Chromosomal Instability in Chromosome Band 12P13

Leukemia (2001) 15, 1193–1202  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Chromosomal instability in chromosome band 12p13: multiple breaks leading to complex rearrangements including cytogenetically undetectable sub-clones Y Sato1, H Kobayashi2, Y Suto3, HJ Olney4, EM Davis4, H Gill Super4, R Espinosa III4, MM Le Beau4 and JD Rowley4

1Division of Molecular Cytogenetics, Department of Clinical Pathology, Research Institute of International Medical Center of Japan, Tokyo; 2Hematologic Clinic, Saitama Cancer Center Hospital, Saitama; 3Department of Pediatrics, Aomori Municipal Hospital, Aomori, Japan; and 4University of Chicago, Department of Medicine, Section of Hematology/Oncology, Chicago, IL, USA

During fluorescence in situ hybridization (FISH) analysis of 12p12–13 are also detected in non-small cell lung cancer, metaphase cells from 70 patients with lymphoid and myeloid breast cancer, and ovarian cancer, where the common hematologic malignancies and chromosomal rearrangements involving band 12p13, we identified nine patients (four with deleted region again includes the TEL to KIP1 interval. In an lymphoid malignancies, four with myeloid malignancies and attempt to clone potential tumor suppressor genes (TSGs) on one with biphenotypic leukemia) who showed more compli- this region, Baens et al20 further refined the common deleted cated rearrangements than we had expected from conventional segment to 600 kb between TEL and D12S358, excluding cytogenetic study. In six patients, multiple breaks occurred in KIP1. small segments of 12p with subsequent translocations and While examining the breakpoint in 23 patients with bal- insertions of these segments into other chromosomes, some- 21 times to unexpected regions. In three patients additional chro- anced translocations involving 12p13 and defining the com- mosome breaks resulted in a sub-clone which was cytogenet- monly deleted region in 47 patients with unbalanced translo- ically indistinguishable from the main clone in each patient cations or deletions involving band 12p13 with FISH,19 we based on the cytogenetic analysis. These subtle molecular found nine patients with multiple chromosomal breaks in 12p. events were detected exclusively in a region covering Similar molecular events have been reported in a few TEL/ETV6 and KIP1/CDKN1B. Seven of nine had a previous his- patients.22,23 tory of chemo/radiotherapy; all the patients showed complex karyotypes, even though they were newly diagnosed with leu- In this report, we tried to delineate clinical and cytogenetic kemia. Survival data were available in five patients, and all sur- characteristics of these patients in comparison with previously vived less than 6 months. These findings suggest that the reported patients. Whereas such events have been reported 12p13 region, especially the above-mentioned region, is geneti- only in myeloid malignancies so far, our patient series cally unstable and fragile. It is likely that multiple chromosome included four lymphoid malignancies, four myeloid malig- breaks were induced through mutagens used in chemo/ nancies and one biphenotypic leukemia. In six patients, these radiotherapy, and are associated with a sub-group of patients with an extremely bad prognosis. Leukemia (2001) 15, 1193– multiple chromosomal breaks resulted in a deletion of a small 1202. segment and/or translocation of this segment to another chro- Keywords: 12p13 rearrangements; hematologic malignancies; mosome, sometimes to unexpected regions. In three patients, FISH; chromosomal instability; TEL/ETV6; KIP1/CDKN1B additional chromosome breaks occurred in some of the cells; the karyotype of the sub-clone was indistinguishable at the standard chromosome analysis level from that of the main Introduction clone in each patient. These molecular events were exclus- ively found in a region covering TEL and KIP1, whereas others Rearrangements of the short arm of chromosome 12 (12p) were found in the telomeric region to TEL.22 These findings have been found in a wide spectrum of hematologic malig- suggest that this region is genetically unstable and fragile, nancies and solid tumors.1–5 Although each band of 12p leading to these complex genetic events. All the patients had a (12p11, 12p12, and 12p13) is rearranged in various types of complex karyotype, even though they were newly diagnosed. abnormalities including balanced or unbalanced trans- Survival data were available in five, and all survived less than locations, insertions, inversions and deletions, rearrangements 6 months. Thus, patients with multiple chromosomal breaks of band 12p13 are most frequently observed.6 TEL/ETV6 and in this region have an extremely bad prognosis. KIP1/CDKN1B are located in this band. TEL is a transcription factor identified by Golub et al7 as a partner gene of PDGFRB in a patient with a t(5;12)(q33;p13). Since then, TEL has been Materials and methods reported to fuse to several tyrosine kinases (ABL,8,9 ARG,10 JAK211,12 and TRKC13–16), a transcription factor (AML117) and Patients unrelated transcription factors. To date, 41 chromosomal bands have been demonstrated to be involved in TEL translo- Patients were referred to