EPA/600/R-10/095F I July 2012 I www.epa.gov
United States
Environmental Protection
Agency
Lymphohematopoietic Cancer
Induced by Chemicals and Other Agen
Overview and Implications for Risk Assessment
^ °
I Center for Environmental Assessmen
f Research and Development
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EPA/600/R-10/095F
July 2012
Lymphohematopoietic Cancers Induced
by Chemicals and Other Agents:
Overview and Implications for Risk Assessment
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
ABSTRACT
The objective of this report is to provide an overview of the types and mechanisms
underlying the lymphohematopoietic cancers induced by chemical agents and radiation in
humans, with a primary emphasis on acute myeloid leukemia and some of the known agents that
induce this type of cancer. Following a brief discussion of hematopoiesis and leukemogenesis,
an overview of the major classes of leukemia-inducing agents—radiation, chemotherapeutic
alkylating agents, and topoisomerase II inhibitors—is presented along with information on
plausible mechanisms by which these leukemias occur. The last section focuses on how
mechanistic information on human leukemia-inducing agents can be used to better inform risk
assessment decisions. It is evident that there are different types of leukemia-inducing agents that
may act through different mechanisms. Even though most have been concluded by IARC to act
through mutagenic or genotoxic mechanisms, leukemia-inducing agents may have different
potencies and associated risks, which appear to be significantly influenced by the specific
mechanisms involved in leukemogenesis. Identifying the specific types of cancer-causing agents
with their associated mechanisms and using this information to inform key steps in the risk
assessment process remains an ongoing challenge.
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CONTENTS
LIST OF TABLES v
LIST OF FIGURES v
LIST OF ABBREVIATIONS vi
PREFACE vii
AUTHORS, CONTRIBUTORS, AND REVIEWERS viii
EXECUTIVE SUMMARY xi
1. INTRODUCTION TO LYMPHOHEMATOPOIETIC CANCERS 1
1.1. OVERALL INCIDENCE AND TRENDS 12
2. HEMATOPOIESIS 14
3. ORIGINS OF LYMPHOHEMATOPOIETIC NEOPLASIA 19
4. LEUKEMIA-AND LYMPHOMA-INDUCING AGENTS 27
5. OVERVIEW OF THE MAJOR CLASSES OF LEUKEMIA-INDUCING AGENTS 38
5.1. IONIZING RADIATION 38
5.2. CHEMOTHERAPEUTIC AGENTS 39
5.2.1. Alkylating Agent-Related Leukemias 40
5.2.2. Topoisomerase II Inhibitor-Related Leukemias 41
5.2.3. Other Likely Leukemia-Inducing Therapeutic Agents 44
6. MECHANISMS INVOLVED IN THERAPY-RELATED ACUTE MYELOID
LEUKEMIA (T-AML) 45
7. FACTORS CONFERRING AN INCREASED RISK OF INDUCED LEUKEMIA 49
7.1. MYELOSUPPRESSION 49
7.2. GENETIC POLYMORPHISMS 50
8. RISK ASSESSMENT IMPLICATIONS 54
8.1. HAZARD IDENTIFICATION 54
8.1.1. Utility of Short-Term Genotoxicity Tests and Human Biomonitoring 55
8.1.2. Usefulness of Animal Bioassays 56
8.1.3. Combining Different Types of Lymphohematopoietic Cancers for
Analysis 57
8.1.4. Potential Influence of Latency Period in Identifying Leukemogens 60
8.1.5. Metabolism and Bioactive Dose at the Target Organ 61
8.1.6. DNA-Adduct Type, Metabolism, and Repair 62
8.1.7. Age-Related and Individual Susceptibility 63
8.1.8. Summary 64
9. REFERENCES 65
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CONTENTS (continued)
APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
COMMENTS AND DISPOSITION 81
IV
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LIST OF TABLES
1. Simplified classification of the major lymphohematopoietic neoplastic diseases in
humans based largely on the French-American-British (FAB) classification 2
2. WHO classification of myeloid and related neoplasms 4
3. WHO classification of lymphoid neoplasms 7
4. Cytogenetic comparisons of de novo leukemias andt-AMLa 22
5. Frequency of molecular mutations in de novo AML and t-MDS/t-AML 24
6. Gene mutations observed in the Copenhagen series of 140 patients with t-MDS
(w = 89)ort-AML(w = 51) 26
7. Characteristics of selected known and probable human leukemia- and
lymphoma-inducing agents 28
8. Likely mechanisms involved in the carcinogenesis of selected known and
probable human leukemia- and lymphoma-inducing agents 31
LIST OF FIGURES
1. Simplified model of hematopoiesis showing lineages of major types of
hematopoietic cells 15
2. Hierarchical stem cell origins of leukemia and related cancers 20
3. Genetic pathways of t-MDS and t-AML based on 140 cases from the Copenhagen
study group 46
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LIST OF ABBREVIATIONS
ALL acute lymphoblastic leukemia
AML acute my el old leukemia
ANLL acute nonlymphocytic leukemia
CLL chronic lymphocytic leukemia
CML chronic myeloid leukemia
ENU TV-nitroso-jV-ethylurea
EPA U.S. Environmental Protection Agency
FAB French-American-British
IARC International Agency for Research on Cancer
MALT mucosa-associated lymphoid tissues
MDS myelodysplastic syndromes
MM multiple myeloma
NCI National Cancer Institute
NHL non-Hodgkin lymphoma
NI no information
NK natural killer
NQO1 NADPH quinone oxidoreductase 1
RARa retinoic acid receptor alpha
SCA structural chromosomal aberrations
t-AML therapy-related acute myeloid leukemia
TCDD Tetrachlorodibenzo-p-dioxin
UNSCEAR United Nations Scientific Committee on the Effects of Atomic Radiation
WHO World Health Organization
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PREFACE
This report represents the update and expansion of an earlier U.S. Environmental
Protection Agency (EPA) document entitled "Chemical and Radiation Leukemogenesis in
Humans and Rodents and the Value of Rodent Models for Assessing Risks of
Lymphohematopoietic Cancers" (EPA/600/R-97/090, May 1997). This report provides an
overview of chemically induced leukemias and lymphomas with a primary emphasis on acute
myeloid leukemia (AML). It is intended to provide insights into how mechanistic information
on AML-inducing agents may be used in risk assessment.
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
PROJECT MANAGER AND CONTRIBUTORS
Nagalakshmi Keshava, PhD
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Babasaheb Sonawane, PhD
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
AUTHOR
David A. Eastmond, PhD
University of California, Riverside
Riverside, CA
INTERNAL REVIEWERS
Glinda Cooper, PhD
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Maureen Gwinn, PhD
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
George Woodall, PhD
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
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Chao Chen, PhD
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Jennifer Jinot, PhD
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Brian Pachkowski, PhD
Oak Ridge Institute for Science and Education Postdoctoral Fellow
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Cheryl Scott, MS
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Suryanarayana Vulimiri, BVSc, MVSc, PhD, DABT
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
EXTERNAL PEER REVIEWERS
James E. Klaunig, PhD
School of Public Health
Indiana University
Bloomington, IN
David Ross, PhD
School of Pharmacy
University of Colorado Denver
Aurora, CO
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Martha S. Sandy, MPH, PhD
Office of Environmental Health Hazard Assessment
California Environmental Protection Agency
Oakland, CA
Martyn T. Smith, PhD
School of Public Health
University of California
Berkeley, CA
Roel Vermeulen, PhD
Utrecht University
Utrecht, The Netherlands
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EXECUTIVE SUMMARY
Lymphohematopoietic neoplasias represent a heterogeneous group of clonal
hematopoietic and lymphoid cell disorders and are one of the most common types of cancer
induced by environmental and therapeutic agents. Leukemia is a lymphohematopoietic cancer
originating in the bone marrow that affects cells of either the myeloid lineages (myeloid
leukemia) or the lymphoid lineage (lymphoid or lymphoblastic leukemia). These can be further
categorized as acute or chronic depending upon the rate of clonal expansion or stage of
differentiation of the cancer cell. The objective of this report is to provide an overview of the
types and mechanisms underlying the lymphohematopoietic cancers induced by chemical agents
and radiation in humans, with a primary emphasis on AML and agents that induce this type of
cancer. Following a brief discussion of hematopoiesis and leukemogenesis, a review of the
major classes of leukemia-inducing agents—radiation, chemotherapeutic alkylating agents, and
topoisomerase II inhibitors—is presented along with information on the mechanisms by which
these leukemias occur. The last section focuses on how mechanistic information on human
leukemia-inducing agents can be used to better inform risk assessment decisions. A brief
overview of the major points in the report is presented below.
It is widely recognized that lymphohematopoietic neoplasms originate through multistep
processes involving a series of genetic and epigenetic alterations that transform a normal
hematopoietic or lymphoid cell into a malignant tumor. The various lymphohematopoietic
cancers are believed to originate in specific types of pluripotent or lineage-restricted cells at
different stages in hematopoiesis and immune cell development. Current evidence indicates that
the acute and chronic myeloid leukemias (CMLs) as well as precursor lymphoid neoplasms,
including the acute lymphoblastic leukemias (ALLs)—B lymphoblastic leukemia/lymphoma and
T lymphoblastic leukemia/lymphoma, originate in hematopoietic stem or progenitor cells;
whereas, other lymphomas and chronic lymphocytic leukemia (CLL) have their origins in mature
lymphoid cells. An understanding of the origin of these cancers as well as morphologic,
cytochemical and immunophenotypic features of the neoplastic cells provide valuable
information for grouping various lymphohematopoietic cancers and for identifying the cell types
targeted by carcinogenic agents.
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Lymphohematopoietic neoplasia represents one of the most common cancers induced by
chemical, physical, and infectious agents. To date, the International Agency for Research on
Cancer has identified over 100 agents as human carcinogens. Of these, approximately 25% have
been shown to induce either leukemias or lymphomas in humans. Many of these identified
human carcinogens are antineoplastic drugs, but a variety of other agents including industrial
chemicals, various forms of radiation, immunosuppressive drugs, and infectious agents have also
been shown to induce lymphohematopoietic cancers in humans. In evaluating the different types
of induced leukemias and lymphomas, a number of general patterns become apparent.
Leukemias are the primary type of cancer induced by chemical agents. Most of these are acute
nonlymphocytic leukemias (synonymous with AML) with relatively short median latency
periods that are formed through the induction of mutations affecting critical cancer-related genes.
Radiation, which is also thought to act through a mutagenic mechanism, is frequently associated
with AML as well as CML and ALL.
Exposure to a variety of infectious agents is causally related to the formation of lymphoid
neoplasms. The induced lymphomas identified to date appear to be primarily associated with
chronic infection with either viruses or Helicobacter bacteria, agents that are
immunomodulating, and/or specifically target lymphoid cells. Two chemical agents,
cyclosporine and azathioprine, which are associated with the development of non-Hodgkin
lymphoma (NHL) are also strongly immunosuppressive, and this immunosuppression is believed
to play a critical role in the development of the associated lymphomas.
Among the induced acute myeloid leukemias, different subtypes with particular
characteristics have shown to be induced by different classes of agents. For example, the
alkylating agent class of chemotherapeutic agents typically induces acute myeloblastic leukemias
that are often preceded by a myelodysplastic phase and are characterized by loss of all or part of
chromosomes 5 or 7. These leukemias generally develop with a median latency period of
5-7 years from the beginning of treatment. In contrast, the leukemias induced by the
epipodophyllotoxin class of topoisomerase II inhibitors develop much sooner with a median
latency period of 2-3 years. These leukemias are typically characterized as monocytic or
myelomonocytic subtypes and exhibit reciprocal translocations involving a specific region on the
long arm of chromosome 11 (Ilq23).
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The last section of the report discusses how mechanistic information on human
leukemia-inducing agents can be used to better assess the risks from exposure to environmental
chemicals. A range of topics is considered including brief discussions on the usefulness of
short-term and chronic animal bioassays; the combining of various tumor types for analysis; the
impact of latency periods on the detection of leukemias; and the modifying influences of
metabolism, pharmacokinetics, DNA-adduct type and repair, as well as individual and
age-related susceptibility factors in assessing the risks associated with leukemia-inducing agents.
In conclusion, it is evident that there are different types of leukemia-inducing agents that
act through different mechanisms. In its evaluations, IARC has concluded that most leukemia-
inducing agents act through mutagenic and/or genotoxic mechanisms, with different potencies
and associated risks, which can be significantly influenced by the specific mechanisms involved
in leukemogenesis. Identifying the specific types of cancer-causing agents with their associated
mechanisms and using this information to inform key steps in the risk assessment process
remains one of the ongoing challenges for researchers and risk assessors. For the alkylating
agent class of carcinogens, an approach such as that described by Vogel and colleagues
supplemented with more recent genomic, proteomic, and biomarker information would appear to
present a reasonable and scientifically valid step towards this objective.
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1. INTRODUCTION TO LYMPHOHEMATOPOIETIC CANCERS
Lymphohematopoietic neoplasia can be described as an uncontrolled proliferation or
expansion of hematopoietic and lymphoid cells that are unable to differentiate normally to form
mature blood cells (Sawyers et al., 1991). These neoplasms represent clonal expansions of
hematopoietic cells, almost always within either the myeloid or lymphoid lineage (Nowell, 1991;
WHO, 2008). Infrequently, some leukemias exhibit both myeloid and lymphoid characteristics
and are known as biphenotypic leukemias (Russell, 1997). The myeloid clones are designated as
chronic or acute leukemias, depending upon the rate of clonal expansion and the stage of
differentiation that dominates the leukemic clone. Lymphoid neoplasms typically manifest
themselves in the blood as chronic or acute lymphoblastic leukemias (ALLs) or remain confined
to lymphoid proliferative sites such as the lymph nodes or spleen and are designated as
lymphomas (Nowell, 1991). Acute leukemias tend to have a rapid onset with a predominance of
immature cells whereas chronic leukemias have a more insidious onset and progress over a
period of months or years to a blast or acute leukemic phase.
Using this basic classification, leukemias can be described as one of four major
types—ALL, acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL), and
chronic myeloid leukemia (CML). Similarly, lymphomas are broadly classified as Hodgkin or
non-Hodgkin lymphomas (NHLs) depending upon the appearance of a specific cancer cell type,
the Reed-Sternberg cell, which is found in Hodgkin lymphomas (ACS, 2009). Within these
larger groupings, there are numerous subtypes involving specific cells that have unique
characteristics, origins, and increasingly recognized clinical significance. These subtypes are
generally classified according to morphologic, cytogenetic, immunophenotypic, and more
recently, molecular characteristics according to the French-American-British (FAB) or World
Health Organization (WHO) classification systems (Head and Pui, 1999; WHO, 2001, 2008;
Haferlach et al., 2005). For convenience, a simplified classification scheme for the leukemias
and lymphomas based largely on the FAB classification system (shown in Table 1 [Sullivan,
1993]) will be the basis for most descriptions used in this document. A more thorough and
complicated classification of the primary types and subtypes of lymphohematopoietic diseases
based on the most recent WHO classification (WHO, 2008; Vardiman et al., 2009) is shown in
Tables 2 and 3 for myeloid and lymphoid neoplasms, respectively. It should be noted that in the
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Table 1. Simplified classification of the major lymphohematopoietic
neoplastic diseases in humans based largely on the French-American-British
(FAB) classification
Neoplasms of multipotent stem cell origin
Chronic myelogenous leukemia
II. Neoplasms possibly originating in the multipotent stem cell
Myelodysplastic syndromes
Refractory anemia
Refractory anemia with ring sideroblasts
Refractory anemia with excess blasts
Chronic myelomonocytic leukemia
Refractory anemia with excess blasts in transformation
Chronic myeloproliferative disorders
III. Neoplasms originating in stem cells or myeloid-committed precursors
Acute myeloid leukemia or acute nonlymphocytic leukemia
Acute myeloblastic leukemia with minimal differentiation
Acute myeloblastic leukemia without maturation
Acute myeloblastic leukemia with maturation
Acute promyelocytic leukemia
Acute myelomonocytic leukemia
Acute monocytic leukemia
Acute erythroleukemia
Acute megakaryoblastic leukemia
Malignant histiocytosis
IV. Neoplasms of lymphoid-committed precursors
Immature phenotype: Acute lymphoblastic leukemia
B-cell lineage
T-cell lineage
Intermediate or mature phenotype: non-Hodgkin lymphoma
Nodal/splenic phase
Leukemic phase
B-cell lineage
non-Burkitt's
Burkitt's
T-cell lineage
Lymphoblastic lymphoma
CML
MDS
RA
RARS
RAEB
CMML
RAEB
AML/ANLL
MO
Ml
M2
M3
M4
M5
M6
M7
ALL, L1,L2
b-ALL
t-ALL
NHL
L3
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Table 1. Simplified classification of the major lymphohematopoietic
neoplastic diseases in humans based largely on the French-American-British
(FAB) classification (continued)
Adult T-cell leukemia/lymphoma
Mature lymphocytic phenotype
Prolymphocytic leukemia
Chronic lymphocytic leukemia CLL
B-cell lineage
T-cell lineage
Hairy cell leukemia
Plasmacytoid phenotype: Marrow-phase predominant
Macroglobulinemia
Heavy chain diseases
Myeloma
Hodgkin lymphoma
Adapted from Sullivan (1993).
