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This report summarizes highlights of the ‘Philadelphia Chromosome Symposium: Past, Present and Future’, held September 28, 2010, to commemorate the 50th anniversary of the discovery of the Philadelphia chromosome. The symposium sessions included presentations by investigators who made seminal contributions concerning the discovery and molecular characterization of the Ph chromosome and others who developed a highly successful therapy based on the specific molecular alteration observed in chronic myelogenous leukemia. Additional presentations highlighted future opportunities for the design of molecularly targeted therapies for various types of cancer. Also included here are reminiscences connected with the discovery of the Ph chromosome by David Hungerford and Peter Nowell, the discovery that the abnormality arises from a chromosomal translocation, by Janet Rowley, and the cloning of the 9;22 translocation breakpoints by Nora Heisterkamp, John Groffen and colleagues.

MicroRNAs (miRNAs) are postulated to be important regulators in cancers. Here, we report a genome-wide miRNA expression analysis in 52 acute myeloid leukemia (AML) samples with common translocations, including t(8;21)/ AML1(RUNX1)-ETO(RUNX1T1) , inv( 16 )/ CBFB-MYH11 , t(15;17)/ PML-RARA , and MLL rearrangements. Distinct miRNA expression patterns were observed for t(15;17), MLL rearrangements, and core-binding factor (CBF) AMLs including both t(8;21) and inv( 16 ) samples. Expression signatures of a minimum of two (i.e., miR-126/126*), three (i.e., miR-224, miR-368, and miR-382), and seven (miR-17–5p and miR-20a, plus the aforementioned five) miRNAs could accurately discriminate CBF, t(15;17), and MLL -rearrangement AMLs, respectively, from each other. We further showed that the elevated expression of miR-126/126* in CBF AMLs was associated with promoter demethylation but not with amplification or mutation of the genomic locus. Our gain- and loss-of-function experiments showed that miR-126/126* inhibited apoptosis and increased the viability of AML cells and enhanced the colony-forming ability of mouse normal bone marrow progenitor cells alone and particularly, in cooperation with AML1-ETO , likely through targeting Polo-like kinase 2 ( PLK2 ), a tumor suppressor. Our results demonstrate that specific alterations in miRNA expression distinguish AMLs with common translocations and imply that the deregulation of specific miRNAs may play a role in the development of leukemia with these associated genetic rearrangements.

The PRDX4 gene located at Xp22 codes for a member of the peroxiredoxin gene family. Genes within this family exhibit thioredoxin-dependent peroxidase activity and have been implicated in cellular functioning, including proliferation and differentiation. Recently, PRDX4 has been identified as a partner gene in an X;21 translocation in a patient with acute myeloid leukemia (AML). To determine whether PRDX4 was involved in other translocations, leukemia cells from fifteen patients with Xp22 abnormalities were screened for the gene’s involvement using fluorescence in situ hybridization (FISH). One sample from a 41 year old female with acute lymphoblastic leukemia (ALL) showed three signals when hybridized with the PRDX4 probe. Cytogenetic analysis of the sample had identified a t(X;18)(p22,q23). Assuming that the three signals indicated a break within the PRDX4 gene, we performed FISH experiments and successfully narrowed the breakpoint on chromosome 18 to a 50 kB region. Subsequent analysis using spectral karyotyping showed that the leukemic cells had undergone multiple rearrangements and that a third X chromosome, albeit rearranged, was present. Additional FISH experiments revealed that the third PRDX4 signal was the result of a third copy of the gene. Analysis of the other rearrangements has helped to characterize the multiple abnormalities within the leukemic cells. Our findings are significant because they underline the importance of using multiple techniques when analyzing complex chromosomal rearrangements in malignant cells.

Specific chromosomal translocations are hallmarks of many human leukemias. The basis for these translocation events is poorly understood, but it has been assumed that spatial positioning of genes in the nucleus of hematopoietic cells is a contributing factor. Analysis of the nuclear 3D position of the gene MLL, frequently involved in chromosomal translocations and five of its translocation partners (AF4, AF6, AF9, ENL and ELL), and two control loci revealed a characteristic radial distribution pattern in all hematopoietic cells studied. Genes in areas of high local gene density were found positioned towards the nuclear center, whereas genes in regions of low gene density were detected closer to the nuclear periphery. The gene density within a 2 Mbp window was found to be a better predictor for the relative positioning of a genomic locus within the cell nucleus than the gene density of entire chromosomes. Analysis of the position of MLL, AF4, AF6 and AF9 in cell lines carrying chromosomal translocations involving these genes revealed that the position of the normal genes was different from that of the fusion genes, and this was again consistent with the changes in local gene density within a 2 Mbp window. Thus, alterations in gene density directly at translocation junctions could explain the change in the position of affected genes in leukemia cells.

The AML1 gene (also known as RUNX1) at 21q22 codes for core binding factor (CBF) α, which forms a heterodimer with CBF β that acts as a transcriptional activating factor. CBF is a critical regulator in the generation and differentiation of definitive hematopoietic stem cells and is frequently disrupted in leukemia through chromosome translocations. We cloned a novel AML1 partner gene, PRDX4, in an X;21 translocation in a 74-year-old male patient diagnosed with acute myeloid leukemia–M2. Chromosome analysis detected a t(X;21)(p22;q22) as the sole abnormality in bone marrow samples. The involvement of AML1 was confirmed by fluorescence in situ hybridization studies. Using 3′ RACE-PCR, we cloned a fusion between exon 5 of AML1 and exon 2 of PRDX4. RT-PCR confirmed the fusion and detected another fusion between exon 6 of AML1 and exon 2 of PRDX4, indicating alternative splicing of exon 6 of AML1 in the fusion transcripts. PRDX4 is one of six peroxiredoxin-family genes that are highly conserved in eukaryotes and prokaryotes and are ubiquitously expressed. Peroxiredoxin genes exhibit thioredoxin-dependent peroxidase activity and have been implicated in a number of other cellular functions such as cell proliferation and differentiation. PRDX4 plays a regulatory role in the activation of the transcription factor NF-κB and is significantly down-regulated in acute promyelocytic leukemia. This is the first example of antioxidant enzyme involvement in a chromosome translocation in leukemia. © 2004 Wiley-Liss, Inc.

The NUP98 gene has been reported to be fused with at least 15 partner genes in leukemias with 11p15 translocations. We report the results of screening of cases with cytogenetically documented rearrangements of 11p15 and the subsequent identification of involvement of NUP98 and its partner genes. We identified 49 samples from 46 hematology patients with 11p15 (including a few with 11p14) abnormalities, and using fluorescence in situ hybridization (FISH), we found that NUP98 was disrupted in 7 cases. With the use of gene-specific FISH probes, in 6 cases, we identified the partner genes, which were PRRX1 (PMX1; in 2 cases), HOXD13, RAP1GDS1, HOXC13, and TOP1. In the 3 cases for which RNA was available, RT-PCR was performed, which confirmed the FISH results and identified the location of the breakpoints in patient cDNA. Our data confirm the previous findings that NUP98 is a recurrent target in various types of leukemia.