the Section of Hematology/Oncology cations.18 at the University of Chicago for chromosome study from 1970 Previously, we reported that the smallest commonly deleted to 1994. Selection of patients was based on diagnosis of a region found in hematologic malignancies with 12p12–13 hematologic malignancy and the presence of 12p12–13 deletions was flanked by TEL at the telomeric side and KIP1 rearrangements in the karyotype. Seventy patients were selec- at the centromeric side.19 Moreover, deletions of bands ted (47 patients with unbalanced translocations or deletions19 and 23 patients with balanced translocations21). These cases included 20 lymphoid malignancies (14 acute lymphocytic leukemia (ALL), one chronic lymphocytic leukemia (CLL) and Correspondence: Y Sato, Division of Molecular Cytogenetics, Depart- five non-Hodgkin’s lymphoma (NHL)), 49 myeloid malig- ment of Clinical Pathology, Research Institute, International Medical Center of Japan, Toyama 1-21-1, Shinjuku-Ku, Tokyo, 162-0052, nancies (four acute leukemia (AL) or acute undifferentiated Japan; Fax: 81-3-5273-8602 leukemia (AUL), 16 acute myelogenous leukemia (AML), five Received 18 January 2001; accepted 17 April 2001 therapy-related AML (t-AML), four chronic myelogenous leu- Chromosomal instability in band 12p13 Y Sato et al 1194 kemia (CML), three myelofibrosis (MF), one polycythemia vera FISH study (PV), 13 myelodysplastic syndrome (MDS), and three t-MDS), and one biphenotypic leukemia. Patient material submitted for The probes were labeled with biotin-11-dUTP or digoxigenin- cytogenetic analysis during this period had the following diag- 11-dUTP using nick translation and were hybridized to chro- nostic breakdown: 918 ALL, 202 CLL/prolymphocytic leuke- mosome preparations as previously described.28 Chromo- mia, 107 hairy cell leukemia, 1566 NHL, 147 unclasified or somal bands were identified using counterstaining with 4Ј 6- biphenotypic leukemia, 2117 AML, 1605 myeloproliferative diamindino-2-phenylindole dihydrochloride (DAPI). The pres- disorders (MPD) (including 880 CML, 143 FM, 185 PV, 58 ence or absence of the FISH signals was scored on an average essential thrombocythemia and 347 other/unclassified MPD), of 10.7 abnormal metaphase cells (range 3–20) per probe per 1454 MDS, and 496 t-AML/MDS cases. All t-AML/MDS patient by two persons blinded to the identity of probes and patients are double counted also being included in either the patients. Images of the hybridizations were captured with a AML or MDS categories; 118 patients had both AML and MDS liquid-cooled, charge-coupled device camera (Photometrics, diagnoses and eight MPD patients have more than one MPD Tucson, AZ, USA). Separate gray-scale images for the DAPI diagnosis included at some point in laboratory records. and the fluorescein isothiocyanate (FITC) or rhodamine fluor- The disease stage when the samples were obtained was as ochromes were acquired. After adjusting the gray levels with follows: among 20 lymphoid malignancies, 14 cases were at the National Institutes of Health (NIH, Bethesda, MD, USA) diagnosis and six at relapse, whereas among 49 myeloid image 1.61 software, the images were merged using Adobe malignancies, 33 were at diagnosis and 16 at relapse. While Photoshop (Adobe System, San Jose, CA, USA) on a Macintosh performing FISH analysis, we identified nine patients who computer (Apple Computer, Cupertino, CA, USA). showed multiple chromosome breaks in 12p13. There were four males and five females aged between 2 and 81. Three were diagnosed as having ALL, three had MDS, and one each Results had NHL, t-AML and biphenotypic leukemia when the chromosomal samples were obtained (Table 1). All of the All the patients showed complex karyotypes patient samples were obtained with informed consent. Clinical data including previous history, treatment and sur- vival and cytogenetic findings of the nine patients are summa- rized in Table 1. All patients were studied a long time ago, Cytogenetic analysis and thus some clinical data were impossible to collect. Two (patients 1 and 9) had newly diagnosed leukemia and for Metaphase cells were prepared for cytogenetic study as pre- whom a previous history of environmental exposure was viously described.24 The karyotypes were described according unknown: the remaining patients had a history of treatment for to the International System for Human Cytogenetic Nomencla- an original leukemia or previous malignancies. Complicated ture (ISCN 1995).25 In the initial cytogenetic study, four chromosomal abnormalities were observed in all the patients (patients 1, 2, 7 and 9) had a balanced translocation, four including two patients with newly diagnosed leukemia. Four (patients 3 to 6) an unbalanced translocation, and one (patient (patients 3, 5, 6 and 8) showed −5/5q− and/or −7/7q− which 8) an interstitial deletion. Patient 9 had a t(12;22) translocation are thought to be associated with a history of previous and two copies of a deletion 12p. chemo/radiotherapy or environmental mutagen exposure. Sur- vival from the present disease was less than 6 months in all the patients for whom data were available. Unfortunately, survival in two newly diagnosed leukemia patients was not FISH probes available.

