¹Division of Molecular Cytogenetics, Department of Clinical Pathology, Research Institute of International Medical Center of Japan, Tokyo, Japan

²Hematologic Clinic, Saitama Cancer Center Hospital, Saitama, Japan

³Department of Pediatrics, Aomori Municipal Hospital, Aomori, Japan

⁴University of Chicago, Department of Medicine, Section of Hematology/Oncology, Chicago, IL, USA

Correspondence to: Y Sato, Division of Molecular Cytogenetics, Department of Clinical Pathology, Research Institute, International Medical Center of Japan, Toyama 1-21-1, Shinjuku-Ku, Tokyo, 162-0052, Japan; Fax: 81-3-5273-8602

During fluorescence in situ hybridization (FISH) analysis of metaphase cells from 70 patients with lymphoid and myeloid hematologic malignancies and chromosomal rearrangements involving band 12p13, we identified nine patients (four with lymphoid malignancies, four with myeloid malignancies and one with biphenotypic leukemia) who showed more complicated rearrangements than we had expected from conventional cytogenetic study. In six patients, multiple breaks occurred in small segments of 12p with subsequent translocations and insertions of these segments into other chromosomes, sometimes to unexpected regions. In three patients additional chromosome breaks resulted in a sub-clone which was cytogenetically indistinguishable from the main clone in each patient based on the cytogenetic analysis. These subtle molecular events were detected exclusively in a region covering TEL/ETV6 and KIP1/CDKN1B. Seven of nine had a previous history of chemo/radiotherapy; all the patients showed complex karyotypes, even though they were newly diagnosed with leukemia. Survival data were available in five patients, and all survived less than 6 months. These findings suggest that the 12p13 region, especially the above-mentioned region, is genetically unstable and fragile. It is likely that multiple chromosome breaks were induced through mutagens used in chemo/ radiotherapy, and are associated with a sub-group of patients with an extremely bad prognosis. Leukemia (2001) 15, 1193-1202.

12p13 rearrangements; hematologic malignancies; FISH; chromosomal instability; TEL/ETV6; KIP1/CDKN1B

Introduction

Rearrangements of the short arm of chromosome 12 (12p) have been found in a wide spectrum of hematologic malignancies and solid tumors.^1,2,3,4,5 Although each band of 12p (12p11, 12p12, and 12p13) is rearranged in various types of abnormalities including balanced or unbalanced translocations, insertions, inversions and deletions, rearrangements of band 12p13 are most frequently observed.⁶ TEL/ETV6 and 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 reported to fuse to several tyrosine kinases (ABL,^8,9 ARG,¹⁰ JAK2^11,12 and TRKC^13,14,15,16), a transcription factor (AML1¹⁷) and unrelated transcription factors. To date, 41 chromosomal bands have been demonstrated to be involved in TEL translocations.¹⁸

Previously, we reported that the smallest commonly deleted region found in hematologic malignancies with 12p12-13 deletions was flanked by TEL at the telomeric side and KIP1 at the centromeric side.¹⁹ Moreover, deletions of bands 12p12-13 are also detected in non-small cell lung cancer, breast cancer, and ovarian cancer, where the common deleted region again includes the TEL to KIP1 interval. In an attempt to clone potential tumor suppressor genes (TSGs) on this region, Baens et al²⁰ further refined the common deleted segment to 600 kb between TEL and D12S358, excluding KIP1.

While examining the breakpoint in 23 patients with balanced translocations involving 12p13²¹ and defining the commonly deleted region in 47 patients with unbalanced translocations or deletions involving band 12p13 with FISH,¹⁹ we found nine patients with multiple chromosomal breaks in 12p. Similar molecular events have been reported in a few patients.^22,23

In this report, we tried to delineate clinical and cytogenetic characteristics of these patients in comparison with previously reported patients. Whereas such events have been reported only in myeloid malignancies so far, our patient series included four lymphoid malignancies, four myeloid malignancies and one biphenotypic leukemia. In six patients, these multiple chromosomal breaks resulted in a deletion of a small segment and/or translocation of this segment to another chromosome, sometimes to unexpected regions. In three patients, 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 clone in each patient. These molecular events were exclusively found in a region covering TEL and KIP1, whereas others were found in the telomeric region to TEL.²² These findings suggest that this region is genetically unstable and fragile, leading to these complex genetic events. All the patients had a complex karyotype, even though they were newly diagnosed. Survival data were available in five, and all survived less than 6 months. Thus, patients with multiple chromosomal breaks in this region have an extremely bad prognosis.

