,XY,t(9;11)(p21;q23),+der(8)t(8;22) 11q23; 9p2? Yes 10 (q24;q12)c[25] 97210/M/50 M5a No 46,XY,t(9;11)(p22;q23)[12] 9p22; 11q23 Yes +57 98079/F/44 M4 No 46,XX,t(10;11)(p12;q23)[25] 10p12;11q23 No 8 97198/F/24 M5a No 46,XX,t(10;11)(p13;q13)[8]/46,XX[2] NE No 20 01424/M/44 M1 No 46,XY,t(11;17)(q23;q2?)[15]/[20] 11q23; 17q2? Yes +24 96259/F/57 M5b No 46,XX,t(11;17)(q23;q24)[20]/46,XX[5] 11q23; 17q25 Yes +77 97212/M/24 M2 No 46,XY,t(11;19)(q23;p13)/47,XY,t(11;19) 11q23; 19p13 Yes +56

(q23;p13),+19/46,XY Downloaded from https://academic.oup.com/ajcp/article/122/2/298/1759501 by guest on 23 September 2021 03535/M/51 M5b No 46,XYt(11;22)(q23;q13)[20] 11q23; 22q13 Yes +1 03589/F/51 M0 Yes (MDS) 46,XX,add(16)(p13)[6] 11q23; 16p13 Yes +4 96334/F/38 M5a No 46,XX,add(11)(q23)[3]/46,XX[4] 6q22; 11q23 No 7 97222/F/41 M5a No 46,XX,add(11)(q23)[12] 10p11-12; 11q23 Yes 12 95078/M/28 M5a No 46,XY,del(11)(q23)[11] 11q23;19p13 No 1 98124/M/51 M5b No — NE NE 1 97025/F/56 M4 No — NE No 1

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 Karyotype Breakpoint CR OS (mo)

95056/M/45 AML-M6 Yes (MDS) 46,XY,t(2;11)(p21;q23)[30] Telomeric to MLL NE 1 98230/F/24 B-cell AL No 46,XX,t(11;16)(q23;p13)[6]/46,XX[4] NE Yes 23 99042/F/62 AML-M0 No 46,XX,t(11;12)(q23;q24)[20]/46,XX[4] Telomeric to MLL Ye s 12 98207/F/14 AML-M1 Yes (CHT) 46,XX,t(11;15)(q23;q12)[15] Telomeric to MLL Ye s 2 4 00383/M/33 AML-M0 No 46,XY,add(1)(q42),del(11)(q13q23),del(12)(p13) Centromeric to MLL Ye s 2 0 02058/F/58 ALL, pre-B cell No 46,XX,t(2;3)(q2?;q3?),add(11)(q23)ish.dup(11) (q23q24)(MLLx1,Tel11q+)[8]/46,XX[3] Telomeric to MLL Ye s 1 95035/M/59 AML-M6 No 46-48,XY,–11,–17,–18,del(4)(q21),inv(6) MLL amplification No 1 (p21.3q13),der(12)t(11;12)(q13;p13), (>30 spots) +(3-5)markers.ish der(11)amp(11)(q23) (WCP11+,MLL>30 )/idem,–Y,–5 02418/M/40 AML-M4 Yes (MDS) 45,XY,–7,dic(12;22)(p13;q10),der(13;13) Centromeric to MLL NE NE (q10;q10),+13 M-FISH: der(8)t(8;11)(p22;q21-22)[20] 02061/F/46 AML-M5a No 46,XX,der(7)add(7)(p10) NE No 1 M-FISH: 46,XX,ish.der(7)t(7;19)(p10;p10) (WCP7+,WCP19+),t(9;11)(p21-22;q23-24) (WCP9+,WCP11+)[15]

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 2.4 months in the favorable, intermediate, and unfavorable t(11;19)(q23;p13), respectively. groups, respectively (P < .001; Figure 1). Patients older than We compared overall survival in patients with MLL+ 60 years (MLL+, 0) or diagnosed with t-AML (MLL+, 2) AML and 122 patients with MLL– AML. The latter group of were excluded from the computation. patients was divided by karyotype into 3 categories: (1) favorable (n = 41), ie, t15;17; t8;21, inv16, or t16;16; (2) Characteristics of Patients With 11q22~25+/MLL– intermediate (n = 61), ie, AML with a normal karyotype or Leukemia with abnormalities not defined as favorable or unfavorable; Of 327 cases analyzed by cytogenetics, 9 showed and (3) unfavorable (n = 20), ie, 5q–/–5, 7q–/–7, inv3/t3;3; rearrangements clustering within the 11q22~25 region t(6;9); t(9;22), 12p–; 9q–; 17p and 21q abnormalities; or without MLL gene splitting (2.8%). In 8 of these cases, the 20q–, complex aberrant karyotype with 3 or more aberra- involved region was within the 11q23~25 bands, and in 1 tions. The median overall survival was 8.2 months in the case, it was within the 11q13~22 bands. Seven rearrange- MLL+ group compared with 35 months, 11.5 months, and ments were detected by conventional cytogenetics and 2 only