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Table 2. WHO classification of myeloid and related neoplasms
Myeloproliferative neoplasms (MPNs)
Chronic myelogenous leukemia, BCR-ABL1 -positive
Chronic neutrophilic leukemia
Polycythemia vera
Primary myelofibrosis
Essential thrombocythemia
Chronic eosinophilic leukemia, not otherwise specified
Mastocytosis
Cutaneous mastocytosis
Systemic mastocytosis
Mast cell leukemia
Mast cell sarcoma
Extracutaneous mastocytoma
Myeloproliferative neoplasms, unclassifiable
Myeloid and lymphoid neoplasms associated with eosinophilia and abnormalities of PDGFRA, PDGFRB, or
FGFR1
Myeloid and lymphoid neoplasms with PDGFRA rearrangement
Myeloid neoplasms with PDGFRB rearrangement
Myeloid and lymphoid neoplasms withFGFRl abnormalities
Myelodysplastic/myeloproliferative neoplasms (MDS/MPN)
Chronic myelomonocytic leukemia
Atypical chronic myeloid leukemia, BCR-ABL 1 -negative
Juvenile myelomonocytic leukemia
Myelodysplastic/myeloproliferative neoplasm, unclassifiable
Refractory anemia with ring sideroblasts and thrombocytosis (provisional entry)
Myelodysplastic syndrome (MDS)
Refractory cytopenia with unilineage dysplasia
Refractory anemia
Refractory neutropenia
Refractory thrombocytopenia
Refractory anemia with ring sideroblasts
Refractory cytopenia with multilineage dysplasia
Refractory anemia with excess blasts
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Table 2. WHO classification of myeloid and related neoplasms (continued)
Myelodysplastic syndrome with isolated del(5q)
Myelodysplastic syndrome, unclassifiable
Childhood myelodysplastic syndrome
Refractory cytopenia of childhood (provisional entry)
Acute myeloid leukemia and related neoplasms
Acute myeloid leukemia with recurrent genetic abnormalities
AML witht(8;21)(q22;q22); RUNX1-RUNX1T1
AML withinv(16)(pl3.1;q22) ort(16;16)(p!3.1;q22); CBFB-MYH11
Acute promyelocytic leukemia witht(15;17)(q22;q!2); PML-RARA
AML with t(9; 11)(p22;q23); MLLT3-MLL
AML witht(6;9)(p23;q34); DEK-NUP214
AML with inv(3)(q21;q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1
AML (megakaryoblastic) witht(l;22)(pl3;q!3); RBM15-MKL1
AML with mutated NPM1 (provisional entry)
AML with mutated CEBPA (provisional entry)
Acute myeloid leukemia with myelodysplasia-related changes
Therapy-related myeloid neoplasms
Acute myeloid leukemia, not otherwise specified
AML with minimal differentiation
AML without maturation
AML with maturation
Acute myelomonocytic leukemia
Acute monoblastic/monocytic leukemia
Acute erythroid leukemia
Pure erythroid leukemia
Erythroleukemia, erythroid/myeloid
Acute megakaryoblastic leukemia
Acute basophilic leukemia
Acute panmyelosis with myelofibrosis
Myeloid sarcoma
Myeloid proliferations related to Down syndrome
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Table 2. WHO classification of myeloid and related neoplasms (continued)
Transient abnormal myelopoiesis
Myeloid leukemia associated with Down syndrome
Elastic plasmacytoid dendritic cell neoplasm
Acute leukemias of ambiguous lineage
Acute undifferentiated leukemia
Mixed phenotype acute leukemia with t(9;22)(q34;ql 1.2); BCR-ABL1
Mixed phenotype acute leukemia with t(v;l Iq23); MLL rearranged
Mixed phenotype acute leukemia, B-myeloid, NOS
Mixed phenotype acute leukemia, T-myeloid, NOS
Natural killer (NK) cell lymphoblastic leukemia/lymphoma (provisional entry)
Source: WHO (2008).
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Table 3. WHO classification of lymphoid neoplasms
Precursor lymphoid neoplasms
B lymphoblastic leukemia/lymphoma
B lymphoblastic leukemia/lymphoma, NOS
B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities
B lymphoblastic leukemia/lymphoma with t(9;22)(q34;qll.2);BCR-ABL 1
B lymphoblastic leukemia/lymphoma witht(v;llq23);MZZ rearranged
B lymphoblastic leukemia/lymphoma with t(12;21)(p!3;q22) TEL-AML1 (ETV6-RUNX1)
B lymphoblastic leukemia/lymphoma with hyperdiploidy
B lymphoblastic leukemia/lymphoma with hypodiploidy
B lymphoblastic leukemia/lymphoma with t(5;14)(q31;q32) IL3-IGH
B lymphoblastic leukemia/lymphoma with t(l;l9)(q23;p!3.3);TCF3-PBXl
T lymphoblastic leukemia/lymphoma
Mature B-cell neoplasms
Chronic lymphocytic leukemia/small lymphocytic lymphoma
B-cell prolymphocytic leukemia
Splenic marginal zone lymphoma
Hairy cell leukemia
Splenic B-cell lymphoma/leukemia, unclassifiable (provisional entry)
Splenic diffuse red pulp small B-cell lymphoma (provisional entry)
Hairy cell leukemia—variant (provisional entry)
Lymphoplasmacytic lymphoma
Waldenstrom macroglobulinemia
Heavy chain diseases
Alpha-heavy chain disease
Gamma-heavy chain disease
Mu heavy chain disease
Plasma cell myeloma
Solitary plasmacytoma of bone
Extraosseous plasmacytoma
Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT-lymphoma)
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Table 3. WHO classification of lymphoid neoplasms (continued)
Nodal marginal zone B-cell lymphoma
Pediatric nodal marginal zone lymphoma (provisional entry)
Follicular lymphoma
Pediatric follicular lymphoma (provisional entry)
Primary cutaneous follicle center lymphoma
Mantle cell lymphoma
Diffuse large B-cell lymphoma (DLBCL), NOS
T-cell/histiocyte-rich large B-cell lymphoma
Primary DLBCL of the CNS
Primary cutaneous DLBCL, leg type
EBVpositive DLBCL of the elderly (provisional entry)
DLBCL associated with chronic inflammation
Lymphomatoid granulomatosis
Primary mediastinal (thymic) large B-cell lymphoma
Intravascular large B-cell lymphoma
ALK-positive large B-cell lymphoma
Plasmablastic lymphoma
Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease
Primary effusion lymphoma
Burkitt lymphoma/leukemia
B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and
Burkett lymphoma
B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and
classical Hodgkin lymphoma
Mature T-cell and NK-cell neoplasms
T-cell prolymphocytic leukemia
T-cell large granular lymphocytic leukemia
Chronic lymphoproliferative disorder ofNK cells (provisional entry)
Aggressive NK-cell leukemia
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Table 3. WHO classification of lymphoid neoplasms (continued)
Systemic EB V-positive T-cell lymphoproliferative disease of childhood
Hydroavacciniforme-like lymphoma
Adult T-cell leukemia/lymphoma
Extranodal NK/T-cell lymphoma, nasal type
Enteropathy-type T-cell lymphoma
Hepatosplenic T-cell lymphoma
Subcutaneous panniculitis-like T-cell lymphoma
Mycosis fungoides
Sezary syndrome
Primary cutaneous CD30-positive anaplastic large cell lymphoma
Lymphomatoid papulosis
Primary cutaneous anaplastic large cell lymphoma
Primary cutaneous gamma-delta T-cell lymphoma
Primary cutaneous CDS positive aggressive epidermotropiccytotoxic T-cell lymphoma (provisional entry)
Primary cutaneous CD4 positive small/medium T-cell lymphoma
Peripheral T-cell lymphoma, NOS (provisional entry)
Angioimmunoblastic T-cell lymphoma
Anaplastic large cell lymphoma, ALK positive
Anaplastic large cell lymphoma, ALK positive (provisional entry)
Hodgkin lymphoma
Nodular lymphocyte predominant Hodgkin lymphoma
Classical Hodgkin lymphoma
Nodular sclerosis classical Hodgkin lymphoma
Lymphocyte-rich classical Hodgkin lymphoma
Mixed cellularity classical Hodgkin lymphoma
Lymphocyte-depleted classical Hodgkin lymphoma
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Table 3. WHO classification of lymphoid neoplasms (continued)
Histiocytic and dendritic-cell neoplasms
Histiocytic sarcoma
Langerhans cell histiocytosis
Langerhans cell sarcoma
Interdigitating dendritic cell sarcoma
Follicular dendritic cell sarcoma
Fibroblastic reticular cell tumor
Indeterminate dendritic cell tumor
Disseminated juvenile xanthogranuloma
Post-transplant lymphoproliferative disorders (PTLDs)
Early lesions
Plasmacytic hyperplasia
Infectious mononucleosis-like PTLD
Polymorphic PTLD
Monomorphic PTLD (B- and T/NK-cell types)
Classical Hodgkin lymphoma type PTLD
Source: WHO (2008).
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more recent WHO classifications (International Agency for Research on Cancer [IARC], 2008c),
lymphocytic leukemias and lymphomas are no longer considered to be different diseases and
should be evaluated accordingly. However, for simplicity and due to the difficulties involved in
reclassifying neoplasms based on studies that were conducted many years ago, the classifications
used in the original studies or in the IARC monographs are used in this document. It should be
recognized, however, that considerable heterogeneity exists among NHL subtypes as classified
according to older classification systems (Morton et al., 2007). In addition, the older schemes do
not consider temporal changes in classifying distinct variants of NHL. For example, lymphomas
of mucosa-associated lymphoid tissues (MALT) and mantle cell lymphoma as classified by
WHO (2008), were previously considered as either pseudolymphomas or benign lymphoid
disorders. Similarly, some forms of T-cell lymphomas and aplastic large-cell lymphomas were
earlier classified as Hodgkin disease (Banks, 1992).
Among the leukemias, the two major diagnostic categories, ALL and AML, can be
further classified based upon cellular features. ALL is subdivided by FAB morphology (LI, L2,
and L3) and by immunophenotype (B-cell, early pre-B, pre-B, and T-cell) (Bhatia et al., 1999).
AML is classified primarily by morphological characteristics into eight different FAB subgroups
(MO-M7) based upon the myeloid lineage and degree of maturation involved. Similarly, the
myelodysplastic syndromes (MDSs), a series of blood disorders characterized by maturation
defects resulting in ineffective hematopoiesis, have also been classified by the FAB and WHO
systems (see Tables 1 and 2, respectively). These are commonly considered to be preleukemic
because a variable, but significant, proportion (1 to 33%) of the various disorders progress to
frank leukemia (Wright, 1995; WHO, 2001, 2008; Hasle et al., 2003).
The objective of this report is to provide an overview of the types of
lymphohematopoietic neoplasia induced by chemical agents and radiation in humans, and to
summarize current information on the mechanisms of chemical leukemogenesis. Much of the
discussion focuses on AML due to the limited and evolving knowledge of chemically induced
lymphoid leukemias and lymphomas that is only now being integrated into ongoing
epidemiological and clinical studies. An overview of the major classes of leukemia-inducing
agents—radiation, the alkylating agents, and topoisomerase II inhibitors is presented followed by
a short discussion of factors influencing chemical leukemogenesis. Lastly, the report ends with a
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discussion of how mechanistic information on human leukemia-inducing agents can be used to
better assess the risks from exposure to environmental chemicals.
1.1. OVERALL INCIDENCE AND TRENDS
Lymphohematopoietic neoplasms are an uncommon, yet significant, cause of
cancer-related deaths. In 2009, it was estimated that leukemia would be diagnosed in
44,790 people in the United States (ACS, 2009). Slightly over half of these will be chronic
leukemias (20,540) with the remainder being acute leukemias (18,570) and others that have not
been clearly identified (5,680). Because of the limitations of current therapies, leukemia
represents the 5th leading cause of cancer deaths among males in the United States and the
7th leading cause among females (ACS, 2009). Furthermore, it was estimated that 74,490 new
cases of lymphoma would be diagnosed in the United States in 2009 (ACS, 2009). Of these,
89% (65,980 cases) were estimated to have been NHL and 11% Hodgkin lymphoma
(8,510 cases). NHL represents the 9* leading cause of cancer-related deaths in males and the
6* in females. It should be noted that more recent cancer incidence and mortality data can be
obtained from the National Cancer Institute (NCI)'s Surveillance Epidemiology and End Results
Web site (http://seer.cancer.gov/statistics/).
While leukemia occurs much more commonly in adults than in children, childhood
leukemia still accounts for approximately 30% of all childhood cancers in the United States and
is a leading cause of disease-related death among children (Smith et al., 2005; ACS, 2006). The
incidence of leukemia in children (36 per million) is similar to that seen in young to middle-aged
adults (ages 20-44) but roughly one-tenth of that of adults aged 45 years and older, where the
annual incidences increase with age from 144 to 545 per million (Xie et al., 2003; ACS, 2006).
In adults, roughly 85% of the acute leukemias are myeloid in origin with the remainder being
lymphoid (Greaves, 1999). In children, the opposite occurs with 80% of the leukemias
originating from lymphoid cells. Moreover, the incidence trends for adult and pediatric
leukemias differ substantially. While the overall trend for adult leukemia has generally declined
with time, the incidence of childhood leukemias in the United States appeared to increase during
the early 1980s with rates increasing from 3.3 cases per 100,000 in 1975 to 4.6 cases per 100,000
in 1985 (NCI, 2008). In the subsequent years, the rates have shown no consistent upward or
downward trend. Over the past 30 years, increases in childhood lymphoid leukemia and
12
-------
lymphoma have also been reported for Europe and in other developed countries (Hrusak et al.,
2002; Steliarova-Foucher et al., 2004). Fortunately, there has been significant progress in
treating childhood leukemias so that the 5-year survival rate for the affected children is now
approximately 80% (ACS, 2006). The survival rate for adult leukemias varies by type with
5-year survival rates of 22% for AML patients, 66% for ALL patients, and 76% for CLL patients
(ACS, 2009).
13
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2. HEMATOPOIESIS
Hematopoiesis is the process by which the cells of the blood are formed. The
development of the hematopoietic system begins in several mesodermal lineages in the
mammalian conceptus with cells migrating from the primitive streak to three blood-forming
tissues: the yolk sac, the para-aortic splanchnopleura/aorta-gonadal-mesonephros region, and the
chorio-allantoic placenta (for review, see Dzierzak and Speck, 2008). Hematopoietic stem cells
capable of conferring complete long-term and multilineage repopulation of hematopoiesis in
irradiated adult recipient mice appear in the aorta-gonadal-mesonephros and other tissues during
the middle of embryogenesis (Embryonic Day 10.5 in the mouse). These cells proceed to
colonize the liver, then the thymus, spleen and bone marrow (by Embryonic Day 15 in the
mouse) where hematopoiesis primarily occurs after birth.
The formation of blood cells originates with the hematopoietic stem cell. As described
by Wilson and Trumpp (2008), stem cells are functionally defined as cells that can both
self-renew (maintain their numbers at a constant level) and give rise to all mature cells in the
tissue in which they reside. The formation of blood cells is supported by a small population of
pluripotent stem cells that exhibit the capacity to self-renew and are capable of extensive
proliferation. The balance between stem cell self-renewal and differentiation is believed to be
controlled by interactions between the stem cells and the adjacent niche-forming stromal cells or
soluble factors produced by the stromal cells (Wilson and Trumpp, 2008). The pluripotent stem
cells can also reconstitute all hematopoietic lineages and are capable of long-term reconstitution
of the hematopoietic system of recipient animals. The primitive pluripotent stem cells are
estimated to comprise 1 in 100,000 bone marrow cells and give rise to multipotent and
committed progenitor cells, which represent approximately 2-5 per 1,000 marrow cells (Mihich
and Metcalf, 1995). Each of these progenitor cells can generate 100,000 or more maturing
progeny. The process of proliferation and differentiation is regulated by more than 25 growth
factors, cytokines, and other regulators that may act directly upon one or more of the major
lineages of blood cells or interact to influence cell growth (Mihich and Metcalf, 1995). A
diagram illustrating the relationships between the major cell types involved in hematopoiesis is
shown in Figure 1 (adapted from Bryder et al., 2006).
14
-------
Hematppoietic
stem cell
(pluripotent)
Multipotent
WVWWJV^K-J-^rV^-^l-S-
progenitor cells
Common myeloid
progenitor cell
Granulocyte
macrophage
progenitor cell
Pro-T; Pro-NK Pro-B
Multi-lymphoid
progenitor cell
Megakaryocyte
erythroid
progenitor cell
Lineage restricted
progenitor cells i
Mature hematppoietic
cells
Platelets
Monocytes/
Macrophages
Erythrocytes Granulocytes^ Dendritic; cells 3 Natural killer cells
T-lymphocytes
B-lymphocytes7
Figure 1. Simplified model of hematopoiesis showing lineages of major types of hematopoietic cells.
-------
With the exception of lymphocytes where maturation also continues in the thymus,
spleen and peripheral tissues, the formation of blood cells in normal human adults occurs almost
exclusively in the bone marrow. All mature circulating blood cells have a finite life with the
majority of cells being terminally differentiated and unable to replicate (Bagby, 1994). In order
to maintain steady-state levels, the formation of cells in the marrow must equal the rate of
cellular senescence and elimination. As a result, the hematopoietic system has a tremendous
proliferative capacity with estimates of cell turnover ranging from 130 to 500 billion cells per
day in a 70-kg man (Jandl, 1996; Bryder et al., 2006). In addition, the hematopoietic system in
the central bone marrow helps respond to a variety of environmental stresses and infection by
increasing the blood cell counts of specific lineages when needed (Bagby, 1994; Jaiswal and
Weissman, 2009). For example, upon exposure to a hypoxic environment, erythrocyte
production will increase without a change in the production of neutrophils. Similar
lineage-specific responses are required following exposure to myelotoxic agents. To maintain
steady-state blood cell levels and to respond to environmental pressures, hematopoiesis, of
necessity, must be a highly regulated process. Historically, the responses to infection and
environmental stresses were believed to occur exclusively within the bone marrow. However, as
described below, it has recently been shown that an immune response to infection can occur at
extramedullary sites due to the homing, proliferation, and differentiation of hematopoietic stem
and progenitor cells at these ecotopic sites within the body (Jaiswal and Weissman, 2009; Schulz
et al., 2009).
As indicated above, the hematopoietic stem cells normally reside within the bone
marrow, a tissue with specialized vasculature and shielding that provides an excellent
environment for the development of blood cells (Papayannopoulou and Scadden, 2008). In
addition to their ability to self-renew, primitive pluripotent hematopoietic stem cells give rise to
a number of multipotent progenitor cells, which, in turn, give rise to oligopotent progenitor cells
(see Figure 1 and Bryder et al., 2006). Among these, the multilymphoid progenitors give rise to
mature B lymphocytes, T lymphocytes, and natural killer (NK) cells whereas the common
myeloid progenitors give rise to granulocyte-macrophage progenitors (which differentiate into
monocytes/macrophages and granulocytes), and megakaryocyte/erythrocyte progenitors (which
differentiate into megakaryocytes/platelets and erythrocytes). Both the myeloid and lymphoid
progenitor cells have also been shown to form monocytes and dendritic cells, which play a role
16
-------
in regulating the immune system (Doulatov et al., 2010; Dorshkind, 2010). In addition to these
major types of blood cells, a number of the cell types, for example, the granulocytes can further
differentiate into more specialized blood cells such as neutrophils, eosinophils, and basophils.
As a result, the number of unique cell types derived from the hematopoietic stem cell is quite
large, and, as indicated above, the total number of individual cells formed per day numbers in the
hundreds of billions.