Materials and methods

Patients

Patients were referred to the Section of Hematology/Oncology at the University of Chicago for chromosome study from 1970 to 1994. Selection of patients was based on diagnosis of a hematologic malignancy and the presence of 12p12-13 rearrangements in the karyotype. Seventy patients were selected (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 five non-Hodgkin's lymphoma (NHL)), 49 myeloid malignancies (four acute leukemia (AL) or acute undifferentiated leukemia (AUL), 16 acute myelogenous leukemia (AML), five therapy-related AML (t-AML), four chronic myelogenous leukemia (CML), three myelofibrosis (MF), one polycythemia vera (PV), 13 myelodysplastic syndrome (MDS), and three t-MDS), and one biphenotypic leukemia. Patient material submitted for cytogenetic analysis during this period had the following diagnostic breakdown: 918 ALL, 202 CLL/prolymphocytic leukemia, 107 hairy cell leukemia, 1566 NHL, 147 unclasified or biphenotypic leukemia, 2117 AML, 1605 myeloproliferative disorders (MPD) (including 880 CML, 143 FM, 185 PV, 58 essential thrombocythemia and 347 other/unclassified MPD), 1454 MDS, and 496 t-AML/MDS cases. All t-AML/MDS patients are double counted also being included in either the AML or MDS categories; 118 patients had both AML and MDS diagnoses and eight MPD patients have more than one MPD diagnosis included at some point in laboratory records.

The disease stage when the samples were obtained was as follows: among 20 lymphoid malignancies, 14 cases were at diagnosis and six at relapse, whereas among 49 myeloid malignancies, 33 were at diagnosis and 16 at relapse. While performing FISH analysis, we identified nine patients who 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 had NHL, t-AML and biphenotypic leukemia when the chromosomal samples were obtained (Table 1). All of the patient samples were obtained with informed consent.

Cytogenetic analysis

Metaphase cells were prepared for cytogenetic study as previously described.²⁴ The karyotypes were described according to the International System for Human Cytogenetic Nomenclature (ISCN 1995).²⁵ In the initial cytogenetic study, four (patients 1, 2, 7 and 9) had a balanced translocation, four (patients 3 to 6) an unbalanced translocation, and one (patient 8) an interstitial deletion. Patient 9 had a t(12;22) translocation and two copies of a deletion 12p.

FISH probes

We used 20 probes for the FISH study: 11 cosmid probes (D12S235, D12S237, D12S134, D12S229, HTY3049c1-7, D12S133, D12S142, D12S119, D12S140, 15a4 (TEL ex.1) and 95h4 (TEL ex.2)); three YAC probes (YAC961a6, YAC771h4 and YAC964c10); a cosmid contig probe (TEL ex.3), a P1 phage contig probe (P27^KIP1),²⁶ three phage probes (D12S54, GDI·D4 and CCND2) and a plasmid probe D12S20. These probes were mapped on 12p13.3 to 12p12.1 and were ordered as previously described.^19,21,27 The YAC 964c10 contains the entire TEL gene and has an internal deletion which includes KIP1/CDKIB gene. The P27^KIP1 probe includes the complete KIP1/CDKIB gene. The location of these probes is shown in Tables 2 and 3.

We used chromosome painting probes and centromere-specific probes (Vysis, Naperville, IL, USA) to clarify the origin of the marker chromosomes. To clarify the translocation of the short arm of chromosome 9 in patient 8, we used the C55B, C173 and C262 cosmid probes (location: 9p22) which were isolated from the chromosome 9 cosmid library derived from the cell line J64051 (a gift from Oncor, Gaithersburg, MD, USA) by one of us (HGS).

FISH study

The probes were labeled with biotin-11-dUTP or digoxigenin-11-dUTP using nick translation and were hybridized to chromosome preparations as previously described.²⁸ Chromosomal bands were identified using counterstaining with 4' 6-diamindino-2-phenylindole dihydrochloride (DAPI). The presence or absence of the FISH signals was scored on an average of 10.7 abnormal metaphase cells (range 3-20) per probe per patient by two persons blinded to the identity of probes and patients. Images of the hybridizations were captured with a liquid-cooled, charge-coupled device camera (Photometrics, Tucson, AZ, USA). Separate gray-scale images for the DAPI and the fluorescein isothiocyanate (FITC) or rhodamine fluorochromes were acquired. After adjusting the gray levels with the National Institutes of Health (NIH, Bethesda, MD, USA) image 1.61 software, the images were merged using Adobe Photoshop (Adobe System, San Jose, CA, USA) on a Macintosh computer (Apple Computer, Cupertino, CA, USA).

