Ann. N.Y. Acad. Sci. ISSN 0077-8923
A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S Issue: The Bone Marrow Niche, Stem Cells, and Leukemia: Impact of Drugs, Chemicals, and the Environment
Cytogenetics in benzene-associated myelodysplastic syndromes and acute myeloid leukemia: new insights into a disease continuum Richard D. Irons1,2,3 and Patrick J. Kerzic3 1
Department of Hematology, Huashan Hospital, Fudan University, Shanghai, China. 2 Anshutz Medical Center, University of Colorado, Aurora, Colorado. 3 Cinpathogen, Inc., Boulder, Colorado Address for correspondence: Richard D. Irons, Cinpathogen, Inc., 4800 Baseline Rd. E104 PMB253, Boulder, CO 80303. [email protected]
Hematopoiesis in health and disease results from complex interactions between primitive hematopoietic stem cells (HSCs) and the extrinsic influences of other cells in the bone marrow (BM) niche. Advances in stem cell biology, molecular genetics, and computational biology reveal that the immortality, self-renewal, and maintenance of blood homeostasis generally attributed to individual HSCs are functions of the cells’ behavior in the normal BM environment. Here we discuss how these advances, together with results of outcomes-based clinical epidemiology studies, provide new insight into the importance of epigenetic events in leukemogenesis. For the chemical benzene (Bz), development of myeloid neoplasms depends predominantly on alterations within the microenvironments in which they arise. The primary persistent disease in Bz myelotoxicity is myelodysplastic syndrome, which precedes cytogenetic injury. Evidence indicates that acute myeloid leukemia arises as a secondary event, subsequent to evolution of the leukemia-initiating cell phenotype within the altered BM microenvironment. Further explorations into the nature of chemical versus de novo disease should consider this mechanism, which is biologically distinct from previous models of clonal cytogenetic injury. Understanding alterations of homeostatic regulation in the BM niche is important for validation of models of leukemogenesis, monitoring at-risk populations, and development of novel treatment and prevention strategies. Keywords: AML; MDS; benzene; cytogenetics
Perhaps 90% of what we know about hematopoietic stem cell (HSC) regulation we have learned over the last two decades. Therefore, it is not surprising that many historical descriptions of the role of benzene (Bz) in the pathogenesis of hematopoietic disease lack today’s sophisticated context. Recently, we have made significant progress in reconciling the clinical and molecular genetic characteristics of myeloid neoplasia associated with Bz exposure with models of altered homeostatic regulation of HSCs in the microenvironment of the bone marrow (BM) niche. The history of Bz toxicity Bz is the simplest aromatic solvent and a major constituent in the manufacture of numerous chemicals. Occurring naturally in petroleum, Bz
is an unavoidable trace contaminant of gasoline and a universal by-product of combustion. Owing largely to its use as a solvent in the first half of the 20th century, Bz myelotoxicity became synonymous with BM suppression. BM failure following chronic occupational exposure to Bz was recognized before 1900,1 and anecdotal studies repeatedly implicated Bz as a cause of pancytopenia, BM failure, and aplastic anemia (AA).2,3 Early appreciation that an agent producing BM suppression might also increase BM cellularity, as observed in leukemia, emerged in the 1940s. Recognition of the increased complexity of hematopathology associated with chronic Bz poisoning is attributed to Francis Hunter, who described cases of myeloid hyperplasia, hypereosinophilia, and leukemia superimposed on the then familiar picture of BM doi: 10.1111/nyas.12336
84
C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1310 (2014) 84–88
Benzene AML closely resembles de novo AML
Irons & Kerzic
failure following occupational exposure to high levels of Bz.4 Young described the BM failure associated with Bz exposure to be intermediate between that observed following treatment with cytotoxic chemotherapy and idiosyncratic drug reactions, and was among the first to draw attention to the potential overlap between AA and hypoplastic myelodysplastic syndrome (MDS).5 Gross et al. finally brought closure to the anecdotal evidence for a relationship between AA and Bz and confirmed a quantitative increase in the risk of AA associated with chronic Bz exposure in 2010.6 Acute myeloid leukemia (AML) and MDS The successful introduction of radiation and alkylating chemotherapy in the 1970s for the treatment of certain cancers, for example, Hodgkin’s lymphoma, led to recognition