Hematopoietic stem and progenitor cells are commonly identified based on the presence
or absence of lineage markers or antigen expression on the cell surface or in the cytoplasm. As
presented by Bryder and associates, the most primitive hematopoietic stem cell identified to date
in humans exhibits the cell surface markers, Lin"CD90+CD38"CD34+ meaning that it is negative
for the lineage differentiation surface antigen Lin, namely B'G'MT" (B220 for B cells, Gr-1 for
granulocytes, Mac-1 for myelomonocytic cells, and CD4 and CDS for T cells), negative for the
surface antigen CD38 and positive for the surface antigens CD90 and CD34 (Bryder et al., 2006;
Li and Li, 2006). As described by Iwasaki and Akashi (2007) and Bryder et al. (2006), the
lineage markers and differentiation pathways differ in notable ways between the mouse and the
human.
While hematopoietic stem and progenitor cells normally reside in specialized niches in
the bone marrow, they can be mobilized into the peripheral blood either at low levels
spontaneously or in large numbers as a result of cytokine or chemical treatment (Levesque et al.,
2007; Schulz et al., 2009). These mobilized cells remain in circulation for only short periods of
time (minutes to hours) before homing to another peripheral tissue or, for a small to very small
percentage of cells under normal conditions, returning to the bone marrow (Wright et al., 2001;
Abkowitz et al., 2003; McKinney-Freeman and Goodell, 2004). As a result, there appears to be
a constitutive recirculation of hematopoietic stem and progenitor cells between the bone marrow,
extramedullary tissues, and the lymphoid compartments (Schulz et al., 2009). Consequently, it is
theoretically possible for DNA damage or other types of potentially leukemogenic alterations to
affect hematopoietic stem cells while they circulate in the blood and extramedullary spaces.
Upon exposure to mobilizing agents such as specific chemokines, toxicants, antibodies, or
growth factors, or under conditions of stress or myelosuppression, large numbers of these stem
and progenitor cells can be released into the peripheral blood (Levesque et al., 2007). Clinically,
these mobilized cells can be harvested for use in bone marrow transplantation. The natural role
17
-------
of mobilization is not fully known, but there is evidence that the circulating hematopoietic stem
and progenitor cells may help replenish tissue-residing myeloid cells such as specific monocytes,
macrophages, and dendritic cells or help in rapidly responding to tissue injury and infection
(Jaiswal and Weissman, 2009; Schulz et al., 2009).
18
-------
3. ORIGINS OF LYMPHOHEMATOPOIETIC NEOPLASIA
As with other cancers, leukemogenesis and lymphomagenesis are multistep processes
involving a series of genetic and epigenetic alterations involved in the transformation of a normal
cell into a malignant cell. The various lymphohematopoietic cancers are believed to originate in
specific types of pluripotent or lineage-restricted cells at different stages in hematopoiesis (see
Figure 2; Greaves, 1999). As illustrated in the figure, leukemias and lymphomas can originate
within many types of hematopoietic or hematopoietic-forming cells. With a few rare exceptions,
most leukemias and lymphomas originate at the hematopoietic stem cell stage or at later
progenitor or lineage-restricted stages. For example, most adult leukemias of myeloid origin
(AML, MDS, and CML) as well as adult ALL are believed to originate at the pluripotent stem or
progenitor cell stage whereas childhood leukemias are believed to originate during a subsequent
stage of differentiation at either the lineage-restricted lymphoid or myeloid stem cell stage. For
most types of adult AML, the key leukemic transformations appear to occur in hematopoietic
stem cells (Warner et al., 2004). However, for acute promyelocytic leukemia, the key
transformative event may occur at the committed myeloid progenitor stage (Passegue et al.,
2003; Warner et al., 2004). For a few others, such as those possessing the MLL-ENL fusion
gene, the transformative event can occur either at the pluripotent or the committed stem cell
stage (Cozzio et al., 2003). In contrast, many lymphomas (NHL, Hodgkin lymphoma, Burkitt
lymphoma) and all myelomas, as well as, several rare leukemias/lymphomas (adult T-cell
leukemia, prolymphocytic leukemia, hairy cell leukemia) and one common leukemia (CLL) are
believed to originate in mature lymphoid cells (Greaves, 1999; Harris et al., 2001). An
understanding of the origin of these cancers can be useful for grouping various
lymphohematopoietic cancers and can provide insight into the cell types targeted by carcinogenic
agents.
Consistent with the multistep nature of leukemogenesis, over 300 different genetic
alterations and mutations have been identified (Kelly and Gilliland, 2002). Indeed, a recent
examination of alterations affecting the MLL gene, a cancer-related gene located at band 1 Iq23,
in pediatric and adult leukemias, identified a total of 87 different rearrangements, primarily
translocations, involving this one gene (Meyer et al., 2006). While most of the detected
alterations are rare, certain translocations and genes are more prevalent and are typically
19
-------
Germ
line
Early embryonic
stem celts
Haemopoietic
(lympho-myelold) stem
cells
Childhood ALL
Cut. T lymphoma
ATI, T-PLL
0©
Mature
Lymphocylc
subsets
Myeiotd
stem cells
B-IMHL
B-CLL. PLL, HCL
Myeloma
Figure 2. Hierarchical stem cell origins of leukemia and related cancers. The
arrows denote the likely level of clonal selection for the majority of the leukemia
subtypes listed.
Abbreviations: AL = acute leukemia; Cut. T lymphoma = cutaneous T-cell
lymphoma; ATL = adult T-cell leukemia; T-PLL = T-cell prolymphocytic
leukemia; B-cell non-Hodgkin lymphoma; PLL = prolymphocytic leukemia;
HCL = hairy cell leukemia. For other abbreviations, see Table 1.
Source: Greaves (1999). Reprinted by permission from Elsevier.
20
-------
associated with specific leukemic subtypes (Bhatia et al., 1999; Greaves and Wiemels, 2003).
Many of these genetic alterations can only be detected at the molecular level. However, others
have been detected using cytogenetic, molecular cytogenetic, and genomic approaches.
Nonrandom chromosomal alterations are detected in the neoplastic cells of a majority of patients
with leukemias or lymphomas, and the identification of genes involved in these alterations has
provided valuable insights into leukemogenesis and lymphomagenesis in humans (Chen and
Sandberg, 2002; Mrozek et al., 2004; Pedersen-Bjergaard et al., 2006; Qian et al., 2009). It is
also likely that in addition to genetic alterations, other processes such as epigenetic modifications
(altered DNA methylation, dysregulation of miRNA, etc.) play an important role in
leukemogenesis (Pedersen-Bjergaard et al., 2006; Nervi et al., 2008; Liu et al., 2011). Because
much more is known about the genetic alterations in carcinogenesis and their recognized
importance, the overview presented below focuses primarily on genetic changes in the
development of myeloid leukemia.
Common clonal cytogenetic changes seen in leukemias include alterations in
chromosome number such as loss or gain of one or more chromosomes (e.g., monosomy 7 and
Trisomy 8, respectively), balanced and unbalanced chromosome translocations, deletions, or
inversions involving specific chromosomal regions as well as complex arrangements involving
combinations of the above. In some cases, these changes are a reflection of the genetic
instability that is common in many types of cancers. However in many cases, there is evidence
that these have originated from a single ancestral cell (i.e., clonal), and are highly specific
involving genes that are directly involved in the cancer process. For example, the translocation
between chromosomes 15 and 17, (t[15;17][q22;ql2-21J) that is characteristic of the
promyelocytic form of AML (FAB M3), results in a fusion gene involving the PML gene on
chromosome 15 and the retinoic acid receptor alpha (RARa) gene on chromosome 17. This
hybrid gene blocks differentiation of the developing myeloid cells at the promyelocytic stage
(Downing, 1999), a characteristic feature of the disease. As seen in Table 4, significant
differences have been seen in the karyotypes of patients who have developed leukemia following
therapy and those who have no history of exposure to chemotherapy or other leukemogenic
agents. The former are called t-AML and the latter, de novo leukemias. In addition to these
microscopically visible chromosomal alterations, mutations at the molecular level affecting
specific cancer-related genes such as RAS, FTL3, GATA1, and TP53 have also been seen in
21
-------
Table 4. Cytogenetic comparisons of de novo leukemias and t-AMLa
Cytogenetic features
AML
de novo
n = 3,649 (%)
t-AML
n = 581 (%)
Number of anomalies
1 anomaly
2 anomalies
>3 anomalies
2,186(60)
641 (18)
822 (23)d
242 (42)
104 (18)
235 (40)
Ploidy level
hypodiploid
pseudodiploid
hyperdiploid
tri-/tetraploid
unknown ploidy
Unbalanced anomalies
3p-
-5
5q-
-7
-7 (sole)
7q-
der(l;7)
loss of 5 and/or 7
+8
+8 (sole)
llq-
der(12p)
13q-
-17
der(17p)
-18
20q-
-21
Balanced anomalies
t(l;3)(p36;q21)
813 (22)
1,843 (51)
974 (27)d
13 (0.4)
6 (0.2)
2,734 (75)d
33 (0.9)d
152 (4.2)d
249 (6.8)d
340 (9.3)d
114(3.1)d
147 (4.Q)d
8 (0.2)d
717 (20)d
614 (17)
269 (7.4)d
82 (2.2)
153 (4.2)d
32 (0.9)
172 (4.7)d
104 (2.9)d
129 (3.5)d
45 (1.2)
92 (2.5)d
1,713 (47)d
2(0.1)d
224 (39)
242 (42)
108(19)
7 (1.2)
0
491 (85)
17 (2.9)
73 (13)
77(13)
167 (29)
51 (8.8)
36 (6.2)
12(2.1)
284 (49)
84 (14)
19(3.3)
17 (2.9)
37 (6.4)
7 (1.2)
53(9.1)
36 (6.2)
40 (6.9)
13 (2.2)
41(7.1)
215 (37)
3 (0.5)
22
-------
Table 4. Cytogenetic comparisons of de novo leukemias and t-AMLa
(continued)
Cytogenetic features
inv(3)(q21q26)b
t(6;ll)(q27;q23)
t(6;9)(p23;q34)
t(8;16)(pll;pl3)
t(8;21)(q22;q22)
t(9;ll)(p22;q23)
t(9;22)(q34;qll)
t(ll;19)(q23;pl3)
t(llq23)
t(15;17)(q22;ql2)
inv(16)(p!3q22)c
t(21q22)
AML
de novo
n = 3,649 (%)
26 (0.7)
7 (0.2)
18 (0.5)
10 (0.3)
335 (9.2)d
64 (1.8)d
52 (1.4)d
16 (0.4)d
144 (3.9)
388(ll)d
144 (3.9)d
375 (10)d
t-AML
n = 581 (%)
1 (0.2)
2 (0.3)
1 (0.2)
2 (0.3)
11(1.9)
35 (6.0)
0
14 (2.4)
72 (12)
16 (2.8)
4 (0.7)
20 (3.4)
Includes 'unselected' cases from the Mitelman Database of Chromosome Aberrations in Cancer.
Includes also cases witht(3;3)(q21;q26).
Includes also cases witht(16;16)(p!3;q22).
dDiffers significantly (p < 0.025) from the t-AML group.
Source: Mauritzson et al. (2002). Reprinted by permission from the Nature Publishing Group.
t-AML and de novo leukemias at varying frequencies (see Table 5). For example, mutations in
genes of the receptor tyrosine kinase/RAS-BRAF signal transduction pathway are reported to be
present in over 50% of de novo AML (Christiansen et al., 2005). These occur through many
mechanisms including base pair substitutions, frame shifts, internal tandem duplications, gene
fusions, and splicing errors (for examples in AML1, see [Roumier et al., 2003]). In addition,
epigenetic alterations such as changes in the methylation patterns in leukemia-related genes or
their promoter regions have also been seen in many types of leukemia, including t-AML (Zheng
et al., 2004; Pedersen-Bjergaard et al., 2006; Nervi et al., 2008).
In recent years, researchers have begun to identify patterns among the myriad of
translocations, gene arrangements, and point mutations involved in myeloid leukemias and have
23
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Table 5. Frequency of molecular mutations in de novo AML and
t-MDS/t-AMLa
Mutated gene
FLT3 (ITDb)
FLT3 (TKDC)
NRAS
KITD816
MIL (ITDb)
RUNX1
TP53
PTPN11
NPM1
CEBPA
JAK2V617F
AML de novo (%)
35
9
10-15
~5
3
10-15
10
~2
35-50
6-15
2-5
t-MDS/t-AML (%)
0
<1
10
NAd
2-3
15-30
25-30
3
4-5
Rare
2-5
aFrom Qian et al. (2009). Reprinted with permission from Elsevier.
bITD = internal tandem duplication.
°TKD = tyrosine kinase-domain.
created a model for leukemogenesis (Deguchi and Gilliland, 2002; Kelly and Gilliland, 2002).
According to the model proposed by (Kelly and Gilliland, 2002),
AML is the consequence of collaboration between at least two broad classes of
mutations. Class I mutations, exemplified by constitutively activated tyrosine
kinases or their downstream effectors, such as BCR/ABL, TEL/PDGF|3R,
N-RAS, or K-RAS mutants, or constitutively activated FLT3, confer a
proliferative or survival advantage to hematopoietic cells. When expressed alone,
these mutant genes confer a CML-like disease characterized by leukocytosis with
normal maturation and function of cells. Class II mutations result in loss of
function of transcription factors that are important for normal hematopoietic
differentiation and include the AML1/ETO, CBFp/SMMHC, PML/RARa, and
NUP98/HOXA9 fusions as well as point mutations in hematopoietic transcription
factors such as AML1 and C/EBPa. These mutations would also be predicted to
impair subsequent apoptosis in cells that do not undergo terminal differentiation.
When expressed alone, these mutations may confer a phenotype like most MDS.
Regardless of the timing or order of acquisition of mutations, individuals who
accrue both Class I and Class II mutations have a clinical phenotype of AML
characterized by a proliferative and/or survival advantage to cells and by impaired
hematopoietic differentiation.
24
-------
A list of the gene mutations seen in patients with t-MDS or t-AML separated into these
two mutation classes is shown in Table 6.
More recently, additional interrelationships between new and previously identified
genetic alterations and leukemogenic agents, and types of leukemia have been reported
(Christiansen et al., 2001, 2004; Harada et al., 2003; Zheng et al., 2004; Klymenko et al., 2005;
Rege-Cambrin et al., 2005; Wiemels et al., 2005). As illustrated by Pedersen-Bjergaard et al.,
2008, the interactions between these various genes can become quite complicated, and the
outcome of these interactions is not fully understood. It should also be noted that some genes
such as TP53 have cellular functions that are quite different from those involved in the Class I
and II mutation pathways and may, therefore, represent a third class of mutated genes that
contribute to malignant transformation in t-AML and t-MDS. These results suggest that multiple
types of genetic changes are likely to be necessary for the conversion of a normal hematopoietic
stem or progenitor cell into a fully transformed leukemic cell (Pedersen-Bjergaard et al., 2008).
25
-------
Table 6. Gene mutations observed in the Copenhagen series of 140 patients
with t-MDS (« = 89) or t-AML (« = 51)a
t-MDS
t-AML
Class I mutations
Tyrosine kinases
FLT3 ITD+ point mutations
KIT point mutations
FMS point mutations
JAK2 point mutations
1
0
0
2
10
2
0
0
Genes in the RAS/BRAF pathway
KRAS or NRAS point mutations
BRAF point mutations
PTPN11 point mutations
7
0
2
7b
3b
2
Class II mutations
Transcription factors
AML1/CBFB chimerically rearranged
AML1 point mutations
MLL chimerically rearranged
MLL ITD
RARA chimerically rearranged
EVI1 chimerically rearranged
CEBPA point mutations
NPM1 point mutations
3
20
0
1
0
3
0
3
7
2
11
1
2
1
0
7
Tumor suppressor gene
TP53 point mutations
Total 131 mutations observed
25
67
9
64
aFrom Pedersen-Bjergaard et al. (2008). Reprinted with permission from the Nature Publishing
Group.
bOne patient had a mutation of KRAS together with a mutation of BRAF possibly in different
subclones.
26
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4. LEUKEMIA- AND LYMPHOMA-INDUCING AGENTS
While most leukemias occur in individuals with no obvious exposure to radiation or
chemical carcinogens (de novo leukemias), a significant number, known as secondary leukemias
develop in individuals who have previously been exposed to radiation, industrial chemicals, or
chemotherapeutic agents. Approximately 40 years ago, the IARC began evaluating chemicals
(and later other types of agents) for their ability to induce cancer in humans. Since 1971, IARC
has evaluated more than 900 agents (including exposure circumstances) and identified more than
100 as being carcinogenic to humans (classified as Group 1: Carcinogenic to Humans [IARC,
2010]). Another 66 have been classified in Group 2A, which indicates agents that are Probably
Carcinogenic to Humans. Among the 100+ agents in Group 1, approximately 25% have been
established as causing a lymphohematopoietic cancer (see Table 7). These include
eight therapeutic drugs or mixtures, five industrial chemicals or contaminants,
two immunosuppressive drugs, six forms of radiation, two occupational or lifestyle exposures,
and six infectious agents. Thus, leukemias and lymphomas represent one of the most common
types of cancer induced by a wide variety of carcinogens. In addition, three other Group 1
carcinogens (see Table 7), which were classified based on the induction of cancer at one or more
other sites, are also thought to be likely to induce either a leukemia or lymphoma. Of the
66 Group 2A agents, there is evidence based either on human studies, animal bioassays, or
structural similarities that at least 10 of these chemicals are also likely to cause leukemia in
humans. Table 7 lists the Group 1 and 2A agents that have been established, or are likely to
cause leukemia or lymphoma, as well some of their characteristics. The same list of agents with
key information related to the likely mechanisms responsible for the lymphohematopoietic
cancers is shown in Table 8.
In examining the information in Tables 7 and 8, a number of general patterns become
apparent. Leukemias are the primary type of cancer induced by the chemical agents, and most of
these are acute nonlymphocytic leukemias (ANLLs, synonymous with AML). These were
almost always induced by agents that are either directly DNA-reactive or that can be metabolized
(or otherwise converted) into DNA-reactive species. Many of the leukemogens are bifunctional
alkylating agents; chemicals that form adducts on DNA bases involved in base-pairing such as
the O6 of guanine, or induce chromosomal damage through the inhibition of topoisomerase II.
