This toxicologic pathology presentation covers basic principles and lexicon of carcinogenesis with representative photomicrographs and case presentations for evaluating potential cancer bioassay tumor responses.
Histopath of Carcinogenesis
View as PDFIndex of Slides
1. Histopathology of Carcinogenesis R. R. Maronpot maronpot@me.com
2. Outline • Overview of carcinogenesis • Lexicon of neoplasia (speaking the language) • Basics of carcinogenesis • Identifying & predicting potential carcinogens • Interpreting tumor bioassay data
3. Overview of Carcinogenesis • Complex disease with multiple causes • Influenced by multiple intrinsic and extrinsic factors • Multistep progressive process at the genetic and phenotypic level
4. Overview of Carcinogenesis
5. Lexicon of Carcinogenesis • Neoplasia (neoplasm, tumor, cancer) • Hyperplasia – Physiological – Pathological • Metaplasia • Anaplasia – Differentiation • Dysplasia
6. Neoplasia “….abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of normal tissue and persists in the same excessive manner after cessation of the stimuli which evoked the change” Willis 1952.
7. Hyperplasia = increase in number of cells in an organ or tissue •Increased volume of the organ or tissue •Usually associated with hypertrophy e.g., hormone-induced uterine hyperplasia (increase in number of smooth muscle and epithelial cells and increased size of these cells)
8. Two categories of hyperplasia Physiological hyperplasia Hormonal – mammary gland proliferation at puberty Compensatory – myth of Prometheus Pathological hyperplasia Excessive (potentially reversible) hormonal stimulation Excessive (but controlled) growth factor stimulation
9. Two categories of hyperplasia Physiological hyperplasia Hormonal – mammary gland proliferation at puberty Compensatory – myth of Prometheus
10. Two categories of hyperplasia Physiological hyperplasia Hormonal – mammary gland proliferation at puberty Compensatory – myth of Prometheus
11. Pathological hyperplasia Excessive (potentially reversible) hormonal stimulation Excessive (but controlled) growth factor stimulation May be associated with concurrent toxicity
12. Mechanisms of physiological hyperplasia Increased local growth factors and/or receptors Activation of intracellular signaling pathways Transcription factors turn on specific genes Cell cycle genes ~70 other genes
13. Proliferation of existing cells and also stem cells Hepatectomy – paracrine stimulation from cytokines & polypeptide growth factors
14. Mechanisms of pathological hyperplasia Exaggerated response to growth factors and hormonal stimulation Hormone imbalance – excessive androgens & benign prostatic hyperplasia Wound healing – a specific form of hyperplasia where parenchymal cells are replaced by scar tissue Viral infections – papilloma virus-induced growth factors leading to skin warts and mucosal epithelial hyperplasias Chronic hepatitis – stem cells proliferate since the capacity of hepatocytes to proliferate is compromised
15. Metaplasia – one mature adult cell type replaced by another mature adult cell type Adaptive process – more sensitive cells replaced by cells less sensitive cells to an adverse environment Frequently – columnar to squamous (epithelial cells) Cigarette smoke Vitamin A deficiency Loss of mucus secretion and mucociliar escalator function Mesenchymal metaplasia – connective tissue osseous tissue Squamous metaplasia Squamous epithelium Normal columnar epithelium Reserve cells If stimulus persists – malignant transformation of the metaplastic cells can occur
16. Mechanisms of metaplasia Differentiation of stem cells along a new pathway Cytokines, growth factors, and extracellular matrix components induce transcription factors that trigger phenotypic-specific genes Vitamin A affects differentiation pathways of stem cells Some cytostatic drugs disrupt DNA methylation with potential to lead to metaplasia 6-Mercaptopurine Methotrexate Dacarbazine Procarbazine Carbopltin
17. Differentiation and Anaplasia •Differentiation in neoplasia refers to morphological and functional similarity to normal •Anaplasia is lack of differentiation •Benign tumors are typically well-differentiated •Malignant tumors range from differentiated to anaplastic with at least some loss of differentiation present •Anaplasia is a hallmark of malignancy •Anaplasia = “to form backward” “reverse differentiation” vs. stem cell theory of carcinogenesis
18. Morphological aspects of anaplasia •Pleomorphism = variation in size and shape •Abnormal nuclear morphology •Hyperchromatism •Karyomegaly •Large nucleoli •Mitoses tend to be increased in malignancy •Giant cells and multinucleated cells