of therapy-related AML (t-AML) and therapy-related MDS (t-MDS) as serious adverse side effects. Both t-AML and t-MDS are distinguished by an extremely high prevalence of clonal cytogenetic abnormalities involving chromosomes 5 and 7, as well as aneuploidy.7–9 Due largely to widespread acknowledgement of t-AML developing secondary to chemotherapy, the risk of AML associated with chronic Bz exposure has been recognized.10,11 The technologies supporting cytogenetic analysis for use in clinical epidemiology were in their infancy and not widely applied, and many scientists surmised that the pathogenesis of Bz-AML would be similar to t-AML described following treatment with alkylating chemotherapy.12–14 For the most part, quantitative retrospective epidemiology studies have confirmed an increased risk of AML following occupational exposure to Bz; however, comparable studies to evaluate the risk of MDS associated with previous exposure to Bz were not performed until after the classification of MDS as a neoplastic disease by the WHO Classification of Tumors of Hematopoietic and Lymphoid Tissue in 2001.15 This was the result of a number of factors. First, unlike AML’s dramatic and life-threatening onset, MDS is insidious and even now is often diagnosed only incidental to routine physical examination. Further, MDS was not uniformly reported as a neoplastic disease until its inclusion in WHO beginning in 2001.15 Finally, the definition, biological scope, and pathogenesis of MDS, which continue to evolve, were expanded in 2008 and remain controversial today.16
The results of subsequent quantitative epidemiology studies suggest that widespread emphasis on the increased risk of AML associated with Bz exposure may have been exaggerated, and that the dose-dependent risk of MDS following Bz exposure is actually more robust than that observed for AML.17–19 Bz toxicology in the modern era The last two decades have witnessed extraordinary growth in molecular biology, genetics, and medical technology and a revolution in the application of evidence-based methods in modern medicine. Formally heralded by the introduction of WHO 2001, the definition and classification of distinct clinical entities has been expanded from a retrospective description of morphology to a combined criteria of morphology, immunophenotype, molecular genetics, and clinical features. The success of this approach has highlighted the importance and power of interrogating the causes and pathogenesis of individual diseases in situ, that is, within the living environment of the host. It is in this context that advances in stem cell biology, molecular genetics, and computational biology have refined our current understanding of the HSC. Previously, the variety of characteristics and capabilities of HSCs could only be indirectly inferred through the use of clonal functional assays, and were consequently attributed to a single cell. The capabilities traditionally attributed to individual HSCs are now recognized as resulting from the collaboration of a community of cells, including primitive, progenitor, and stromal cells composing a feedback network within the microenvironment of the BM niche. Our group characterized hematopoietic and lymphoid diseases for 2,923 consecutive patients prospectively diagnosed in our laboratory in Shanghai utilizing WHO criteria. We described the clinical, phenotypic, and molecular genetic characteristics of MDS developing in 649 patients, 80 of whom were documented to have some Bz exposure, and 722 cases of AML, including 78 with documented Bz exposure.18,20,21 Inasmuch as t-MDS and t-AML are considered to be overlapping entities with a common pathogenesis, we were surprised to discover that MDS presenting in individuals with the highest chronic exposure to Bz did not exhibit the pattern of cytogenetic, phenotypic, or clinical
C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1310 (2014) 84–88
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Benzene AML closely resembles de novo AML
Irons & Kerzic
Table 1. Prevalence (%) and relative risk of cytogenetic features in Bz-AML and de novo AML in Shanghai, China, and t-AML from a pooled series of international studies
Prevalence (%)
Aneuploidy –5/5q– –7/7q– 11q23 t(8;21) t(15;17)
Bz-AML (n = 78)
De novo AML (n = 644)
t-AML (n = 178)
RR (95% CI)
23 5.13 7.69 2.56 14.10 23.08
23 6.52 6.06 4.97 8.54 15.58
NA 23.03 24.72 7.29 6.18 3.37
NA 0.22 (0.10–0.52) 0.31 (0.15–0.64) 0.35 (0.08–1.52) 2.28 (1.05–4.97) 6.85 (3.21–14.62)
Note: RR is the risk of clonal abnormalities in the Shanghai Bz-exposed AML series compared to other center-based series of t-AML in which clonal cytogenetic abnormalities were analyzed.