27
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Table 7. Characteristics of selected known and probable human leukemia- and lymphoma-inducing agents
Agents carcinogenic to humans
CAS
1° Lymphohematopoietic
cancer
2° limited
evidence
Myelotoxicity
Chromosomal
aberrations"
Source(s)
DNA-reactive
1,3 -Butadiene
1,4-Butanediol dimethanesulfonate (Busulfan,
Myleran)
Chlorambucil
(l-(2-)Chlorethyl)-3-(4-methylcyclohexyl)
nitrosurea (Methyl-CCNU, Semustine)
Cyclophosphamide
Ethylene Oxide
Formaldehyde
Melphalan
MOPP therapy
Treosulfan
Thio-TEPA (tris(l-aziridinyl)-phosphine)
55-98-1
305-03-3
13909-09-6
50-18-0
75-21-8
50-00-0
148-82-3
299-75-2
52-24-4
Lympho-hematopoietic
cancers
ANLL
ANLL
ANLL
ANLL
Myeloid leukemias
ANLL
ANLL
ANLL
Leukemia
NHL, MM,
CLL
b
+
+
+
+
-
+/-
+
+
+
+
-
SCA
SCA
NT
SCA
SCA
+/-
SCA
SCA
NI
SCA
IARC, 2008a, 2009; Baan
et al., 2009
Grosse et al., 2009; IARC,
1987b
Grosse et al., 2009; IARC,
1987b
Grosse et al., 2009
Grosse et al., 2009; IARC,
1987b
IARC, 2009, 2008b; Baan
et al., 2009
IARC, 2009, 2006
Grosse et al., 2009; IARC,
1987b
Grosse et al., 2009; IARC,
1987b
Grosse et al., 2009; IARC,
1987b
Grosse et al., 2009; IARC,
1987b
Topoisomerase II-inhibitor
Etoposide
33419-42-0
ANLL
+
MN
Grosse et al., 2009; IARC,
2000a
Immunosuppressive agents
Cyclosporine
Azathioprine
79217-60-0
446-86-6
NHL
NHL
-
+
SCA
SCA
Grosse et al., 2009; IARC,
1990c
Grosse et al., 2009; IARC,
1987b
to
oo
-------
Table 7. Characteristics of selected known and probable human leukemia- and lymphoma-inducing agents
(continued)
Agents carcinogenic to humans
CAS
1° Lymphohemato-poetic
cancer
2° limited
evidence
Myelotoxicity
Chromosomal
aberrations"
Source(s)
Other
Benzene
2,3,7,8-TCDD
X- and Gamma-radiation
Neutron radiation
rhorium-232 and its decay products
Phosphorus-32, as phosphate
Fission products including strontium-90
Tobacco smoking and tobacco smoke
Tobacco smoking (parental exposure)
Rubber manufacturing occupation
Painting occupation (maternal exposure)
71-43-2
1746-01-6
ANLL
ANLL, CML, ALL
ANLL, CML, ALL
ANLL
Leukemia (non-CLL)
ANLL
Leukemia, Lymphoma
NHL, ALL,
CLL, MM
NHL
Leukemia
Childhood
Leukemia
(ALL)
Childhood
leukemia
+
-
+
NI
NI
+
NI
NI
NI
NI
NI
SCA
-
SCA
SCA
SCA
SCA
NI
SCA
NI
SCA
NI
IARC, 2009;Baanetal.,
2009
IARC, 2009
[ARC, 2000b
[ARC, 2000b
ElGhissassietal.,2009;
[ARC, 2001
[ARC, 2001
ElGhissassietal.,2009;
Krestinina etal., 2010
IARC, 2004
Secretan et al., 2009
[ARC, 2009
IARC, 2009
Infectious agents
Epstein-Barr virus
Human immunodeficiency virus Type 1
Human T-cell lymphotrophic virus Type 1
Hepatitis C virus
Helicobacter pylori
Burkitt's lymphoma, NHL
NK/T-cell lymphoma,
Hodgkin lymphoma
NHL, Hodgkin lymphoma
Adult T-cell leukemia and
lymphoma
NHL
low-grade B-cell MALT
+
-
-
-
NI
NI
NI
NI
NI
Bouvard et al., 2009; IARC,
1997
Bouvard et al., 2009; IARC,
1996a
Bouvard et al., 2009; IARC,
1996b
Bouvard etal., 2009
Bouvard etal., 2009
to
VO
-------
Table 7. Characteristics of selected known and probable human leukemia- and lymphoma-inducing agents
(continued)
Agents carcinogenic to humans
Kaposi's sarcoma herpes virus
CAS
1° Lymphohemato-poetic
cancer
1 "effusion lymphoma
Selected asents probably carcinogenic to humans (IARC Group 2A)
2° limited
evidence
Myelotoxicity
-
Chromosomal
aberrations"
NI
Source(s)
Bouvardetal.,2009
DNA-reactive
Bischloroethyl nitrosourea (BCNU;
carmustine)
1 -(2-)Chlorethyl-3 -cyclohexyl- 1 -nitrosurea
(CCNU; lomustine)
/V-Ethyl-jV-nitrosourea
Cisplatin
Nitrogen Mustard (Mechlorethamine)
Procarbazine
Chlorozotocin
154-93-8
13010-47-4
759-73-9
15663-27-1
51-75-2
671-16-9
54749.90-5
ANLL
ANLL
ANLL?
leukemia
ANLL
ANLL
ANLL
+
+
NI
+
+
+
+
NId
NI
NId
NId
SCA
NId
NI
[ARC, 1987b
IARC, 1987b
[ARC, 1987b
IARC, 1987b
IARC, 1987b
IARC, 1987b
IARC, 1990b
Topoisomerase II-inhibitor
Adriamycin
Teniposide
25316-40-9
29767-20-2
ANLL
ANLL
+
+
SCA
NId
[ARC, 1987b
IARC, 2000c
Other
Azacytidine
Chloramphenicol
320-67-2
56-75-7
Leukemia
ANLL
+
+
NId
NI
[ARC, 1990a
IARC, 2000c
aSCA = structural chromosome aberrations; MN = micronuclei; MM = multiple myeloma; TCDD = Tetrachlorodibenzo-p-dioxin; NI = no information.
bMyelotoxicity is either not seen or infrequently seen.
°No information located for humans.
Increases seen in the blood or bone marrow of experimental animals.
-------
Table 8. Likely mechanisms involved in the carcinogenesis of selected known and probable human leukemia-
and lymphoma-inducing agents
Agent
Cancer
Class
Activation
1° Mechanism
Other
1° Source
Agents carcinogenic to humans (IARC Group 1 or similar)
1,3 -Butadiene
Busulfan (Myleran;
1,4-Butanediol
dimethanesulfonate)
Chlorambucil
Semustine (Methyl-CCNU;
l-(2-Chlorethyl)-3-
(4-methylcyclohexyl) 1 -
nitrosurea)
Cyclophosphamide
Ethylene oxide
Formaldehyde
Melphalan
Various
ANLL
ANLL
ANLL
ANLL
Various
ANLL
ANLL
Industrial
chemical
Therapeutic
agent
Therapeutic
agent
Therapeutic
agent
Therapeutic
agent
Industrial
chemical
Industrial
chemical
Therapeutic
agent
Bioactivated to mono-
and bifunctional
alkylating agents
Direct-acting
bifunctional alkylating
agent
Direct-acting
bifunctional alkylating
agent
Degrades to direct-
acting alkylating and
carbamoylating agents
Bioactivated to
bifunctional alkylating
agent and acrolein
Direct-acting
alkylating agent
Direct-acting, forms
DNA-protein
crosslinks
Direct-acting
bifunctional alkylating
agent
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
Unknown, possibly
mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
Metabolized into
diepoxybutane
IARC, 2008a, 2009;
Baan et al., 2009
Grosse et al., 2009
Grosse et al., 2009
Grosse et al., 2009
Grosse et al., 2009
IARC, 2008b, 2009;
Baan et al., 2009
IARC, 2006, 2009;
Baan et al., 2009
Grosse et al., 2009
-------
Table 8. Likely mechanisms involved in the carcinogenesis of selected known and probable human leukemia-
and lymphoma-inducing agents (continued)
Agent
MOPP therapy
Treosulfan
Thio-TEPA(tris(l-
aziridinyl)-phosphine)
Etoposide
Cyclosporine
Azathioprine
Cancer
ANLL
ANLL
Leukemia
ANLL
NHL
NHL
Class
Combination of
therapeutic
agents
Therapeutic
agent
Therapeutic
agent
Therapeutic
agent
Therapeutic
agent
Therapeutic
agent
Activation
A direct-acting
bifunctional and an
indirect
mono functional
alkylating agent, a
microtubule inhibitor,
and a glucocortocoid
Converts to a mono-
and bifunctional
alkylating agent
Direct-acting
trifunctional alkylating
agent. Also,
metabolized to
monofunctional
alkylating agent
aziridine
Topoisomerase II-
poison
Inhibition of
transcription factors
that regulate inducible
cytokine expression
Metabolized into
nucleotide analog
1° Mechanism
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
chromosomal breakage
and translocations
Immunosuppression
Immunosuppression
Other
Converts into
diepoxybutane, an
(intrastrand?)
crosslinking agent
Results in modified
transcription factor
Also genotoxic
1° Source
Grosse et al., 2009
Hartley et al., 1999;
Grosse et al., 2009
Maanen et al., 2000;
Grosse et al., 2009
Grosse et al., 2009
Grosse et al., 2009
Grosse et al., 2009
to
-------
Table 8. Likely mechanisms involved in the carcinogenesis of selected known and probable human leukemia-
and lymphoma-inducing agents (continued)
Agent
Benzene
2,3,7,8-TCDD
X- and Gamma-radiation
Alpha and beta particle
emitters
Tobacco smoking and
tobacco smoke
Tobacco smoking (parental)
Cancer
ANLL
NHLa
ANLL
CML
ALL
ANLL
(also CML
and
ALL for
Th-32)
ANLL
Childhood
ALL
Class
Industrial
chemical and
environmental
agent
Environmental
contaminant
Therapeutic,
energy and
military uses
Therapeutic,
energy and
military uses
Lifestyle use
Parental use
Activation
Metabolized into
reactive protein and
DNA-binding species
Receptor-mediated
effects modifying
cellular replication and
apoptosis
Direct and indirect
DNA damage
Direct and indirect
DNA damage
Direct and indirect
DNA damage
Direct and indirect
DNA damage
1° Mechanism
Unknown, likely
mutation resulting from
either DNA binding,
topoisomerase II-
inhibition, and/or
oxidative damage
Unknown, likely
immunosuppression
Mutation resulting from
DNA damage and/or
chromosomal alterations
Mutation resulting from
DNA damage and/or
chromosomal alterations
Unknown, likely
mutation resulting from
DNA binding and/or
chromosomal alterations
Unknown, assumed
mutation resulting from
DNA binding and/or
chromosomal alterations
occurring in germ cells
or in utero
Other
Likely multiple
metabolites and
modes of action
involved
Also can lead to
DNA damage
through oxidative
stress
Epigenetic changes
could also
contribute
1° Source
Baan et al., 2009;
IARC, 2009
Baan et al., 2009;
IARC, 2009;
Holsapple et al.,
1996
IARC, 2000b;
El Ghissassi et al.,
2009
IARC, 2001; El
Ghissassi etal., 2009
IARC, 2004;
Secretan etal., 2009
IARC, 2004;
Secretan etal., 2009
-------
Table 8. Likely mechanisms involved in the carcinogenesis of selected known and probable human leukemia-
and lymphoma-inducing agents (continued)
Agent
Rubber manufacturing
occupation
Painting occupation
Epstein-Barr vims
Human immunodeficiency
vims Type 1
Human T-cell lymphotrophic
vims Type 1
Cancer
Leukemia
Lymphoma
Childhood
leukemia3
Burkitt's
lymphoma
NHL
NK/T-cell
lymphoma
Hodgkin
lymphoma
NHL
Adult T-cell
leukemia and
lymphoma
Class
Occupational
exposure
Occupational
exposure
Infectious agent
Infectious agent
Infectious agent
Activation
Unknown but DNA-
reactive chemicals are
used
Unknown but DNA-
reactive chemicals are
used
Viral infection and
expression of viral
proteins leading to
lymphocyte
transformation
Viral infection and
expression of viral
proteins leading to loss
of CD 4+ T
lymphocytes
Viral infection and
expression leading to
lymphocyte
transformation
1° Mechanism
Unknown, assumed to be
mutation resulting from
DNA binding and/or
chromosomal alterations
and/or
immunosuppression
Unknown, assumed
mutation resulting from
DNA binding and/or
chromosomal alterations
occurring in germ cells
or in utero
Alteration in normal
B -lymphocyte function
leading to cell
proliferation, inhibition
of apoptosis, genomic
instability, and cell
migration
Immunosuppression (as
an indirect effect)
Immortalization and
transformation of T cells
Other
Other mechanisms
are also likely
1° Source
IARC, 2009
IARC, 2009
Hjalgrim and Engels,
2008;Bouvardetal.,
2009
Hjalgrim and Engels,
2008;Bouvardetal.,
2009
Hjalgrim and Engels,
2008;Bouvardetal.,
2009
-------
Table 8. Likely mechanisms involved in the carcinogenesis of selected known and probable human leukemia-
and lymphoma-inducing agents (continued)
Agent
Hepatitis C vims
Helicobacter pylori
Kaposi's sarcoma herpes
virus
Cancer
NHL
Low-grade B-
cell MALT
1° Effusion
lymphoma
Class
Infectious agent
Infectious agent
Infectious agent
Activation
Viral infection and
expression of viral
proteins leading to
chronic immune
stimulation
Inflammation leading
to cellular alterations
Viral infection and
expression of viral
proteins
1° Mechanism
Chronic immune
stimulation
Oxidative stress, altered
cellular turnover and
gene expression,
methylation, and
mutation
Cell proliferation,
inhibition of apoptosis,
genomic
instability, cell migration
Other
Chronic immune
stimulation
1° Source
Hjalgrim and Engels,
2008;Bouvardetal.,
2009
Hjalgrim and Engels,
2008;Bouvardetal.,
2009
Bouvard et al., 2009
Selected agents probably carcinogenic to humans (IARC Group 2A)
Bischloroethyl nitrosourea
(BCNU; carmustine)
(l-(2-)Chlorethyl)-3-
cyclohexyl- 1 -nitrosurea
(CCNU; lomustine)
7V-Ethyl-7V-nitrosourea
Cisplatin
ANLL
ANLL
ANLL?
Leukemia
Therapeutic
agent
Therapeutic
agent
Experimental
reagent
Therapeutic
agent
Direct-acting
bifunctional alkylating
agent
Direct-acting
bifunctional alkylating
agent
Direct-acting
alkylating agent
Direct-acting
bifunctional DNA
binding agent
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
IARC, 1987b; Vogel
etal., 1998
IARC, 1987b; Vogel
etal., 1998
IARC, 1987b; Vogel
etal., 1998
IARC, 1987b; Vogel
etal., 1998
-------
Table 8. Likely mechanisms involved in the carcinogenesis of selected known and probable human leukemia-
and lymphoma-inducing agents (continued)
Agent
Nitrogen Mustard
(Mechlorethamine)
Procarbazine
Chlorozotocin
Adriamycin
Teniposide
Azacytidine
Chloramphenicol
Cancer
Leukemia
ANLL
ANLL
ANLL
ANLL
Leukemia
ANLL
Class
Therapeutic
agent
Therapeutic
agent
Therapeutic
agent
Therapeutic
agent
Therapeutic
agent
Therapeutic
agent
Therapeutic
agent
Activation
Direct-acting
bifunctional alkylating
agent
Bioactivated to a
monorunctional
alkylating agent
Direct-acting
bifunctional alkylating
agent
Topoisomerase II-
poison and redox-
cycling agent
Topoisomerase II-
poison
DNA-
methyltransferase
inhibitor through
metabolism and
incorporation into
DNA
Binds to ribosomal
subunit blocking
protein synthesis in
mitochondria.
Metabolite may also
induce DNA damage
1° Mechanism
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
DNA binding and/or
chromosomal alterations
Mutation resulting from
chromosomal breakage
and translocations
Mutation resulting from
chromosomal breakage
and translocations
Alters DNA methylation
and gene expression. Is
also genotoxic
Unknown, presumed to
be mutation resulting
from DNA damage
and/or chromosomal
alterations
Other
1° Source
IARC, 1987b; Vogel
etal., 1998
IARC, 1987b; Vogel
etal., 1998
IARC, 1987b; Vogel
etal., 1998
IARC, 1987b
IARC, 2000c
NTP, 2005;
Stresemann and
Lyko, 2008
NTP, 2005
"Limited evidence.
-------
The primary mode of action for these agents is through the induction of mutations, either gene
mutations or chromosomal mutations (IARC, 2012). Consistent with their proposed mutagenic
mechanisms, most of the leukemia-inducing agents have been reported to induce structural
chromosome aberrations in the peripheral blood of exposed humans. Myelotoxicity is also
commonly seen in humans (and animals) exposed to these leukemogenic agents. Exposure to
radiation in various forms was frequently associated with ALL and CML, in addition to ANLL.
These are also likely due to the effects of ionizing radiation on DNA, either directly or indirectly,
and are believed to occur through mutagenic and epigenetic mechanisms (IARC, 2012).
In contrast to these DNA-damaging agents, exposure to a variety of infectious agents is
causally related to the formation of lymphoid neoplasms. The induced lymphomas appear to be
primarily associated with chronic infection with either viruses or Helicobacter bacteria that are
immunomodulating and/or specifically target lymphoid cells. These agents are believed to act as
either direct carcinogens acting on the cellular DNA, indirect carcinogens acting through chronic
inflammation, or through immune-suppression (IARC, 2012). The two chemical agents,
cyclosporine and azathioprine, which also induce NHL, are also strongly immunosuppressive. In
addition, Tetrachlorodibenzo-p-dioxin (TCDD), which may induce NHL, has been shown in
animal studies to be immunosuppressive (Holsapple et al., 1996), so this could be the mechanism
underlying its lymphoma-inducing effects. Although not reviewed by IARC and, hence, not
presented in Tables 7 and 8, autoimmune disorders and other diseases associated with immune
stimulation and inflammation are increasingly recognized from large consortia studies as risk
factors for lymphoma (Vajdic et al., 2009, Ekstrom Smedby et al., 2008; Cocco et al., 2008;
Meters et al., 2006).
Similar to the Group 1 agents, the Group 2A leukemogens are primarily associated with
the induction of ANLL. For chemicals for which information is available, these agents are also
myelotoxic and clastogenic and are believed to most likely induce their leukemogenic effects
through a mutagenic mechanism (see Table 8).
In summary, based on the IARC evaluations, the vast majority of the leukemia-inducing
agents are believed to act through a mutagenic or genotoxic mechanism(s) whereas the
lymphoma-inducing agents most likely act through immunomodulation and related effects. An
overview of the major classes of leukemia-inducing agents is presented below.