Results

All the patients showed complex karyotypes

Clinical data including previous history, treatment and survival and cytogenetic findings of the nine patients are summarized in Table 1. All patients were studied a long time ago, and thus some clinical data were impossible to collect. Two (patients 1 and 9) had newly diagnosed leukemia and for whom a previous history of environmental exposure was unknown: the remaining patients had a history of treatment for an original leukemia or previous malignancies. Complicated chromosomal abnormalities were observed in all the patients including two patients with newly diagnosed leukemia. Four (patients 3, 5, 6 and 8) showed -5/5q- and/or -7/7q- which are thought to be associated with a history of previous chemo/radiotherapy or environmental mutagen exposure. Survival 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 available.

Three patients had a sub-clone with additional chromosomal breaks but with no detectable change in the karyotype from that of the main clone

FISH results of three patients (patients 1 to 3) are summarized in Table 2. Representative karyotypes with the FISH signals are shown in Figure 1A to F.

Patient 1: The FISH signals for the telomeric probes, D12S237 and D12S134, were found on the der(22) in all the cells analyzed and most of the signals for the more centromeric probes, from YAC961a6 to P27^KIP1, were found on the der(12), suggesting that the breakpoint in the t(12;22) is between D12S134 and YAC961a. However, with the D12S133 probe, the signals were found on der(22) in three cells (Figure 1A), whereas they were seen on the der(12) (Figure 1B) in nine cells on the same slide. This indicates existence of a sub-clone with two more chromosomal breaks (between HTY3049c1-7 and D12S133, and between D12S133 and YAC964c10), resulting in translocation of a small region including D12S133 to the der(22).

Patient 2: The D12S237 and D12S134 probes hybridized to the der(2) in all the cells analyzed and most of the signals for YAC961a6 to P27^KIP1 probes were found on the der(12), suggesting that the breakpoint of t(2;12) is between D12S134 and YAC961a. However, with the HTY3049c1-7 probe, the signals were found on the der(2) in two cells, and with the D12S133 probe, they were split between the der(2) and the der(12) in five cells (Figure 1C). With the same probes on the same slide, the signals were found on the der(12) in the remaining cells analyzed, in 18 cells with HTY3049c1-7 and in nine cells with D12S133 (Figure 1D). This indicates the existence of a sub-clone which has two additional chromosomal breaks (between D12S229 and HTY3049c1-7, and in D12S133), resulting in translocation of a small region including HTY3049c1-7 and D12S133 to the der(2).

Patient 3: The signals of D12S235 to YAC964c10 probes were found on the der(2) in all the cells analyzed and signals of D12S142 to D12S20 were not detected, indicating that this region was deleted. However, the signals of the TEL ex.3 and P27^KIP1 probes were found on the der(12) in five cells (Figure 1E) and two cells, respectively. With the same probe on the same slide, the signals were found on the der(2), in five cells (Figure 1F). This indicates that the sub-clone has an additional break in the telomeric region of TEL ex.3 with the retention of the region including TEL ex.3 and P27^KIP1 on the der(12).

The signals of 12p probes were always found on the normal 12p in these three patients. On re-review of the G-banded metaphase cells from these patients, we could not detect any difference in the karyotype between the each main clone and the sub-clone.

Six patients showing multiple chromosomal breaks resulting in complex rearrangements

FISH results of six patients (patients 4 to 9) are summarized in Table 3. Representative karyotypes with the FISH signals are shown in Figure 2A to F. A schematic diagram illustrates how these complex translocations or deletions in the 12p13 region could occur (Figure 3A to F).

Patient 4: Except for YAC 964c10, none of the signals of the telomeric probes (D12S235 to P27^KIP1 ) were found on the der(12) or on any other chromosome, whereas the signals of the centromeric probes (D12S142 to D12S20) were found on the der(12), suggesting that the 12p13.1 to 12p13.3 region was deleted. However, the YAC964c10 probe hybridized to the end of the long arm (qter) of the marker chromosome (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), whereas a small region including YAC 964c10 was translocated to the qter of the marker chromosome (Figure 3A).