We used 20 probes for the FISH study: 11 cosmid probes (D12S235, D12S237, D12S134, D12S229, HTY3049c1–7, Three patients had a sub-clone with additional D12S133, D12S142, D12S119, D12S140, 15a4 (TEL ex.1) chromosomal breaks but with no detectable change in and 95h4 (TEL ex.2)); three YAC probes (YAC961a6, the karyotype from that of the main clone YAC771h4 and YAC964c10); a cosmid contig probe (TEL ex.3), a P1 phage contig probe (P27KIP1),26 three phage probes FISH results of three patients (patients 1 to 3) are summarized (D12S54, GDI·D4 and CCND2) and a plasmid probe D12S20. in Table 2. Representative karyotypes with the FISH signals These probes were mapped on 12p13.3 to 12p12.1 and were are shown in Figure 1A to F. ordered as previously described.19,21,27 The YAC 964c10 con- tains the entire TEL gene and has an internal deletion which includes KIP1/CDKIB gene. The P27KIP1 probe includes the Patient 1: The FISH signals for the telomeric probes, complete KIP1/CDKIB gene. The location of these probes is D12S237 and D12S134, were found on the der(22) in all the shown in Tables 2 and 3. cells analyzed and most of the signals for the more centro- We used chromosome painting probes and centromere-spe- meric probes, from YAC961a6 to P27KIP1, were found on the cific probes (Vysis, Naperville, IL, USA) to clarify the origin der(12), suggesting that the breakpoint in the t(12;22) is of the marker chromosomes. To clarify the translocation of the between D12S134 and YAC961a. However, with the short arm of chromosome 9 in patient 8, we used the C55B, D12S133 probe, the signals were found on der(22) in three C173 and C262 cosmid probes (location: 9p22) which were cells (Figure 1A), whereas they were seen on the der(12) isolated from the chromosome 9 cosmid library derived from (Figure 1B) in nine cells on the same slide. This indicates exist- the cell line J64051 (a gift from Oncor, Gaithersburg, MD, ence of a sub-clone with two more chromosomal breaks USA) by one of us (HGS). (between HTY3049c1–7 and D12S133, and between