302 Am J Clin Pathol 2004;122:298-306 © American Society for Clinical Pathology 302 DOI: 10.1309/RX27R8GJQM330C22 Hematopathology / ORIGINAL ARTICLE

A B MLL probe MLL probe

t (2;11)(p21;q23) t (11;15)(q23;q12) der(11)

11 Downloaded from https://academic.oup.com/ajcp/article/122/2/298/1759501 by guest on 23 September 2021

11 der(11)

C D 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 double- color 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 myelodysplasia 12 (UPN, 02418) and 20 (UPN, 95056) months to MLL, in 2 it was centromeric to MLL, in 1 case the MLL before the leukemia outbreak; in 1 patient, AML recurred gene was amplified, and in the remaining 2 cases, no after chemotherapy and allotransplantation for previous AML metaphase was available to assess MLL status. In 3 cases, the that at disease onset showed a normal karyotype (UPN, 11q rearrangement was associated with a complex karyotype 98207). Of 7 patients with AML, clinical outcome could be (3 or more aberrations), in 4 it was the sole chromosomal evaluated for 6, and 1 was lost to follow-up. Two died during change, and in 2 it was associated with another structural the chemotherapy induction phase, 3 achieved CR, and the abnormality (Table 3). remaining 1 had resistant disease (Table 3). All 6 patients died Of 9 patients, 7 had AML and were a median age of 42 (median survival, 10 months; range, 1-24 months). years (range, 14-62 years). The FAB subtypes represented The remaining 2 patients with 11q22~25+/MLL– were were as follows: AML-M0, 2; AML-M1, 1; AML-M4, 1; characterized as having pre-B-cell ALL (UPN, 02058) and AML-M5a, 1; and AML-M6, 2 (Table 3). Three patients B-cell AL with T-cell and myeloid markers (UPN, 98230); (33%) had secondary AML: 2 were diagnosed with both cases were de novo leukemias. Pre-B-cell ALL was