Patient 5: D12S235 to D12S133 hybridized to the der(18), whereas the signals of most probes in the region of 12p13.1-12p12.1 except for the YAC 964C10 probe were deleted. The YAC probe unexpectedly labeled the qter of a normal appearing chromosome 2 (indicated as 'der(2)' in Figure 2B), indicating that a small segment including the YAC964c10 was translocated to chromosome 2 (Figure 3B).

Patient 6: 12S235, D12S134 and D12S133 hybridized to the der(2) (qter) whereas the YAC 964C10, D12S142, D12S119 and D12S20 probes were deleted. Unexpectedly, the P27^KIP1 probe hybridized to the short arm of the der(12) and near to the centromere of the der(2) (Figure 2C). These findings showed that the telomeric part of 12p13 was involved in the t(2;12)(q37;p13) translocation and that a small segment including the KIP1 gene was split into two parts, being translocated to the end of the short arm of the der(12) and near the centromere of the der(2) (Figure 3C).

Patient 7: The signals of all the probes on 12p13.3 and D12S133 were found on the marker chromosome, whereas signals of the 12p12.3-12p12.1 region probes were found on the der(12). The signals of the YAC964c10 and the TEL ex.3 probe were found on the marker and normal appearing chromosome 21 (indicated as 'der(21)' in Figure 2D), indicating that the chromosomal break occurred within the TEL gene, and a small segment including exon 3 of TEL was translocated to the chromosome 21, whereas the distal part of the YAC964c10 was translocated to the marker chromosome. Moreover, the signals of the P27^KIP1 probe hybridized only to the normal 12p, indicating that this segment was deleted from the der(12) (Figure 3D). Despite our efforts, we could not identify the origin of the marker chromosome, only confirming that it was not chromosomes 21 or 22.

Patient 8: The telomeric probes D12S235 to D12S133 were not used because of lack of material. The YAC964c10 probe hybridized to the der(9), the marker chromosome 1 (mar1) and the der(12) (Figure 2E), whereas TEL ex.1 and TEL ex.2 probes hybridized only to mar1. Signals of the TEL ex.3 and P1 TEL probes were found on mar1 and the der(12). These results suggest that a break occurred within TEL exon 3, and the telomeric portion was translocated to mar1 with the centromeric portion remaining on the der(12). Another chromosomal break seemed to occur within the YAC964c10 probe and the telomeric portion of this probe was translocated to the der(9). Moreover, the P27^KIP1 probe labeled only the normal 12p, indicating that this segment was deleted from the der(12). We were not able to determine the extent of this centromeric deletion on the der(12). With the chromosome 12 painting probe (WCP12, Vysis), we confirmed that chromosome 12 was on pter of the der(9) chromosome. With the cosmid probes located on 9p22 (C55B, C173 and C262), we demonstrated that the 9p22 region was translocated to the qter of mar1. Therefore, this patient appears to have had a three-way translocation involving 9p, 12p and the long arm of the mar1 (Figure 3E); we do not know whether it was a stepwise or simultaneous three-way translocation. We tried to identify this marker chromosome with painting probes but we could only confirm that it was not chromosome 7 or the X chromosome.

Patient 9: Probes including D12S235 to HTY3049c1-7 all labeled the der(22), whereas D12S133, YAC964c10, P27^KIP1 and 12p12.3 to 12p12.1 region probes remained on the der(12). Only the TEL ex.3 probe signal was split between the der(12) and der(22) (Figure 2F). These findings suggest that a chromosomal break occurred in the TEL ex.3 probe, and an undefined segment of TEL was translocated to the der(22) (Figure 3F). Probes including D12S235 to TEL ex.3 labeled both del(12), and probes including P27^KIP1 to D12D20 were deleted. We did not detect any difference between two copies of del(12) as to deleted region.

The FISH signals for each probe were always found on the normal 12p in metaphase cells in all six patients.

Discussion

In this report, we describe nine patients: three with a sub-clone having an additional translocation or insertion, and six with more complicated and cryptic chromosomal rearrangements including multiple chromosome breaks and non-contiguous deletions which were not expected from standard chromosome analysis. Some data for patients 1, 2, 8 and 9 have been described elsewhere²¹ (see footnote in Table 1). FISH studies of patients 3, 5 and 6 were performed previously.²⁹ However, the findings presented here were not identified, because the probes between D12S142 and D12S133 (P27^KIP1, P1 TEL, TEL ex.3, 95h4, 15a4, YAC964c10) were not used at that time.