abnormalities typically observed in t-MDS. In fact, these subjects were found to have a slightly lower prevalence of clonal cytogenetic abnormalities (24%) when compared to unexposed (i.e., de novo) MDS cases (30%; n = 569). No increase in clonal deletions involving all or part of chromosomes 5 or 7 were demonstrated in Bz-exposed MDS relative to unexposed MDS cases, and no case involving –5/5q– as the sole abnormality was observed for Bzexposed MDS.21 As in previous historical accounts, the predominant morphologic pattern in BM was hypocellular with eosinophilic dysplasia. This was accompanied by persistent immune-mediated inflammation and monoclonal or oligoclonal expansion of activated cytotoxic T cell populations.21,22 Indicative of an autoimmune process, cases of Bz-exposed MDS demonstrate positive clinical responses to immunosuppressive therapy and exhibit characteristics essentially identical to those described in evolving MDS with immune features.23–25 Cell-specific toxicity to myeloid cells by a variety of agents, including Bz, involves peroxidation mediated by myeloperoxidase (MPO), which catalyzes the formation of hypochlorous acid.26 MPO expression is the defining marker of myeloid differentiation in hematopoiesis and is first expressed in the subset of CD34+ CD38+ CD33+ myeloid progenitor cells (MPCs) that are committed to producing granulocytic and monocytic lineages.27,28 The emergence of cytotoxic T cell inflammation in the BM following chronic Bz myelotoxicity may be explained, in part, by the observation that MPO, normally expressed by myeloid cells in the BM, directly blocks antigen presentation and suppresses T cell–driven inflammatory response.29 This raises the distinct 86
possibility that early nonclonal dysplastic changes associated with chronic MPC dysfunction following exposure to Bz may be driven by cytotoxic T cells generated against dysplasia-related antigens originally expressed in MPC, as a consequence of MPOdependent cytoxicity. Consistent with the lack of clonal cytogenetic abnormalities in Bz-MDS, AML cases with previous Bz exposure do not exhibit an increased frequency of aneuploidy, numerical cytogenetic lesions commonly found in t-AML series such as –5/5q–, –7/7q–, or lesions involving 11q23.20 On the other hand, Bz-AML cases are more likely to possess karyotypes typically found in de novo AML, that is, normal karyotypes or recurrent abnormalities with increases in balanced translocations, such as t(8;21) and t(15;17) (Table 1). The pathogenesis of myeloid neoplasms Both BM failure and leukemogenesis find common origins in the evolution of the myelodysplastic processes occurring in the BM. One question of great importance concerns the origin and pathogenesis of recurrent balanced translocations involving t(8;21) and t(15;17) observed in AML associated with Bz exposure. Leukemia-initiating cells (LICs) in AML with t(8;21) are markedly less prevalent and more primitive than MPCs, do not form colonyforming units in semisolid medium, and possess lineage-negative phenotypes that do not express MPO.30–33 Although the identity of LICs in acute promyelocytic leukemia with t(15;17) is less certain, multiple lines of evidence suggest a primitive lineage-negative cell of origin in most cases.34–37 Inasmuch as MPO-dependent myelotoxicity
C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1310 (2014) 84–88
Benzene AML closely resembles de novo AML
Irons & Kerzic
implicates agent-specific targeting of committed MPCs, it is not consistent with direct targeting of primitive lineage-negative hematopoietic cells. A possible explanation involves disruption of the extrinsic homeostatic feedback mechanisms normally provided by MPCs that govern proliferation versus quiescence, self-renewal, and differentiation of HSCs in the BM niche.38–40 The mechanical disruption of homeostatic feedback networks alone in committed MPCs has been demonstrated to result in uncontrolled proliferation of HSCs. This appears to be both necessary and sufficient to undermine the replicative integrity of these primitive cells and result in the emergence of a leukemic stem cell phenotype.41 During his personal odyssey of discovery in the 1970s and 1980s, Bruce Ames first suggested that the risk of human cancer equated with mutagenesis;42 however, he subsequently concluded that loss of control over target-cell proliferation was the defining risk.43 Today, this observation finds renewed meaning in the maintenance of HSC quiescence in the BM niche, which is thought to be of paramount importance in preserving the genomic integrity of these primitive cells.40 Because our observations reveal a pattern of abnormalities in AML following exposure to Bz that more closely resembles de novo than therapy-related disease, they may have broad implications for understanding the pathogenesis of de novo AML and, consequently, its detection, treatment, and prevention. The disruption of feedback signaling by MPCs, not themselves clonal targets of leukemic transformation, leads to unregulated proliferation in primitive HSCs. Consequently, recurrent cytogenetic abnormalities, including balanced translocations, but not aneuploidy, emerge as predictable events leading to leukemic transformation in Bz-related neoplasms. Conflicts of interest This work was supported by the authors themselves with no third-party funds. References 1. Santesson, C. 1897. Uber chronische vergiftung mit steinkohlenteerbenzin: vier todesfalle. Arch. Hyg. Berl. 31: 336–376. 2. Aksoy, M. et al. 1972. Details of blood changes in 32 patients with pancytopenia associated with long-term exposure to benzene. Br. J. Ind. Med. 29: 56–64.