19. Another example of multinucleated giant hepatocytes. Chronic exposure to chlordane in a mouse.
20. Dysplasia = disordered growth Primarily an epithelial change Constellation of changes Loss of polarity Loss of uniformity Pleomorphism Nuclear abnormalities Squamous metaplasia Squamous epithelium Normal columnar epithelium Reserve cells Dysplastic epithelium Normal forestomach Forestomach dysplasia If marked and involves the entire thickness of the epithelium but is confined there = carcinoma in situ
21. Normal Mouse Trachea
22. Normal mouse trachea
23. 90-Day Formaldehyde Inhalation Study in Mice
24. 90-Day Formaldehyde Inhalation Study in Mice
25. 90-Day Formaldehyde Inhalation Study in Mice
26. 90-Day Formaldehyde Inhalation Study in Mice
27. 90-Day Formaldehyde Inhalation Study in Mice
28. Neoplasia = “….abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of normal tissue and persists in the same excessive manner after cessation of the stimuli which evoked the change” Willis 1952. Growth Rate Cell production vs cell loss Malignant neoplasms grow faster than benign (oversimplified) Growth rate is not constant Hormones Adequacy of blood supply Other factors Anticancer agents tend to work on fast-growing tumors Cells in proliferative phase If a low percentage (~5%) of the cells are in the proliferative phase = slow-growing tumor that is refractory to treatment Debulking tumor with surgery surviving cells enter the cell cycle (leave G0) and become susceptible to anticancer agent treatment
29. Essential alterations for malignancy Self-sufficient growth (don’t require external stimulation) Ability to synthesize growth factors Insensitive to growth inhibitory signals Evasion of apoptosis Defects in DNA repair Limitless replication – maintain telomere length and function Sustained angiogenesis Ability to invade and metastasize Hepatocellular carcinoma Pulmonary metastases
30. Hyperplasia • -plasia = formation • Neoplasia – new formation • Hyperplasia – enhanced formation • Metaplasia – changed formation • Anaplasia – backward formation • Dysplasia – abnormal formation SUMMARY Lexicon Of Carcinogenesis
31. Outline • Overview of carcinogenesis • Lexicon of neoplasia (speaking the language) • Basics of carcinogenesis • Carcinogenic agents • Identifying & predicting potential human carcinogens • Interpreting actual tumor bioassay data
32. Basics of Carcinogenesis • Molecular factors • Morphologic factors • Modulators and modifiers
33. From Robbins and Cotran Pathologic Basis Of Disease, 7th Edition, 2004.
34. From Robbins and Cotran Pathologic Basis Of Disease, 7th Edition, 2004.
35. Growth Factors • Normal development – Embryogenesis • Normal cell function – Locomotion, contractility • Regeneration – E.g., hepatectomy • Repair – Wound healing – Scar tissue formation From Robbins and Cotran Pathologic Basis Of Disease, 7th Edition, 2004.
