Insights into Leukemogenesis from Therapy-Related Leukemia
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《新英格兰医药杂志》
Therapy-related acute myeloid leukemia (AML), often presenting as therapy-related myelodysplasia, is the most serious long-term complication of cancer chemotherapy. This disease offers a unique opportunity to study leukemogenesis by relating specific cytogenetic and genetic abnormalities to the biologic effects of cytostatic agents. Two types of drugs, alkylating agents and topoisomerase II inhibitors, have been shown to induce therapy-related leukemia.
High risks of therapy-related myelodysplasia and AML were first reported in patients with multiple myeloma who had been treated with melphalan.1 Subsequently, almost all other alkylating agents in clinical use have been shown to be leukemogenic in patients treated for a wide spectrum of diseases (Table 1). The actuarial risk of therapyrelated AML varied from study to study, but an increase in the risk of AML of 0.25 to 1.0 percent per year, beginning two years after the start of chemotherapy and lasting five to seven years after its cessation, was generally observed.2 The risk was dose-dependent and increased exponentially with age after the age of 40 years,2 paralleling the risk of primary AML in the general population. Differences in the ages of the patients and in the cumulative dose of alkylating agents can explain the wide variations in risk from study to study.
Table 1. Diseases and Drugs with a Reported Risk of Treatment-Related Myelodysplasia or AML.
In addition to alkylating agents, topoisomerase II inhibitors have been shown to be leukemogenic, as first demonstrated in children who received epipodophyllotoxins for acute lymphoblastic leukemia.3 Subsequently, the anthracyclines, mitoxantrone, and the dioxypiperazine derivatives razoxane and bimolane, all of which also inhibit topoisomerase II, were found to be associated with AML after their use in the treatment of various types of primary cancers (Table 1). Accurate assessment of the risk of AML associated with therapy with these drugs has been hampered because patients with cancer often receive alkylating agents, platinum derivatives, and other drugs simultaneously, making it difficult to ascribe an increased risk to a specific agent. In these studies of topoisomerase II inhibitors, the actuarial risk of therapy-related AML varied widely, but in general, it was of the same order of magnitude as the risk after treatment with alkylating agents. Some cases of AML related to topoisomerase II inhibitors had a short latent period — one year or less from the start of therapy. The risk of epipodophyllotoxin-induced AML increased with twice-weekly administration. However, the possibility of dose dependency has been questioned, and the risk has not been shown to increase with the age of the patient. In fact, many cases occur in children.
Chromosomal abnormalities of the same types that are seen in primary AML occur in most cases of therapy-related myelodysplasia and AML. Deletions or loss of chromosome arm 5q or 7q or loss of the entire chromosome (5q–/–5 and 7q–/–7) are characteristic of myelodysplasia and AML that arise after treatment with alkylating agents.4 The karyotypic abnormalities in AML related to topoisomerase II inhibitors, by contrast, are characteristically balanced aberrations with a rearrangement, but no gain or loss, of chromosomal material.4
The chromosomal abnormalities of therapy-related myelodysplasia and AML characterize at least eight genetic pathways.5 Cases with the abnormality 7q–/–7 but normal chromosomes 5, belong to the first pathway, and cases with 5q–/–5, with or without abnormalities of chromosome 7, belong to the second pathway; hematopoietic cells from these two subtypes have different gene-expression profiles.6 The abnormal 5q–/–5 karyotype is closely associated with a point mutation of the p53 gene, and in such cases, there are many additional chromosomal abnormalities and highly rearranged chromosomes, sometimes with gene amplification. The abnormality 7q–/–7, by contrast, is associated with a less complicated karyotype, and a point mutation of the AML1 gene is common.5 Despite considerable research, the critical molecular events underlying 5q–/–5 and 7q–/–7 have not been identified.
Balanced chromosomal aberrations involving the MLL, AML1/CBFB, RARA, or NUP98 genes characterize four other genetic pathways of therapy-related myelodysplasia and AML.5 As in primary AML, these abnormalities result in rearrangements between genes coding for hematopoietic transcription factors and their various partner genes. The result is a loss of function and the development of oncogenes.