The mixed lineage leukemia gene (MLL, also known as HRX, ALL-1 and Htrx) located at 11q23 is involved in translocations with over 40 different chromosomal bands in a variety of leukemia subtypes. Here we report our analysis of a rare but recurring translocation, t(11;15)(q23;q14). This translocation has been described in a small subset of cases with both acute myeloblastic leukemia and ALL. Recent studies have shown that MLL is fused to AF15q14 in the t(11;15). Here we analyse a sample from another patient with this translocation and confirm the presence of an MLL-AF15q14 fusion. However, we have also identified and cloned another fusion transcript from the same patient sample. In this fusion transcript, MLL is fused to a novel gene, MLL partner containing FYVE domain (MPFYVE). Both MLL-AF15q14 and MLL-MPFYVE are in-frame fusion transcripts with the potential to code for novel fusion proteins. MPFYVE is also located on chromosome 15, approximately 170 kb telomeric to AF15q14. MPFYVE contains a highly conserved motif, the FYVE domain which, in other proteins, has been shown to bind to phosphotidyl-inositol-3 phosphate (PtdIns(3)P). The MLL-MPFYVE fusion may be functionally important in the leukemia process in at least some patients containing this translocation.

Although it has been clear for more than a century that the chromosomes in human tumour cells are often wildly abnormal, there has been controversy as to whether these changes are primary events or are merely secondary epiphenomena that reflect the genomic instability of these cells. The prevailing view for most of this period was that chromosome changes were secondary events. What happened to change this view?

Improved techniques in identifying the chromosome changes and the affected genes that are involved in acute leukemias have led to improved treatments for these diseases. Identification of consistent chromosomal changes has allowed us to target the location of particular genes and has enabled us to focus our treatments more specifically to certain subtypes of leukemia. Translocations, in particular, are common cytogenetic abnormalities in human leukemia, and the prevalence of certain types of translocations varies with age. Cancers, lymphomas and leukemias are now known to be genetic diseases and it is recognized that genotype-specific therapies should be used that take into account the genetic alterations of the particular leukemia.

Cell lines derived from patients with leukemia are used in many molecular biology studies. Here we report the cytogenetic analysis of the THP-1 cell line using G-banding, fluorescence in situ hybridization (FISH), and spectral karyotyping (SKY), and the molecular characterization of the MLL-AF9 rearrangement by RT-PCR. The THP-1 cell line was established from the peripheral blood of a 1-year-old boy with acute monocytic leukemia (AML-M5). THP-1 is near-diploid and consists of two related subclones with a number of aberrations, including the t(9;11), associated with AML M5. The use of FISH allowed us to identify and characterize otherwise hidden cytogenetic rearrangements, which include duplication of the 3' portion of MLL in the derivative 9 chromosome and a deletion of the 5' portion of the AF9 gene involved in the translocation. In addition to confirming the FISH results, SKY allowed for a more precise characterization of the karyotype of THP-1 and allowed us to identify other abnormalities in this cell line, including der(1)t(1;12), der(20)t(1;20), deletions 6p, 12p, and 17p, trisomy 8, and monosomy 10. Sequencing of the RT-PCR product showed a direct in-frame fusion product on the derivative chromosome 11 between exon 6 (exon 9) of MLL and exon 5 of AF9, which is most commonly involved in MLL-AF9 translocations. This study demonstrates that combining different techniques to achieve a more precise characterization of the THP-1 cell line provides important information that will be valuable for understanding the critical events required for leukemogenesis.

The t(10;11)(p12-p13;q14-q21) observed in a subset of patients with either acute lymphoblastic leukemia or acute myeloid leukemia has been shown to result in the fusion of AF10 on chromosome 10 with CALM (also named CLTH) on chromosome 11. AF10 was originally identified as a fusion partner of MLL in the t(10;11)(p12-p13;q23) observed in myeloid leukemia. CALM is a newly isolated gene, cloned as the fusion partner of AF10 in the monocytoid cell line, U937. In order to understand the relationship between MLL, AF10, CALM and the leukemic process, fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction were used to study a series of nine leukemia patients with a t(10;11). Six had myeloid leukemia (AML-M0, AML-M1, AML-M4 and AML-M5) and three had T cell lymphoblastic leukemia. We identified four different CALM/AF10 fusion products in five patients and AF10/CALM reciprocal message in one. We conclude that fusion of CALM and AF10 is a recurring abnormality in both lymphoid and myeloid leukemias of various types including AML-M5, and that the breakpoints in the two types of leukemia do not differ. Our data indicate that the CALM/AF10 fusion product on the der(10) chromosome is critical to leukemogenesis. Leukemia (2000) 14, 100-104.

The inv(16) and t(16;16) characterize a subgroup of acute myelomonocytic leukemia (AML) with distinct morphological features and a favorable prognosis. Both cytogenetic abnormalities result in a fusion of CBF beta at 16q22 and MYH11 gene at 16p13, whose detection by PCR and fluorescence in situ hybridization (FISH) is useful for diagnosis and monitoring of the disease. Variant translocations of inv(16)/t(16;16) are very rare and whether they are also associated with a favorable prognosis is unknown. We report a patient presenting with typical AML-M4Eo and a three-way translocation of inv(16) involving 16p13, 16q22, and 3q22. FISH studies on bone marrow (BM) chromosomes using CBFB and MYH11 DNA probes revealed a fusion of CBFB and MYH11 on 16q of the der(16), as well as a signal from MYH11 on 16p but not from CBFB; normal signals for both probes were present on the normal 16. Neither of these labeled probes was on the der(3), but the translocation between the der(3) and der(16) was confirmed by using a chromosome 16 painting probe. Molecular analysis of BM cells using RT-PCR identified a CBFB-MYH11 fusion transcript type D. After achieving complete remission, the patient relapsed. We conclude that FISH and PCR are feasible tools to distinguish cases with variant abnormalities of inv(16) from cases with other chromosome 16 abnormalities. Variant abnormalities of inv(16) may be not associated with favorable prognosis.

We describe a patient with acute myeloblastic leukemia (AML-M0) whose cells had a t(2;11)(p21;q23). Fluorescence in situ hybridization analysis with a probe for MLL showed that it was split, hybridizing to both the derivative 2 and 11 chromosomes. Nineteen other patients with 2p;11q translocations have been described; breakpoints in 14 of these are the same as in the case we describe. The phenotype of these patients is quite variable, with 14 patients having myelodysplastic syndrome which evolved to AML in six. Four patients had AML and two had acute lymphoblastic leukemia. MLL status has been studied in two other patients; one had MLL rearranged and one did not.