36. Molecular Factors in Carcinogenesis • Non-lethal genetic damage • Alteration of normal regulatory genes – Growth promoting protooncogenes – Growth inhibiting cancer suppressor genes – Genes that regulate programmed cell death (apoptosis) • Alteration of genes that regulate DNA repair • Epigenetic changes (methylation, imprinting) • Multistep cascade of events
37. Multiple Roles of Proto-oncogenes • Participate in functions related to cell growth and proliferation • Encode proteins that function as: – Growth factor ligands – Growth factor receptors – Signal transducers – Transcription factors – Cell cycle components
38. From Robbins and Cotran Pathologic Basis Of Disease, 7th Edition, 2004.
39. From Robbins and Cotran Pathologic Basis Of Disease, 7th Edition, 2004.
40. From Robbins and Cotran Pathologic Basis Of Disease, 7th Edition, 2004.
41. Proto-oncogene Activation Growth Factor (Proto-oncogene)
44. Multistage Hepatocarcinogenesis normal focus of altered hepatocytes hepatocellular adenoma hepatocellular carcinoma H-ras activation altered Brca1 altered TGFa Cathepsins Osteopontin Goliath MIG MHC class II B-catenin apoptosis c-fos cyr61
45. Basics of Carcinogenesis • Molecular factors • Morphologic factors • Modulators and modifiers
46. NORMAL PATHOLOGICAL HYPERPLASIA AND PRENEOPLASIA ADENOMA CARCINOMA
47. Thyroid hypertrophy, hyperplasia and adenoma secondary to liver enzyme induction Normal thyroid Follicular cell hyperplasia and hypertrophy Follicular cell adenoma
48. Hepatic Foci of Cellular Alteration Eosinophilic Focus Clear Cell Focus Basophilic Focus Mixed Cell Focus
49. Hepatocellular Adenoma
50. Hepatocellular Adenoma
51. Hepatocellular Adenoma
52. Hepatocellular Carcinoma
53. Hepatocellular Carcinoma
54. Hepatocellular Carcinoma
55. Carcinoma arising in Adenoma
56. Carcinoma arising in Adenoma
57. Carcinoma arising in Adenoma
58. Hepatoblastoma arising in adenoma
59. Essential alterations for malignancy Self-sufficient growth (don’t require external stimulation) Ability to synthesize growth factors Insensitive to growth inhibitory signals Evasion of apoptosis Defects in DNA repair Limitless replication – maintain telomere length and function Sustained angiogenesis Ability to invade and metastasize
60. Progression of Proliferative Liver Lesions Basophilic Focus Hepatocellular adenoma Metastatic carcinoma Hepatocellular carcinoma
61. Progression of Proliferative Forestomach Lesions
62. From Robbins and Cotran Pathologic Basis Of Disease, 7th Edition, 2004.
63. From Robbins and Cotran Pathologic Basis Of Disease, 7th Edition, 2004.
64. Basics of Carcinogenesis • Molecular factors • Morphologic factors • Modulators and modifiers
65. Modifying Factors • Cell proliferation & apoptosis • Enzyme induction • Methylation & imprinting • Oncogenes & tumor suppressor genes • Hormones • Diet & body weight • Intercellular communication
66. promotioninitiation progression
67. Outline • Overview of carcinogenesis • Lexicon of neoplasia (speaking the language) • Basics of carcinogenesis • Identifying & predicting potential carcinogens • Interpreting tumor bioassay data
68. Identifying potential carcinogens Genotoxic vs non-genotoxic agents Rodent bioassays History & evolution Pathology evaluation of bioassay Peer review (previous Hardisty presentation) Predicting carcinogenic hazard Using toxicity study data
69. Carcinogenic agents Chemical carcinogens Radiant energy UV, ionizing radiation Oncogenic DNA viruses Papillomavirus Epstein-Barr virus Hepatitis B virus Oncogenic RNA viruses Human T-cell leukemia virus Type 1
70. 1700’s 1950 1960 1970 1980 1990 2000 2010 • Bernardino Ramazzini – 1713 • John Hill – 1761 • Percival Pott – 1775 • Elmslie -1866 • Jonathon Hutchinson – 1888 • Rehn – 1895 • Yamagiwa & Ichikawa – 1918 • Murphy & Sturm – 1925 • Cook et al. – 1932 • Sasaki & Yoshida – 1935 • Berenblum – 1941 • Magee & Barnes – 1956 • Realization that chemicals, environmental factors, and aspects of life style cause cancer • Concept of the cancer bioassay