diagnosed in a 58-year-old woman who died of disease recur- the majority of multicenter clinical trials still base their data rence after achieving CR that lasted 420 days. B-cell AL was only on karyotype results.14-17 In the present study, conven- diagnosed in a 24-year-old woman who died of infection tional cytogenetics was combined with FISH analysis19,28 during the course of aploidentical stem cell transplantation. using a commercial probe that should permit the identifica- In 5 patients with 11q22~25+/MLL– leukemia, 5 tion of all MLL rearrangements (not MLL-PTD) and of the different balanced translocations cytogenetically indistin- translocated partner chromosome.34 The overall incidence of guishable (Table 3) from typical 11q23+/MLL+ were MLL+ cases in patients with AML (4.6%) is comparable to observed: (1) The t(2;11)(p21;q23) was found in a case of that recently reported by Schoch et al.9 MLL+ cases were not AML-M6 evolved after myelodysplastic syndrome. detected in the 47 cases of ALL and 4 cases of B-cell AL t(2;11)(p21;q23) has been described in t-AML and in analyzed by FISH. We attribute this finding to the low myelodysplastic syndrome.29 The MLL gene rearrangement number of cases studied. Notably 2 (4%) of these 51 patients Downloaded from https://academic.oup.com/ajcp/article/122/2/298/1759501 by guest on 23 September 2021 was shown at the molecular level in 2 cases and excluded in had 11q23+ without MLL rearrangement. 1 patient. (2) The t(11;16)(q23;p13) was found in a young Compared with FISH, the sensitivity of conventional patient with a B-cell AL with T and myeloid markers. To our cytogenetics was 73%: FISH permitted the identification of 4 knowledge this is the only reported case of t(11;16)(q23;p13) additional MLL+ samples (15/378 vs 11/378; Table 1). Of without MLL+ leukemia.30,31 (3) The t(11;12)(q23;q24) was the 15 cases, 2 (13%) were truly cryptic MLL+ cases, which found. By studying printed research, we found only 1 other is similar to the rate reported in adult AML.9,10 In infant and case with a similar translocation, but it showed an MLL split- childhood AL,19,20 the incidence of cryptic MLL+ cases ting pattern by FISH analysis.9 (4) The t(11;15)(q23;q12) might be higher. A recently published series showed that was observed in a patient with AML at the time of leukemia 25% of MLL+ cases were missed by conventional cytoge- recurrence. t(11;15)(q23;q12~15) is a rare 11q23v. In a few netic analysis,19 and several of these cases had insertion of cases studied at the molecular level, MLL rearrangement has the MLL gene. We found only 1 case of cryptic MLL inser- been ascertained and a partner gene named AF15 has been tion in a patient with the t(10;11)(p12;q13) (UPN, 97198). cloned.32 (5) The t(9;11)(p21-22;q23-24) was identified inci- Extensive FISH studies have shown that t(10;11) is a dentally by M-FISH and confirmed by whole chromosome complex translocation that implies inversion of translocated painting. This case was previously reported,30 and to our chromosomes with multiple breaks.35 Furthermore, in knowledge, no other such case has been described. patients with the t(10;11), the possibility of AF10/CALM The remaining 4 patients had various unbalanced gene fusion without MLL gene involvement should be ruled 11q22~25 translocations always associated with other chro- out. AF10/CALM is a nonrandom translocation described in mosomal changes: (1) The del(11)(q13q23) was associated AML, ALL, and non-Hodgkin lymphoma.36,37 with 2 additional aberrations. (2) An unbalanced Not infrequently, reciprocal MLL translocation appears t(8;11)(p21~22; q13~22) was identified through M-FISH in a at the chromosomal level as an unbalanced rearrangement complex karyotype. (3) The add(11)(q23) was identified by and is referred to as add(11)(q23) or del(11)(q23).38 In the conventional cytogenetic analysis; it was revised after FISH present series, FISH permitted the revision of 3 (20%) of 15 (using MLL and 11q telomeric probes) to dup(11)(q23~25). cases reported as unbalanced 11q23+ aberrations. A (4) FISH analysis showed MLL amplification33 on the deriva- del(11)(q23) was revised to t(11;19)(q23;p13), and 2 cases tive markers of chromosome 11 within a complex karyotype with add(11)(q23) were reclassified as t(6;11)(q22;q23)39 also bearing a t(11;12)(q13;p13) without MLL rearrangement. and t(10;11)(p12;q23), respectively. The 9 11q22~25+/MLL– cases were clinically and cytoge- Although the analysis of clinical outcome is beyond the netically heterogeneous, and statistical consideration regarding scope of this work, we briefly report that the overall survival survival is not feasible. Nevertheless, the overall outcome was of patients with de novo MLL+ AML was comparable to that rather poor; no patients survived more than 24 months. 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 sensi- Discussion tivity of FISH. Conversely, except for sporadic articles,7,19 In the forthcoming era of tailored, targeted therapy, the little emphasis has been given to the incidence of cases identification of genetic aberration in leukemia will become displaying 11q22~25 aberrations without MLL rearrangement more and more important for assigning patients to more in AL. Only recently, Tanaka et al8 reported a series of specific or more intensive treatment. The combining of 11q+/MLL– cases in Japanese patients affected by various conventional cytogenetics and molecular and FISH methods hematologic malignant neoplasms. They identified several greatly increases the accuracy of information; nevertheless, restricted breakpoint sites involved in translocations, deletions,