3. Young, N.S. 1988. “Drugs and chemicals as agents of bone marrow failure.” In Hematopoiesis: Long Term Effects of Chemotherapy and Radiation. N.G. Testa & R.P. Gale, Eds. Pg. 131. New York: Marcel Dekker, Inc. 4. Hunter, F. 1939. Chronic exposure to benzene (benzol) II: the clinical effects. J. Indust. Hyg. Toxicol. 21: 331–354. 5. Young, N.S. & B.P. Alter. 1994. Aplastic Anemia: Acquired and Inherited. Philadelphia: W.B. Saunders Co. 6. Gross, S. et al. 2010. A hospital-based case control study of aplastic anemia in Shanghai, China. Chem. Biol. Interact. 184: 165–173. 7. Rowley, J.D. et al. 1981. Nonrandom chromosome abnormalities in acute leukemia and dysmyelopoietic syndromes in patients with previously treated malignant disease. Blood 58: 759–767. 8. Le Beau, M.M. et al. 1986. Clinical and cytogenetic correlations in 63 patients with therapy-related myelodysplastic syndromes and acute nonlymphocytic leukemia: further evidence for characteristic abnormalities of chromosomes no. 5 and 7. J. Clin. Oncol. 4: 325–345. 9. Smith, S.M. et al. 2003. Clinical-cytogenetic associations in 306 patients with therapy-related myelodysplasia and myeloid leukemia: the University of Chicago series. Blood 102: 43–52. 10. Aksoy, M. et al. 1971. Haematological effects of chronic benzene poisoning in 217 workers. Br. J. Ind. Med. 28: 296– 302. 11. Infante, P.F. et al. 1977. Leukaemia in benzene workers. Lancet 2: 76–78. 12. Irons, R.D. & W.S. Stillman. 1996. The process of leukemogenesis. Environ. Health Perspect. 104: 1239–1246. 13. Smith, M.T. & L.P. Zhang. 1998. Biomarkers of leukemia risk: benzene as a model. Environ. Health Perspect. 106(Suppl. 4): 937–946. 14. Zhang, L. et al. 2002. The nature of chromosomal aberrations detected in humans exposed to benzene. Crit. Rev. Toxicol. 32: 1–42. 15. WHO. 2001. Pathology and Genetics of Tumours of the Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press. 16. WHO. 2008. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press. 17. Hayes, R.B. et al. 1997. Benzene and the dose-related incidence of hematologic neoplasms in China. J. Natl. Cancer Inst. 89: 1065–1071. 18. Wong, O. et al. 2010. A hospital-based case-control study of acute myeloid leukemia in Shanghai: analysis of environmental and occupational risk factors by subtypes of the WHO classification. Chem. Biol. Interact. 184: 112–128. 19. Schnatter, A.R. et al. 2012. Myelodysplastic syndrome and benzene exposure among petroleum workers: an international pooled analysis. J. Natl. Cancer Inst. 104: 1724–1737. 20. Irons, R.D. et al. 2013. Acute myeloid leukemia following exposure to benzene more closely resembles de novo than therapy related-disease. Genes Chromosomes Cancer 52: 887– 894. 21. Irons, R.D. et al. 2010. Integrating WHO 2001–2008 criteria for the diagnosis of myelodysplastic syndrome (MDS): a case-case analysis of benzene exposure. Chem. Biol. Interact. 184: 30–38.