Leukemia Chromosomal instability in band 12p13 Y Sato et al 1195

h 1mo 2mo Ͻ Ͻ is not known. Patient 9 is patient 11 ,-13, ,-17,-18, 6 mo 19 . ,-17,-18, omplicated rearrangements in et al e diagnosis was changed after mar[13]/ lost der(?)t(?;6)(?;p21), mar2[3]/46,XX[6] mar3[3]/46,XX[6] mar2[10]/45, + + + + 18[8]/unrelated 1 mo , + + [17]/46,XX[7] lost del(12)(p11p13) ,-22, 16,add(16)(q24), mar1, mar2, ),del(3)(p14p23);-4,del(5) lost syndrome in refractory anemia with excess mar1, + + + + , p13 der(12)t(10;12)(q11;p13) ,add(18)(q23)[21] lost )(q37; t(9;12;mar1)(p2?2;p13;q?) 12 12,add(14)(q32), ,-5,-10, + , mar[14]/46,XX[6] 4 mo t(12;22)(p13;q11) + 8,add(8)(p21),add(11)(p1?3), mar[6]/46,XY[3] mar1[4]/46,idem, mar2[4]/46,idem, + + + + 18, Karyotype Survival + 16, + , ,t(8;11), 10[2]46,XY,idem,add(13)(p11),add(22)(q13),r(22), der(18)t(12;18)(p13;p11) t(12;22)(p13;q11) + t(2;12)(p11;p13) r[7]/45,idem, r[7]/45,idem, ,-17, ,mar3[3]/43,idem,add(8)(p2?3), del(12)(p11p13),t(12;22)(p13;q13) + + + + der(12)del(12)(p11p13)t(2;12)(q37;p13) : 44,XX,del(5)(q22q35),-7,add(9)(p2?2), : 44,XX,del(5)(q22q35),-7, mar2, + t(5;12)(q?15;p13) mar1[2]/46,idem, mar1, . + + ␣ Y,t(1;14)(q21;q32), 8[3]/45,X,-Y,add(2)(q3?4),-5,-7, + del(12)(p11p13), der(12)t(10;12)(q11;p13) + der(2)del(2)(q21q31)t(2;12)(q37;p13) respectively. mar1[2] mar2[3]/46,XY[11] der(19)t(7;19)(p12;p13), revised karyotype der(19)t(7;19)(p12;p13), + + der(12)t(12;17)(p13;q11) idem,add(10)(p13),-add(11),-mar2[5] -1721,-22, -mar3[13]/46,XX[3] original karyotype 29 ,

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Patients 4–9 are patientset 27, al 28, 37, 31, 34, and 32, respectively, in Sato 21 . BM 46,XX,t(5;8;14)(q11;q24;q32),

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(duration) 3 yr) 47,XY,del(6), Ͼ −− CLL, colon Ca(10 yr 9 10 courses mo) (11 yr) clones[2]/46,XY[8] (q13q34 or q35),-6,-7, (4 yr) (

c

b

disease

the present malignancy , no previous chemotherapy; COPP, cyclophosphamide, vincristin, procarbazine, prednisolone; chemo, chemotherapy was done, although the content and patients 3, 5 and 6 are patients 1, 3, and 2 in Kobayashi − leukemia 27 ,

et al Clinical and cytogenetic findings of nine patients with hematologic malignancies who had multiple chromosomal breaks resulting in the formation of c

Age/Sex Diagnosis of Stage

a Diagnosis: NHL-DL, non-Hodgkin’s lymphoma-diffuse large cell; t-AML, therapy-related acute myeloblastic leukemia; MDS-RAEB, myelodysplastic Previous malignancy: NHL-DI, NHL-diffuse intermediate cells. Source: BM, bone marrow; LN, lymph node; BC, bone core. Patients 1, 2, 8 and 9 are patients 19, 20, 10 and 21, respectively, in Sato Stage: Dx, diagnosis; 2nd RL, second relapse. Duration shows the period from the time the first malignancy occurred to theSurvival: time duration the from present the malignancy time occurred. the present disease occurred to the time of death. Previous treatment: Table 1 the band 12p13 1 60F ALL-L2 Dx a b c d e f g h rad, irradiation; NA, information is not available; HD-Ara C, high-dose cytarabine; INF blasts; t-MDS-RAEBT, t-MDSre-review. in RAEB in transformation. Patients 8 and 9 were previously reported to have AML-M6 and ALL-L3, respectively, however, th in Kobayashi 2 74M NHL-DL Dx NHL-DI, COPPx LN 49,XY, 3 79M t-AML Dx ANLL, NHL chemo, rad BM 45,XY,der(2)add(2)(p13)del(2)(q2?3q3?5)t(2; 9 21F Biphenotypic Dx – – BM 47,XX, 4 2F ALL 2nd RL ALL chemo BM 49,XX, 5 68M MDS-RAEB Dx NA NA BM 47,XY, 6 81F MDS-RAEB Dx NA NA BM 43,XX, 7 15M ALL 2nd RL ALL chemo BM 46,XY,del(6)(q1?5q2?3),t(8;11)(q24;p13), Patient 8 53F t-MDS-RAEBT Dx NA HD-Ara C, BC