37
-------
5. OVERVIEW OF THE MAJOR CLASSES OF LEUKEMIA-INDUCING AGENTS
5.1. IONIZING RADIATION
As a result of its widespread medical, military, and energy-related uses, large numbers of
people have been exposed to ionizing radiation, and its adverse effects have been extensively
documented (IARC, 2000b, 2001; UNSCEAR, 2000a, b; Ron, 2003; NRC, 2006). Exposure to
ionizing radiation has been shown to result in numerous types of cancer including several types
of leukemia as well as adverse effects that are likely to be involved in leukemogenesis including
myelotoxicity, immune suppression, and genetic damage leading to chromosomal and gene
mutations. The earliest association between radiation and cancer was seen for leukemia, and it
has been repeatedly seen in numerous population studies including those exposed through
medical, military, and environmental exposures (IARC, 2000b, 2001; UNSCEAR, 2000b; Ron,
2003; NRC, 2006). It should be noted that there are a variety of types of radiation and
radioactive materials. Most of the studies that have been conducted have investigated the effects
of ionizing radiation, which for simplicity; will be considered as one individual agent in this
section. Assessing the risks associated with radiation exposure is challenging and is influenced
by the type of radiation, the tissue dose, the proportion of the body exposed, the extent of cell
killing, and the DNA-repair capacity of the individual or tissue (Curtis et al., 1994). Host factors
such as age, sex, genetic composition, and health of the exposed individuals can also influence
the risk of radiation-induced leukemia.
All age groups are affected, but children, in particular, have been shown to be at an
elevated risk for radiation-induced leukemia as seen in the Life Span Study of the atomic bomb
survivors in Hiroshima and Nagasaki. Among the survivors who were exposed to both gamma,
and to a lesser extent, neutron radiation, the highest leukemia risks were seen in children
(Preston et al., 1994, 2004). Significant dose-related increases were seen for ALL, AML, and
CML, with the excess risks for ALL and CML being approximately 100% higher in the exposed
males than in the females. When the leukemias were reclassified using the FAB classification
scheme, all AML subtypes were present with a predominance of myeloblastic leukemias with
and without maturation (Ml and M2) (Matsuo et al., 1988). Among the ALL cases, most cases
exhibited the L2 subtype, but a significant portion also showed the LI subtype. Latency periods
were shorter for those under 15 years of age with a leukemia peak at 5-7 years post exposure as
38
-------
compared to later peaks for those exposed at later ages (Kamada and Tanaka, 1983). The
leukemia risk also decreased more rapidly in children as compared to those in older age groups.
Overall, the risks to children were highest during the period from 1950 to 1965 and returned to
near background levels thereafter (Preston et al., 1994).
Similarly, the use of radiation for medical purposes has been associated with increases in
leukemia. Increased risk of leukemia has been reported following the use of radiation for
diagnostic tests, for the treatment of benign diseases, and following radiotherapy for cancer
(Ron, 1998, 2003). For hematopoietic and lymphoid tissues that are diffuse, a significant portion
of the tissue must be irradiated to increase the incidence of neoplasia (Storer et al., 1982). The
leukemia risks from medical irradiation appear to have diminished in recent years due to a use of
lower and more restricted doses as well as changes in therapeutic strategy in which high doses
are applied within limited fields. High radiation doses (above 3-4 Gray) result in extensive
killing of marrow-containing progenitor cells and have been associated with a reduced risk of
developing leukemia (Boice et al., 1987; NRC, 1990; Curtis et al., 1994).
5.2. CHEMOTHERAPEUTIC AGENTS
Following the initiation of intensive chemotherapy with genotoxic agents, increases in
therapy-related leukemias began to appear (Seiber and Adamson, 1975; Kantarjian and Keating,
1987; Levine and Bloomfield, 1992; Leone et al., 1999). These leukemias, also known as
treatment-related or secondary leukemias, consisted primarily of AML, although a small increase
in therapy-related ALL has also been seen (Hunger et al., 1992; Andersen et al., 2001). With
increased periods of follow-up, increases in other types of solid tumors have also been observed
(Tucker et al., 1988; Loescher et al., 1989; Boffetta and Kaldor, 1994; van Leeuwen et al., 1994;
Vega-Stromberg, 2003; Travis et al., 2005; Hodgson et al., 2007).
Over time, as new agents and therapeutic strategies have been employed; additional
agents have been recognized as inducing leukemia in humans. Indeed, as seen in Table 7,
chemotherapeutic drugs comprise the largest group of agents generally recognized as human
leukemia-inducing agents. Currently, therapy-related leukemias constitute 10 to 20% of the
leukemia cases seen at major medical institutions (Deschler and Lubbert, 2006;
Pedersen-Bjergaard et al., 2007).
39
-------
The identification of specific agents involved in leukemogenesis and the interpretation of
many of the studies can be challenging due to the use of multiple therapeutic agents, varying
dosing regimens, concurrent use of radiotherapy, and variable periods of patient follow-up. Over
time, two main classes of leukemogenic therapeutic drugs have been identified—alkylating
agents and topoisomerase II inhibitors (Pedersen-Bjergaard and Philip, 1991; Pedersen-Bjergaard
and Rowley, 1994; Pedersen-Bjergaard et al., 2006). An overview of the AMLs (including
myelodysplastic syndromes) induced by these two classes of chemotherapeutic drugs is briefly
discussed in the following sections.
5.2.1. Alkylating Agent-Related Leukemias
A large number of studies have demonstrated that patients treated with alkylating
agent-based chemotherapy are at an increased risk of MDS and AML (Levine and Bloomfield,
1992; Pedersen-Bjergaard and Rowley, 1994; Smith et al., 1994). These risks have been seen for
both children and adults and are strongly related to the cumulative dose of the alkylating agent
(Pedersen-Bjergaard and Philip, 1987; Tucker et al., 1987; Hunger et al., 1992;
Pedersen-Bjergaard et al., 2000; Pyatt et al., 2005). The administered agents also exhibit varying
degrees of hematopoietic toxicity, immunotoxicity, and genotoxicity, which can also be affected
by the agent, dose, and route of administration (Gale, 1988; Ferguson and Pearson, 1996;
Sanderson and Shield, 1996). The incidence of therapy-related MDS and AML (t-MDS/t-AML)
in adults treated with antineoplastic drugs has been reported to range from 1 to >20% (Felix,
1998; Leone et al., 2001). The increases in this type of therapy-related leukemia generally
appear 1 to 2 years after treatment and may remain elevated for 8 or more years following the
completion of chemotherapy (Pedersen-Bjergaard and Philip, 1987; Pedersen-Bjergaard and
Philip, 1991; Davies, 2000; Schonfeld et al., 2006).
In adults, leukemias induced by alkylating agents exhibit characteristics that generally
allow them to be distinguished from de novo leukemias and those induced by topoisomerase II
inhibitors (Pedersen-Bjergaard and Rowley, 1994; Eastmond et al., 2005). These leukemias are
typically myeloblastic with or without maturation (FAB Ml and M2 subtypes) and are
characterized by trilineage dysplasia and clonal unbalanced chromosome aberrations, most
commonly involving loss of the entire chromosome or part of the long arms of
chromosomes Sand 7 (-7, 7q-, -5, 5q-). These t-AML have a modal latency of 4-7 years, and
40
-------
the onset of the actual leukemia is often preceded by myelodysplasia. It should be noted that
AML induced by ionizing radiation tends to exhibit similar features (MDS, clonal unbalanced
chromosomal aberrations, etc.) although the range of the FAB subtypes induced by radiation
tends to be broader (Matsuo et al., 1988; Philip and Pedersen-Bjergaard, 1988; Gundestrup et al.,
2000).
Using structure-activity relationships as well as other predictive approaches with in vivo
rodent and Drosophila data, Vogel et al. (1998) were able to identify three major categories of
DNA-reactive chemical and therapeutic agents. As described by the authors, Category 1
consisted of mono-functional alkylating agents such as ethylene oxide and methyl methane
sulfonate, which primarily react at the N7 and N3 moieties of purines in DNA. Efficient DNA
repair was the major protective mechanism against the relatively weak genotoxic effects of these
agents, which might not be detectable in repair-competent cells. High doses were generally
needed for the induction of tumors in rodents. Strong target site specificity for the adverse
effects was seen, and this appeared to be related to DNA repair capacity. Category 2 agents such
as procarbazine and TV-nitroso-jV-ethylurea (ENU), induce both O-alkyl adducts and 7V-alkyl
adducts in DNA. In general, the induced O-alkyl adducts appeared to be slowly repaired, or not
repaired, which made these agents potent carcinogens and germ cell mutagens. The inefficient
repair of the O-alkyl-pyrimidines, in particular, was responsible for their potent mutagenic
activity. Category 3 agents such as melphalan and busulfan induced structural aberrations
through their ability to cross-link DNA, and this was the major factor contributing to their high
genotoxic potency, which appeared to be related to the number of DNA crosslinks per target
dose unit that were induced. The genotoxic effects for the Category 3 agents occurred at, or
near, toxic levels. For all three categories of genotoxic agents, strong correlations were observed
between their carcinogenic potency, acute toxicity, and germ-cell specificity.
5.2.2. Topoisomerase II Inhibitor-Related Leukemias
In the late 1980s, a new type of therapy-related leukemia was recognized that exhibited
unusual features that differed from those previously seen following treatment with alkylating
agents and radiation (Pui et al., 1989; Pui and Relling, 2000). The affected patients had been
treated with etoposide or teniposide, two of a newly developed epipodophyllotoxin class of
chemotherapeutic agents (Pedersen-Bjergaard and Philip, 1991). In the ensuing years, these
41
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epipodophyllotoxins have become widely used, especially for treating childhood cancers, and a
large number of studies have been published establishing an association between treatment with
these drugs and the subsequent development of leukemia (Haupt et al., 1993; Smith et al., 1994;
Pedersen-Bjergaard et al., 1995; Pui and Relling, 2000; Leone et al., 2001; Hijiya et al., 2009).
The leukemia risk following epipodophyllotoxin therapy was reported to be very high in
early studies, with cumulative incidences approaching 19% in some treatment groups (Pui et al.,
1989). Interestingly, in some cases, the risk of a treatment-related cancer appeared to be more
closely related to the treatment regimen than to total dose (Pui et al., 1991; Pui and Relling,
2000). Patients receiving epipodophyllotoxins on a weekly or twice-weekly basis had much
higher cumulative risks (12.4%) than those receiving the drugs on a biweekly schedule (1.6%).
Similar results have been seen in more recent studies (Smith et al., 1999) although others have
seen a correlation between cumulative dose and epipodophyllotoxin-induced t-AML (Neglia
et al., 2001; Le Deley et al., 2003). With implementation of newer treatment protocols, the risk
of t-AML has been reduced substantially and is now within the range of that seen with alkylating
chemotherapeutic agents (Smith et al., 1993; Pui and Relling, 2000). There is also evidence that
the combined treatment of topoisomerase II inhibitors with alkylating agents (or cisplatin)
confers a greater risk of t-AML than seen with either type of chemotherapeutic agent alone
(Sandoval et al., 1993; Smith et al., 1994; Blatt, 1995). In most reported cases, the incidence of
t-AML induced by these drugs is estimated to range from 2 to 12% (Felix, 1998; Leone et al.,
2001).
The epipodophyllotoxins exhibit moderate myelosuppression, identifiable chromosomal
damage, and their leukemogenic effects by inhibiting topoisomerase II through a process that
involves stabilization of the DNA-enzyme complex (Smith et al., 1994; Ferguson and Pearson,
1996). Topoisomerase II is a nuclear enzyme involved in a wide variety of cellular functions,
including DNA replication, transcription, and chromosome segregation (Anderson and Berger,
1994; Nitiss, 2009). Leukemias induced following treatment with epipodophyllotoxin-type
topoisomerase inhibitors generally appear from 10 months to 8 years following the initiation of
chemotherapy, with a median latency of 2 to 3 years (Smith et al., 1994; Leone et al., 2001). The
induced AMLs are primarily of the monocytic or myelomonocytic subtypes (M4 and M5) and
are rarely preceded by a myelodysplastic phase, a pattern that differs significantly from
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alkylating agent or radiation-induced leukemias (Pedersen-Bj ergaard and Rowley, 1994; Smith
et al., 1994; Leone et al., 2001; Pedersen-Bj ergaard et al., 2006).
Infrequently, ALL has been reported in patients following treatment with both alkylating
agents and topoisomerase II-inhibiting drugs (Hunger et al., 1992; Andersen et al., 2001;
Hijiya et al., 2009). Although t-ALLs occur infrequently, they seem to be more common in
children under the age of 15 who have previously been treated with a topoisomerase II inhibitor
(Andersen et al., 2001).
One of the unique features of t-AML, and to a lesser extent t-ALL, induced by the
epipodophyllotoxin-type of topoisomerase II inhibitors, is the presence of clonal-balanced
translocations involving the MLL gene (also known as ALL-1, HRX, and HTRX-1) located on
the long arm of chromosome 11 (1 Iq23) in the leukemic cells. Cytogenetic studies of patients
with leukemias induced by these agents have shown that in over 50% of the cases, the leukemic
clone involved a balanced translocation affecting the 1 Iq23 region and another chromosomal
partner, usually t(6;l 1), t(9;l 1), and t(l 1;19) (Pedersen-Bjergaard and Rowley, 1994; Smith
et al., 1994; Canaani et al., 1995). In children previously treated with topoisomerase inhibitors,
up to 90% of the treatment-related leukemias have an 1 Iq23 alteration (Canaani et al., 1995). As
indicated previously, as of 2006, 87 rearrangements involving the MLL gene have been
identified, and 51 of the translocation partner genes had been characterized at the molecular level
(Meyeret al., 2006). The four most common MLL translocation partner genes (i.e., AF4, AF9,
ENL, and AF10) encode nuclear proteins that are part of a protein network involved in histone
H3K79 methylation (Meyer et al., 2006) indicating a potentially important role for this pathway
in epipodophyllotoxin leukemogenesis.
Numerous lines of evidence indicate that topoisomerase II as well as DNA-repair
enzymes such as those involved in nonhomologous end joining play an important role in the
formation of the 1 Iq23 translocations. These have been summarized in a series of reviews
(Greaves and Wiemels, 2003; Apian, 2006; Felix et al., 2006; Zhang and Rowley, 2006). The
presence of topoisomerase II recognition sites has been found in proximity to the translocation
breakpoints as have recombinase recognition sites, Alu sequences, DNase hypersensitive sites,
and scaffold attachment regions, and suggests that multiple types of damage and repair probably
contribute to the generation of the observed translocations (Pui and Relling, 2000; Felix et al.,
2006; Zhang and Rowley, 2006). Interestingly, in a recent report, Le et al. (2009) reported that
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the translocation breakpoints within the MLL gene occurred in a highly specific fashion at the
base of a secondary DNA structure formed from a palindrome. A stringent topoisomerase II
consensus binding site was located at the apex of the secondary DNA structure. The authors
proposed a model in which topoisomerase II facilitates the formation of a secondary structure
that results in site-specific DNA strand breakage that is efficiently processed into translocations
(Le et al., 2009). This processing most likely occurs through a mechanism involving
nonhomologous end joining (Greaves and Wiemels, 2003; Zhang and Rowley, 2006). It should
also be noted that the MLL gene has been shown to be prone to breakage during apoptosis, and it
has been proposed that the observed translocations occur in hematopoietic cells rescued at an
early, reversible stage during the apoptotic process (Stanulla et al., 1997; Betti et al., 2005;
Vaughan et al., 2005; Basecke et al., 2006). A role for apoptosis has also been proposed in the
formation of other leukemia-related translocations (Eguchi-Ishimae et al., 2001).
5.2.3. Other Likely Leukemia-Inducing Therapeutic Agents
In recent years, evidence has accumulated that other inhibitors of topoisomerase II, such
as the anthracycline, anthracenedione, and bisdioxopiperazine derivatives, can also induce
treatment-related leukemias (Xue et al., 1992; Zhang et al., 1993; Blatt, 1995; Andersen et al.,
1998; Le Deley et al., 2003; Mistry et al., 2005; Mays et al., 2010). The leukemias induced by
these agents are similar to the epipodophyllotoxins in that they have short latency periods and are
infrequently preceded by myelodysplasia. However, they typically exhibit different types of
clonal-balanced rearrangements (e.g., t[8;21], t[15;17], and inv[16]) and different FAB subtypes
(M2 and M3).
Although azathioprine, 6-thioguanine, and 6-mercaptopurine are primarily associated
with immunosuppression and the induction of NHL, a number of studies have suggested that
patients treated with these 6-thioguanosine monophosphate-producing drugs are at an increased
risk of developing ANLL (Bo et al., 1999; Karran, 2006; Yenson et al., 2008). It has been
suggested that the risks are higher in patients with low thiopurine S-methyltransferase activity
and may involve aberrant mismatch repair and microsatellite instability (Bo et al., 1999; Karran,
2006). Interestingly, the leukemias of many of these patients also show loss of chromosome 7 or
part of chromosomes 5 or 7 (5q- or 7q-) (Yenson et al., 2008).
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6. MECHANISMS INVOLVED IN THERAPY-RELATED ACUTE MYELOID
LEUKEMIA (T-AML)
For more than 20 years, Pedersen-Bjergaard and colleagues have collected information
on patients with t-AML and t-MDS who have been treated at their clinic in Copenhagen,
Denmark. Based on the genetic alterations seen in this cohort of 140 patients as well as
information in the literature, they have identified eight separate pathways that appear to lead
either directly to t-AML or lead to t-MDS and then to t-AML. These pathways are illustrated in
Figure 3 (from [Pedersen-Bjergaardet al., 2006]). The pathways have been classified into
three groups largely based upon the therapeutic agent that was likely responsible for the
leukemia. Each of the pathways is described below based on descriptions extracted from their
2006 article (i.e., Pedersen-Bjergaard et al., 2006).
Pathway I in the figure was characterized by patients who developed t-MDS prior to
t-AML and exhibited either loss of chromosome 7 or part of the long arm of chromosome (7q-).
These patients also did not exhibit the recurrent balanced translocations that are associated with
AML. Most of these patients (35/39) had been treated with alkylating agents, and most (35/39)
presented with t-MDS prior to t-AML. Methylation of the pi 5 promoter, considered a late event
in leukemogenesis, was seen in 84% of the patients. In addition to the chromosome 7 changes,
the leukemias of a significant portion (15/39 or 38%) of these patients had point mutations in the
AML1 (also known as RUNX1) gene. A number of these patients also had mutations in either
the TP53 tumor suppressor gene or an activating mutation in the RAS oncogene.