About 1 m of double-stranded DNA is packed into the nucleus of a normal cell in a form called supercoiled DNA. To make this intricate tangle accessible to transcription factors and other DNA-binding proteins, supercoiled DNA must be unwound and unknotted by topoisomerases. Topoisomerase II is an ATP-dependent enzyme that cuts both strands of supercoiled DNA, making double-stranded breaks and thereby changing the topology of DNA (Figure 1). The breaks are ultimately repaired when homologous chromosome fragments realign. An important question is whether the balanced chromosomal aberrations of primary AML develop when topoisomerase II–induced double-stranded breaks are incorrectly repaired by crossover recombination — illegitimate joints made between two unrelated (nonhomologous) chromosomes. A related question is whether treatment with topoisomerase II inhibitors, which interferes with ligation of the enzyme-induced breaks in DNA, enhances this mechanism of inducing chromosomal abnormalities.
Figure 1. The Effects of Topoisomerase II Inhibitors.
Normally, topoisomerase II relaxes supercoiled DNA. After cleaving of the G segment of double-stranded DNA (red), strand passage of the unbroken T segment (green) occurs through the break (arrow), and religation of the G segment takes place (Panels A and B). Exposure to a topoisomerase II inhibitor, such as mitoxantrone, is thought to interfere with the strand passage and religation process, thus increasing and stabilizing the number of cleaved complexes (Panel C). After a double-strand break, also of the T segment (Panel D), there is an increased likelihood of crossover recombination with nonhomologous end-joining between the two DNA strands, leading to the development of a balanced chromosomal translocation (Panel E).
Answers to these questions have been sought by examining the genomic breakpoints of the balanced chromosomal aberrations. The breakpoints for translocations to chromosome band 11q23 with rearrangements of the MLL gene,7,8,9 the t(8;21) with rearrangement between the AML1 and the ETO genes,10 and the t(11;20) with rearrangement between the NUP98 and the TOP1 genes11 have been examined, but no specific motifs in nucleic acid sequences were identified at these junctions. However, all studies showed that the breakpoints of the involved genes correlated with cleavage sites in DNA for topoisomerase II.7,8,9,10,11 In two of these studies, the cleavage sites colocalized with DNase-hypersensitive sites that have an open chromatin structure.9,10
In this issue of the Journal, Mistry et al. report on studies of acute promyelocytic leukemia (APL) characterized by t(15;17), which rearranges the PML and RARA genes. The studies included cases of APL that were related to treatment with mitoxantrone or other agents and primary cases of APL.12 With the addition of the study by Mistry et al., all four genetic pathways with balanced chromosomal aberrations have been investigated. The researchers elegantly demonstrated microhomologies at the translocation breakpoints in mitoxantrone-related APL,12 which indicates that nonhomologous end-joining of the broken DNA strands occurred (Figure 1E). In therapy-related AML involving translocations to 11q23, the breakpoints within the MLL gene cluster differently from those of primary AML.9 Likewise, in mitoxantrone-related APL, Mistry et al. observed a clustering of breakpoints within a short 8-bp region of intron 6 of the PML gene, whereas the breakpoints for other therapy-related cases of APL and cases of primary APL were more dispersed within the intron.12 The specific 8-bp region of PML was a particular "hot spot" for mitoxantrone-induced topoisomerase II cleavage.
The development of balanced chromosomal aberrations in AML has turned out to be a complicated process. Despite the results of the new studies, many questions remain. Is topoisomerase II inevitably involved? To what extent are regions of the chromosomal scaffold implicated (the scaffold consists of protein fibers that remain after removal of histones)?9 Do apoptosis-inducing DNases participate?10,11 Are the many cases of therapy-related AML with translocations to 11q23 arising in children who received epipodophyllotoxins and the many cases of APL in women who received mitoxantrone for breast cancer the results of a different targeting of DNA by the two drugs, as suggested by Mistry et al., or are they related to other factors — the patient's age, for instance? After more than 30 years of comprehensive research on the risk of, risk factors for, and pathological and cytogenetic findings in therapy-related myelodysplasia and AML, these diseases now provide new insights into the molecular biology of myelodysplasia and AML in general.
Source Information
From the Cytogenetics Laboratory, Rigshospitalet, Copenhagen.
References
Kyle RA, Pierre RV, Bayrd ED. Multiple myeloma and acute myelomonocytic leukemia: report of four cases possibly related to melphalan. N Engl J Med 1970;283:1121-1125.
Pedersen-Bjergaard J, Specht L, Larsen SO, et al. Risk of therapy-related leukaemia and preleukaemia after Hodgkin's disease: relation to age, cumulative dose of alkylating agents, and time from chemotherapy. Lancet 1987;2:83-88.