71. 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP CANCER BIOASSAY TIMELINE
72. 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP FDA OECD IARC EPA ICH VICH EUROTOX IUTOX SOT DST BTS STP BSTP ASIATOX ESTPCANCER BIOASSAY TIMELINE
73. 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP CANCER BIOASSAY TIMELINE 50 Male and 50 female F344 rats & B6C3F1 Mice Maximum tolerated dose & lower doses Routes: feed, gavage, drinking water, inhalation, dermal Test duration of 2 years Diet: NIH-07 and NTP-2000 Extensive histopathology & peer review “Current” Testing Paradigm
74. Positive Aspects of the Bioassay • Standardized (informative databases) • Yields positive results for known human carcinogens • Trans-species carcinogens • Identification of important variables & modulators • Informative for chronic toxicity • Appreciation of benefits of historical controls • Reproducible • Search for alternatives
75. Limitations of the Bioassay • Resource intensive • Inherent insensitivity for detecting weak or moderate carcinogens • Not ideal for determining if an agent has carcinogenic potential under actual human exposure conditions • Single chemical exposure vs “real world” • Historical inertia • Debate regarding relevance –Rodent-specific mechanisms –High doses
76. Search for alternatives 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP CANCER BIOASSAY TIMELINE • A viable alternative needs a champion • A successful alternative needs to be validated • An ideal alternative should be less expensive and faster than the conventional bioassay
77. Model! Model! Who’s Got the Model?
78. • Genotoxicity batteries • Strain A mouse • Two-stage liver model • Neonatal mouse model • Ito medium-term model • Genetically engineered mouse models • Rat mammary gland • Local subcutaneous injection • Guppy & Medaka • Hamster cheek pouch • Structure-activity relationships & AI • Genomics & proteomics Model! Model! Who’s Got the Model?
79. Identifying potential carcinogens Genotoxic vs non-genotoxic agents Rodent bioassays History & evolution Pathology evaluation of bioassay Peer review (previous Hardisty presentation) Predicting carcinogenic hazard Using toxicity study data
80. Pathology Evaluation An iterative process for identification of subtle differences among groups of experimental animals
81. Defining Diagnostic Criteria • What is hyperplasia versus neoplasia in the broad context of toxicologic pathology – There is a range of change – Diagnoses determined by training, published literature, and experience – The greater the experience, the broader the ranges of non-neoplastic and benign NORMAL PATHOLOGICAL HYPERPLASIA AND PRENEOPLASIA ADENOMA CARCINOMA
82. Personal Diagnostic Judgment • Inexperienced pathologists tend to overdiagnose neoplastic changes • Thousands of tissues later, the number of tumors diagnosed is decreased • Result of increased familiarity with spectrum of hyperplasia and neoplasia in laboratory animals, increased confidence
83. Drift Over Time • Professional drift – changing criteria for a given lesion • Personal drift – Increased familiarity with a given lesion with greater exposure
84. Reasons for a Pathology Peer Review • Routine peer reviews • Assure consistency in terminology and grading • Increase confidence in the study data • Ensure data meets requirements of regulatory agencies • Confirm target tissues/lesions • Confirm NOEL • Non-routine peer reviews • Target tissue reviews • Pathology Working Groups
85. Identifying potential carcinogens Genotoxic vs non-genotoxic agents Rodent bioassays History & evolution Pathology evaluation of bioassay Peer review (previous Hardisty presentation) Predicting carcinogenic hazard Using toxicity study data