Pathway II predominated in patients that exhibited either loss of all (-5) or part of the
long arm of chromosome 5 (5q-). Approximately half of the patients in this group also had -7
or 7q- alterations. As with Pathway I, onset of this disease is closely related to the use of
alkylating agent chemotherapy (27/34) and an initial presentation as t-MDS (26/34).
Seventy-seven percentage of these patients exhibited mutations in TP53. The patients also often
present with a complex karyotype or loss of part of the short arm of chromosome 17 (17p-) or
showed an amplification or duplication of chromosome bands 1 Iq23 or 21q22. Methylation of
the pi 5 promoter occurred in 80-90% of the patients of this group.
Pathway III was characterized by balanced translocations involving the 1 Iq23
chromosome band and one of many partner chromosomes. These translocations frequently
occurred in patients who had been previously treated with topoisomerase II inhibitors and
45
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Pathway t-MDS
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Figure 3. Genetic pathways of t-MDS and t-AML based on 140 cases from
the Copenhagen study group. The black triangle indicates significant
association with transformations from t-MDS to t-AML.
Source: Pedersen-Bjergaard et al. (2006). Reprinted by permission from
Macmillan Publishers Ltd.
46
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exhibited AML of the FAB subtypes M4 or M5. Three of the 11 patients in this group had a
RAS mutation, and another 3 had a BRAF mutation. Methylation of the pi 5 promoter was seen
in 50% of the patients.
Pathway IV occurred in patients with balanced translocations involving chromosome
bands 21q22 or 16q22 leading to chimeric rearrangements involving the core binding factor
genes AML1 or CBFB. These chromosomal changes are generally associated with previous
treatment with topoisomerase II inhibitors, most commonly of the anthracycline class. While
patients in this group with a t(3;21)(q26;q22) often presented as t-MDS, most of the others
presented directly with t-AML. Chromosome 7 alterations were also seen in five of nine patients
with 21q22 translocations. Point mutations in KIT (c-Kit) and PTPN11 were seen in a few
patients. Methylation of the pi 5 promoter was seen in 83% of the patients.
Pathway V occurred in only two patients and is characterized by a translocation between
chromosomes 15 and 17 involving the PML and RARA genes. Therapy-related promyelocytic
leukemia (M3) that exhibits the characteristic t(15; 17) has been reported to occur in patients
treated with doxorubicin or mitoxanthrone. One of the patients in this group also had a mutation
due to FLT3 internal tandem duplication.
Pathway VI is an uncommon pathway in which t-AML patients exhibit balanced
translocations involving the NU98 gene at chromosome band 1 Ipl5. None of the 140 patients in
the Copenhagen series exhibited this pattern.
Pathway VII exhibits a normal karyotype, and most often presents directly as t-AML.
Approximately 17% of the patients in the Copenhagen series fell within this group and have not
been consistently associated with any previous type of chemotherapy. The mutations seen are
also commonly seen in de novo leukemias suggesting that these may represent sporadic cases of
de novo leukemia occurring in these patients. Methylation of the pi 5 promoter was only seen in
50% of these patients.
Pathway VIII is composed of patients that exhibit unique or unusual chromosome
alterations and represents 14% of the Copenhagen cohort. These cases do not show an
association to any specific type of previous therapy and may also represent cases of de novo
leukemia. Methylation of the pi 5 promoter was also uncommon, occurring in 40% of the cases.
Consistent with the description presented above, patients with t-AML with leukemic cells
exhibiting loss of all or part of chromosomes 5 or 7, frequently present initially with t-MDS and
47
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have often been previously treated with alkylating agent chemotherapeutic drugs. In contrast,
t-AMLs exhibiting certain specific reciprocal translocations such as t(l Iq23) and t(21q22) occur
in patients that were previously treated with a topoisomerase II-containing chemotherapeutic
regimen and develop without a preceding MDS.
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7. FACTORS CONFERRING AN INCREASED RISK OF INDUCED LEUKEMIA
7.1. MYELOSUPPRESSION
Myelosuppression and immunotoxicity frequently accompany exposure to
leukemia-inducing agents, particularly those such as the chemotherapeutic drugs, ionizing
radiation, and benzene, for which leukemogenicity has been clearly established (Ferguson and
Pearson, 1996; Eastmond, 1997). The number of individuals affected and the magnitude of
toxicity are influenced by the agent and dose-related factors such as total dose, dose per
treatment, schedule, and route of administration as well as individual host factors (age, genetic
susceptibility, prior therapy, health status, etc.) (Gale, 1988). A brief overview of the
myelosuppressive effects of cancer therapeutic drugs has been written by Gale (1988) and is the
basis for the following description. The severity of myelotoxicity induced by antineoplastic
drugs varies considerably by chemical class. For drugs associated with t-AML, moderate-to-
severe effects are generally seen. Epipodophyllotoxins, cisplatin, and procarbazine typically
produce more moderate toxicity whereas severe effects are more common for the alkylating
agents, the anthracyclines, and the nitrosoureas.
The period between dosing and the onset or appearance of the myelosuppression is also
related to the class of agent. For some agents such as ionizing radiation, the onset of
myelotoxicity occurs within 0 to 48 hours after exposure. For others, longer periods are
required. The onset of myelosuppression by the alkylating agents and anthracyclines occurs 1 to
3 weeks following exposure and is believed to be due to the effect of these agents on immature
hematopoietic cells that becomes more evident as the more mature blood cells die and require
replacement. Myelosuppression induced by the nitrosoureas and mitomycin C is less frequent
and occurs 4 to 8 weeks after treatment. This delayed effect is believed to be due to a relatively
selective effect on immature stem cells. However, the onset of this delayed effect is
dose-dependent. A two- to threefold increase in dose reduces the onset of myelotoxicity to
1 week. For some drugs such as busulfan, the manifestation of the myelotoxic effects is
considered to be latent and may only be manifested under stress-related conditions.
In most cases, the induced myelotoxicity is transient with the blood cell counts returning
to normal following the cessation of treatment or exposure (Hendry and Feng-Tong, 1995; Irons
et al., 2005). However, in some cases, the induced myelosuppression can be more persistent and
49
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progress to pancytopenia or infrequently to aplastic anemia, a condition that confers a much
greater risk of developing leukemia (-10%) (Aksoy et al.,1984; Jandl, 1987; Ohara et al., 1997;
Imashuku et al., 2003). Increased risks have also been seen for those who have previously
exhibited less severe forms of bone marrow toxicity. Studies of benzene-exposed workers have
shown that the leukemia mortality rate was much higher for workers who had previously been
diagnosed with bone marrow poisoning (700 per 106 person-years) as compared to those exposed
to, but not poisoned by, benzene (14 per 106 person-years), and particularly as compared to the
general population (2 per 106 person-years) (Yin and Li, 1994). In one report, 36% of the
benzene leukemia cases had a history of benzene poisoning with leukopenia or pancytopenia
(Yin et al., 1994). However, this also indicates that for most cases, clinically detectable
myelotoxicity (e.g., leucopenia or pancytopenia) may not be observed. It should also be noted
that at lower exposure levels, the decreases in cell counts occurring in exposed groups may fall
within what is considered the normal clinical range (Qu et al., 2002; Lan et al., 2004). This
highlights one of the challenges in using myelotoxicity as a biomarker, as the normal range
varies considerably in adults. For example, the mean white blood cell count in adults is
7,200 x 103/|iL with a 95% range from 3,900 to 10,900 x 103/|iL (Jandl, 1996).
7.2. GENETIC POLYMORPHISMS
Inherited polymorphisms in genes involved in xenobiotic metabolism and other cellular
processes have been associated with increased risks of myelotoxicity or leukemia in numerous
studies of patients or workers exposed to leukemogenic agents. In some instances, similar
associations have been seen in follow-up studies by other investigators. However, in a
significant number of cases, the results have either not been repeated or have not been
reproducible. This is likely due to the limited nature of the studies that have been conducted to
date. Consequently, it is difficult to make firm conclusions about many of the reported
polymorphisms. Several recent reviews on polymorphisms and leukemia have been published
that the reader may want to refer to for further details (Cheok and Evans, 2006; Cheok et al.,
2006; Sinnett et al., 2006; Seedhouse and Russell, 2007). The following is a brief overview of
some of the genetic polymorphisms involved in DNA metabolism, repair, and xenobiotic
metabolism that have been repeatedly associated with altered risks of developing leukemia,
particularly induced acute myeloid leukemias.
50
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Because most leukemogens are genotoxic and exert their effects by damaging DNA, the
DNA repair capacity of the bone marrow is likely to have a significant influence on the risk of
t-AML. Indeed, individuals with inherited deficiencies in enzymes that are involved in DNA
repair such as ataxia telangiectasia, Bloom syndrome, and Fanconi anemia are at significantly
higher risk for developing leukemia and other lymphohematopoietic cancers (Segel and
Lichtman, 2004). The associated risks can to be very high with up to 25% of the affected
individuals developing leukemia/lymphoma during their lifetime. Individuals with these and
other uncommon genetic syndromes such as neurofibromatosis 1 are also at an increased risk of
t-AML following treatment with radiation, alkylating agents, and/or topoisomerase II inhibitors
(Maris et al., 1997; Seedhouse and Russell, 2007).
Genetic polymorphisms in DNA repair genes that occur more frequently in the
population can also confer an increased risk of t-AML. Defective mismatch repair is often
manifested by microsatellite instability in cancer cells. Microsatellite instability is frequently
seen in leukemias, particularly in t-AML, where it has been reported to occur in -50% of the
cases (Karran et al., 2003; Seedhouse and Russell, 2007). In contrast, less than 5% of de novo
leukemias exhibit microsatellite instability. While the basis for this instability is still unknown,
factors that may contribute are deficiencies in DNA-mismatch repair enzymes. In a fairly recent
report, the hMSH2 mismatch repair variant was shown to be significantly overrepresented in
t-AML patients that had previously been treated with 06-guanine-forming alkylating agents
including cyclophosphamide and procarbazine, as compared with controls (Worrillow et al.,
2003). Similarly, polymorphisms in genes involved in DNA repair (e.g., WRN, TP53, hOGGl,
XRCC1, ERCC3, and BRCA2) have been reported by a number of investigators to be associated
with a decrease in white blood cell counts or an increase in chromosomal damage in
benzene-exposed workers (Shen et al., 2006; Kim et al., 2008; Wu et al., 2008; Hosgood et al.,
2009; Sun et al., 2009).
Polymorphisms affecting DNA metabolism have also been reported to confer an
increased risk of t-AML. Thiopurine methyltransferase catalyzes the ^-methylation of thiopurine
medications such as 6-mercaptopurine and 6-thioguanine, which are commonly used as
chemotherapeutic agents. As summarized from Cheok and Evans (2006), the thiopurine
methyltransferase pathway is the primary mechanism for inactivation of thiopurines in
hematopoietic tissues. The link between thiopurine methyltransferase polymorphisms and
51
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mercaptopurine toxicity has been extensively investigated, and studies have shown a strong
relationship between thiopurine methyltransferase deficiency polymorphisms and hematopoietic
toxicity. Three variant alleles are responsible for >95% of the cases with low or intermediate
thiopurine methyltransferase activity. Patients homozygous or heterozygous for the low activity
alleles are inefficient at detoxifying the mercaptopurines and accumulate high concentrations in
their hematopoietic tissues. If their administered doses are not modified, they are at high risk for
severe hematopoietic toxicity. Inherited thiopurine methyltransferase deficiency has also been
associated with a higher risk of t-AML, particularly in ALL patients treated with topoisomerase
II inhibitors (Thomsen et al., 1999; Gadner et al., 2006). It has been postulated that the increased
risk may be due to an interference of 6-thioguanine or methylated 6-mercaptopurine with DNA
repair after DNA damage has been induced by other chemotherapeutic agents.
The influence of polymorphisms in other xenobiotic metabolizing genes on the incidence
of leukemia has been the subject of many investigations (Cheok et al., 2006; Leone et al., 2007;
Seedhouse and Russell, 2007; Guillem and Tormo, 2008; Leone et al., 2010). In many cases,
there was no difference between the frequencies seen in t-AML patients as compared to de novo
leukemia patients or a control cohort. A number of studies have indicated that individuals with
genes coding for nonfunctional or less active copies of various glutathione-^-transferases,
CYP3A4, and NADPH quinone oxidoreductase 1 (NQO1) have an increased risk for developing
t-AML following chemotherapy. However, these results have not consistently been seen. The
relationship between NQO1 and leukemia is presented below as an example. However, it should
be noted that these genes may have other important cellular functions in addition to their roles in
xenobiotic metabolism. For example, NQO1 has recently been reported to also influence cellular
signaling in the bone marrow niche (Ross et al., 2010).
Polymorphisms in NQO1, an enzyme involved with the reduction of quinones and
protection against oxidative stress, have been repeatedly associated with the development of
leukemia. Studies have reported that an inactivating polymorphism in NQO1 (the C609T, a
functionally null variant) was overrepresented in patients with t-AML (Larson et al., 1999), in
those with de novo AML (particularly those with translocations or an inv [16] clonal aberration
[Smith et al., 2001]), in infant leukemias with a 1 Iq23 karyotype, and in infants and children
with the t(4;l 1) form of ALL (Wiemels et al., 1999; Smith et al., 2002). While these initial
studies indicated a consistent association with a number of different leukemia types, more recent
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studies have been less consistent with many not showing an association (Blanco et al., 2002;
Sirma et al., 2004; Eguchi-Ishimae et al., 2005; Malik et al., 2006).
Genetic polymorphisms affecting NQO1 have also been associated with increased
myelotoxicity and may confer an increased risk of leukemia. For example, benzene-exposed
individuals who were rapid CYP2E1 metabolizers and had the C609T null variant for NQO1 had
a 7.6-fold increased risk of benzene poisoning as compared to exposed individuals with the slow
CYP2E1 metabolizer phenotype who had one or two of the wild type NQO1 alleles (Rothman
et al., 1997). In another study by this research group, different NQO1 polymorphisms, as well as
a polymorphism in myeloperoxidase, an enzyme implicated in the bioactivation of benzene's
quinone metabolites in the bone marrow, were associated with lower blood cell counts in
benzene-exposed workers (Lan et al., 2004). A similar association between NQO1 and
chromosomal damage in the peripheral blood lymphocytes of workers exposed to benzene (as
well as other potentially confounding chemicals) was also recently reported by another research
group (Kim et al., 2008).
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8. RISK ASSESSMENT IMPLICATIONS
As indicated above, approximately 25% of the agents established by IARC as human
carcinogens induce lymphohematopoietic cancers, and approximately 22% are associated with
the induction of leukemia, primarily AML. These agents have a wide variety of uses, and given
their established carcinogenic effects, they have been the focus of many risk assessments and
safety evaluations. The sections below focus on how mechanistic information on these
carcinogenic agents can be used to inform risk assessment decisions. The following discussion
is not intended to be comprehensive but rather will focus on some key issues related to
contemporary risk assessment discussions.
8.1. HAZARD IDENTIFICATION
Hazard identification is the process by which hazardous substances—carcinogenic agents
in this case—are identified and characterized. According to the IARC Preamble (IARC, 2008d),
which describes the IARC groupings and the weight-of-evidence decision-making process, the
Group 1 category "is used when there is Sufficient Evidence of Carcinogenicity in humans.
Exceptionally, an agent may be placed in this category when evidence of carcinogenicity in
humans is less than sufficient but there is Sufficient Evidence of Carcinogenicity in experimental
animals and strong evidence in exposed humans that the agent acts through a relevant
mechanism of carcinogenicity."
The Group 2A agents represent chemicals that are probable human carcinogens for which
there "is Limited Evidence of Carcinogenicity in humans and Sufficient Evidence of
Carcinogenicity in experimental animals. In some cases, an agent may be classified in this
category when there is Inadequate Evidence of Carcinogenicity in humans and Sufficient
Evidence of Carcinogenicity in experimental animals and strong evidence that the carcinogenesis
is mediated by a mechanism that also operates in humans. Exceptionally, an agent may be
classified in this category solely on the basis of Limited Evidence of Carcinogenicity in humans.
An agent may be assigned to this category if it clearly belongs, based on mechanistic
considerations, to a class of agents for which one or more members have been classified in
Group 1 or Group 2A." Most of the Group 2A agents listed in Table 8 have mechanisms of
action such as mutagenicity that are commonly associated with carcinogenesis. However, they
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have not been adequately studied in humans, or for many of the chemotherapeutic agents, they
were administered in combination therapy often with other carcinogenic agents so that the
carcinogenic effects for the agent being investigated cannot be separately identified.
For most of the evaluations, studies of cancer in humans and animals have played the
primary role in the IARC classification. In the past, information on the mechanism of action has
typically only played a supportive role. More recently, however, the role of mechanism of action
has increased such that insight into human relevance of observable mechanisms is more
frequently used to reduce or elevate a carcinogenicity hazard characterization. As a result, it is
reasonable to examine information on the mechanisms of action of the Group 1 and 2A
carcinogens to help identify carcinogens and improve the hazard identification process. Below
are several observations about how mechanistic information has and may be used in hazard
identification for leukemia-inducing agents.
8.1.1. Utility of Short-Term Genotoxicity Tests and Human Biomonitoring
As seen in Table 8, most of the agents identified by IARC as human carcinogens are
likely to act through a mutagenic or genotoxic mechanisms. The majority of the Group 1 and 2A
carcinogens are alkylating agents, but other classes of genotoxic agents such as topoisomerase II
inhibitors or nucleotide analogs are also present. The majority of the Group 1 and 2A leukemia-
and lymphoma-inducing agents are active in short-term tests both in vitro (with metabolic
activation as needed) and in vivo (IARC, 1987a). This is not surprising given their likely
mechanisms of action. The exceptions to this tend to be the infectious agents and the
immunosuppressive agents that act through nongenotoxic or indirect genotoxic mechanisms of
action.
As also shown in Table 7, most of the leukemia-inducing agents have been shown to
induce chromosomal aberrations or micronuclei in the peripheral blood lymphocytes of exposed
humans and animals. There is increasing evidence that elevated frequencies of structural
chromosomal aberrations and micronuclei in human lymphocytes can serve as predictive
indicators (biomarkers) of cancer risk (Hagmar et al., 1998; Liou et al., 1999; Smerhovsky et al.,
2001; Hagmar et al., 2004; Boffetta et al., 2007; Bonassi et al., 2007; Bonassi et al., 2008). In
the recent Bonassi et al. (2008) study, the association between the frequency of chromosomal
aberrations and the risk of lymphohematopoietic neoplasia, while elevated, did not achieve
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statistical significance. The agents listed in Table 7 also cause significant bone marrow toxicity
with noticeable decreases in blood cell counts of the exposed individuals. There are a few
Group 1 agents that are exceptions such as ethylene oxide and 1,3-butadiene and do not appear to
manifest these particular patterns. In summary, while hematotoxicity can be induced by most of
the Group 1 and 2A leukemogens, this is not uniformly the case and clinically detectable
hematotoxity does not need to occur in order for an agent to be classified as a human
leukemogen.