Pui C-H, Behm FG, Raimondi SC, et al. Secondary acute myeloid leukemia in children treated for acute lymphoid leukemia. N Engl J Med 1989;321:136-142.
Pedersen-Bjergaard J, Rowley JD. The balanced and the unbalanced chromosome aberrations of acute myeloid leukemia may develop in different ways and may contribute differently to malignant transformation. Blood 1994;83:2780-2786.
Pedersen-Bjergaard J, Andersen MK, Christiansen DH, Nerlov C. Genetic pathways in therapy-related myelodysplasia and acute myeloid leukemia. Blood 2002;99:1909-1912.
Qian Z, Fernald AA, Godley LA, Larson RA, Le Beau MM. Expression profiling of CD34+ hematopoietic stem/progenitor cells reveals distinct subtypes of therapy-related acute myeloid leukemia. Proc Natl Acad Sci U S A 2002;99:14925-14930.
Stanulla M, Wang J, Chervinsky DS, Thandla S, Aplan PD. DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during initial stages of apoptosis. Mol Cell Biol 1997;17:4070-4079.
Felix CA, Lange BJ, Hosler MR, Fertala J, Bjornsti MA. Chromosome band 11q23 translocation breakpoints are DNA topoisomerase II cleavage sites. Cancer Res 1995;55:4287-4292.
Strissel PL, Strick R, Rowley JD, Zeleznik-Le NJ. An in vivo topoisomerase II cleavage site and a DNase I hypersensitive site colocalize near exon 9 in the MLL breakpoint cluster region. Blood 1998;92:3793-3803.
Zhang Y, Strissel P, Strick R, et al. Genomic DNA breakpoints in AML1/RUNX1 and ETO cluster with topoisomerase II DNA cleavage and DNase I hypersensitive sites in t(8;21) leukaemia. Proc Natl Acad Sci U S A 2002;99:3070-3075.
Ahuja HG, Felix CA, Aplan PD. Potential role for DNA topoisomerase II poisons in the generation of t(11;20)(p15;q11) translocations. Genes Chromosomes Cancer 2000;29:96-105.
Mistry AR, Felix CA, Whitmarsh RJ, et al. DNA topoisomerase II in therapy-related acute promyelocytic leukemia. N Engl J Med 2005;352:1529-1538.(Jens Pedersen-Bjergaard, )
High risks of therapy-related myelodysplasia and AML were first reported in patients with multiple myeloma who had been treated with melphalan.1 Subsequently, almost all other alkylating agents in clinical use have been shown to be leukemogenic in patients treated for a wide spectrum of diseases (Table 1). The actuarial risk of therapyrelated AML varied from study to study, but an increase in the risk of AML of 0.25 to 1.0 percent per year, beginning two years after the start of chemotherapy and lasting five to seven years after its cessation, was generally observed.2 The risk was dose-dependent and increased exponentially with age after the age of 40 years,2 paralleling the risk of primary AML in the general population. Differences in the ages of the patients and in the cumulative dose of alkylating agents can explain the wide variations in risk from study to study.
Table 1. Diseases and Drugs with a Reported Risk of Treatment-Related Myelodysplasia or AML.
In addition to alkylating agents, topoisomerase II inhibitors have been shown to be leukemogenic, as first demonstrated in children who received epipodophyllotoxins for acute lymphoblastic leukemia.3 Subsequently, the anthracyclines, mitoxantrone, and the dioxypiperazine derivatives razoxane and bimolane, all of which also inhibit topoisomerase II, were found to be associated with AML after their use in the treatment of various types of primary cancers (Table 1). Accurate assessment of the risk of AML associated with therapy with these drugs has been hampered because patients with cancer often receive alkylating agents, platinum derivatives, and other drugs simultaneously, making it difficult to ascribe an increased risk to a specific agent. In these studies of topoisomerase II inhibitors, the actuarial risk of therapy-related AML varied widely, but in general, it was of the same order of magnitude as the risk after treatment with alkylating agents. Some cases of AML related to topoisomerase II inhibitors had a short latent period — one year or less from the start of therapy. The risk of epipodophyllotoxin-induced AML increased with twice-weekly administration. However, the possibility of dose dependency has been questioned, and the risk has not been shown to increase with the age of the patient. In fact, many cases occur in children.