We found these nine patients among 70 patients (9/70 = 12.9%) with 12p13 rearrangements, four in 20 lymphoid malignancies, four in 49 myeloid malignancies and one in one biphenotypic leukemia. Although other investigators have reported similar complicated molecular events including non-contiguous segments,^22,23 all of the cases were myeloid malignancies. Thus, this is the first report that identified such events in lymphoid malignancies. Interestingly, it is likely that the frequency of cases with such events is much higher in lymphoid malignancies (4/20 = 20.0%) than in myeloid malignancies (4/49 = 8.2%). One may think that this higher frequency is associated with the higher rate of mutagen-exposed patients in the lymphoid malignancy group. However, when we compared the ratio of patients at diagnosis and at relapse between lymphoid and myeloid malignancy groups, the ratio is not significantly different (14 at diagnosis/six at relapse in 20 lymphoid malignancies, vs 33 at diagnosis/16 at relapse in 49 myeloid malignancies), although we do not know the history of potential environmental exposure in each group.

In our series, five patients (patients 2-4, 7, 8) obviously had a previous history of chemo/radiotherapy, although two (patients 1 and 9) did not, and in the remaining two (patients 5 and 6), we had no informative data. Nevertheless, all the patients including the new leukemia patients (patients 1 and 9) showed a complex karyotype which is thought to be associated with poor prognosis, especially when -5/5q- and/or -7/7q- are present.³⁰ Actually, four patients (patients 3, 5, 6 and 8) showed -5/5q- and/or -7/7q-, and two of them (patients 3 and 8) had a previous history of chemo/ radiotherapy. As other investigators have reported,^22,23 prognosis in our patients was also extremely bad among those for whom data were available (patients 2, 4-6, 8). We do not know which chemotherapeutic reagent is most closely associated with inducing multiple chromosome breaks. However, taken together, it seems that multiple chromosome breaks are associated with a bad prognosis.

The sub-clone found in the first three patients appears to be derived from the main clone. It is likely that after additional chromosome breaks occurred, some genetic material was translocated from the original site to the other site in the sub-clone, although this genetic event was too subtle to be detected by standard cytogenetic analysis. In such a sub-clone, an additional breakpoint might involve a gene which is broken and fused to another gene. In our previous paper,²¹ we suggested that in the telomeric region to TEL, there were at least three small breakpoint cluster regions (sbcr) among the 12p13 translocations where unidentified genes might be 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 reported one patient to have such a sub-clone. Their patient 14 had two clones; one with a del(12p) telomeric to KRAS except for retention of VWF, and the other with the del(12p) involved in a translocation with 3q. This observation shows that a critical genetic event can occur in the leukemic clone without detectable karyotypic change. Of interest is that patient 1 had newly diagnosed leukemia. This suggests that some patients could have such a sub-clone from the beginning of the disease. Clinical relevance of this observation is important, because this could explain the fact that during the clinical course of leukemia, the response to chemotherapy may have altered although the observed karyotype is not changed.

In six patients, it is likely that multiple chromosome breaks occurred followed by translocation and/or deletion of a small segment. This small chromosome fragment was sometimes translocated to the partner derivative chromosome involved in the original translocation, and sometimes to an unexpected chromosomal region as shown in Figure 3. Similar molecular events were reported in three myeloid leukemia patients by La Starza et al:²² in their patient 1(AML) with add(12p), FISH signals with three probes telomeric to TEL were found not only on add(12p) but also on the marker chromosome (mar)-2; in patient 2 (AML-M1) with ring(12), FISH signals of 12p probes were separately found on ring(12), add(4) and der(17); and in patient 3 (t-AML-M2) with der(12)t(12;14)(p13;q11), FISH signals with two probes centromeric to TEL were unexpectedly found on a mar. In their three patients, multiple chromosome breaks occurred in the telomeric region to TEL, and in patients 2 and 3, the TEL region was deleted. However, in our patients, multiple chromosome breaks occurred exclusively in a region covering TEL and KIP1 as shown in patients 4-9. To determine the precise breakpoint is somewhat difficult in some patients (patients 4-6), because they had a large deleted region with some retaining region within it as shown in Figure 3. However, in patients 7-9, the breakpoint should be within TEL ex.3 probe, although we²¹ and others²² previously reported that the 12p13 translocation breakpoints were frequently found in the telomeric region to TEL.