8.1.2. Usefulness of Animal Bioassays
There is substantial evidence to indicate that chronic animal bioassays are effective in
detecting human carcinogens. As described in the IARC Preamble (IARC, 2008d), "all known
human carcinogens that have been studied adequately for carcinogenicity in experimental
animals have produced positive results in one or more animal species." However, as further
noted in the Preamble, "although this association cannot establish that all agents that cause
cancer in experimental animals also cause cancer in humans, it is biologically plausible that
agents for which there are Sufficient Evidence of Carcinogenicity in experimental animals also
present a carcinogenic hazard to humans." Accordingly, the vast majority of the Group 1
leukemia- and lymphoma-inducing agents shown in Table 7 have been reported previously to be
carcinogenic in rodent bioassays (Eastmond, 1997). Indeed, many of them have been shown to
induce leukemias or lymphomas in mice and in rats. Most of these have been shown to occur in
mice and were T-cell leukemias or lymphomas, rather than the ANLL, which is prevalent in
humans. As a result, there appears to be fundamental differences between the types of
lymphohematopoietic tumors induced in humans and rodents. The reason for this remains
unknown but may be related to species differences in hematopoiesis and/or immune surveillance
(Eastmond, 1997).
The good correlation seen for animals and humans mentioned above was based primarily
on the results for chemical carcinogens and probably does not hold for many of the infectious
agents, which are likely to only infect humans and closely related species. In addition, it is not
clear at this point if this correlation will hold true for the topoisomerase II inhibitors, as most of
them have not been adequately tested in experimental animals. While the fusion gene products
have clearly been shown to affect hematopoiesis and can cause leukemias in mice (Corral et al.,
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1996; Dobson et al., 1999; Lavau et al., 2000; Forster et al., 2003; So et al., 2003), it is not
certain if the genes are located in regions in the rodent genomes that will allow the same
chimeric genes to be formed. It should also be noted that the Group 2A carcinogens are also
positive in rodent bioassays; however, this is to be expected, as this characteristic is one of the
primary reasons that the chemicals have been listed.
8.1.3. Combining Different Types of Lymphohematopoietic Cancers for Analysis
In evaluating epidemiological studies, a decision has to be made about which specific
types of disease should be combined for the analysis. Often the reporting of leukemias has been
grouped into the four common categories of AML, CML, ALL, and CLL. Similarly, the
lymphomas and myelomas have been grouped into NHL (lymphosarcoma, reticulosarcoma, and
other malignant neoplasms of lymphoid and histocytic tissue), Hodgkin disease, and multiple
myeloma (MM). More recent case-control studies of lymphoma have expanded the NHL
grouping to include CLL, or to group lymphomas by cell type or by specific International
Classification of Disease-Oncology category (e.g., diffuse lymphatic B-cell lymphoma, follicular
lymphoma, etc.). However, many different types of groupings have been used in analyzing
epidemiological data, primarily reflecting the classification system in use at the time of diagnosis
or cause of death. An illustration of the various types of groupings used for studies of benzene
has been described by Savitz and Andrews, 1997. In addition, evaluations of the induction of
lymphohematopoietic diseases by an agent over time can be challenging due to changes in
diagnosis, names of diseases, and classification schemes, as well as lack of detailed case and
exposure information. Other issues such as misclassification errors and insufficient information
on death certificates can also influence the outcome of the studies. The decision about how to
analyze the data can also have a significant influence on the outcome of the analysis and the
conclusions of the study.
As illustrated in Tables 1-3, there are a large number of distinct lymphohematopoietic
cancers. By analyzing each separately, a study would only have the power to detect cancers that
were very strongly associated with exposure to an agent, if detection was even possible. In
addition, the large number of comparisons could lead to some associations being labeled as
significant due simply to random chance. As a result, it is common to combine specific
lymphohematopoietic cancers for analysis. Indeed, due to the low incidence of individual
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lymphohematopoietic cancers, it is often necessary to combine subtypes to achieve the statistical
power to detect exposure-related increases in disease. The key question, which has not fully
been answered, is "Which categories of lymphohematopoietic cancer should be combined for
analysis?" In theory, only the relevant disease entities would be combined for analysis. This
might include combining specific types of hematopoietic cancer that share an underlying
mechanism, that originate from a common cell, or that share key biological (morphologic,
genetic, immunologic, etc.) features. However, in practice this becomes challenging, because all
myeloid and lymphoid cells originate in the hematopoietic stem cells of the bone marrow, and it
is often not clear at what stage in maturation the critical genetic and epigenetic events occurred.
In addition, the delineation between myeloid and lymphoid lineages and the separation between
lymphoid leukemias and lymphomas are not as distinct or dichotomous as once thought (WHO,
2008; IARC, 2008c; Doulatov et al., 2010; Dorshkind, 2010; Kawamoto et al., 2010).
The value of combining uncommon lymphohematopoietic cancers has been demonstrated
for benzene (Savitz and Andrews, 1996, 1997). Benzene exposure is strongly associated with
AML. However, its association with lymphoid cancers has been a source of ongoing discussion.
Savitz and Andrews (1996, 1997) showed that by analyzing non-AML hematopoietic cancers
together, a significant association between benzene exposure and these cancers could also be
shown. IARC, in its most recent evaluation, has given more credence to an association between
benzene and other lymphohematopoietic cancers as it concluded that there was evidence, albeit
limited, for an association between benzene and ALL, CLL, NHL, and multiple myeloma (Baan
et al., 2009; IARC, 2009). More recently, a similar association between benzene exposure and
an increased incidence of lymphoid cancers (MM, ALL, and CLL) was seen in a metaanalysis of
occupational benzene studies (Vlaanderen et al., 2011). These results suggest that the critical
events induced by benzene that result in the development of leukemias and lymphomas occur
either in the bone marrow hematopoietic stem cell and/or progenitor cells that give rise to both
the myeloid and lymphoid lineages or that benzene can target both the hematopoietic stem and
progenitor cells as well as the more mature lymphoid cells.
In addition to combining specific types of cancers for analysis during individual studies,
it may be informative to see how various authoritative bodies such as IARC, the National
Academies of Sciences, Biological Effects of Ionizing Radiation committee, and the United
Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) have combined
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the lymphohematopoietic cancers in making decisions about risk or for weight of evidence
determinations for various leukemogenic agents. As indicated above for benzene, IARC chose
to evaluate the major types of cancer separately. However, it is likely that the association
between benzene and one type of cancer provided supportive evidence that benzene could be
associated with another type of lymphohematopoietic cancer. This type of supporting evidence
appears to have played an important role in the evaluations of butadiene where associations with
CML, CLL, and to some degree, NHL, were combined in concluding that butadiene exposure
caused cancer of the "haematolymphatic organs." Similarly, information on associations with
different types of lymphohematopoietic cancers contributed to the recent IARC evaluation on
formaldehyde (AML and CML) and ethylene oxide (NHL, MM, and CLL). It should be noted
that for butadiene and probably benzene, supportive evidence came from associations with both
myeloid and lymphoid tumors.
In evaluations of ionizing radiation, it has been common to use all leukemias or combine
the results for ALL, AML, and CML in modeling the data and assessing cancer risks
(UNSCEAR, 2000a, b; NRC, 2006 and references therein). Increases in CLL have not been seen
in radiation-exposed study groups. When presenting the study results, the radiation-induced
leukemias are frequently described in various documents as simply leukemias or non-CLL
leukemias.
In contrast, in its recent review of U.S. Environmental Protection Agency's (EPAs) draft
Integrated Risk Information System risk assessment for formaldehyde, a National Research
Council review committee concluded that the broad grouping of "all lymphohematopoietic
cancers" included at least 14 biologically distinct diagnoses in humans and should not be used in
determinations of causality (NRC, 2011). The committee recommended that the EPA focus on
the most specific diagnoses available in the epidemiological data.
Thus, as illustrated in the evaluation of the various cancer-inducing agents by the
different authoritative groups, there is a range of opinions and no strict consensus on how
specific lymphohematopoietic cancers should be combined for analysis and weight-of-evidence
determinations. As discussed above, a variety of combinations including those combining acute
and chronic myeloid neoplasms as well as lymphoid and myeloid neoplasms have been used in
recent evaluations. With an increased understanding of the biology and mechanisms underlying
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de novo and induced lymphohematopoietic cancers, a more accurate identification of the
appropriate groupings for these cancers should be achievable.
8.1.4. Potential Influence of Latency Period in Identifying Leukemogens
The period between the initial exposure to a carcinogenic agent and the onset of cancer is
known as the latency period. Latency periods for acute myeloid leukemias induced by
chemotherapeutic agents tend to be much shorter than those seen for other induced cancers such
as lung cancer induced by tobacco smoke, which has a median latency period of approximately
30 years (Weiss, 1997). As indicated above, the latency period for topoisomerase II inhibitors is
quite short with median latency periods of 2-3 years. For radiation and chemical chemotherapy
agents, the induced leukemias first appear 1-2 years after the beginning of treatment. The
median incidence peaks at about 4-7 years, and by 10-15 years after the beginning of treatment;
the incidence has frequently declined to control or near control levels (Casciato and Scott, 1979;
Pedersen-Bjergaard et al., 1987; Kaldor et al., 1990; Davies, 2000; Schonfeld et al., 2006). This
indicates that for leukemogens such as the chemotherapeutic alkylating agents, the risk of
developing t-AML will most likely peak at -4-7 years after the beginning of exposure and will
begin to decline with additional follow-up. Continuing follow-up for many years after treatment
would be expected to significantly reduce associations between exposure and leukemia and could
mask risks that might be present and detectable at earlier times. Hence, adding person-years
after exposure ends or is substantially reduced, as sometimes occurs in occupational studies, may
significantly weaken true associations between exposure and the incidence of leukemia. The
influence of latency or time since first exposure has been clearly demonstrated for benzene
(Rinsky et al., 2002; Silver et al., 2002; Triebig, 2010) and has been postulated to have had an
influence on recent formaldehyde results (Beane Freeman et al., 2009). Failure to account for
latency period for weak carcinogens or under conditions of modest exposure for potent
carcinogens could reduce the association so that the agent or exposure would no longer be
considered carcinogenic. Two notes of caution are warranted: (1) The described latency periods
have typically been seen with high doses of the leukemia-inducing agents. There is evidence that
the latency period can be significantly influenced by the administered dose with higher doses
producing shorter latency periods and lower doses producing longer latency periods (Cadman
et al., 1977); (2) It should be emphasized that while latency periods are often reported as the
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median or average number of years since first exposure, induced leukemias can occur many
years after the reported median latency periods, and many years after exposure has ended (see
Triebig, 2010 for examples). Similarly, treatment-related lymphomas have been reported to
occur many years following radio- and/or chemotherapy with latency periods that are generally
longer than those reported for t-AML (Krishnan and Morgan, 2007).
8.1.5. Metabolism and Bioactive Dose at the Target Organ
One of the fundamental principles of toxicology is that the toxicological response is
related to the dose of the bioactive agent in the target tissue. However, the tissue dose of the
bioactive agent may differ significantly from the external exposure dose. For many carcinogens,
even if exposure occurs, the chemical may not arrive at the target organ in a form that enables it
to exert a toxic or mutagenic effect due to pharmacokinetic or metabolic reasons. In fact, this is
a fundamental difference between the risk assessment of ionizing radiation and chemical
leukemogens. Because of its ability to directly penetrate tissues, radiation risk estimates are
almost always based on the dose of radiation that reaches the bone marrow. In contrast, risks for
chemical leukemogens such as benzene, butadiene, or ethylene oxide are typically based on the
exposure dose and may not accurately reflect the bioactive dose that reaches the bone marrow.
Studies have also shown that pharmacokinetic and metabolic factors can significantly
influence the risks from leukemia-inducing agents. For example, the peak plasma concentrations
of melphalan have been shown to vary by over 50-fold after oral dosing due to variability in
absorption, first pass metabolism, and rapid hydrolysis (GlaxoSmithKline, 2007). Indeed, for a
few patients, detectable plasma levels of melphalan were not seen even after the administration
of a high dose (1.5 mg/kg) of the drug (Choi et al., 1989). The differences in plasma
concentration would undoubtedly have an effect on its efficacy as well as its risk of inducing
t-AML.
Metabolism has also been shown to exert a major effect on the mutagenic and
carcinogenic risks of leukemia-inducing agents. For example, metabolic activation by the
cytochrome P450 enzymes (CYP450) has been shown to play an important role in the toxic and
genotoxic effects of benzene and cyclophosphamide (Snyder, 2004; Rooney et al., 2004). Other
xenobiotic metabolizing enzymes such as the epoxide hydrolase, myeloperoxidase, glutathione
transferases, and other detoxification enzymes, as well as efficient repair of the induced adducts,
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may also influence the toxicity and carcinogen!city of leukemogenic agents. In addition,
population variation in metabolism may also have contributed to the mixed results reported in
epidemiological studies (Doughety et al., 2008).
8.1.6. DNA-Adduct Type, Metabolism, and Repair
The type of DNA adducts formed and their repair can have a major impact on the
toxicity, mutagenicity, and probably the cancer risk of leukemia-inducing agents. As indicated
earlier, three major classes of alkylating agents have been identified that have significantly
different mutagenic and carcinogenic potencies (Vogel et al., 1998). Category 1 agents are
mono-functional alkylating agents such as ethylene oxide and methyl methane sulfonate, which
primarily react at the N7 and N3 moieties of purines in DNA. These adducts are efficiently
repaired, and as a result, these agents tend to be relatively weak mutagens. Category 2 agents
such as procarbazine and ENU induce (9-alkyl adducts and TV-alky! adducts in DNA, which are
often involved in base pairing and are slowly repaired. These agents are generally potent
mutagens and carcinogens. Category 3 agents, such as melphalan and busulfan induce DNA
breaks and structural aberrations through their ability to cross-link DNA (Vogel et al., 1998).
They are highly toxic, mutagenic, and carcinogenic. For example, butadiene can be metabolized
to diepoxybutane, a cross linking agent, so it has been classified as a Category 3 agent.
However, its potency for rats and probably humans has been reported to be much lower than the
other Category 3 agents. This might be due to the need for two separate bioactivation steps to
form diepoxybutane as indicated above as well as the nature of the adducts formed by butadiene.
The butadiene crosslinks tend to be intrastrand and are more efficiently repaired. Consistent
with this explanation is the fact that diepoxybutane is believed to be the reactive intermediate
formed from treosulfan, a chemotherapeutic agent that appears to be somewhat less potent in
inducing t-AML in humans than other Category 3 chemotherapeutic agents when considered on
a mg/kg-basis (Kaldor et al., 1990). Interestingly, it should be noted that treosulfan
administration to humans has been associated with AML whereas, as described above,
occupational exposure to 1,3-butadiene has been associated with CLL and CML. One possible
explanation is that the epoxybutene metabolite may play a more important role than
diepoxybutane in inducing the butadiene-related leukemias in humans. It should be noted,
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however, that there is also considerable overlap in the carcinogenic potency of chemicals in each
of the three categories.
8.1.7. Age-Related and Individual Susceptibility
Many factors such as age, sex, genetic composition, and general health status have been
shown to influence susceptibility to leukemia-inducing agents. For example, age has been
shown to be a factor in susceptibility to radiation-induced leukemia. Studies of the atomic bomb
survivors, who were exposed to both gamma, and to a lesser extent, neutron radiation, indicated
that the highest leukemia risks were seen in children (Preston et al., 1994, 2004). The leukemia
risk also decreased more rapidly in children as compared to those in older age groups (Preston
et al., 1994). Interestingly, a similar increased susceptibility has not been seen for children
treated with alkylating agent-based chemotherapy as their leukemia risks do not appear to be
greater than those seen in adults, and in some cases, may be less (Levine and Bloomfield, 1992;
Pyatt et al., 2005, 2007). However, direct comparisons are difficult because in most studies, the
children and adults have been treated with different therapeutic regimens. For the nontherapeutic
classes of leukemia-inducing agents, little is known about the relative susceptibility of children
to leukemia, as the critical studies have almost always been conducted on adults. However, a
number of studies on related biomarkers have indicated that children or adolescents are also
susceptible to the toxic and genotoxic effects of these leukemogenic chemicals (Aksoy et al.,
1974; Aksoy, 1988; Niazi and Fleming, 1989; Neri et al., 2006).
Genetic polymorphisms in genes coding for xenobiotic metabolizing enzymes,
DNA-metabolizing or repair enzymes, and drug transporter proteins are other factors that have
been implicated as significantly influencing an individual's risk of developing leukemia when
exposed to genotoxic agents. A considerable number of studies have been conducted to identify
enzymes involved in the bioactivation and the inactivation of carcinogenic agents (for reviews,
see Cheoket al., 2006; Leone et al.,2007, 2010, and Guillem and Tormo,2008). Individuals with
genes coding for nonfunctional or less active copies of various glutathione-S-transferases,
CYP3A4, and NQO1 have been reported to be at increased risk for developing t-AML following
chemotherapy. Similar increased risks have been seen for individuals that lack efficient
DNA-metabolizing or repair enzymes such as enzymes involved in nucleotide excision repair or
mismatch repair. However, further research in this area is needed as similar associations for the
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metabolizing enzymes and DNA-repair enzymes have not been seen in other studies. As
discussed by Lutz and colleagues, from a population risk perspective, the increased susceptibility
associated with the various genetic polymorphisms can have a significant impact on cancer risks,
particularly among those exposed to lower doses (Lutz, 1990, 2001).
8.1.8. Summary
As reported in Table 8 and from the above discussion, leukemia-inducing agents act
through different mechanisms to induce their carcinogenic effects. While most of these have
been determined by IARC to likely act through mutagenic or genotoxic mechanisms, different
leukemogens have different potencies and different associated risks, which appear to be
significantly influenced by the specific mechanisms involved. Even among the alkylating
agents, different chemicals can have different potencies that are likely due to the nature of the
DNA adducts formed and their repair as well as metabolic and pharmacokinetic factors. In
addition, polymorphisms in genes related to xenobiotic metabolism and DNA repair can lead to
increased susceptibility among groups in the population. Furthermore, identifying the specific
types of cancer-causing agents with their associated mechanism and using that information to
inform key steps in the risk assessment process remains one of the ongoing challenges.