Chromosomal abnormalities of the same types that are seen in primary AML occur in most cases of therapy-related myelodysplasia and AML. Deletions or loss of chromosome arm 5q or 7q or loss of the entire chromosome (5q–/–5 and 7q–/–7) are characteristic of myelodysplasia and AML that arise after treatment with alkylating agents.4 The karyotypic abnormalities in AML related to topoisomerase II inhibitors, by contrast, are characteristically balanced aberrations with a rearrangement, but no gain or loss, of chromosomal material.4
The chromosomal abnormalities of therapy-related myelodysplasia and AML characterize at least eight genetic pathways.5 Cases with the abnormality 7q–/–7 but normal chromosomes 5, belong to the first pathway, and cases with 5q–/–5, with or without abnormalities of chromosome 7, belong to the second pathway; hematopoietic cells from these two subtypes have different gene-expression profiles.6 The abnormal 5q–/–5 karyotype is closely associated with a point mutation of the p53 gene, and in such cases, there are many additional chromosomal abnormalities and highly rearranged chromosomes, sometimes with gene amplification. The abnormality 7q–/–7, by contrast, is associated with a less complicated karyotype, and a point mutation of the AML1 gene is common.5 Despite considerable research, the critical molecular events underlying 5q–/–5 and 7q–/–7 have not been identified.
Balanced chromosomal aberrations involving the MLL, AML1/CBFB, RARA, or NUP98 genes characterize four other genetic pathways of therapy-related myelodysplasia and AML.5 As in primary AML, these abnormalities result in rearrangements between genes coding for hematopoietic transcription factors and their various partner genes. The result is a loss of function and the development of oncogenes.
About 1 m of double-stranded DNA is packed into the nucleus of a normal cell in a form called supercoiled DNA. To make this intricate tangle accessible to transcription factors and other DNA-binding proteins, supercoiled DNA must be unwound and unknotted by topoisomerases. Topoisomerase II is an ATP-dependent enzyme that cuts both strands of supercoiled DNA, making double-stranded breaks and thereby changing the topology of DNA (Figure 1). The breaks are ultimately repaired when homologous chromosome fragments realign. An important question is whether the balanced chromosomal aberrations of primary AML develop when topoisomerase II–induced double-stranded breaks are incorrectly repaired by crossover recombination — illegitimate joints made between two unrelated (nonhomologous) chromosomes. A related question is whether treatment with topoisomerase II inhibitors, which interferes with ligation of the enzyme-induced breaks in DNA, enhances this mechanism of inducing chromosomal abnormalities.
Figure 1. The Effects of Topoisomerase II Inhibitors.
Normally, topoisomerase II relaxes supercoiled DNA. After cleaving of the G segment of double-stranded DNA (red), strand passage of the unbroken T segment (green) occurs through the break (arrow), and religation of the G segment takes place (Panels A and B). Exposure to a topoisomerase II inhibitor, such as mitoxantrone, is thought to interfere with the strand passage and religation process, thus increasing and stabilizing the number of cleaved complexes (Panel C). After a double-strand break, also of the T segment (Panel D), there is an increased likelihood of crossover recombination with nonhomologous end-joining between the two DNA strands, leading to the development of a balanced chromosomal translocation (Panel E).
Answers to these questions have been sought by examining the genomic breakpoints of the balanced chromosomal aberrations. The breakpoints for translocations to chromosome band 11q23 with rearrangements of the MLL gene,7,8,9 the t(8;21) with rearrangement between the AML1 and the ETO genes,10 and the t(11;20) with rearrangement between the NUP98 and the TOP1 genes11 have been examined, but no specific motifs in nucleic acid sequences were identified at these junctions. However, all studies showed that the breakpoints of the involved genes correlated with cleavage sites in DNA for topoisomerase II.7,8,9,10,11 In two of these studies, the cleavage sites colocalized with DNase-hypersensitive sites that have an open chromatin structure.9,10
In this issue of the Journal, Mistry et al. report on studies of acute promyelocytic leukemia (APL) characterized by t(15;17), which rearranges the PML and RARA genes. The studies included cases of APL that were related to treatment with mitoxantrone or other agents and primary cases of APL.12 With the addition of the study by Mistry et al., all four genetic pathways with balanced chromosomal aberrations have been investigated. The researchers elegantly demonstrated microhomologies at the translocation breakpoints in mitoxantrone-related APL,12 which indicates that nonhomologous end-joining of the broken DNA strands occurred (Figure 1E). In therapy-related AML involving translocations to 11q23, the breakpoints within the MLL gene cluster differently from those of primary AML.9 Likewise, in mitoxantrone-related APL, Mistry et al. observed a clustering of breakpoints within a short 8-bp region of intron 6 of the PML gene, whereas the breakpoints for other therapy-related cases of APL and cases of primary APL were more dispersed within the intron.12 The specific 8-bp region of PML was a particular "hot spot" for mitoxantrone-induced topoisomerase II cleavage.