86. Rodent Liver Toxicity •Cytomegaly •Hypertrophy •Necrosis •Bile duct hyperplasia •Hepatocellular degeneration (rats) •Liver weight Cytomegaly Hypertrophy Necrosis Bile duct hyperplasia Degeneration Toxicologic Pathology 39: 393-401 (2004)
87. Summary from Allen et al., 2004 Mouse • A chemical showing a positive response for hypertrophy, cytomegaly and necrosis has a high likelihood of producing liver neoplasia • Failed to identify more than 1/3 of the liver carcinogens • Inclusion of increased liver weight increased sensitivity but decreased specificity of the prediction Rat • No single lesion was a strong predictor • Hepatocellular hypertrophy was the strongest predictor • Bile duct hyperplasia and hepatocellular degeneration did not contribute • Grouping hypertrophy, cytomegaly, and necrosis correctly identified 7 of 11 liver carcinogens but doubled the number of false positives
88. Toxicological Sciences 80: 225-229 (2004)
89. Toxicological Sciences 2005 88(1):18-23 Prediction of 2-Year Carcinogenicity Study Results for Pharmaceutical Products: How Are We Doing? Abigail Jacobs1 Center for Drug Evaluation and Research, USFDA, 9201 Corporate Blvd, Rm N212, Rockville, Maryland 20850 Received May 4, 2005; accepted June 24, 2005 Some have proposed that 2-year carcinogenicity studies may not be necessary if the material is a direct-acting DNA mutagen,induces liver enzymes, causes hyperplasia or toxicity in particular organs, causes cell proliferation, is cytotoxic, causes hormonal perturbations, or if one has QSAR analyses or ‘omics information. Safety pharmacology data, pharmacologic activity, metabolismdata, and results of 13-week dose ranging studies (with organ weight data, clinical chemistry data, hematologic data, clinical signs and histopathologic findings) were compared with resultsof 2-year carcinogenicity studies reviewed by the Center for Drug Evaluation and Research (CDER)/FDA. The experience with the ICH genetic toxicology battery and alternative carcinogenicity models was also reviewed. It appears that the information available from short- term studies is not currently sufficient to accurately and reliably predict the outcome of long-term carcinogenicity studies.
90. SOT Annual Meeting Salt Lake City, UT March 9, 2010 Preneoplastic lesions not predictive A completely negative 12-month rat toxicity study don’t need to do a carcinogenicity study Liver response appears generically predictive even for other target tissues
91. • Core set of mechanistic assays – DNA adducts, repair & reactivity – DNA crosslinking – Genotoxicity – Receptor-mediated assays – Microtubule inhibition – Intercellular communication – Enzyme induction – Cell cycle perturbations – Endocrine disruption – Altered methylation – Oxidative stress; free radicals – Immunosuppression – Serum biochemistry – Genomics/proteomics – Hormone activity • Abnormal phenotype • Toxicologic pathology A N C H O R I N G • Biologically plausible • Computational/Informatics – SAR & other alerts – Artificial intelligence – Modeling, including PBPK – Database mining – Focused epidemiology C A N C E R
92. The Way Forward • Search for alternatives – Multiple inbred strains – “Humanized” mouse • Continued “refinement and improvement” of the conventional bioassay – Stop studies – In utero and neonatal exposures • Develop predictive strategies to minimize the need for long term in vivo testing • Embracing each new approach and each new promising technology – Systems biology – “Omics” and biological pathways – Comparative genomics – Multimodality molecular and functional imaging – In situ molecular methods – New biomarkers
93. Interpreting Tumor Bioassay Data
94. Purpose of interpreting bioassay = detect differences that may be directly or indirectly related to exposure to the test agent