Chimeric RNAs, transcribed from malignancy-associated chromosomal translocations, can also arise from RNA trans-splicing in normal cells.

CBFA2(AML1) has emerged as a gene critical in hematopoiesis; its protein product forms the DNA-binding subunit of the heterodimeric core-binding factor (CBF) that binds to the transcriptional regulatory regions of genes, some of which are active specifically in hematopoiesis. CBFA2 forms a fusion gene with ETO andMDS1/EVI1 in translocations in myeloid leukemia and withETV6(TEL) in the t(12;21) common in childhood pre-B acute lymphoblastic leukemia. We have analyzed samples from 30 leukemia patients who had chromosome rearrangements involving 21q22 by using fluorescence in situ hybridization (FISH). Our analysis showed that 7 of them involved CBFA2 and new translocation partners. Two patients had a t(17;21)(q11.2;q22), whereas the other 5 had translocations involving 1p36, 5q13, 12q24, 14q22, or 15q22. Five of these novel breakpoints in CBFA2 occurred in intron 6; this same intron is involved in the t(3;21). One breakpoint mapped to the t(8;21) breakpoint region in intron 5, and 1 mapped 5′ to that region. All 7 CBFA2 rearrangements resulted from balanced translocations. All 7 patients had myeloid disorders (acute myeloid leukemia or myelodysplastic syndrome); 2 were de novo and 5 had treatment histories that included topoisomerase II targeting agents. The association of therapy-related disorders with translocations involving CBFA2 was significant by Fisher’s exact test (P < .003). These results provide further evidence that this region of CBFA2 is susceptible to breakage in cells exposed to topoisomerase II inhibitors. © 1998 by The American Society of Hematology.

Many chromosome abnormalities, especially translocations or inversions, are closely associated with a particular morphologic or phenotypic subtype of leukemia, lymphoma, or sarcoma. Cloning the genes at the breakpoints of these rearrangements has provided critical tools for more-precise diagnosis; in some cases the particular diagnosis has prognostic implications. In addition, many of the genes had not been previously identified; their discovery has had a major impact on our understanding of the molecular biology of cancer. One such gene is MLL (myeloid-lymphoid or mixed-lineage leukemia), which is located at chromosome band 11q23. This gene is involved in the 4;11 and 11;19 (p13.3) translocations in acute lymphoblastic leukemia and in the 6;11, 9;11, and 11;19 (p13.1) translocations in acute myeloblastic leukemia. It is also involved in most translocations in infants (under 1 year of age) with acute leukemia and in patients with acute leukemia who were previously treated with drugs that inhibit toposiomerase II. The target gene of MLL is unknown at present, but because of its homology to the trithorax gene in Drosophila, and based on experimental data from mice, it appears to be involved in maintaining the function of some of the homeobox genes. The development of cytogenetic and molecular probes for MLL rearrangements has confirmed that translocations involving MLL are associated with a very poor prognosis. Thus physicians can identify patients with MLL involvement and can institute treatment for these high-risk patients. An increasing understanding of MLL should lead to more-effective targeted therapy.

A new cell line with megakaryoblastic features, designated UoC-M1, was established from the malignant cells of a 68-year-old patient with acute myeloid leukemia. The patient's leukemic cells reacted with alpha-naphthyl acetate esterase and acid phosphatase and expressed CD7, CD24, CD34, CD38, CD45, HLA-DR and CD61. Cytogenetic analysis of the patient's malignant cells (and of the UoC-M1 cells) showed a human, male hypodiploid karyotype with many chromosome rearrangements and marker chromosomes. Spectral karyotyping (SKY) analysis complemented the G-banded karyotyping and clarified several chromosomal translocations and identified the marker chromosomes. Fluorescence in situ hybridization (FISH) and SKY analysis demonstrated that one marker chromosome contained three segments of chromosome 9 interspersed with three segments of chromosome 11, as well as a portion of chromosome 19. FISH analysis with a probe for MLL revealed that the UoC-M1 cells contained four copies of the MLL gene. Southern blot analysis determined that the MLL gene had a germline profile while Northern and Western analyses showed that the MLL mRNAs and protein were of the appropriate sizes. This is the first report of amplification of the MLL gene which may be an additional mechanism of leukemogenesis or disease progression.

The MOLT-16 cell line was established from the leukemic cells of a patient with T-cell acute lymphoblastic leukemia and contains a t(8;14)(q24;q11) resulting in juxtaposition of sequences downstream of the MYC gene on chromosome 8 and the J region of the T-cell receptor alpha chain gene (TCRA) on chromosome 14. The reciprocal translocation involved a complex rearrangement with two chromosome breakpoints within the TCRA J region on chromosome 14, resulting in inversion of a 1.4 kb DNA fragment between the two breakpoints. The 5′ border of the inversion joins with another segment of chromosome 14, whereas the 3′ border joins with a region of chromosome 8 located at least 257 kb downstream of MYC. Extensive deletions have occurred on both chromosomes 8 and 14 in conjunction with the translocation. To investigate the possible involvement of the V(D)J recombinase in this translocation, we analyzed the nucleotide sequences surrounding the translocation breakpoints. The breakpoint on chromosome 14 occurs between a segment coding for a TCRA J sequence and its heptamer-nonamer signal. Heptamer-nonamer consensus sequences are also identified on chromosome 8 adjacent to the breakpoint. Inserted N and P nucleotides are observed at the breakpoint junctions. Genes Chromosomes Cancer 20:363–371, 1997. © 1997 Wiley-Liss, Inc.

The MLL gene at chromosome 11, band q23, is involved in translocations with as many as 40 different chromosomal bands. Virtually all breakpoints occur within an 8.3 kb BamHI fragment and result in 5' MLL fused to partner genes in a 5'-3' orientation. The translocation t(9;11)(p22;q23), which results in the fusion of MLL to AF9, is the most common of the 11q23 chromosomal abnormalities observed in de novo acute myeloid leukemia (AML), in therapy related leukemia (t-AML), and rarely in acute lymphoblastic leukemia (ALL). We have studied 24 patients with a t(9;11) and an MLL rearrangement, including 19 patients with AML, four with t-AML, and one with ALL. To understand the mechanisms of this illegitimate recombination, we cloned and sequenced the t(9;11) translocation breakpoint junctions on both derivative chromosomes from one AML patient and from the Mono Mac 6 (MM6) cell line, which was derived from a patient with AML. Two different complex junctions were noted. In the AML patient, both chromosome 11 and 9 breaks were staggered, occurred in Alu DNA sequences, and resulted in a 331 bp duplication. In the MM6 cell line, breaks in chromosomes 11 and 9 were also staggered, but, in contrast to the finding in the AML patient, the breaks did not involve Alu DNA sequences and resulted in a 664 bp deletion at the breakpoints. Using reverse transcriptase (RT-) PCR, we analyzed 11 patient samples, including the two just described, for MML-AF9 fusions. The fusion occurred in six of seven AML patients, two of two t-AML patients, one patient with ALL, and in the MM6 cell line. Interestingly, all of the breaks within the AF9 gene in AML patients occurred in the central AF9 exon, called Site A by others, whereas in the single ALL patient the breakpoint mapped to a more 3' region of the AF9 gene. Our data, when combined with those of others, suggest that the fusion point within the AF9 gene, and thus the amount of AF9 material included in the MLL-AF9 fusion gene product, may influence the phenotype of the resulting leukemia. This further supports the proposal that the MML translocation partner genes play a critical role in the leukemogenic process.