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APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
COMMENTS AND DISPOSITION
This report on "Lymphohematopoietic Cancers Induced by Chemicals and Other Agents:
Overview and Implications for Risk Assessment" has undergone an external peer review
performed by expert scientists in accordance with the EPA guidance on peer review (U.S. EPA,
2006). The external peer reviewers were tasked with providing written answers regarding their
general impression of the document as well as specific charge questions. A summary of
significant comments made by the external peer reviewers and EPA's response to these
comments arranged by charge question follow. In many cases, the comments of the individual
reviewers have been synthesized and paraphrased in the development of Appendix A.
The public comments and responses have been summarized in the subsequent section following
the external peer review comments and disposition.
EPA determined not to include Section 8, examples of specific environmental agents
inducing leukemia, because of ongoing chemical assessments (ethylene oxide and formaldehyde)
and previously completed assessment (1,3-butadiene). Therefore, any external reviewer's or
public comments on these chemical agents as well as melphalan are not addressed in this final
revised report.
A.l. EXTERNAL PEER REVIEW COMMENTS
The reviewers have made editorial and sentence modification suggestions to clarify
specific portions of the text. These changes were incorporated in the document as
appropriate and are not discussed further. When the external peer reviewers have similar
comments on multiple charge questions, the comments were organized under the most
appropriate charge question.
A.1.1. General Comments
Comments: Reviewers considered the overall report to be of excellent quality,
well-written, well organized, comprehensive, thoughtful, and insightful and of
exceptional accuracy. The reviewers commented that the clarity of presentation was
excellent and the document had compiled a huge amount of epidemiological and
mechanistic information and synthesized into a comprehensible and succinct framework.
The reviewers also thought that the report addressed an important question on how
mechanistic information could be used in the assessment of risk for lymphohematopoietic
cancers. One reviewer had a concern that there was little discussion of de novo
myelodysplastic syndromes (MDS) which are an increasingly prevalent type of
hematopoietic cancer.
Response: EPA thanks the reviewers for their constructive comments. With respect to
the myelodysplastic syndrome, it should be noted that the focus of this document is on
treatment-related MDS rather than de novo MDS. The discussion of t-MDS has been
integrated with the t-AML discussion throughout the document (e.g., see Section 2,
Tables 5 and 6, Figure 3).
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A.1.2. Specific Charge Questions
1. Evaluate and comment on the draft report organization, content, and clarity of the
presentation. Please make specific suggestions on how to improve the draft report.
Comment: Most reviewers thought that the report was well organized and the contents in
the report were clear, complete and the presentation was excellent. One reviewer
suggested inclusion of a section on benzene as an example of a specific leukemia
inducing chemical. This reviewer also suggested that the authors should consider data
from both myeloid and lymphoid origins. A comment was made to differentiate
individual susceptibility from age related susceptibility. A suggestion was also made for
adding a table for easy reference which would summarize the DNA alkylating agent
categorization scheme of Vogel and colleagues. Reorganization of text under Section 8
was also suggested. The same reviewer also commented that the report mostly focuses
on leukemia and limited information was provided on lymphoma and that the mechanism
of immunomodulation was not comprehensively described. Discussion of hematoxicity
and other biological observations was suggested to be included in the report. One
reviewer suggested that we delete Table 9. There were other minor editorial changes
recommended.
Response: The chemicals discussed for illustrative purposes were selected from among
those that had been recently reviewed by the International Agency for Research on
Cancer or similar authoritative body. Such a recent review was not available for benzene.
However, as noted, benzene is used as an example in many places throughout the
document. Sections 4 and 5 provide the background on leukemia and
lymphoma-inducing agents and as such serve as introductions to Section 6. Additional
information on the mechanisms underlying induced lymphomas to Section 4 is now
included. In response to one of the reviewer's comment, rather than create a separate
section on age-related susceptibility, EPA has changed the heading of Section 8.1.7 to
note this difference.
With respect to summarizing the DNA alkylating agent categorization, given that
it would be difficult to include all of the relevant information in a table, EPA prefers not
to include one. Furthermore, regarding comments on Section 8 of the external peer
review draft (examples of Specific Leukemia-inducing Agents), Section 8 has not been
included in the revised document so as to not have the appearance of certifying or further
debating controversial topics associated with WHO hazard characterizations of the
exampled agents. In reference to the comment on additional information on lymphomas,
while there is an introductory material regarding lymphohematopoietic cancers in
general, the primary emphasis of the document is on myeloid leukemias where most
information on chemical etiology is available. The summary and introduction have been
modified to emphasize this point. Similarly, with respect to addition of hematotoxicity,
the document is intended as an overview, such a detailed discussion is outside the scope
of this document.
2. Within the goals of the draft report, evaluate and comment on the description of the
types of induced lymphohematopoietic neoplasms, their characteristics and the
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mechanisms underlying their development as well as the general implications of the
presented information for risk assessment.
Comment: Overall, the comments by the reviewers were "excellent description of the
types of lymphohematopoietic neoplasms and defines to the average scientific reader and
toxicologist the origins of these neoplasms and the characteristics and mechanisms that
are similar as well as different in their development. Both the text and the figures that
follow within the document are excellent, easy to read, and provide, in exceptional
clarity". One reviewer appreciated the simplified scheme of hematopoietic neoplasm
classification shown in Table 1. Another reviewer endorsed this report to be an excellent
overview that would serve as a useful resource for use in the identification and risk
assessment of chemicals with the potential to cause LHP cancer. Specific comments on
this charge question included clarity on the issue of latency as it might be applied to risk
assessment. Additional references were provided for inclusion in the document, a
suggestion was made to elaborate on the implications of risk assessment as it pertains to
chemical specific metabolism, DNA adduct type, metabolism and repair, discussion on
DNA reactive metabolites of butadiene. One reviewer thought there was missing
information on MDS.
Response: EPA thanks the peer reviewers for the positive and constructive comments.
The section on latency has been revised in response to reviewers' comments.
Information as well as references about other mechanisms that are likely to contribute to
induced leukemogenesis has been added. The text has been modified to address the
comment on risk assessment implications regarding certain chemical-specific
metabolism, implications on DNA adduct type are addressed and information on other
metabolism and repair has been addressed in other sections. The focus of this document
is on treatment-related MDS rather than de novo MDS. The discussion of t-MDS has
been integrated with the t-AML discussion throughout the document (e.g., Section 2,
Tables 5 and 6, Figure 3). Other editorial changes recommended have been incorporated.
3. Evaluate and comment on the quality and completeness of the information and
literature discussed in the draft report. Please identify any additional relevant
published information in the peer-reviewed literature, which may enhance the
quality of the draft report. Please justify their inclusion in relevance to the goals of
the draft report.
Comment: Overall, the reviewers were impressed with the quality and completeness of
information in the document. Reviewers thought that EPA had compiled a huge amount
of data and literature into the document in an efficient and meaningful way, appropriately
focused on the stated purpose and goals. Specific comments included additional
description of bone marrow niches and stem cell cycling, potential influence of stroma
and styromal factors on leukemic clones, consideration of stereochemistry in butadiene
metabolism and relationship to DNA damage, evidence of systemic genotoxic effects of
formaldehyde, genotoxic actions of LHP cancer-inducing agents, and discussion on
lymphomas.
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Response: Again, EPA thanks the reviewers for the positive and constructive comments.
Specific comments are addressed in the revised document. For example, additional
information on stem cell cycling has been added, although EPA believes that a detailed
description of the bone marrow niche is outside the scope of this document; mention of
styromal cells and factors has been added, additional explanation has been provided
regarding why p53 does not easily fit into the classification scheme, text has been
changed to address the polymorphism issue. A full discussion regarding the genotoxic
actions of LHP cancer-inducing agents that don't seem to be myelotoxic and the
implications of these data for indentifying chemicals with similar properties and actions
is outside the scope of this document. Again, a sentence has been added to indicate the
agents do not have to exhibit hematotoxicity in humans to be considered human
leukemogens. A clarifying statement on lymphomas has been incorporated to specify
this information. Suggested and appropriate references have been added and editorial
comments have been considered.
4. Considering the stated goals and objectives, and scope of the draft report, were the
goals of the draft report met? If not, what specific recommendations would help
meet the goals of the draft report?
Comment: Most reviewers agreed that the report met the stated goals and objectives.
They agreed that this was an excellent overview of what is know about the mechanisms
of LHP carcinogenesis. One reviewer thought that the report falls short of making strong
conclusions and recommendations.
Response: EPA believes that the strength of the recommendations and conclusions are
appropriate for this type of "overview" report. This is not a guidance or policy report, but
an overview of the existing literature.
A.2. PUBLIC COMMENTS
Several public comments were received during the public comment period including
American Chemistry Council, International Institute of Synthetic Rubber Producers, Dr.
Richard Albertini, and Sielken and Associated Consulting. Following the public
comment period, the report was subject to external peer review including several experts
in the field of leukemia, mode of action and general toxicology.
A.2.1. General Comments:
Comments: Most comments by the public focused on three specific chemical examples
(1,3-butadiene, ethylene oxide and formaldehyde) discussed in Section 8 of the external
peer review draft. Other comments included providing additional information and/or
clarification on different physiological functions of many lymphoid and myeloid cells,
mechanism of lymphoid malignancies, developmental differences between myeloid and
lymphoid lineage, information quality act etc.
Response: EPA, when appropriate, has considered the public comments and made
necessary changes to the document. EPA considers a detailed discussion of
lymphopoiesis, full discussion of stem cell mobilization and homing, different
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physiological functions of the many lymphoid and myeloid cells is outside the intended
scope of this document. The objective of this document is to provide an overview of
lymphohematopoietic cancer; in particular, the document focuses primarily on acute
myeloid leukemias.
A.2.2. Specific Comments:
EPA has determined not to include Section 8 of the external peer review draft,
i.e., examples of specific environmental agents inducing leukemia, because of ongoing
chemical assessments (ethylene oxide and formaldehyde) and previously completed
assessment (1,3-butadiene). Therefore, public comments on these chemical agents as
well as melphalan are not addressed in this final revised report. Response to information
quality act has been provided.
Comment: Detailed description of different types of physiological functions of the
different cell types that lead to different leukemia endpoints was suggested. Furthermore,
extensive information on origin of spontaneous/non-xenobiotic-induced leukemias was
recommended. Grouping or combining various lymphohematopoietic subtypes was
suggested.
Response: As indicated in the abstract and introduction, the objective of the document is
to provide an overview of lymphohematopoietic cancers. In particular, the document
focuses primarily on acute myeloid leukemias induced by chemical, and to a lesser
degree, radiation, where the majority of information in induced leukemias is available.
The abstract, the executive summary and the introduction have been modified to clarify
this point. Correspondingly, a detailed description of the different physiological
functions of the many lymphoid and myeloid cells is outside the intended scope of this
document. Similarly, detailed information on the origin of
spontaneous/nonxenobiotic-induced leukemias, both lymphoid and myeloid, where there
is also extensive information, is outside the intended scope of the document. As
described in the report, there is currently no consensus on how lymphohematopoietic
cancers should be grouped. In specific instances, different authoritative bodies have used
different groupings or have made different recommendations. In response to the public
and peer reviewers' comments, EPA has made substantial changes to current
Section 8.1.3 to discuss in more detail issues related to the combining various
lymphohematopoietic subtypes. In addition, EPA has also added the recent 2011
National Research Council's recommendation on this subject with regard to
formaldehyde.
Comment: A comment suggested considering lymphoid malignancies and its
physiological mechanisms of DNA double-strand breaks, translocations and potential
malignant transformation and to expand such information in the document.
Response: Recent information on the mechanisms involved in the development of
induced leukemias is presented for all of the IARC Group 1 and Group 2A carcinogens in
presented in Table 8. In addition, detailed information on specific classes of agents is
presented in Sections 6-8. While there is a large database on the role of DNA
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double-strand breaks, translocations and potential malignant transformation in
spontaneous lymphoid malignancies or those of infectious origins, there is much less
information on those induced by chemical agents. As indicated above, the primary focus
of this document is on chemical-induced leukemias. Because of the paucity of
mechanistic information on chemically induced lymphoid leukemias, this topic was not
selected as an example in the document. As clearly documented in Table 8 of the
document and discussed in the text, most leukemia-inducing agents induce AML through
a mechanism involving genotoxicity and/or mutagenicity. In contrast, most
lymphoma-inducing agents are immunomodulating and/or specifically target lymphoid
cells. These agents are believed to act as either direct carcinogens acting on the cellular
DNA, indirect carcinogens acting through chronic inflammation, or through
immune-suppression. To supplement the information found in Table 8, additional
information on these lymphoma-inducing agents has been added to Section 4.
Comment: A detailed discussion of developmental differences between myeloid
and lymphoid lineage cells is recommended. Furthermore, a discussion of
migration of pluripotent stem cells from the bone marrow to the blood is
suggested. The public comment also suggested including description and
discussion of the myelodysplastic syndrome and its relationship to myeloid
leukemia. Detailed description of the genotoxic mechanism (MO A) of
lymphohematopoietic malignancies is suggested. Furthermore, public comments
suggest addressing the potential implications on the dose response phase of risk
assessment for this MOA.
Response: While there is an introduction of lymphohematopoietic cancers in
general, the primary emphasis of the document is on myeloid leukemias where the
most information on chemical etiology is available. The summary and
introduction have been modified to emphasize this point. A detailed discussion of
lymphopoiesis, full discussion of stem cell mobilization and homing is outside the
scope of this document. However, the discussion of mobilization has been
modified to reflect the observation that only a small to very small percentage of
the mobilized stem cells appear to return to the bone marrow. The focus of this
document is on treatment-related MDS rather than de novo MDS. The discussion
of t-MDS has been integrated with the t-AML discussion throughout the
document (e.g., Section 2 and 6, Tables 5 and 6, Figure 3). As indicated
previously, the primary focus of this document is on chemically induced acute
myeloid leukemia. As shown in Table 8 and throughout the text, the vast majority
of the IARC Group 1 leukemogens listed are believed to cause leukemia through
a mechanism involving mutation. These can be chromosomal mutations or point
mutations. As described in Sections 4-6, the major genetic changes in t-AML
induced by chemotherapeutic alkylating agents are interstitial deletions of the
long arms of chromosomes 5 and/or 7. These chromosomal mutations are likely
the result of DNA alkylation by the alkylating agent leading to chromosome
deletion rather than the result of endogenous processes. Only a small portion of
t-AML exhibit reciprocal translocations and these are due to an inhibition of the
enzyme topoisomerase II and are not likely to be the direct result of endogenous
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processes. The focus of the Section 8 risk assessment discussion is on hazard
identification and not dose-response assessment.
Comment: A comment on EPA report that certain sections were extracted from
respective IARC monographs, thus resulting in misunderstandings of relevant
literature was mentioned. This comment was particularly on a section that
mentions "a chemical that induces structural chromosome aberrations causes
cancer". This comment was focused on publications by Bonassi et al. (2008).
Response: EPA appreciates the comment in bringing the recent Bonassi et al. (2008)
paper to its attention. A citation to this paper as well as the descriptor "nonspecific" has
been added to the discussion of chromosomal aberrations in Section 8.1. However, the
Agency disagrees with the reviewer's interpretation of the ramifications of the Bonassi
results. The Bonassi study indicated that the association between chromosomal
aberrations and cancer was independent of the origin of the chromosomal aberrations.
Whether the chromosomal aberrations were formed from exposure to a genotoxic agent
or whether the aberrations were formed by genetic or endogenous factors, the risk is
similar. As a result, the results of this study clearly support the association between
exposure to genotoxic agents and cancer risk that is indicated in the document. In the
Bonassi et al. (2008) article, the association between the frequency of chromosomal
aberrations and lymphohematopoietic diseases, while elevated, did not achieve statistical
significance. This is not surprising given the uncommon nature of the
lymphohematopoietic cancers, the heterogeneity of exposures and the populations
studied, and the imprecision in scoring chromosomal aberrations.
Comment: Use of rodent T-cell leukemias/lymphomas as evidence that a
particular agent causes LHC cancer in humans while it is known fact that mice
harbor viruses in these cells that themselves are leukemogenic was questioned.
Also, a public comment suggested that EPA better define the statements that
'chemicals that cause leukemia or lymphomas in humans do so by a mutagenic
mode of action'.
Response: In spite of the potential influence of murine viruses, T-cell leukemias
and lymphomas induced by radiation and chemical agents in mice are considered
by authoritative bodies to provide useful information for assessing the risk of
chemical agents. The observed site concordance is mentioned in the document as
being notable, but is not considered necessary for the animal data to be useful for
risk assessment. Furthermore, the statements regarding the involvement of
mutations or genotoxicity in the mechanism of action of the listed carcinogens is
largely based on the conclusions of the recent IARC Volume 100 review.
However, to avoid possible policy implications associated with using "mutagenic
mode of action", EPA has changed the descriptions throughout the document.
The basis for this conclusion with reference can be found in Table 8 of the
document. As indicated above, the wording has been changed.
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Comment: The final report should fully comport with the explicit information quality act
(IQA) guidelines by assessing the best available science and using a weight-of-evidence
approach as indicated in the OMB guidelines and EPA-issues IQA guidelines. The
document should undergo independent scientific peer review.
Response: EPA has followed OMB Information quality Act Guidelines as well as
EPA guidelines for ensuring and maximizing the quality, objectivity, utility and
integrity of information. The objective of this report is to provide an overview of
the types and mechanisms underlying the lymphohematopoietic cancers induced
by chemical agents and radiation in humans, with a primary emphasis on acute
myeloid leukemia and agents that induce this type of cancer. A weight of
evidence approach has been used including giving the most weight to the
peer-reviewed publications, evaluations and monographs which are consensus
documents that have been written and reviewed by knowledgeable experts in the
field. EPA conducted an independent peer-review of the external review draft
and the peer-reviewers comments were considered and the revised final document
reflects response to their comments (see Appendix A).
Comment: Extensive information on polymorphism in metabolic genes as its relevance
for human susceptibility to butadiene was provided. Furthermore, information was
provided on "estimating an upper bound on the added number of leukemia mortalities in
2010 possibly due to butadiene exposure to an ambient concentration of 0.6ppb rather
than Oppb"
Response: EPA thanks the public for the in depth analysis of mouse-human
interspecies comparison and human intraspecies differences by genotypes and
also quantitative risk assessment information for butadiene exposure. The
information provided is of interest, though beyond the scope of this document.
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