The development of balanced chromosomal aberrations in AML has turned out to be a complicated process. Despite the results of the new studies, many questions remain. Is topoisomerase II inevitably involved? To what extent are regions of the chromosomal scaffold implicated (the scaffold consists of protein fibers that remain after removal of histones)?9 Do apoptosis-inducing DNases participate?10,11 Are the many cases of therapy-related AML with translocations to 11q23 arising in children who received epipodophyllotoxins and the many cases of APL in women who received mitoxantrone for breast cancer the results of a different targeting of DNA by the two drugs, as suggested by Mistry et al., or are they related to other factors — the patient's age, for instance? After more than 30 years of comprehensive research on the risk of, risk factors for, and pathological and cytogenetic findings in therapy-related myelodysplasia and AML, these diseases now provide new insights into the molecular biology of myelodysplasia and AML in general.
Source Information
From the Cytogenetics Laboratory, Rigshospitalet, Copenhagen.
References
Kyle RA, Pierre RV, Bayrd ED. Multiple myeloma and acute myelomonocytic leukemia: report of four cases possibly related to melphalan. N Engl J Med 1970;283:1121-1125.
Pedersen-Bjergaard J, Specht L, Larsen SO, et al. Risk of therapy-related leukaemia and preleukaemia after Hodgkin's disease: relation to age, cumulative dose of alkylating agents, and time from chemotherapy. Lancet 1987;2:83-88.
Pui C-H, Behm FG, Raimondi SC, et al. Secondary acute myeloid leukemia in children treated for acute lymphoid leukemia. N Engl J Med 1989;321:136-142.
Pedersen-Bjergaard J, Rowley JD. The balanced and the unbalanced chromosome aberrations of acute myeloid leukemia may develop in different ways and may contribute differently to malignant transformation. Blood 1994;83:2780-2786.
Pedersen-Bjergaard J, Andersen MK, Christiansen DH, Nerlov C. Genetic pathways in therapy-related myelodysplasia and acute myeloid leukemia. Blood 2002;99:1909-1912.
Qian Z, Fernald AA, Godley LA, Larson RA, Le Beau MM. Expression profiling of CD34+ hematopoietic stem/progenitor cells reveals distinct subtypes of therapy-related acute myeloid leukemia. Proc Natl Acad Sci U S A 2002;99:14925-14930.
Stanulla M, Wang J, Chervinsky DS, Thandla S, Aplan PD. DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during initial stages of apoptosis. Mol Cell Biol 1997;17:4070-4079.
Felix CA, Lange BJ, Hosler MR, Fertala J, Bjornsti MA. Chromosome band 11q23 translocation breakpoints are DNA topoisomerase II cleavage sites. Cancer Res 1995;55:4287-4292.
Strissel PL, Strick R, Rowley JD, Zeleznik-Le NJ. An in vivo topoisomerase II cleavage site and a DNase I hypersensitive site colocalize near exon 9 in the MLL breakpoint cluster region. Blood 1998;92:3793-3803.
Zhang Y, Strissel P, Strick R, et al. Genomic DNA breakpoints in AML1/RUNX1 and ETO cluster with topoisomerase II DNA cleavage and DNase I hypersensitive sites in t(8;21) leukaemia. Proc Natl Acad Sci U S A 2002;99:3070-3075.
Ahuja HG, Felix CA, Aplan PD. Potential role for DNA topoisomerase II poisons in the generation of t(11;20)(p15;q11) translocations. Genes Chromosomes Cancer 2000;29:96-105.
Mistry AR, Felix CA, Whitmarsh RJ, et al. DNA topoisomerase II in therapy-related acute promyelocytic leukemia. N Engl J Med 2005;352:1529-1538.(Jens Pedersen-Bjergaard, )