Using probes located between 12p12.1 and 12p13.3, we performed fluorescence in situ hybridization (FISH) analysis and identified an inv(12)(p13.1q11) in a patient with acute myeloid leukemia (AML-M6). Standard cytogenetic analysis had identified the rearranged chromosomes 12 as del(12) (p11p13). Although deletions and translocations involving band 12p13 are fairly common chromosomal abnormalities observed in a broad spectrum of hematologic malignancies, inv(12) is a rather rare abnormality. We compare the clinical and cytogenetic findings with those of the previous cases reported in the literature.

The t(14;19)(q32.3;q13.1) is a recurring translocation found in the neoplastic cells of some patients with chronic lymphocytic leukemia (CLL) or other B-lymphocytic neoplasms. We previously cloned the translocation breakpoint junctions present in the leukemic cells from three such patients and identified a gene, BCL3, whose transcription is increased as a result of the translocation. In the present paper, we describe three additional patients with the t(14;19), one with lymphoma and two with CLL, and report the cloning and sequencing of the breakpoint junction in one of these patients as well as in a previously reported patient. We and others have found that the breakpoints on chromosome 14, with one exception, fall within the switch region upstream of the immunoglobulin heavy chain C alpha 1 or C alpha 2 sequences. Several of the breaks within chromosome 19 fall immediately upstream of the BCL3 gene, but several others are more than 16 kb 5' of the gene. Most patients with CLL and the t(14;19) also show trisomy 12.

ETV6 (TEL) is rearranged in various types of hematologic malignancies. The B-cell precursor acute lymphoblastic leukemia (ALL) cell line SUP-B2 has a t(6;12)(q23;p13) involving ETV6 at 12p13 and a submicroscopic deletion of the other ETV6 allele. The reciprocal translocation results in the fusion of ETV6 to a previously unknown gene at 6q23, which we named STL (six-twelve leukemia gene). Both reciprocal fusion transcripts can be detected: On the der(6) chromosome, the ETV6/STL mRNA shows an apparently out of frame fusion of ETV6 at nucleotide 187 to STL, which would result in the addition of 14 amino acids to the first 54 amino acids of ETV6. On the der(12) chromosome three different variants of the STL/ETV6 fusion mRNA could be detected; variable size segments were inserted at the breakpoint between STL and ETV6 exon 3. One of these variants could give rise to a protein in which the first 54 amino acids of ETV6 are replaced by 12 amino acids from one of the STL short open reading frames. Sequence analysis of a 1.4 kb STL cDNA clone from a skeletal muscle library revealed no long open reading frames. This cell line will be very useful in studying the different mechanisms by which alterations of ETV6 contribute to leukemogenesis and in testing the hypothesis that ETV6 might act as a tumor suppressor gene. Genes Chromosom. Cancer 18:254–268, 1997. © 1997 Wiley-Liss, Inc.

Leukemia and lymphoma are characterized by various chromosome abnormalities, including translocation, inversion, deletion and duplication, and variable genomic imbalances. Conventional cytogenetics, fluorescence in situ hybridization (FISH) and expression and genomic microarray are the specific techniques used to detect disease-related chromosome aberrations, genomic imbalances and gene mutations and deregulation. Detection of these genetic abnormalities is significant in clinical practice because many chromosome translocations, inversion and deletions, and gene mutations are associated with subtypes of leukemia and lymphoma. For instance, the t(9;22) in CML and ALL, t(15;17) in APL, t(8;21) and inv(16)/t(16;16) in CBF leukemia, as well as t(2;5) in ALCL. Moreover, chromosome abnormalities are one of the most reliable predictors of disease prognosis, and instruct treatment choice. Molecular studies have revealed the mechanisms of certain chromosome translocations and other abnormalities in leukemia and lymphoma, and have led to the discovery of gene and mutation specific therapeutic options. The detection of the t(9;22), characterization of the BCR/ABL fusion in CML, and the discovery of Imatinib have elegantly reflected the success of our research efforts in leukemia and other cancers. With many powerful techniques including microarray and next-generation sequencing, we will soon discover more genetic abnormalities and understand how various gene mutations and epigenetic deregulation cooperate to induce leukemia and lymphoma; these insights will eventually lead to personalized medicine.

The recently identified ETV6/CBFA2 (formerly known as TEL/AML1) fusion gene occurs as a result of the t(12;21)(p12;q22). Initial reports have indicated that the fusion transcript occurs in up to 30% of children diagnosed with B-cell precursor (CD10+, CD19+) acute lymphoblastic leukemia (ALL). In order to characterize the incidence of the t(12;21) at both the chromosomal level as well as the RNA transcript level, we have used a combination of classical cytogenetics, reverse transcriptase-polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH) to examine the bone marrow of 34 children diagnosed with B-cell precursor ALL. Nine of the 34 patient samples expressed the ETV6/CBFA2 transcript. When the results of RT-PCR were compared with the conventional karyotype, the fusion was present in 3 of 10 (33%) with chromosome 12 abnormalities, none of whom had an obvious t(12;21). The transcript was also detected in 5 of the 12 (41%) bone marrow samples with other abnormalities and in 1 of 12 (8%) samples with a normal karyotype. Seven of the 9 RT-PCR positive patient samples were studied with FISH. Of the 7, FISH confirmed the ETV6/CBFA2 fusion in 6. One other patient with a 12p abnormality had evidence for the fusion using FISH which was not detected by RT-PCR. Our results not only confirm that the frequency of the t(12;21) is unusually high in childhood B-cell precursor ALL, but also that none of the translocations in our series was detected with conventional cytogenetic techniques.