95. Considerations in Interpretation of Bioassay Data Neoplasia • Modifying factors • Dose relationships • Trans-sex & trans-species • Common vs. unique lesions • Lesion progression • Species/strain susceptibility • Controls • Lumping & Splitting • Direct vs. indirect causality • Benign vs. malignant • Latency • Multiplicity • Levels of evidence of carcinogenicity Non-neoplasia • Modifying factors • Dose relationships • Trans-sex & trans-species • Common vs. unique lesions • Lesion progression • Species/strain susceptibility • Controls • Lumping & Splitting • Direct vs. indirect causality • Adaptive vs. adverse • Severity • MTD, NOEL and NOAEL
96. Modifying Factors • Diet & body weight • Cell proliferation & apoptosis • Enzyme induction • Methylation & imprinting • Oncogenes & tumor suppressor genes • Hormones • Intercellular communication
97. Dose and Dose Relationships
98. Considerations • Trans-sex & trans-species • Common vs. unique lesions – Common lesions will tend to have a higher background (spontaneous) incidence – Unique (rare) lesions typically show marginal increases compared to control • Lumping & Splitting – Relates to how the pathologist categorizes his or her findings • Direct vs. indirect causality – Determination if observed effect is secondary to something other than a direct response to the test agent
99. Considerations in Interpretation of Bioassay Data Neoplasia • Modifying factors • Dose relationships • Trans-sex & trans-species • Common vs. unique lesions • Lesion progression • Species/strain susceptibility • Controls • Lumping & Splitting • Direct vs. indirect causality • Benign vs. malignant • Latency • Multiplicity • Levels of evidence of carcinogenicity Non-neoplasia • Modifying factors • Dose relationships • Trans-sex & trans-species • Common vs. unique lesions • Lesion progression • Species/strain susceptibility • Controls • Lumping & Splitting • Direct vs. indirect causality • Adaptive vs. adverse • Severity • MTD, NOEL and NOAEL
100. Progression of Proliferative Liver Lesions Basophilic Focus Hepatocellular adenoma Metastatic carcinoma Hepatocellular carcinoma
101. Progression of Proliferative Forestomach Lesions
102. Considerations • Species/strain susceptibility – Gallbladder adenoma/carcinoma – Hepatoblastoma – Stellate cell tumor Hepatoblastoma Stellate cell tumor Gallbladder Adenoma
103. Lung Colon Liver Skin 1.0 0.8 0.6 0.4 0.2 0 Relative Susceptibility of Inbred Mouse Strains to Chemically Induced Carcinogenesis Drinkwater & Bennett 1991
104. Male Mouse Liver Tumors (Spontaneous Frequency) King-Herbert & Thayer – 2006
105. Relative susceptibilities of selected strains to liver tumor induction High susceptibility Intermediate susceptibility Relatively resistant C3H C57BR/cdJ BALB/c CBA FVB C57BL/6 B6C3F1 SM/J C57BL/10 DBA/2 (infant model) P/J 129 Tif:MAGf CE/J DBA/2 (> 5 weeks old) C3H x CBA LP SWR CBA x C57BL/10 AKR/J A C3H x A/J CD-1 IF DBA/2 x CE/J NMRI RF LP x 129 A x C57BL/6 LP x DBA/2 C57BL x A LP x C57BL/10 A x C57BL/10 129 x DBA/2 C57BL/6 x BALB/c
106. Historical Control Incidences
107. Considerations • Neoplasia • Benign vs. malignant • Latency • Multiplicity
108. Considerations in Interpretation of Bioassay Data Neoplasia • Modifying factors • Dose relationships • Trans-sex & trans-species • Common vs. unique lesions • Lesion progression • Species/strain susceptibility • Controls • Lumping & Splitting • Direct vs. indirect causality • Benign vs. malignant • Latency • Multiplicity • Levels of evidence of carcinogenicity Non-neoplasia • Modifying factors • Dose relationships • Trans-sex & trans-species • Common vs. unique lesions • Lesion progression • Species/strain susceptibility • Controls • Lumping & Splitting • Direct vs. indirect causality • Adaptive vs. adverse • Severity • MTD, NOEL and NOAEL
109. NTP Levels of Evidence of Carcinogenicity • Clear evidence • Some evidence • Equivocal evidence • No evidence – no chemically related increases in malignant or benign neoplasms • Inadequate study – because of major limitations, cannot be interpreted as valid for showing either the presence or absence of carcinogenic activity