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22. Irons, R.D. et al. 2005. Chronic exposure to benzene results in a unique form of dysplasia. Leuk. Res. 29: 1371–1380. 23. Song, Y. et al. 2010. Immunosuppressive therapy of cyclosporin A for severe benzene-induced hameatopoietic disorders and a 6-month follow-up. Chem. Biol. Interact. 186: 96–102. 24. Kordasti, S.Y. et al. 2009. IL-17-producing CD4+ T cells, pro-inflammatory cytokines and apoptosis are increased in low risk myelodysplastic syndrome. Br. J. Haematol. 145: 64–72. 25. Aggarwal, S. et al. 2011. Role of immune responses in the pathogenesis of low-risk MDS and high-risk MDS: implications for immunotherapy. Br. J. Haematol. 153: 568–581. 26. Schattenberg, D.G. et al. 1994. Peroxidase activity in murine and human hematopoietic progenitor cells. Potential relevance to benzene-induced toxicity. Mol. Pharmacol. 46: 346– 351. 27. Strobl, H. et al. 1993. Myeloperoxidase expression in CD34+ normal human hematopoietic cells. Blood 82: 2069–2078. 28. Vlasova, II et al. 2011. Myeloperoxidase-dependent oxidation of etoposide in human myeloid progenitor CD34+ cells. Mol. Pharmacol. 79: 479–487. 29. Odobasic, D. et al. 2013. Neutrophil myeloperoxidase regulates T-cell-driven tissue inflammation in mice by inhibiting dendritic cell function. Blood 121: 4195–4204. 30. Bonnet, D. & J.E. Dick. 1997. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 3: 730–737. 31. Wittebol, S. et al. 1998. In AML t(8;21) colony growth of both leukemic and residual normal progenitors is restricted to the CD34+ lineage-negative fraction. Leukemia 12: 1782–1788.
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32. Jordan, C.T. 2002. Unique molecular and cellular features of acute myelogenous leukemia stem cells. Leukemia 16: 559– 562. 33. Warner, J.K. et al. 2004. Concepts of human leukemic development. Oncogene 23: 7164–7177. 34. Edwards, R.H. et al. 1999. Evidence for early hematopoietic progenitor cell involvement in acute promyelocytic leukemia. Am. J. Clin. Pathol. 112: 819–827. 35. Grimwade, D. et al. 2002. The T-lineage-affiliated CD2 gene lies within an open chromatin environment in acute promyelocytic leukemia cells. Cancer Res. 62: 4730–4735. 36. Grimwade, D. & T. Enver. 2004. Acute promyelocytic leukemia: where does it stem from? Leukemia 18: 375–384. 37. Puccetti, E. & M. Ruthardt. 2004. Acute promyelocytic leukemia: PML/RARa and the leukemic stem cell. Leukemia 18: 1169–1175. 38. Wilson, A. & A. Trumpp. 2006. Bone marrow hematopoietic stem cell niches. Nat. Rev. Immunol. 6: 93–106. 39. Wilson, A. et al. 2008. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homoestasis and repair. Cell 135: 1118–1129. 40. Pietras, E. et al. 2011. Cell cycle regulation in hematopoietic stem cells. J. Cell Biol. 195: 709–720. 41. Kirouac, D. et al. 2009. Cell-cell interaction networks regulate blood stem and progenitor cell fate. Mol. Syst. Biol. 5: 293–299. 42. Ames, B.N. et al. 1973. Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. U. S. A. 70: 2281–2285. 43. Ames, B.N. et al. 1987. Ranking possible carcinogenic hazards. Science 236: 271–280.