BNORMALITIES of the short arm of chromosome 12 A are relatively common in childhood acute lymphoblastic leukemia (ALL). Approximately 5% to 7% of children with ALL have cytogenetic evidence of a translocation involving 12p, whereas 3% to 5% have deletions that suggest the presence of a tumor-suppressor gene at this location.’ Through the use of sensitive molecular techniques, 12p or 12~12-13 loss of heterozygosity (LOH) has been shown in approximately 25% of childhood ALL cases.z4 Fluorescence in situ hybridization (FISH) mapping has detected a minimum region of overlap for the 12p deletions between the TEL. and CDKNIB (KIpl) genes.’ Recently, chromosomal translocations involving the TEL gene at 12~13 have been cloned in several hematopoietic In ALL cells with the t(12;21)(p13;q22), the 5’ part of TEL is fused with the AMLl gene.’.” Abnormalities of 12p, especially the t(12;21), are more reliably detected by FISH than by classical cytogenetics because the translocated portions of 12p and 21q are virtually identical cytogenetically.’* When FISH was combined with Southern blotting and reverse transcriptasepolymerase chain reaction (RT-PCR), the t(12;21) was identified as a recurring chromosomal abnormality in 16% to 25% of childhood B-lineage Despite the identification of the fusion partners in the t(12;21), the actual function of the TEL-AML1 and AMLl-TEL fusion proteins in promoting malignant transformation is unclear. Whether the TEL-AMLl fusion alone is necessary and sufficient for malignant transformation in ALL and whether TEL inactivation has a role in leukemogenesis is currently unknown. Loss of the CDKN2 (p16) gene at 9p21 is a common genetic abnormality in ALL and a variety of other malignancies. Homozygous CDKN2 deletions have been detected in approximately 15% of B-lineage ALL and 75% of T-lineage ALL The p16 protein functions as an inhibitor of cyclin-dependent kinase 4, and normally acts to stop cell cycle progres~ion.’~**~ Whether loss of p16 alone is necessary and sufficient for malignant transformation in childhood ALL has not been determined. In this report, 6 cell lines with the TEL-AMLl fusion transcript are described. Although only 1 of the 6 cell lines translocated allele of EL and 5 cell lines lacked expression of CDKN2. Moreover, in 2 patients (1 with the TEL-AMLI transcript and 1 without), EL exprwsion was lost with disease progression; ie, EL was expressed in the initial cell lines (established at diagnosis or first relapse) whereas 7Ef was not expressed in the cell lines established from these patients in late-stage disease. These data show the coexistence of multiple genetic defects in childhood B-lineage ALL. Cell lines with t(12;21) will faciliate the study of TEL-AML1 and AMLl-TEL fusion proteins as well as EL and CDKM gene inactivation in leukemia transformation and progression.

The t(6;11)(q27;q23) is one of the most common translocations observed in patients with acute myeloid leukemia (AML). The translocation breakpoint involves the MLL gene, which is the human homolog of the Drosophila trithorax gene, at 11q23 and the AF6 gene at 6q27. Reverse transcriptase-polymerase chain reaction (RT-PCR) using an MLL sense primer and an AF6 antisense primer detected the MLL/AF6 fusion cDNA from three leukemia patients with the t(6;11) [two AML and one T-acute lymphoblastic leukemia (ALL)] and one cell line. The fusion point in the AF6 cDNA from these cases is identical, regardless of the leukemia phenotype. The ML-2 cell line, which was established from a patient with AML that developed after complete remission of T-cell lymphoma, has retained an 11q23–24 deletion from the lymphoma stage and has acquired the t(6;11) with development of AML. The ML-2 cells have no normal MLL gene on Southern blot analysis, which indicates that an intact MLL gene is not necessary for survival of leukemic cells. Genes Chromosom Cancer 15:206–216 (1996). © 1996 Wiley-Liss, Inc.

A major unresolved question for 11q23 translocations involving MLL is the chromosomal mechanism(s) leading to these translocations. We have mapped breakpoints within the 8.3-kb BamHI breakpoint cluster region in 31 patients with acute lymphoblastic leukemia and acute myeloid leukemia (AML) de novo and in 8 t-AML patients. In 23 of 31 leukemia de novo patients, MLL breakpoints mapped to the centromeric half (4.57 kb) of the breakpoint cluster region, whereas those in eight de novo patients mapped to the telomeric half (3.87 kb). In contrast, only two t-AML breakpoints mapped in the centromeric half, whereas six mapped in the telomeric half. The difference in distribution of the leukemia de novo breakpoints is statistically significant (P = .02). A similar difference in distribution of breakpoints between de novo patients and t-AML patients has been reported by others. We identified a low- or weak-affinity scaffold attachment region (SAR) mapping just centromeric to the breakpoint cluster region, and a high-affinity SAR mapping within the telomeric half of the breakpoint cluster region. Using high stringency criteria to define in vitro vertebrate topoisomerase II (topo II) consensus sites, one topo II site mapped adjacent to the telomeric SAR, whereas six mapped within the SAR. Therefore, 74% of leukemia de novo and 25% of t-AML breakpoints map to the centromeric half of the breakpoint cluster region map between the two SARs; in contrast, 26% of the leukemia de novo and 75% of the t-AML patient breakpoints map to the telomeric half of the breakpoint cluster region that contains both the telomeric SAR and the topo II sites. Thus, the chromatin structure of the MLL breakpoint cluster region may be important in determining the distribution of the breakpoints. The data suggest that the mechanism(s) leading to translocations may differ in leukemia de novo and in t-AML.

We performed fluorescence in situ hybridization (FISH) on bone marrow or peripheral blood cells thought to contain a del(12p) or an unbalanced 12p11-12 translocation from 17 patients who had various hematologic malignant diseases. We used 11 cosmid, phage, and plasmid probes which we had previously ordered on 12p. Cells from three patients with myeloid disorders were shown to have 12p13 translocations that involved chromosome 2 in two of them. Moreover, in all patients, FISH showed that the translocations were associated with proximal interstitial deletions which contributed to the difficulty in identifying these translocations. Our data suggest that some rearrangements of 12p which have been described previously as deletions or unbalanced translocations may, in fact, represent 12p13 translocations accompanied by an interstitial deletion.

Two general types of clonal chromosome abnormality are observed in de novo acute myeloid leukemia (AML): the unbalanced aberrations with visible gain or loss of chromosome material and the balanced aberrations without such visible gain or loss. AML can be induced by therapy with cytostatic drugs and radiation. The alkylating agents reacting directly with DNA induce AML which often presents as myelodysplasia with unbalanced aberrations, primarily loss of chromosome material. Cytostatic agents targeting DNA-topoisomerase II, frequently administered together with alkylating agents or cisplatin, induce the same type of leukemia. In addition, they often induce another type with a more rapid onset and with specific balanced chromosome aberrations rarely observed after therapy with alkylating agents alone. All of the most important chromosome aberrations found in de novo AML are now also found in therapy-related AML (t-AML); thus, t- AML may serve as a model in the search for mechanisms leading to the development of AML in general. Unbalanced chromosome aberrations with partial deletions or with loss of whole chromosomes may develop as a result of alkylation of DNA or other cellular targets. Balanced chromosome aberrations, on the other hand, may develop as illegitimate recombinations related to the activity of DNA-topoisomerase II. The balanced translocations contribute to malignant transformation by the formation of abnormal chimeric genes, whereas deletions may contribute by the loss of putative tumor suppressor genes. In either situation, the chromosome changes provide the altered cells with a proliferative advantage compared with normal cells.