Leukemia Chromosomal instability in band 12p13 Y Sato et al 1196 Table 2 Results of FISH study using 12p12 to p13 probes in three patients who showed a subclone with different breakpoints but the same karyotype

Tel Location Patient 3 der(2) add(2)(p13)del(2) (q2?313?5)t(2;12)(q37;p13) Patient 1 Patient 2 and der(12)del(12)(p11p13) t(12;22)(p13;q11) t(2;12)(p11;p13) t(2;12)(q37;p13)

Main clone No. of Main clone No. of Main clone No. of cells cells cells analyzed analyzed analyzed

D12S235 p13.3 der(2) 13 D12S237 p13.3 der(22) 13 der(2) 14 D12S134 p13.3 der(22) 15 der(2) 12 der(2) 10 →→ 10 YAC961a6 p13.3 der(12) der(12) 11 YAC771h4 p13.3 der(12) 11 der(12) 7 HTY3049 sub-clone 20 c1–7 p13.3 der(12) → der(12) 15 der(2) 2 18

sub-clone der(2) D12S133 p13.1 → der(12) → der(12) der(2) 10 der(22) der(12) 3 9 12 5 914 YAC964 p13.1 → der(12) 15 der(2) 8 18 c10 der(12) 15a4 p13.1 (TEL ex.1) der(12) 15 U 95h4 p13.1 (TEL ex.2) der(12) 14 U sub-clone TEL ex.3 p13.1 der(12) 15 der(12) 15 → der(12) der(2) 5 510 p27KIP1 p13.1 der(12) 15 der(12) 15 der(12) der(2) 2 57 → D12S142 p12.3 deleted 13 D12S119 p12.3 deleted 10 GDI·D4 p12.3 deleted 8 D12S140 p12.2–12.1 deleted 8 D12S20 p12.2–12.1 deleted 10

Bold letters indicate the sub-clone found on the same slide in each patient. U: unsuccessful hybridization.

D12S133 and YAC964c10), resulting in translocation of a D12S133), resulting in translocation of a small region includ- small region including D12S133 to the der(22). ing HTY3049c1–7 and D12S133 to the der(2).

Patient 2: The D12S237 and D12S134 probes hybridized Patient 3: The signals of D12S235 to YAC964c10 probes to the der(2) in all the cells analyzed and most of the signals were found on the der(2) in all the cells analyzed and signals for YAC961a6 to P27KIP1 probes were found on the der(12), of D12S142 to D12S20 were not detected, indicating that this suggesting that the breakpoint of t(2;12) is between D12S134 region was deleted. However, the signals of the TEL ex.3 and and YAC961a. However, with the HTY3049c1–7 probe, the P27KIP1 probes were found on the der(12) in five cells (Figure signals were found on the der(2) in two cells, and with the 1E) and two cells, respectively. With the same probe on the D12S133 probe, they were split between the der(2) and the same slide, the signals were found on the der(2), in five cells der(12) in five cells (Figure 1C). With the same probes on the (Figure 1F). This indicates that the sub-clone has an additional same slide, the signals were found on the der(12) in the break in the telomeric region of TEL ex.3 with the retention remaining cells analyzed, in 18 cells with HTY3049c1–7 and of the region including TEL ex.3 and P27KIP1 on the der(12). in nine cells with D12S133 (Figure 1D). This indicates the The signals of 12p probes were always found on the normal existence of a sub-clone which has two additional chromo- 12p in these three patients. On re-review of the G-banded somal breaks (between D12S229 and HTY3049c1–7, and in metaphase cells from these patients, we could not detect any

Leukemia Chromosomal instability in band 12p13 Y Sato et al 1197 del(12) a → — der(12) der(22)der(22) del(12) der(12) del(12) der(22) del(12) der(12) del(12) der(12) deleted der(12) deleted formation of complicated → → mar1 mar1 mar1 mar1 der(12) der(12) der(22) der(12) →→→ →→→→ 21 mar1 21 mar der(9) mar der(12) der(12) deleted deleted deleted der(12) deleted →→ and deleted cen of der(2) pter of der(12)

(p13;p11) t(2;12)(q37;p13)

der(18)t(12;18) der(2)del(2)(q11q31) →→ →→→ →→ →→ mar 2qter

(q11;p13) (p11;q11) and t(10;12)(q11;p13) and (p13;q11) (p13;p2?2;q?) del(12)(p11p13)x2 12.2 12.2 deleted 12.2 der(12) deleted deleted der(12) der(12) deleted – – – p13.3 der(18) p13.1 p13.1p12.3 deleted deleted 8) – Results of FISH analysis using 12p12 to p13 probes in six patients with hematologic malignancies who had multiple chromosomal breaks resulting in the ) indicate the points where chromosomal breaks occurred. → ex.1) ex.2) ex.3 7 p13.3 – ex.3 p13.1 deletedD4 deleted nd