C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1310 (2014) 84–88
A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S Issue: The Bone Marrow Niche, Stem Cells, and Leukemia: Impact of Drugs, Chemicals, and the Environment
Cytogenetics in benzene-associated myelodysplastic syndromes and acute myeloid leukemia: new insights into a disease continuum Richard D. Irons1,2,3 and Patrick J. Kerzic3 1
Department of Hematology, Huashan Hospital, Fudan University, Shanghai, China. 2 Anshutz Medical Center, University of Colorado, Aurora, Colorado. 3 Cinpathogen, Inc., Boulder, Colorado Address for correspondence: Richard D. Irons, Cinpathogen, Inc., 4800 Baseline Rd. E104 PMB253, Boulder, CO 80303. [email protected]
Hematopoiesis in health and disease results from complex interactions between primitive hematopoietic stem cells (HSCs) and the extrinsic influences of other cells in the bone marrow (BM) niche. Advances in stem cell biology, molecular genetics, and computational biology reveal that the immortality, self-renewal, and maintenance of blood homeostasis generally attributed to individual HSCs are functions of the cells’ behavior in the normal BM environment. Here we discuss how these advances, together with results of outcomes-based clinical epidemiology studies, provide new insight into the importance of epigenetic events in leukemogenesis. For the chemical benzene (Bz), development of myeloid neoplasms depends predominantly on alterations within the microenvironments in which they arise. The primary persistent disease in Bz myelotoxicity is myelodysplastic syndrome, which precedes cytogenetic injury. Evidence indicates that acute myeloid leukemia arises as a secondary event, subsequent to evolution of the leukemia-initiating cell phenotype within the altered BM microenvironment. Further explorations into the nature of chemical versus de novo disease should consider this mechanism, which is biologically distinct from previous models of clonal cytogenetic injury. Understanding alterations of homeostatic regulation in the BM niche is important for validation of models of leukemogenesis, monitoring at-risk populations, and development of novel treatment and prevention strategies. Keywords: AML; MDS; benzene; cytogenetics
Perhaps 90% of what we know about hematopoietic stem cell (HSC) regulation we have learned over the last two decades. Therefore, it is not surprising that many historical descriptions of the role of benzene (Bz) in the pathogenesis of hematopoietic disease lack today’s sophisticated context. Recently, we have made significant progress in reconciling the clinical and molecular genetic characteristics of myeloid neoplasia associated with Bz exposure with models of altered homeostatic regulation of HSCs in the microenvironment of the bone marrow (BM) niche. The history of Bz toxicity Bz is the simplest aromatic solvent and a major constituent in the manufacture of numerous chemicals. Occurring naturally in petroleum, Bz
is an unavoidable trace contaminant of gasoline and a universal by-product of combustion. Owing largely to its use as a solvent in the first half of the 20th century, Bz myelotoxicity became synonymous with BM suppression. BM failure following chronic occupational exposure to Bz was recognized before 1900,1 and anecdotal studies repeatedly implicated Bz as a cause of pancytopenia, BM failure, and aplastic anemia (AA).2,3 Early appreciation that an agent producing BM suppression might also increase BM cellularity, as observed in leukemia, emerged in the 1940s. Recognition of the increased complexity of hematopathology associated with chronic Bz poisoning is attributed to Francis Hunter, who described cases of myeloid hyperplasia, hypereosinophilia, and leukemia superimposed on the then familiar picture of BM doi: 10.1111/nyas.12336
84
C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1310 (2014) 84–88
Benzene AML closely resembles de novo AML
Irons & Kerzic
failure following occupational exposure to high levels of Bz.4 Young described the BM failure associated with Bz exposure to be intermediate between that observed following treatment with cytotoxic chemotherapy and idiosyncratic drug reactions, and was among the first to draw attention to the potential overlap between AA and hypoplastic myelodysplastic syndrome (MDS).5 Gross et al. finally brought closure to the anecdotal evidence for a relationship between AA and Bz and confirmed a quantitative increase in the risk of AA associated with chronic Bz exposure in 2010.6 Acute myeloid leukemia (AML) and MDS The successful introduction of radiation and alkylating chemotherapy in the 1970s for the treatment of certain cancers, for