Two genes have been implicated in leukemias of patients with abnormalities of chromosome 3, band q26: EVI1, which can be activated over long distances by chromosomal rearrangements involving 3q26, and EAP, a ribosomal gene that fuses with AML1 in a therapy-related myelodysplasia patient with a t(3;21)(q26.2;q22). AML1 was identified by its involvement in the t(8;21)(q22;q22) of acute myeloid leukemia. Here we report the consistent identification of fusion transcripts between AML1 and EAP or between AML1 and previously unidentified sequences that we named MDS1 (MDS-associated sequences) in the leukemic cells of four patients with therapy-related myelodysplasia/acute myeloid leukemia and in one patient with chronic myelogenous leukemia in blast crisis, all of whom had a t(3;21). In addition, we have identified a third chimeric transcript, AML1/EVI1, in one of the therapy-related acute myeloid leukemia patients. Pulsed-field gel electrophoresis established the order of the genes as EAP, the most telomeric, and EVI1, the most centromeric, gene. The results indicate that translocations could involve multiple genes and affect gene expression over long distances.

Translocations involving chromosome band 11q23 are found in both lymphoid and myeloid leukemias as well as in lymphomas, and we have recently reported that rearrangement of the MLL gene is involved in these translocations. The chromosomes most frequently involved in reciprocal translocations include chromosomes 4, 6, 9, and 19, and we and others have reported that chromosomes 1, 2, 10, 15, 17, 18, 22, and X are also involved. In the cell line Karpas 45, which has a t(X;II) (q13;q23) translocation, we report here that the MLL gene is rearranged and that there are two altered transcripts of MLL that come from the der(11) chromosome. Genes Chrom Cancer 9:221-224 (1994). © 1994 Wiley-Liss, Inc.

Rearrangements of chromosome band 11q23 are common in infant leukemias, comprising more than 70% of the observed chromosome abnormalities in children less than 1 year of age. The MLL gene, which is located at the 11q23 breakpoint in infant, childhood, and adult acute leukemias, has been cloned and has homology to the Drosophila trithorax gene. The breakpoints in MLL are restricted to an 8.3-kilobase pair (kb) region of the gene that is involved in translocations with as many as 29 other chromosomal regions in a number of phenotypically distinct acute leukemias. We have detected an identical, clonal, nonconstitutional rearrangement of the MLL gene in peripheral blood cells from a pair of female infants twins with acute lymphoblastic leukemia (ALL) and a t(11;19)(q23;p13.3). The detection of nonidentical IGH rearrangements suggests that the MLL rearrangement took place in a B-cell precursor or hematopoietic stem cell in one twin which was transferred in utero to the other fetus resulting in ALL with an identical aneuploid karyotype in both infants. We speculate that the other MLL-related infant leukemias may also develop in utero, and that the rearrangements may occur consistently in stem cells or early precursor cells, accounting for the frequency of mixed-lineage leukemia in infants.

The translocation between chromosomes 8 and 21, t(8;21)(q22;q22), is the most frequent abnormality seen in approximately 46% of patients with acute myeloid leukemia with French-America-British (FAB)-M2 morphology and an aneuploid karyotype. The breakpoints in this translocation have been characterized at the molecular level, and the genes involved are AML1 on chromosome 21 and ETO (eight twenty one) on chromosome 8. AML1 has homology to the alpha subunit of the murine polyoma enhancer binding protein, pebp2, and to the segmentation gene, runt, of Drosophila melanogaster. ETO, also called MTG8 (myeloid translocation gene on 8) has no overall homology to known proteins, but it contains two DNA-binding zinc finger motifs and several regions that are proline- and serine-rich. Both AML1 and ETO are thought to be transcription factors because the motifs they contain are found in other transcription factors. Both genes are transcribed from telomere to centromere, and cytogenetic analysis of variant translocations has shown that the critical junction always conserved is on the derivative 8 chromosome. The rearrangement between the two chromosomes results in a fusion gene that contains the 5' region of AML1 including that homologous to runt fused to almost all of ETO. The fusion transcript from the der(8) chromosome is consistently detected in patients with the t(8;21). The translocation can be detected at the molecular level with selected genomic DNA probes from chromosome 21 and from chromosome 8 near the breakpoint in 80-100% of the t(8;21) patients at diagnosis and in relapse, and with reverse transcriptase-polymerase chain reaction (RT-PCR) in all of the patients at diagnosis and in long-term remission. These results indicate that leukemic clones are still circulating in patients who have been in remission for as long as 8 years.

Recurring chromosome changes, particularly those closely associated with specific subtypes of tumors, have provided critical clues to the location of the genes involved in the genesis of these tumors. These, in turn, have led to the cloning of these genes, especially those located at translocation breakpoints in leukemias, lymphomas, and sarcomas. The immediate consequence of the cloning of these genes is to provide DNA probes for various diagnostic tests, including standard Southern blot analysis, the polymerase chain reaction, and fluorescence in situ hybridization. Because many of these chromosome rearrangements are associated with either an improved or a poorer survival, karyotypic abnormalities provide prognostic information. In the future, more sophisticated understanding of the genes involved and of the nature of their altered function should lead to improved therapy.

Chromosome band 11q23 is frequently involved in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) de novo, as well as in myelodysplastic syndromes (MDS) and lymphoma. Five percent to 15% of patients treated with chemotherapy for a primary neoplasm develop therapy-related AML (t-AML) that may show rearrangements, usually translocations involving band 11q23 or, less often, 21q22. These leukemias develop after a relatively short latent period and often follow the use of drugs that inhibit the activity of DNA-topoisomerase II (topo II). We previously identified a gene, MLL (myeloid-lymphoid leukemia or mixed-lineage leukemia), at 11q23 that is involved in the de novo leukemias. We have studied 17 patients with t-MDS/t-AML, 12 of whom had cytogenetically detectable 11q23 rearrangements. Ten of the 12 t-AML patients had received topo II inhibitors and 9 of these, all with balanced translocations of 11q23, had MLL rearrangements on Southern blot analysis. None of the patients who had not received topo II inhibitors showed an MLL rearrangement. Of the 5 patients lacking 11q23 rearrangements, some of whom had monoblastic features, none had an MLL rearrangement, although 4 had received topo II inhibitors. Our study indicates that the MLL gene rearrangements are similar both in AML that develops de novo and in t-AML. The association of exposure to topo II- reactive chemotherapy with 11q23 rearrangements involving the MLL gene in t-AML suggests that topo II may play a role in the aberrant recombination events that occur in this region both in AML de novo and in t-AML.