It is well established that a small segment deletion sometimes occurs together with an 'apparent' reciprocal translocation, for example, in the 3' region of MLL in 11q23 translocations³¹ and in the 3' region of MYH11 in 16p13 translocations.^32,33 Moreover, recently, a large deletion has been reported to frequently occur adjacent to the breakpoint of t(9;22)³⁴ and t(8;21) translocations.³⁵ Tanaka et al³⁶ reported that small chromosomal segments including the ABL gene and/or MLL gene were translocated to multiple other chromosomal regions in a single cell, resulting in formation of partial tri-, tetra- or pentasomy of these regions. They called this 'segmental jumping translocation', and assumed that it was one of the mechanisms for gene amplification. However, our observation differs from their report, because the small segment is not amplified, but rather is translocated or deleted, presumably as an isolated event in a single cell containing a non-contiguous small segment in 12p13. On the other hand, Beans et al²⁰ further defined the common deleted segment to 600 kb between TEL and D12S358 at 12p13.3 with FISH using contig probes. Unfortunately, we did not study whether or not this region was deleted in our patients, because we lacked the appropriate probes.

The frequent occurrence of multiple chromosome breaks in a region covering TEL and KIP1 suggests that this region is genetically unstable and fragile. An important question is whether these events would occur preferentially in the 12p13 region, or whether the events could happen in any other regions, especially adjacent to the breakpoint of disease-specific translocations or deletions, without being detected. Extensive FISH studies should be done to answer this question in other translocation regions.

In summary: (1) we detected a total of nine patients who had complicated genetic events including multiple chromosome breaks, not only in myeloid malignancies but also in lymphoid malignancies with higher frequency, which is the largest study of patients with such genetic events; (2) these complicated genetic events were frequently observed in a region covering TEL and KIP1, suggesting that this region is genetically unstable and fragile; (3) all the patients had complex karyotypes, and most patients showed a history of chemo/radiotherapy and an extremely short survival, suggesting that multiple chromosome breaks were induced through chemo/radiotherapy or mutagen, and are associated with a sub-group of patients with a bad prognosis, possibly through mechanism of additional activation of oncogenes or loss of TSGs.

Acknowledgements

We thank Marjorie Isaacson for gathering the data on these patients and Shalini Reshmi for technical assistance. This work was supported by a Grant-in-Aid for International Exchange from Japan Clinical Pathology Foundation (YS), NIH grants CA42557 (JDR) and CA40046 (JDR and MML), The Spastic Paralysis Foundation of the Illinois-Eastern Iowa District of Kiwanis International (JDR) and the G Harold and Leila Y Mathers Charitable Foundation (JDR).

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Figure 1 Representative cells with FISH signals of patients 1 to 3. The left column (A, C and E) shows the sub-clone, and the right column (B, D and F) shows the main clone in each patient. The signals for each probe were always observed on the normal 12p. Patient 1: With D12S133 probe, FISH signals were seen on the der(22) in three cells (A), but were also found on the der(12) in nine cells (B) on the same slide. Patient 2: With D12S133 probe, FISH signals were seen to be split between the der(2) and the der(12) in five cells (C), but were also found only on the der(12) in nine cells (D) on the same slide. Patient 3: With P27^KIP1 probe, FISH signals were seen on the der(12) in two cells (E), but were also found on the der(2) in five cells (F) on the same slide.

Figure 2 Representative cells with FISH signals of patients 4 to 9. The signals for each probe were always observed on normal 12p. Patient 4: (A) The signal of the YAC964c10 probe was seen on the marker chromosome. Patient 5: (B) The signal of the YAC964c10 probe was seen on the terminus of the long arm of the normal appearing chromosome No. 2 (indicated as 'der(2)'). Patient 6: (C) The signal of the P27^KIP1 probe was seen on the terminus of the short arm of the der(12) and in the vicinity of the centromere of the der(2). Patient 7: (D) The signal of the YAC964c10 probe was seen on the marker and normal appearing No.21 (indicated as 'der(21)'). Patient 8: (E) The signal of the 964c10 probe was seen on the add(9), the marker chromosome 1 and del(12). Patient 9: (F) The signal of the TEL ex.3 probe was split between the der(12) and der(22).

Figure 3 (A-F) The diagram explains how the translocations or deletions might have occurred in the 12p13 region in patients 4 to 9.

Table 1 Clinical and cytogenetic findings of nine patients with hematologic malignancies who had multiple chromosomal breaks resulting in the formation of complicated rearrangements in the band 12p13

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

Table 3 Results of FISH analysis using 12p12 to p13 probes in six patients with hematologic malignancies who had multiple chromosomal breaks resulting in the formation of complicated rearrangements in the band 12p13