example, Hodgkin’s lymphoma, led to recognition of therapy-related AML (t-AML) and therapy-related MDS (t-MDS) as serious adverse side effects. Both t-AML and t-MDS are distinguished by an extremely high prevalence of clonal cytogenetic abnormalities involving chromosomes 5 and 7, as well as aneuploidy.7–9 Due largely to widespread acknowledgement of t-AML developing secondary to chemotherapy, the risk of AML associated with chronic Bz exposure has been recognized.10,11 The technologies supporting cytogenetic analysis for use in clinical epidemiology were in their infancy and not widely applied, and many scientists surmised that the pathogenesis of Bz-AML would be similar to t-AML described following treatment with alkylating chemotherapy.12–14 For the most part, quantitative retrospective epidemiology studies have confirmed an increased risk of AML following occupational exposure to Bz; however, comparable studies to evaluate the risk of MDS associated with previous exposure to Bz were not performed until after the classification of MDS as a neoplastic disease by the WHO Classification of Tumors of Hematopoietic and Lymphoid Tissue in 2001.15 This was the result of a number of factors. First, unlike AML’s dramatic and life-threatening onset, MDS is insidious and even now is often diagnosed only incidental to routine physical examination. Further, MDS was not uniformly reported as a neoplastic disease until its inclusion in WHO beginning in 2001.15 Finally, the definition, biological scope, and pathogenesis of MDS, which continue to evolve, were expanded in 2008 and remain controversial today.16
The results of subsequent quantitative epidemiology studies suggest that widespread emphasis on the increased risk of AML associated with Bz exposure may have been exaggerated, and that the dose-dependent risk of MDS following Bz exposure is actually more robust than that observed for AML.17–19 Bz toxicology in the modern era The last two decades have witnessed extraordinary growth in molecular biology, genetics, and medical technology and a revolution in the application of evidence-based methods in modern medicine. Formally heralded by the introduction of WHO 2001, the definition and classification of distinct clinical entities has been expanded from a retrospective description of morphology to a combined criteria of morphology, immunophenotype, molecular genetics, and clinical features. The success of this approach has highlighted the importance and power of interrogating the causes and pathogenesis of individual diseases in situ, that is, within the living environment of the host. It is in this context that advances in stem cell biology, molecular genetics, and computational biology have refined our current understanding of the HSC. Previously, the variety of characteristics and capabilities of HSCs could only be indirectly inferred through the use of clonal functional assays, and were consequently attributed to a single cell. The capabilities traditionally attributed to individual HSCs are now recognized as resulting from the collaboration of a community of cells, including primitive, progenitor, and stromal cells composing a feedback network within the microenvironment of the BM niche. Our group characterized hematopoietic and lymphoid diseases for 2,923 consecutive patients prospectively diagnosed in our laboratory in Shanghai utilizing WHO criteria. We described the clinical, phenotypic, and molecular genetic characteristics of MDS developing in 649 patients, 80 of whom were documented to have some Bz exposure, and 722 cases of AML, including 78 with documented Bz exposure.18,20,21 Inasmuch as t-MDS and t-AML are considered to be overlapping entities with a common pathogenesis, we were surprised to discover that MDS presenting in individuals with the highest chronic exposure to Bz did not exhibit the pattern of cytogenetic, phenotypic, or clinical
C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1310 (2014) 84–88
85
Benzene AML closely resembles de novo AML
Irons & Kerzic
Table 1. Prevalence (%) and relative risk of cytogenetic features in Bz-AML and de novo AML in Shanghai, China, and t-AML from a pooled series of international studies
Prevalence (%)
Aneuploidy –5/5q– –7/7q– 11q23 t(8;21) t(15;17)
Bz-AML (n = 78)
De novo AML (n = 644)
t-AML (n = 178)
RR (95% CI)
23 5.13 7.69 2.56 14.10 23.08
23 6.52 6.06 4.97 8.54 15.58
NA 23.03 24.72 7.29 6.18 3.37
NA 0.22 (0.10–0.52) 0.31 (0.15–0.64) 0.35 (0.08–1.52) 2.28 (1.05–4.97) 6.85 (3.21–14.62)
Note: RR is the risk of clonal abnormalities in the Shanghai Bz-exposed AML series compared to other center-based series of t-AML in which clonal cytogenetic abnormalities were analyzed.