Translocations involving chromosome band 11q23 are very frequent in both acute lymphoblastic and acute myeloid leukemias and are the most common genetic alteration in infants with leukemia. In all age groups and all phenotypes of leukemia, an 11q23 translocation carries a poor prognosis. A major question has been whether one or several genes on band 11q23 are implicated in these leukemias. Previously, we identified the chromosomal breakpoint region in leukemias with the common 11q23 translocations and subsequently cloned a gene named MLL that spans the 11q23 breakpoint.We isolated a 0.74-kb BamHI fragment from a complementary DAN (cDNA) clone of the MLL gene. To determine the incidence of MLL rearrangements in patients with 11q23 abnormalities, we analyzed DNA from 61 patients with acute leukemia, 3 cell lines derived from such patients, and 20 patients with non-Hodgkin's lymphoma and 11q23 aberrations.The 0.74-kb cDNA probe detected DNA rearrangements in the MLL gene in 58 of the patients with leukemia, in the 3 cell lines, and in 3 of the patients with lymphoma. All the breaks occurred in an 8.3-kb breakpoint cluster region within the MLL gene. The probe identified DNA rearrangements in all 48 patients with the five common 11q23 translocations involving chromosomes 4, 6, 9, and 19, as well as in 16 patients with uncommon 11q23 aberrations. Twenty-one different chromosomal breakpoints involving the MLL gene were detected.MLL gene rearrangements were detected with a single probe and a single restriction-enzyme digest in all DNA samples from patients with the common 11q23 translocations as well as in 16 patients or cell lines with other 11q23 anomalies. The ability to detect an MLL gene rearrangement rapidly and reliably, especially in patients with limited material for cytogenetic analysis, should make it possible to identify patients who have a poor prognosis and therefore require aggressive chemotherapy or marrow transplantation.

In the 8;21 translocation, the AML1 gene, located at chromosome band 21q22, is translocated to chromosome 8 (q22), where it is fused to the ETO gene and transcribed as a chimeric gene. AML1 is the human homolog of the recently cloned mouse gene pebp2 alpha B, homologous to the DNA binding alpha subunit of the polyoma enhancer factor pebp2. AML1 is also involved in a translocation with chromosome 3 that is seen in patients with therapy-related acute myeloid leukemia and myelodysplastic syndrome and in chronic myelogenous leukemia in blast crisis. We have isolated a fusion cDNA clone from a t(3;21) library derived from a patient with therapy-related myelodysplastic syndrome; this clone contains sequences from AML1 and from EAP, which we have now localized to band 3q26. EAP has previously been characterized as a highly expressed small nuclear protein of 128 residues (EBER 1) associated with Epstein-Barr virus small RNA. The fusion clone contains the DNA binding 5' part of AML1 that is fused to ETO in the t(8;21) and, in addition, at least one other exon. The translocation replaces the last nine codons of AML1 with the last 96 codons of EAP. The fusion does not maintain the correct reading frame of EAP and may not lead to a functional chimeric protein.

Fluorescence in situ hybridization (FISH) was performed on bone marrow or peripheral blood cells thought to contain a del(11)(q23q25) from four patients who had acute leukemia or myelodysplasia. Cells from all patients were shown to contain translocations that involved chromosome 6 in three of them. Our data suggest that a large proportion of presumptive del(11)(q23) or del(11)(q23q25) chromosomes may represent previously unidentified translocations that can be detected by FISH. © 1993 Wiley-Liss, Inc.

The translocation between chromosomes 8 and 21, t(8;21) (q22;q22), is the most frequent abnormality in acute myeloid leukemia (AML) with French-American-British type M2 (FAB-M2) morphology. The breakpoints in this translocation have been characterized at the molecular level, and the genes involved are AML1 on chromosome 21 and ETO on chromosome 8. The rearrangement of the two chromosomes results in a fusion gene and in the production of a consistent fusion transcript on the der(8) chromosome. We have used oligonucleotide primers derived from both sides of the fusion cDNA junction and reverse transcription-polymerase chain reaction (RT-PCR) to analyze six AML-M2 patients with a t(8;21) during various stages of their disease. Two patients studied at diagnosis and one studied at first relapse are alive off therapy and in continuous complete remission for 83 to 94 months. We have detected the AML/ETO fusion transcript in recent peripheral blood samples from each of them. Three other patients also had a fusion transcript detected after 1 to 4 months in remission. Two of these patients subsequently relapsed and died whereas the third patient is alive and in continuous complete remission 70 months later. Thus, our preliminary data suggest that cells with the translocation are still circulating in t(8;21) patients in long-term remission. This finding raises serious questions regarding the interpretation of positive results obtained only with this technique that may not be suitable to decide appropriate further treatment for patients in clinical remission.

/1p;&-3qChromosome 11, band q23, is the frequent site of recurring cytogenetic rearrangements in human leukemia. We have cloned and sequenced the breakpoint junctions from a patient who had null-cell acute lymphoblastic leukemia (ALL) with a t(11;14)(q23;q11). The chromosome 14 breakpoints occurred within the TCRD locus, close to two diversity segments. The chromosome 11 breakpoint occurred between two head-to-head heptamer sequences, and junctional diversity was evident at both derivative junctions, suggesting involvement of the V(D)J recombinase. The TCRA/D locus on the normal chromosome 14 had undergone a Vδ2-Dδ3-ΨJα joining. Two phage clones with this VDJ rearrangement were isolated; one of these contained an intra-Jα region deletion. Two clones with the derivative 11 junction were isolated; one of these had a similar, but not identical, deletion. A heptamer-nonamer recognition sequence (located ∼70 kb 5′ to Cα), not associated with a TCR gene coding segment, was found in the immediate vicinity of both 5′ breakpoints. We have designated this sequence 5′del for 5′ deleting element. An intra-Jα region deletion involving this heptamer-nonamer was previously identified in the leukemia cells recovered from a patient who had T-cell ALL. Fifty kilobases of DNA on 11q23 surrounding the breakpoint were cloned and analyzed. No CpG islands or conserved sequences were identified within this region. Fluorescence in situ hybridization analysis showed that this 11q23 breakpoint mapped distal to the MLL gene associated with the recurring breakpoints in the 4;11, 9;11, and 11;19 translocations, distal to the RCK gene associated with an 11;14 translocation, and proximal to the ETSI gene, which is located at 11q24. © 1993 Wiley-Liss, Inc.