304 Am J Clin Pathol 2004;122:298-306 © American Society for Clinical Pathology 304 DOI: 10.1309/RX27R8GJQM330C22 Hematopathology / ORIGINAL ARTICLE or both. The candidate targets of these rearrangements might 2. Pui CH, Chessells JM, Camitta B, et al. Clinical heterogeneity be a few genes known to map at the 11q23 locus that are in childhood acute lymphoblastic leukemia with 11q23 rearrangements. Leukemia. 2003;17:700-706. implicated in hematopoietic malignant neoplasms.40-46 3. Vardiman JW, Harris NL, Brunning RD. The World Health Overall in our series, 9 (45%) of 20 11q22~25+ cases Organization (WHO) classification of the myeloid neoplasms. showed no translocation of the MLL gene. Seven of these Blood. 2002;100:2292-2302. were identified by conventional cytogenetic analysis, and 2 4. Armstrong SA, Staunton JE, Silverman LB, et al. MLL were observed incidentally in a group of 11 AML cases translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet. 2002;30:41-47. analyzed by M-FISH.47,48 Cryptic rearrangement of 11q in 5. Dohner K, Tobis K, Ulrich R, et al. Prognostic significance of the form of balanced or unbalanced translocation has been partial tandem duplications of the MLL gene in adult patients reported in published series of M-FISH and spectral kary- 16 to 60 years old with acute myeloid leukemia and normal otyping.47,48 Our findings further strengthen the notion that cytogenetics: a study of the Acute Myeloid Leukemia Study Group Ulm. J Clin Oncol. 2002;20:3254-3261. Downloaded from https://academic.oup.com/ajcp/article/122/2/298/1759501 by guest on 23 September 2021 wider use of these technologies could give a relevant hint 6. Mitelman F. Catalog of Chromosome Abnormalities in Cancer about the chromosomal changes in AL. and Leukemias. 6th ed. New York, NY: Wiley-Liss; 1998. A better characterization of 11q22~25+/MLL– 7. Giugliano E, Rege-Cambrin G, Scaravaglio P, et al. Two new leukemias is relevant for the identification of new recurrent translocations involving the 11q23 region map outside the MLL translocations, cloning of genes, and elucidation of the path- locus in myeloid leukemias. Haematologica. 2002;87:1014-1020. ogenic mechanism involved in AL. Moreover, these cases 8. Tanaka K, Eguchi M, Eguchi-Ishimae M, et al. Restricted chromosome breakpoint sites on 11q22-q23.1 and 11q25 in should be readily distinguished from 11q23+/MLL+ cases. various hematological malignancies without MLL/ALL-1 gene Beyond FISH, MLL rearrangement also can be detected rearrangement. Cancer Genet Cytogenet. 2001;124:27-35. by Southern blot and RT-PCR. Southern blot is sensitive and 9. Schoch C, Schnittger S, Klaus M, et al. AML with 11q23/MLL capable of identifying all MLL translocations and MLL-PTD abnormalities as defined by the WHO classification: incidence, partner chromosomes, FAB subtype, age distribution, and but is not used routinely because it is laborious and unable to prognostic impact in an unselected series of 1897 cytogenetically discriminate different MLL rearrangements. RT-PCR is the analyzed AML cases. Blood. 2003;102:2395-2402. most sensitive approach for detecting specific subtypes of MLL 10. Haferlach T, Schoch C, Schnittger S, et al. Distinct genetic rearrangements. The main drawback of this method is that the patterns can be identified in acute monoblastic and acute monocytic leukaemia (FAB AML M5a and M5b): a study of partner gene needs to be known. To overcome this limitation, a 124 patients. Br J Haematol. 2002;118:426-431. 49 multiplex RT-PCR approach has been devised. This method 11. Pui CH, Evans WE. Acute lymphoblastic leukemia. N Engl J is useful because it identifies, in a single reaction, the most Med. 1998;339:605-615. common MLL translocations: (4;11)(q21;q23) (MLL/AF4); 12. Cimino G, Elia L, Mancini M, and the GIMEMA Group. t(6;11)(q27;q23) (MLL/AF6); t(9;11)(p21-22;q23) (MLL/AF9); Clinico-biologic features and treatment outcome of adult pro- B-ALL patients enrolled in the GIMEMA 0496 study: t(10;11)(p11-13;q23) (MLL/AF10); t(11;19)(q23;p13.1) absence of the ALL1/AF4 and of the bcr/abl fusion genes (MLL/ELL); and t(11;19)(q23;p13.3) (MLL/ENL). correlates with a significantly better clinical outcome. Blood. Whatever the method for determining MLL gene status, 2003;102:2014-2020. the diagnosis of 11q23+/MLL+ leukemia should not be done 13. Ishizawa S, Slovak ML, Popplewell L, et al. High frequency of pro-B acute lymphoblastic leukemia in adults with secondary without confirmation by molecular or FISH methods. In the leukemia with 11q23 abnormalities. Leukemia. 2003;17:1091- light of our increased knowledge of the complexity of genetic 1095. aberrations in leukemias, this translocation is a good para- 14. Grimwade D, Walker H, Oliver F, et al. The importance of digm of the need for common criteria for genetic diagnosis. diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. Blood. 1998;7:2322-2333. From the Departments of 1Hematology and 2Anatomic Pathology, 3 15. Slovak M, Kopecky KJ, Cassileth PA, et al. Karyotypic S’Eugenio Hospital, University of Tor Vergata and Alte analysis predicts outcome of preremission and postremission Specialità, S’Eugenio, Hospital, Rome, Italy. therapy in adult acute myeloid leukemia: a Southwest Ministero della Salute (Ricerca Finalizzata), MIUR (FIRB Oncology Group/Eastern Cooperative Oncology Group Study. Blood. 2000;96:4075-4083. Project) and AIRC. Address reprint requests to Dr Cox: UOC Ematologia, P.le 16. Byrd JC, Mrozek K, Dodge RK, and the Cancer and Leukemia Group B (CALGB 8461). Pretreatment cytogenetic Umanesimo 10, 00144 Rome, Italy. abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia References Group B (CALGB 8461). Blood. 2002;100:4325-4336. 17. Mrozek K, Heinonen K, Lawrence D, et al. Adult patients 1. Dessen P, Huret JL. Chromosome 11. Atlas Genet Cytogenet with de novo acute myeloid leukemia and t(9;11)(p22;q23) Oncol Haematol. 2002. Available at have a superior outcome to patients with other translocations http://www.infobiogen.fr/services/chromcancer/Indexbychrom involving band 11q23: a cancer and leukemia group B study. /idx_11.html. Accessed May 21, 2004. Blood. 1997;90:4532-4538.