abnormalities typically observed in t-MDS. In fact, these subjects were found to have a slightly lower prevalence of clonal cytogenetic abnormalities (24%) when compared to unexposed (i.e., de novo) MDS cases (30%; n = 569). No increase in clonal deletions involving all or part of chromosomes 5 or 7 were demonstrated in Bz-exposed MDS relative to unexposed MDS cases, and no case involving –5/5q– as the sole abnormality was observed for Bzexposed MDS.21 As in previous historical accounts, the predominant morphologic pattern in BM was hypocellular with eosinophilic dysplasia. This was accompanied by persistent immune-mediated inflammation and monoclonal or oligoclonal expansion of activated cytotoxic T cell populations.21,22 Indicative of an autoimmune process, cases of Bz-exposed MDS demonstrate positive clinical responses to immunosuppressive therapy and exhibit characteristics essentially identical to those described in evolving MDS with immune features.23–25 Cell-specific toxicity to myeloid cells by a variety of agents, including Bz, involves peroxidation mediated by myeloperoxidase (MPO), which catalyzes the formation of hypochlorous acid.26 MPO expression is the defining marker of myeloid differentiation in hematopoiesis and is first expressed in the subset of CD34+ CD38+ CD33+ myeloid progenitor cells (MPCs) that are committed to producing granulocytic and monocytic lineages.27,28 The emergence of cytotoxic T cell inflammation in the BM following chronic Bz myelotoxicity may be explained, in part, by the observation that MPO, normally expressed by myeloid cells in the BM, directly blocks antigen presentation and suppresses T cell–driven inflammatory response.29 This raises the distinct 86
possibility that early nonclonal dysplastic changes associated with chronic MPC dysfunction following exposure to Bz may be driven by cytotoxic T cells generated against dysplasia-related antigens originally expressed in MPC, as a consequence of MPOdependent cytoxicity. Consistent with the lack of clonal cytogenetic abnormalities in Bz-MDS, AML cases with previous Bz exposure do not exhibit an increased frequency of aneuploidy, numerical cytogenetic lesions commonly found in t-AML series such as –5/5q–, –7/7q–, or lesions involving 11q23.20 On the other hand, Bz-AML cases are more likely to possess karyotypes typically found in de novo AML, that is, normal karyotypes or recurrent abnormalities with increases in balanced translocations, such as t(8;21) and t(15;17) (Table 1). The pathogenesis of myeloid neoplasms Both BM failure and leukemogenesis find common origins in the evolution of the myelodysplastic processes occurring in the BM. One question of great importance concerns the origin and pathogenesis of recurrent balanced translocations involving t(8;21) and t(15;17) observed in AML associated with Bz exposure. Leukemia-initiating cells (LICs) in AML with t(8;21) are markedly less prevalent and more primitive than MPCs, do not form colonyforming units in semisolid medium, and possess lineage-negative phenotypes that do not express MPO.30–33 Although the identity of LICs in acute promyelocytic leukemia with t(15;17) is less certain, multiple lines of evidence suggest a primitive lineage-negative cell of origin in most cases.34–37 Inasmuch as MPO-dependent myelotoxicity
C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1310 (2014) 84–88
Benzene AML closely resembles de novo AML
Irons & Kerzic
implicates agent-specific targeting of committed MPCs, it is not consistent with direct targeting of primitive lineage-negative hematopoietic cells. A possible explanation involves disruption of the extrinsic homeostatic feedback mechanisms normally provided by MPCs that govern proliferation versus quiescence, self-renewal, and differentiation of HSCs in the BM niche.38–40 The mechanical disruption of homeostatic feedback networks alone in committed MPCs has been demonstrated to result in uncontrolled proliferation of HSCs. This appears to be both necessary and sufficient to undermine the replicative integrity of these primitive cells and result in the emergence of a leukemic stem cell phenotype.41 During his personal odyssey of discovery in the 1970s and 1980s, Bruce Ames first suggested that the risk of human cancer equated with mutagenesis;42 however, he subsequently concluded that loss of control over target-cell proliferation was the defining risk.43 Today, this observation finds renewed meaning in the maintenance of HSC quiescence in the BM niche, which is thought to be of paramount importance in preserving the genomic integrity of these primitive cells.40 Because our observations reveal a pattern of abnormalities in AML following exposure to Bz that more closely resembles de novo than therapy-related disease, they may have broad implications for understanding the pathogenesis of de novo AML and, consequently, its detection, treatment, and prevention. The disruption of feedback signaling by MPCs, not themselves clonal targets of leukemic transformation, leads to unregulated proliferation in primitive HSCs. Consequently, recurrent cytogenetic abnormalities, including balanced translocations, but not aneuploidy, emerge as predictable events leading to leukemic transformation in Bz-related neoplasms. Conflicts of interest This work was supported by the authors themselves with no third-party funds. References 1. Santesson, C. 1897. Uber chronische vergiftung mit steinkohlenteerbenzin: vier todesfalle. Arch. Hyg. Berl. 31: 336–376. 2. Aksoy, M. et al. 1972. Details of blood changes in 32 patients with pancytopenia associated with long-term exposure to benzene. Br. J. Ind. Med. 29: 56–64.
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