The (8;21)(q22;q22) translocation is a frequent karyotypic abnormality seen in approximately 40% of patients with acute myeloid leukemia subtype M2 (AML-M2) and an abnormal karyotype. The translocation interrupts two genes, AML1 on chromosome 21 and ETO on chromosome 8, that are consequently fused in the der(8) chromosome to produce a novel chimeric gene and message. Selected genomic DNA probes from chromosome 21 and from chromosome 8 near the breakpoint junction detect rearrangements in the DNA of about 80% of the patients with the rearrangement at diagnosis and in relapse. We analyzed the DNA of 20 patients with t(8;21) AML by standard Southern blot with probes originating from chromosomes 21 and 8 near the breakpoint junction, and we identified rearranged bands in 17 of the 20 patients at diagnosis and in relapse. We also used the polymerase chain reaction (PCR) with appropriate primers from the AML1 and ETO genes to amplify the cDNAs from a cell line with the t(8;21) and from seven AML patients with the t(8;21). We detected a fused transcript in the cell line and in all of the patients analyzed, including three patients who did not show any rearrangement by Southern blot analysis and one patient in hematologic remission, who later relapsed. Combining the results from Southern blot and PCR analysis, we could detect the t(8;21) in all of the patients tested. These results indicate that, whereas several DNA probes used as genetic markers do detect the t(8;21) in most, but not all Southern blots of patients with AML, PCR amplification with primers from AML1 and ETO can be used as a more sensitive and accurate means for detecting this chromosomal abnormality, and for observing the patients' response to therapy.

Recurring chromosomal abnormalities involving translocations at chromosome 11 band q23 are associated with human myeloid and lymphoid leukemia as well as lymphoma. We have identified the gene located at this break-point and have named it MLL (for myeloid-lymphoid, or mixed-lineage, leukemia). The t(4;11), t(6;11), t(9;11), and t(11;19) are among the most common reciprocal translocations in leukemia cells involving this chromosomal band. We now have evidence that the breakpoints in all of these translocations are clustered within a 9-kilobase (kb) BamHI genomic region of the MLL gene. By Southern blot hybridization using a 0.7-kb BamHI cDNA fragment of the MLL gene called MLL 0.7B, we have detected rearrangements of DNA from cell lines and patient material with an 11q23 translocation in this region. Northern blot analyses indicate that this gene has multiple transcripts, some of which appear to be lineage-specific. In normal pre-B cells, four transcripts of 12.5, 12.0, 11.5, and 2.0 kb are detected. These transcripts are also present in monocytoid cell lines with additional hybridization to a 5.0-kb transcript, indicating that expression of different-sized MLL transcripts may be associated with normal hematopoietic lineage development. In a cell line with a t(4;11), the expression of the 12.5-, 12.0-, and 11.5-kb transcripts is reduced, and there is evidence of three other altered transcripts of 11.5, 11.25, and 11.0 kb. Thus, these 11q23 translocations result in rearrangements of the MLL gene and may lead to altered function(s) of MLL and of other gene(s) involved in the translocation.

The partner gene of MLL was identified in a patient with treatment-related acute myeloid leukemia in which the karyotype suggested t(3;11)(q25;q23). Prior therapy included the DNA topoisomerase II inhibitors, teniposide and doxorubicin. Southern blot analysis indicated that the MLL gene was involved in the translocation. cDNA panhandle polymerase chain reaction (PCR) was used, which does not require partner gene-specific primers, to identify the chimeric transcript. Reverse-transcription of first-strand cDNAs with oligonucleotides containing known MLL sequence at the 5′ ends and random hexamers at the 3′ ends generated templates with an intra-strand loop for PCR. In-frame fusions of either MLLexon 7 or exon 8 with the GMPS (GUANOSINE 5′-MONOPHOSPHATE SYNTHETASE) gene from chromosome band 3q24 were detected. The fusion transcript was alternatively spliced. Guanosine monophosphate synthetase is essential for de novo purine synthesis. GMPS is the first partner gene ofMLL on chromosome 3q and the first gene of this type in leukemia-associated translocations.

Although it was definitely not obvious at first, consistent chromosomal translocations are major contributors to cellular transformation in some leukemias, lymphomas, sarcomas, prostate cancer, and other benign and malignant neoplasms. In the 50 years since the discovery of the Ph chromosome, the elucidation of recurring abnormalities has been an ongoing challenge that has evolved as new technologies allowed an ever more accurate definition of the precise changes in DNA resulting from these abnormalities. As we enter a new era of understanding enriched by gene expression studies, we still know little about the changes in the level of critical proteins, which may be the ultimate effectors of the genetic/epigenetic abnormalities in cancer. Despite remarkable progress in identifying both obvious chromosome abnormalities and subtle changes in DNA such as mutations and small copy-number variations, the impact of this knowledge has been variable. The challenge for the future is to enhance our ability to translate these genetic changes into effective therapies for other malignant diseases.

Bone marrow and peripheral blood were studied from 200 patients with acute leukemia [109 with acute myeloid leukemia (AML), 91 with acute lymphoblastic leukemia (ALL)] who had samples cultured for varying times and who had a mixture of chromosomally abnormal and normal cells. The mean percentage of abnormal metaphase cells increased with culture time. The peak was reached at 48 hours and declined slightly after 72 hours in culture for ALL patients. The mean percentage of abnormal cells increased up to 72 hours in culture for AML patients. In 68 patients (31 AML and 37 ALL), cytogenetic data were available from samples processed with both direct preparations and culture methods. The percentage of abnormal cells increased after culture in 49 patients (23 AML and 26 ALL), while it decreased or remained at the same level in 19 patients. For AML patients, the mean percentage of abnormal cells was significantly different between direct (38%) and cultured preparations (63%), (p less than 0.001). Seven of 9 patients with AML who showed a greater than 50% increase in abnormal cells after culture had either a t(8;21), t(15;17), or abnormalities involving 11q23. The two patients who showed a significant decrease in abnormal cells both had a translocation involving 11q13. Compared with ALL, more AML patients showed greater than 80% abnormal bone marrow metaphase cells at diagnosis or at relapse.

It is hard today, in 2008, when genetic changes in cancer and specifically hematologic malignancies are accepted as central players, to look back 50 years and realize how different the landscape was then. In 1957, knowledge of the correct chromosome number of man was 1 year old,[1][1] and that DNA

We have identified an identical reciprocal translocation between the long arms of chromosomes 3 and 21 with breakpoints at bands 3q26 and 21q22, [t(3;21)(q26;q22)], in the malignant cells from five adult patients with therapy-related myelodysplastic syndrome (t-MDS) or acute myeloid leukemia (t-AML). Primary diagnoses were Hodgkin's disease in two patients and ovarian carcinoma, breast cancer, and polycythemia vera in one patient each. Patients had been treated with chemotherapy including an alkylating agent for their primary disease 1 to 18 years before the development of t-MDS or t-AML. We have not observed the t(3;21) in over 1,500 patients with a myelodysplastic syndrome or acute myeloid leukemia arising de novo or in over 1,000 patients with lymphoid malignancies. We have previously reported that the t(3;21) occurs in Philadelphia chromosome-positive chronic myelogenous leukemia (CML). Thus, the t(3;21) appears to be limited to t-MDS/t-AML and CML, both of which represent malignant disorders of an early hematopoietic precursor cell. These results provide a new focus for the study of therapy-related leukemia at the molecular level.