110. NTP Levels of Evidence of Carcinogenic Activity • Clear evidence (CE) – a dose related increase in: a) malignant neoplasms, b) benign and malignant neoplasms, or marked increase in benign neoplasms with ability to progress Stomach – benign NE tumor 0 0 13** 9** Stomach – malignant NE tumor 0 1 12** 26** Combined 0 1 25** 34** Methyleugenol – CE in female rats N = 50
111. Lung – A/B adenoma 5 9 10 16** Lung – A/B carcinoma 2 1 5 3 Combined 7 10 15* 19** Some evidence (SE) – an increase of benign, malignant, or combined in which the strength of the response is less than that required for clear evidence. Ethylbenzene – SE in male mice NTP Levels of Evidence of Carcinogenic Activity N = 50
112. Lung – A/B adenoma 0 0 0 3 Lung – A/B carcinoma 0 1 1 1 Combined 0 1 1 4 • Equivocal evidence (EE) – a marginal increase of neoplasms that may be chemically related Molybdenum Trioxide – EE in male rats NTP Levels of Evidence of Carcinogenic Activity N = 50
113. Spectrum of Esophageal Lesions Normal mucosa Hyperplasia Papilloma Squamous cell carcinoma
114. Esophageal lesions in a two-year rat carcinogenicity study. Male Sprague-Dawley rats. Administration of compound by gavage in water. N= 50/dose. The intended route of human exposure is by oral tablet.
115. Esophageal lesions in a two-year rat carcinogenicity study. Male Sprague-Dawley rats. Administration of compound by gavage in water. N= 60/dose. The intended route of human exposure is by oral tablet. •Laboratory historic control = 0 •No esophageal neoplasms in the females or in mice (males and females). •No forestomach tumors in the rats. No oral cavity tumors in rats. •Compound is irritating. •Esophageal inflammation in a 28-day and 6-month study at higher doses: Control 3/10 versus High dose 8/10 Intended human exposure is by coated tablet that dissolves in the stomach.
116. Evidence of carcinogenic activity (n=290) Liver 57 % Lung 22 % Kidney 22 % Mammary gland 14 % Hematopoeitic 13 % Forestomach 12 % Thyroid 10 % Vascular System 9 %
117. Is There Evidence of Carcinogenicity in the Liver of Male Mice Treated with 2-Butoxyethanol? Liver – Hepatocellular adenoma 22 18 18 17 Liver – Hepatocellular carcinoma 10 11 16 21** Liver – combined 30 24 31 30 N = 50
118. Is There Evidence of Carcinogenicity in the Liver of Male Mice Treated with 2-Butoxyethanol? Liver – Hepatocellular adenoma 22 18 18 17 Liver – Hepatocellular carcinoma 10 11 16 21** Liver – combined 30 24 31 30 Historical control range for Hepatocellular carcinoma 14 to 40% N = 50
119. What might explain the lack of a clear tumor response in the high dose group? Liver tumor response in a 2-year rodent carcinogenicity study
120. What might explain the lack of a clear tumor response in the high dose group? Liver tumor response in a 2-year rodent carcinogenicity study
121. Liver tumor response in a 2-year rodent carcinogenicity study There was no decrease in tumor latency or multiplicity. Survival and body weight gain were similar among the 4 groups. What could explain the statistically significant low dose response? Is this a positive rodent carcinogen?
122. Liver tumor response in a 2-year rodent carcinogenicity study There was no decrease in tumor latency or multiplicity. Survival and body weight gain were similar among the 4 groups. What could explain the statistically significant low dose response? Is this a positive rodent carcinogen?
123. Other types of liver tumors Hemangioma/hemangiosarcoma Histiocytic sarcoma Kupffer cell sarcoma Stellate cell tumor Cholangioma Cholangiocarcinoma Hemangiosarcoma Cholangiocarcinoma Histiocytic sarcoma Stellate cell tumor
124. Liver tumor response in a 2-year rat carcinogenicity study N = 50 What diagnostic entities are legitimate to combine? Would you classify this as a positive carcinogenic response?