18. Ibrahim S, Estey EH, Pierce S, et al. 11q23 abnormalities in 34. von Bergh A, Emanuel B, van Zelderen-Bhola S, et al. A patients with acute myelogenous leukemia and DNA probe combination for improved detection of myelodysplastic syndrome as detected by molecular and MLL/11q23 breakpoints by double-color interphase-FISH in cytogenetic analyses. Am J Clin Pathol. 2000;114:793-797. acute leukemias. Genes Chromosomes Cancer. 2000;28:14-22. 19. Watanabe N, Kobayashi H, Ichiji O, et al. Cryptic insertion and 35. Van Limbergen H, Poppe B, Janssens A, et al. Molecular translocation or nondividing leukemic cells disclosed by FISH cytogenetic analysis of 10;11 rearrangements in acute myeloid analysis in infant acute leukemia with discrepant molecular and leukemia. Leukemia. 2002;16:344-351. cytogenetic findings. Leukemia. 2003;17:876-882. 36. Bohlander SK, Muschinsky V, Schrader K, et al. Molecular 20. Forestier E, Heim S, Blennow E, and the Nordic Society of analysis of the CALM/AF10 fusion: identical rearrangements Paediatric Haematology and Oncology (NOPHO), Swedish in acute myeloid leukemia, acute lymphoblastic leukemia and Cytogenetic Leukaemia Study Group (SCLSG), NOPHO malignant lymphoma patients. Leukemia. 2000;14:93-99. Leukaemia Cytogenetic Study Group (NLCSG). Cytogenetic 37. Asnafi V, Radford-Weiss I, Dastugue N, et al. CALM-AF10 is abnormalities in childhood acute myeloid leukaemia: a a common fusion transcript in T-ALL and is specific to the