KIP1 · TEL TEL TEL TEL ( c1 c10 p13.1 ( ( Only one metaphase seen; signal on normal 12 but not on der(12) or der(22). 15a4 p13.1 D12S229HTY3049 D12S133 p13.3YAC964 p13.1 deleted deleted der(18)D12S142D12S119D12S54 der(2) p12.3 p12.3 p12.1 der(12) der(12) mar mar deleted deleted deleted der(12) deleted der(22) del(12) del(12) der(12) der(12) der(12) deleted deleted Table 3 rearrangements in the band 12p13 D12S235D12S237D12S134 p13.3 p13.3 p13.3 deleted deleted der(18) der(18) der(2) der(2) mar marArrows ( a der(22) der(22) del(12) del(12) P1 95h4 p13.1 P27 D12S140 p12.1 In each case, all of the signals were found on the normal 12p. D12S20 p12.1 Probeorder LocationCCND2 Patient 4 der(12)t(10;12) Patient 5 der(12)t(12;17) der(12) Patient 6TEL t(12;22) Patient 7 t(12;9;mar1)GDI Patient 8 t(12;22)(p13;q13) and CEP12 Patient 9 der(12) der(12) der(12) der(12) del(12) der(12) del(12)

Leukemia Chromosomal instability in band 12p13 Y Sato et al 1198

Figure 2 Representative cells with FISH signals of patients 4 to 9. The signals for each probe were always observed on normal 12p. Figure 1 Representative cells with FISH signals of patients 1 to 3. Patient 4: (A) The signal of the YAC964c10 probe was seen on the The left column (A, C and E) shows the sub-clone, and the right col- marker chromosome. Patient 5: (B) The signal of the YAC964c10 umn (B, D and F) shows the main clone in each patient. The signals probe was seen on the terminus of the long arm of the normal appear- for each probe were always observed on the normal 12p. Patient 1: ing chromosome No. 2 (indicated as ‘der(2)’). Patient 6: (C) The signal With D12S133 probe, FISH signals were seen on the der(22) in three of the P27KIP1 probe was seen on the terminus of the short arm of the cells (A), but were also found on the der(12) in nine cells (B) on the der(12) and in the vicinity of the centromere of the der(2). Patient 7: same slide. Patient 2: With D12S133 probe, FISH signals were seen (D) The signal of the YAC964c10 probe was seen on the marker and to be split between the der(2) and the der(12) in five cells (C), but normal appearing No.21 (indicated as ‘der(21)’). Patient 8: (E) The were also found only on the der(12) in nine cells (D) on the same KIP1 signal of the 964c10 probe was seen on the add(9), the marker chro- slide. Patient 3: With P27 probe, FISH signals were seen on the mosome 1 and del(12). Patient 9: (F) The signal of the TEL ex.3 probe der(12) in two cells (E), but were also found on the der(2) in five cells was split between the der(12) and der(22). (F) on the same slide. the der(12), suggesting that the 12p13.1 to 12p13.3 region was deleted. However, the YAC964c10 probe hybridized to difference in the karyotype between the each main clone and the end of the long arm (qter) of the marker chromosome the sub-clone. (Figure 2A). We could not identify the origin of this marker chromosome due to poor quality and paucity of metaphase cells. Thus, 12p13.3 to 12p13.1 was deleted from the der(12), Six patients showing multiple chromosomal breaks whereas a small region including YAC 964c10 was translo- resulting in complex rearrangements cated to the qter of the marker chromosome (Figure 3A). FISH results of six patients (patients 4 to 9) are summarized in Table 3. Representative karyotypes with the FISH signals Patient 5: D12S235 to D12S133 hybridized to the der(18), are shown in Figure 2A to F. A schematic diagram illustrates whereas the signals of most probes in the region of 12p13.1– how these complex translocations or deletions in the 12p13 12p12.1 except for the YAC 964C10 probe were deleted. The region could occur (Figure 3A to F). YAC probe unexpectedly labeled the qter of a normal appear- ing chromosome 2 (indicated as ‘der(2)’ in Figure 2B), indicat- ing that a small segment including the YAC964c10 was trans- Patient 4: Except for YAC 964c10, none of the signals of located to chromosome 2 (Figure 3B). the telomeric probes (D12S235 to P27KIP1 ) were found on the der(12) or on any other chromosome, whereas the signals of the centromeric probes (D12S142 to D12S20) were found on Patient 6: 12S235, D12S134 and D12S133 hybridized to