125. Liver tumor response in a 2-year rat carcinogenicity study What diagnostic entities are legitimate to combine? Would you classify this as a positive carcinogenic response? N = 50
126. Considerations in Interpretation of Bioassay Data Neoplasia • Modifying factors • Dose relationships • Trans-sex & trans-species • Common vs. unique lesions • Lesion progression • Species/strain susceptibility • Controls • Lumping & Splitting • Direct vs. indirect causality • Benign vs. malignant • Latency • Multiplicity • Levels of evidence of carcinogenicity Non-neoplasia • Modifying factors • Dose relationships • Trans-sex & trans-species • Common vs. unique lesions • Lesion progression • Species/strain susceptibility • Controls • Lumping & Splitting • Direct vs. indirect causality • Adaptive vs. adverse • Severity • MTD, NOEL and NOAEL
127. Summary • Purpose of interpreting bioassay = detect differences that may be directly or indirectly related to exposure to the test agent – In rodent studies we are concerned with effects in a group of animals rather than in individual animals – Dose relationships are very important – Responses are compared to the concurrent control and, in instances where the response is questionable, comparison to historic controls may be appropriate – It is sometimes useful to combine certain lesions to better interpret bioassay results
128. Case 3 – Malignant Lymphoma in female B6C3F1 mice 0 ppm 10 ppm 100 ppm 1000 ppm Incidence (percentage) All organs – malignant lymphoma 3 (6%) 8 (16%) 11* (22%) 13** (26%) Historical control data: mean 15.5%; range 6-32% 50 animals examined per group; *p<0.05; **p<0.01
129. Case 6 – Uterine tumors in female Wistar rats C LD MD HD Number examined 40 49 50 50 Fibromatous Polyp 7 11 12 10 Multiple Fibrous Polyps 1 1 0 2 Adenocarcinoma 6 4 5 7 Papilloma 0 0 1 0 Carcinoma in situ 1 0 0 1 Stromal Sarcoma 0 0 0 2 Poorly Diff. Sarcoma 0 0 0 1
130. Case 2 – Hemangioma in male B6C3F1 mice Hemangioma only 0 ppm 10 ppm 100 ppm 1000 ppm Liver 0 1 0 0 Heart 0 0 1 0 Spleen 0 0 0 0 Subcutis 0 1 0 0 Mesentery 0 0 1 2 All Organs 0 2 2 2
131. Case 2 – Hemangiosarcoma in male B6C3F1 mice Hemangiosarcoma only 0 ppm 10 ppm 100 ppm 1000 ppm Liver 2 5 6 8 Heart 0 0 0 0 Spleen 0 2 2 1 Subcutis 1 3 1 7 Mesentery 0 3 13 7 All Organs 3 13 22 23
132. Case 2 – Hemangioma or Hemangiosarcoma in male B6C3F1 mice Hemangioma/HSA 0 ppm 10 ppm 100 ppm 1000 ppm Liver 2 6 6 8* Heart 0 0 1 0 Spleen 0 2 2 1 Subcutis 1 4 1 7* Mesentery 0 3 14** 9** All Organs 3 15** 24** 25** 50 animals examined per group; *p<0.05; **p<0.01
133. Case 2 – Male B6C3F1 mice Historical Control Data All Sites Rate (%) Range (%) Hemangioma 0.5 0-4 Hemangiosarcoma 5.4 0-12
134. Case 2 – Male B6C3F1 mice Historical Control Data Rate (%) Range (%) Liver – Hemangioma 0.2 0-2 Spleen – Hemangioma 0.1 0-2 Subcutis – Hemangioma 0.0 0.0 Liver – Hemangiosarcoma 2.6 0-6 Spleen – Hemangiosarcoma 2.2 0-8 Subcutis – Hemangiosarcoma 0.7 0-4