Nordic series comprising all children enrolled in the TCRgammadelta lineage. Blood. 2003;102:1000-1006. Downloaded from https://academic.oup.com/ajcp/article/122/2/298/1759501 by guest on 23 September 2021 NOPHO-93-AML trial between 1993 and 2001. Br J Haematol. 2003;121:566-577. 38. Harbott J, Mancini M, Verellen-Dumoulin C, et al, on behalf of European 11q23 Workshop participants. Hematological 21. Bennett JM, Catovsky D, Daniel MT, et al (FAB Cooperative malignancies with a deletion of 11q23: cytogenetic and Group). Proposed revised criteria for the classification of acute clinical aspects. Leukemia. 1998;12:823-827. myeloid leukemia: a report of the French-American-British Cooperative Group. Ann Intern Med. 1985;103:620. 39. Bernard OA, Hillion J, Le Coniat M, et al. A new case of translocation t(6;11)(q21;q23) in a therapy-related acute 22. Bennett JM, Catovsky D, Daniel MT, et al. Proposal for the myeloid leukemia resulting in an MLL-AF6q21 fusion. Genes recognition of minimally differentiated acute myeloid Chromosomes Cancer. 1998;22:221-224. leukaemia (AML-M0). Br J Haematol. 1991;78:325-329. 40. Baud V, Lipinski M, Rassart E, et al. The human homolog of 23. Ludwig WD, Raghavachar A, Thiel E. Immunophenotypic the mouse common viral integration region, FLI1, maps to classification of acute lymphoblastic leukaemia. Baillieres Clin 11q23-q24. Genomics. 1991;11:223-224. Haematol. 1994;7:235-262. 41. Kourlas PJ, Strout MP, Becknell B, et al. Identification of a 24. Annino L, Vegna ML, Camera A, and the GIMEMA Group. gene at 11q23 encoding a guanine nucleotide exchange Treatment of adult acute lymphoblastic leukemia (ALL): factor: evidence for its fusion with MLL in acute myeloid long-term follow-up of the GIMEMA ALL 0288 randomized leukemia. Proc Natl Acad Sci U S A. 2000;97:2145-2150. study. Blood. 2002;99:863-871. 42. Kawasaki K, Suehiro Y, Umayahara K, et al. 11q23-24 loss is 25. Del Principe MI, Del Poeta G, Maurillo L, et al. P- associated with chromosomal instability in endometrial glycoprotein and bcl-2 levels predict outcome in adult acute cancer. Int J Mol Med. 2003;12:727-731. lymphoblastic leukaemia. Br J Haematol. 2003;121:730-738. 43. Martin ES, Cesari R, Pentimalli F, et al. The BCSC-1 locus at 26. Le Beau Michelle M. Cytogenetic Analysis of Hematologic chromosome 11q23-q24 is a candidate tumor suppressor gene. Malignancies. Pacific Palisades, CA: Association of Proc Natl Acad Sci U S A. 2003;100:11517-11522. Cytogenetics Technologists; 1984. 44. Katoh M, Katoh M. Identification and characterization of 27. Mitelman F, ed. ISCN 1995: An International System for TPARM gene in silico. Int J Oncol. 2003;23:1213-1217. Human Cytogenetic Nomenclature 1995. Basel, Switzerland: S Karger; 1995. 45. Katoh M, Katoh M. KIAA1735 gene on human chromosome 11q23.1 encodes a novel protein with myosine-tail 28. Dyson MJ, Talley PJ, Reilly JT, et al. Detection of cryptic homologous domain and C-terminal DIX domain. Int J Oncol. MLL insertions using a commercial dual-color fluorescence in 2003;23:145-150. situ hybridization probe. Cancer Genet Cytogenet. 2003;147:81-83. 46. Gentile M, Ahnstrom M, Schon F, et al. Candidate tumour suppressor genes at 11q23-q24 in breast cancer: evidence of 29. Fleischman EW, Reshmi S, Frenkel MA, et al. MLL is alterations in PIG8, a gene involved in p53-induced involved in a t(2;11)(p21;q23) in a patient with acute apoptosis. Oncogene. 2001;20:7753-7760. myeloblastic leukemia. Genes Chromosomes Cancer. 1999;24:151-155. 47. Zhang FF, Murata-Collins JL, Gaytan P, et al. Twenty-four- color spectral karyotyping reveals chromosome abnormalities 30. Cox MC, Panetta P, Venditti A, et al. Fluorescence in situ in cytogenetically normal acute myeloid leukemia. Genes hybridization and conventional cytogenetics for the diagnosis Chromosomes Cancer. 2000;28:318-328. of 11q23+/MLL+ translocation in leukaemia. Br J Haematol. 2003;121:953-955. 48. Van Limbergen H, Poppe B, Michaux L, et al. Identification of cytogenetic subclasses and recurring chromosomal 31. Rowley JD, Reshmi S, Sobulo O, et al. All patients with the aberrations in AML and MDS with complex karyotypes using t(11;16)(q23;p13.3) that involves MLL and CBP have M-FISH. Genes Chromosomes Cancer. 2002;33:60-72. treatment-related hematologic disorders. Blood. 1997;90:535-541. 49. Andersson A, Hoglund M, Johansson B, et al. Paired 32. Hayette S, Tigaud I, Vanier A, et al. AF15q14, a novel multiplex reverse-transcriptase polymerase chain reaction partner gene fused to the MLL gene in an acute myeloid (PMRT-PCR) analysis as a rapid and accurate diagnostic tool leukaemia with a t(11;15)(q23;q14). Oncogene. for the detection of MLL fusion genes in hematologic 2000;19:4446-4450. malignancies. Leukemia. 2001;15:1293-1300. 33. Dolan M, McGlennen RC, Hirsch B. MLL amplification in myeloid malignancies: clinical, molecular, and cytogenetic findings. Cancer Genet Cytogenet. 2002;134:93-101.

306 Am J Clin Pathol 2004;122:298-306 306 DOI: 10.1309/RX27R8GJQM330C22