Leukemia Chromosomal instability in band 12p13 Y Sato et al 1199

Figure 3 (A–F) The diagram explains how the translocations or deletions might have occurred in the 12p13 region in patients 4 to 9. the der(2) (qter) whereas the YAC 964C10, D12S142, to the chromosome 21, whereas the distal part of the D12S119 and D12S20 probes were deleted. Unexpectedly, YAC964c10 was translocated to the marker chromosome. the P27KIP1 probe hybridized to the short arm of the der(12) Moreover, the signals of the P27KIP1 probe hybridized only to and near to the centromere of the der(2) (Figure 2C). These the normal 12p, indicating that this segment was deleted from findings showed that the telomeric part of 12p13 was involved the der(12) (Figure 3D). Despite our efforts, we could not in the t(2;12)(q37;p13) translocation and that a small segment identify the origin of the marker chromosome, only confirming including the KIP1 gene was split into two parts, being translo- that it was not chromosomes 21 or 22. cated to the end of the short arm of the der(12) and near the centromere of the der(2) (Figure 3C). Patient 8: The telomeric probes D12S235 to D12S133 were not used because of lack of material. The YAC964c10 Patient 7: The signals of all the probes on 12p13.3 and probe hybridized to the der(9), the marker chromosome 1 D12S133 were found on the marker chromosome, whereas (mar1) and the der(12) (Figure 2E), whereas TEL ex.1 and TEL signals of the 12p12.3–12p12.1 region probes were found on ex.2 probes hybridized only to mar1. Signals of the TEL ex.3 the der(12). The signals of the YAC964c10 and the TEL ex.3 and P1 TEL probes were found on mar1 and the der(12). These probe were found on the marker and normal appearing chro- results suggest that a break occurred within TEL exon 3, and mosome 21 (indicated as ‘der(21)’ in Figure 2D), indicating the telomeric portion was translocated to mar1 with the cen- that the chromosomal break occurred within the TEL gene, tromeric portion remaining on the der(12). Another chromo- and a small segment including exon 3 of TEL was translocated somal break seemed to occur within the YAC964c10 probe

Leukemia Chromosomal instability in band 12p13 Y Sato et al 1200 and the telomeric portion of this probe was translocated to 49 myeloid malignancies), although we do not know the his- the der(9). Moreover, the P27KIP1 probe labeled only the nor- tory of potential environmental exposure in each group. mal 12p, indicating that this segment was deleted from the In our series, five patients (patients 2–4, 7, 8) obviously had der(12). We were not able to determine the extent of this cen- a previous history of chemo/radiotherapy, although two tromeric deletion on the der(12). With the chromosome 12 (patients 1 and 9) did not, and in the remaining two (patients painting probe (WCP12, Vysis), we confirmed that chromo- 5 and 6), we had no informative data. Nevertheless, all the some 12 was on pter of the der(9) chromosome. With the patients including the new leukemia patients (patients 1 and cosmid probes located on 9p22 (C55B, C173 and C262), we 9) showed a complex karyotype which is thought to be asso- demonstrated that the 9p22 region was translocated to the qter ciated with poor prognosis, especially when −5/5q− and/or of mar1. Therefore, this patient appears to have had a three- −7/7q− are present.30 Actually, four patients (patients 3, 5, 6 way translocation involving 9p, 12p and the long arm of the and 8) showed −5/5q− and/or −7/7q−, and two of them mar1 (Figure 3E); we do not know whether it was a stepwise (patients 3 and 8) had a previous history of chemo/ or simultaneous three-way translocation. We tried to identify radiotherapy. As other investigators have reported,22,23 prog- this marker chromosome with painting probes but we could nosis in our patients was also extremely bad among those for only confirm that it was not chromosome 7 or the X chromo- whom data were available (patients 2, 4–6, 8). We do not some. know which chemotherapeutic reagent is most closely asso- ciated with inducing multiple chromosome breaks. However, taken together, it seems that multiple chromosome breaks are Patient 9: Probes including D12S235 to HTY3049c1–7 all associated with a bad prognosis. labeled the der(22), whereas D12S133, YAC964c10, P27KIP1 The sub-clone found in the first three patients appears to be and 12p12.3 to 12p12.1 region probes remained on the derived from the main clone. It is likely that after additional der(12). Only the TEL ex.3 probe signal was split between the chromosome breaks occurred, some genetic material was der(12) and der(22) (Figure 2F). These findings suggest that a translocated from the original site to the other site in the sub- chromosomal break occurred in the TEL ex.3 probe, and an clone, although this genetic event was too subtle to be undefined segment of TEL was translocated to the der(22) detected by standard cytogenetic analysis. In such a sub- clone, an additional breakpoint might involve a gene which (Figure 3F). Probes including D12S235 to TEL ex.3 labeled 21 both del(12), and probes including P27KIP1 to D12D20 were is broken and fused to another gene. In our previous paper, deleted. We did not detect any difference between two copies we suggested that in the telomeric region to TEL, there were of del(12) as to deleted region. at least three small breakpoint cluster regions (sbcr) among The FISH signals for each probe were always found on the the 12p13 translocations where unidentified genes might be normal 12p in metaphase cells in all six patients. located. The breakpoint of the sub-clone in patient 1 is located in one of the sbcr (between D12S133 and HTY3049c1–7), although those of the sub-clone of the remaining patients are not. So far, only Wlodarska et al23 have Discussion reported one patient to have such a sub-clone. Their patient 14 had two clones; one with a del(12p) telomeric to KRAS In this report, we describe nine patients: three with a sub- except for retention of VWF, and the other with the del(12p) clone having an additional translocation or insertion, and six involved in a translocation with 3q. This observation shows with more complicated and cryptic chromosomal rearrange- that a critical genetic event can occur in the leukemic clone ments including multiple chromosome breaks and non-con- without detectable karyotypic change. Of interest is that tiguous deletions which were not expected from standard patient 1 had newly diagnosed leukemia. This suggests that chromosome analysis. Some data for patients 1, 2, 8 and 9 some patients could have such a sub-clone from the beginning have been described elsewhere21 (see footnote in Table 1). of the disease. Clinical relevance of this observation is FISH studies of patients 3, 5 and 6 were performed pre- important, because this could explain the fact that during the viously.29 However, the findings presented here were not clinical course of leukemia, the response to chemotherapy identified, because the probes between D12S142 and may have altered although the observed karyotype is not D12S133 (P27KIP1,P1TEL, TEL ex.3, 95h4, 15a4, changed. YAC964c10) were not used at that time. In six patients, it is likely that multiple chromosome breaks We found these nine patients among 70 patients (9/70 = occurred followed by translocation and/or deletion of a small 12.9%) with 12p13 rearrangements, four in 20 lymphoid segment. This small chromosome fragment was sometimes malignancies, four in 49 myeloid malignancies and one in one translocated to the partner derivative chromosome involved biphenotypic leukemia. Although other investigators have in the original translocation, and sometimes to an unexpected reported similar complicated molecular events including non- chromosomal region as shown in Figure 3. Similar molecular contiguous segments,22,23 all of the cases were myeloid malig- events were reported in three myeloid leukemia patients by nancies. Thus, this is the first report that identified such events La Starza et al:22 in their patient 1(AML) with add(12p), FISH in lymphoid malignancies. Interestingly, it is likely that the signals with three probes telomeric to TEL were found not only frequency of cases with such events is much higher in lymph- on add(12p) but also on the marker chromosome (mar)-2; in oid malignancies (4/20 = 20.0%) than in myeloid malig- patient 2 (AML-M1) with ring(12), FISH signals of 12p probes nancies (4/49 = 8.2%). One may think that this higher fre- were separately found on ring(12), add(4) and der(17); and quency is associated with the higher rate of mutagen-exposed in patient 3 (t-AML-M2) with der(12)t(12;14)(p13;q11), FISH patients in the lymphoid malignancy group. However, when signals with two probes centromeric to TEL were unexpec- we compared the ratio of patients at diagnosis and at relapse tedly found on a mar. In their three patients, multiple chromo- between lymphoid and myeloid malignancy groups, the ratio some breaks occurred in the telomeric region to TEL, and in is not significantly different (14 at diagnosis/six at relapse in patients 2 and 3, the TEL region was deleted. However, in our 20 lymphoid malignancies, vs 33 at diagnosis/16 at relapse in patients, multiple chromosome breaks occurred exclusively in

Leukemia Chromosomal instability in band 12p13 Y Sato et al 1201 a region covering TEL and KIP1 as shown in patients 4–9. To References determine the precise breakpoint is somewhat difficult in some patients (patients 4–6), because they had a large deleted 1 Raimondi SC, Shurtleff SA, Downing JR, Rubnitz J, Mathew S, region with some retaining region within it as shown in Figure Hancock M, Pui CH, Rivera GK, Grosveld GC, Behm FG. 12p 3. However, in patients 7–9, the breakpoint should be within abnormalities and the TEL gene (ETV6) in childhood acute lym- TEL ex.3 probe, although we21 and others22 previously phoblastic leukemia. Blood 1997; 90: 4559–4566. reported that the 12p13 translocation breakpoints were 2 Takeuchi S, Seriu T, Bartram CR, Golub TR, Reiter A, Miyoshi I, frequently found in the telomeric region to TEL. Gilliland DG, Koeffler HP. TEL is one of the targets for deletion on 12p in many cases of childhood B-lineage acute lymphoblastic It is well established that a small segment deletion some- leukemia. Leukemia 1997; 11: 1220–1223. times occurs together with an ‘apparent’ reciprocal translo- 3 Andreasson P, Johansson B, Billstrom R, Garwicz S, Mitelman F, cation, for example, in the 3Ј region of MLL in 11q23 translo- Hoglund M. Fluorescence in situ hybridization analyses of hema- cations31 and in the 3Ј region of MYH11 in 16p13 tologic malignancies reveal frequent cytogenetically unrecognized translocations.32,33 Moreover, recently, a large deletion has 12p rearrangements. Leukemia 1998; 12: 390–400. been reported to frequently occur adjacent to the breakpoint 4 Wlodarska I, La Starza R, Baens M, Dierlamm J, Uyttebroeck A, 34 35 36 Selleslag D, Mecucci C, Hagemeijer A, Van den Berghe H, of t(9;22) and t(8;21) translocations. Tanaka et al Marynen P. Fluorescence in situ hybridization characterization of reported that small chromosomal segments including the ABL new translocation involving TEL (ETV6) in a wide spectrum of gene and/or MLL gene were translocated to multiple other hematologic malignancies. Blood 1998; 91: 1399–1406. chromosomal regions in a single cell, resulting in formation 5 Rowley JD. The role of chromosomal translocations in leukemia. of partial tri-, tetra- or pentasomy of these regions. They called Semin Hematol 1999; 36: 59–72. this ‘segmental jumping translocation’, and assumed that it 6 Mitelman F. Catalog of Chromosome Aberrations in Cancer ‘98. Wiley-Liss: New York, 1998. was one of the mechanisms for gene amplification. However, 7 Golub TR, Barker GF, Lovett M, Gilliland DG. Fusion of PDGF our observation differs from their report, because the small receptor ␤ to a novel ets-like gene, tel, in chronic myelomonocytic segment is not amplified, but rather is translocated or deleted, leukemia with t(5;12) chromosomal translocation. Cell 1994; 77: presumably as an isolated event in a single cell containing a 307–316. non-contiguous small segment in 12p13. On the other hand, 8 Golub TR, Goga A, Barker GF, Afar DE, McLaughlin J, Bohlander Beans et al20 further defined the common deleted segment to SK, Rowley JD, Witte ON, Gilliland DG. Oligomerization of the ABL tyrosine kinase by the Ets protein TEL in human leukemia. 600 kb between TEL and D12S358 at 12p13.3 with FISH Mol Cell Biol 1996; 16: 4107–4116. using contig probes. Unfortunately, we did not study whether 9 Papadopoulos P, Ridge SA, Boucher CA, Stocking C, Wiedemann or not this region was deleted in our patients, because we LM. The novel activation of ABL by fusion to an ets-related gene, lacked the appropriate probes. TEL. Cancer Res 1995; 55: 34–38. The frequent occurrence of multiple chromosome breaks in 10 Iijima Y, Ito T, Oikawa T, Eguchi M, Eguchi-Ishimae M, Kamada a region covering TEL and KIP1 suggests that this region is N, Kishi K, Asano S, Sakaki Y, Sato Y. A new ETV6/TEL partner gene, ARG (ABL-related gene or ABL2), identified in an AML-M3 genetically unstable and fragile. An important question is cell line with a t(1;12)(q25;p13) translocation. Blood 2000; 95: whether these events would occur preferentially in the 12p13 2126–2131. region, or whether the events could happen in any other 11 Peeters P, Raynaud SD, Cools J, Wlodarska I, Grosgeorge J, Philip regions, especially adjacent to the breakpoint of disease- P, Monpoux F, Van Rompaey L, Baens M, Van den Berghe H, specific translocations or deletions, without being detected. Marynen P. Fusion of TEL, the ETS-variant gene 6 (ETV6), to the Extensive FISH studies should be done to answer this question receptor-associated kinase JAK2 as a result of t(9;12) in a lymphoid and t(9;15;12) in a myeloid leukemia. Blood 1997; 90: 2535– in other translocation regions. 2540. In summary: (1) we detected a total of nine patients who 12 Lacronique V, Boureux A, Valle VD, Poirel H, Quang CT, Mau- had complicated genetic events including multiple chromo- chauffe M, Berthou C, Lessard M, Berger R, Ghysdael J, Bernard some breaks, not only in myeloid malignancies but also in OA. A TEL-JAK2 fusion protein with constitutive kinase activity in lymphoid malignancies with higher frequency, which is the human leukemia. Science 1997; 278: 1309–1312. largest study of patients with such genetic events; (2) these 13 Eguchi M, Eguchi-Ishimae M, Tojo A, Morishita K, Suzuki K, Sato Y, Kudoh S, Tanaka K, Setoyama M, Nagamura F, Asano S, Kam- complicated genetic events were frequently observed in a ada N. Fusion of ETV6 to neurotrophin-3 receptor TRKC in acute region covering TEL and KIP1, suggesting that this region is myeloid leukemia with t(12;15)(p13;q25). Blood 1999; 93: genetically unstable and fragile; (3) all the patients had com- 1355–1363. plex karyotypes, and most patients showed a history of 14 Knezevich SR, McFadden DE, Tao W, Lim JF, Sorensen PH. A chemo/radiotherapy and an extremely short survival, suggest- novel ETV6-NTRK3 gene fusion in congenital fibrosarcoma. Nat ing that multiple chromosome breaks were induced through Genet 1998; 18: 184–187. 15 Knezevich SR, Garnett MJ, Pysher TJ, Beckwith JB, Grundy PE, chemo/radiotherapy or mutagen, and are associated with a Sorensen PH. ETV6-NTRK3 gene fusions and trisomy 11 establish sub-group of patients with a bad prognosis, possibly through a histogenetic link between mesoblastic nephroma and congenital mechanism of additional activation of oncogenes or loss of fibrosarcoma. Cancer Res 1998; 58: 5046–5048. TSGs. 16 Rubin BP, Chen CJ, Morgan TW, Xiao S, Grier HE, Kozakewich HP, Perez-Atayde AR, Fletcher JA. Congenital mesoblastic nephroma t(12;15) is associated with ETV6-NTRK3 gene fusion: Acknowledgements cytogenetic and molecular relationship to congenital (infantile) fibrosarcoma. Am J Pathol 1998; 153: 1451–1458. We thank Marjorie Isaacson for gathering the data on these 17 Romana SP, Mauchauffe M, Le CM, Chumakov I, Le PD, Berger patients and Shalini Reshmi for technical assistance. This work R, Bernard OA. The t(12;21) of acute lymphoblastic leukemia was supported by a Grant-in-Aid for International Exchange results in a TEL-AML1 gene fusion. Blood 1995; 85: 3662–3670. from Japan Clinical Pathology Foundation (YS), NIH grants 18 Odero M, Carlson K, Calasanz M, Chinwalla V, Rowley J. Identi- CA42557 (JDR) and CA40046 (JDR and MML), The Spastic fication of eight new translocations involving TEL/ETV6 in hemato- logical malignancies using FISH and spectral karyotyping. Blood Paralysis Foundation of the Illinois-Eastern Iowa District of 1999; 94 (Suppl. 1): 495a. Kiwanis International (JDR) and the G Harold and Leila Y 19 Sato Y, Suto Y, Pietenpol J, Golub TR, Gilliland DG, Davis EM, Mathers Charitable Foundation (JDR). Le Beau MM, Roberts JM, Vogelstein B, Rowley JD, Bohlander SK.

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