Histopath of carcinogenesis

Histopathology of Carcinogenesis

R. R. [email protected]

Outline

• Overview of carcinogenesis• Lexicon of neoplasia (speaking the language)• Basics of carcinogenesis• Identifying & predicting potential carcinogens• Interpreting tumor bioassay data

Overview of Carcinogenesis

• Complex disease with multiple causes• Influenced by multiple intrinsic and

extrinsic factors• Multistep progressive process at the

genetic and phenotypic level

Overview of Carcinogenesis

Lexicon of Carcinogenesis• Neoplasia (neoplasm, tumor, cancer)• Hyperplasia

– Physiological– Pathological

• Metaplasia• Anaplasia

– Differentiation• Dysplasia

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.

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 andepithelial cells and increased size of these cells)

Two categories of hyperplasiaPhysiological hyperplasia

Hormonal – mammary gland proliferation at pubertyCompensatory – myth of Prometheus

Pathological hyperplasiaExcessive (potentially reversible) hormonal stimulationExcessive (but controlled) growth factor stimulation





Pathological hyperplasiaExcessive (potentially reversible) hormonal stimulationExcessive (but controlled) growth factor stimulationMay be associated with concurrent toxicity

Mechanisms of physiological hyperplasiaIncreased local growth factors and/or receptorsActivation of intracellular signaling pathways

Transcription factors turn on specific genesCell cycle genes~70 other genes

Proliferation of existing cells and also stem cells

Hepatectomy – paracrine stimulation from cytokines &polypeptide growth factors

Mechanisms of pathological hyperplasia

Exaggerated response to growth factors and hormonal stimulation

Hormone imbalance – excessive androgens & benignprostatic hyperplasia

Wound healing – a specific form of hyperplasia whereparenchymal cells are replaced by scar tissue

Viral infections – papilloma virus-induced growth factors leadingto skin warts and mucosal epithelial hyperplasias

Chronic hepatitis – stem cells proliferate since the capacity ofhepatocytes to proliferate is compromised

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

environmentFrequently – columnar to squamous (epithelial cells)

Cigarette smokeVitamin A deficiencyLoss of mucus secretion and mucociliar escalator function

Mesenchymal metaplasia – connective tissue osseous tissue

If stimulus persists – malignant transformation of the

metaplastic cells can occur

Mechanisms of metaplasiaDifferentiation of stem cells along a new pathwayCytokines, growth factors, and extracellular matrix components

induce transcription factors that trigger phenotypic-specific genes

Vitamin A affects differentiation pathways of stem cellsSome cytostatic drugs disrupt DNA methylation with potential

to lead to metaplasia

6-MercaptopurineMethotrexateDacarbazineProcarbazineCarbopltin

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

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

Another example of multinucleated giant hepatocytes.Chronic exposure to chlordane in a mouse.

Dysplasia = disordered growthPrimarily an epithelial changeConstellation of changes

Loss of polarityLoss of uniformityPleomorphismNuclear abnormalities

Normal forestomach

Forestomachdysplasia

If marked and involves the entire thickness of the epithelium but is confined there = carcinoma in situ

Normal Mouse Trachea

Normal mouse trachea

90-Day Formaldehyde Inhalation Study in Mice





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 RateCell production vs cell lossMalignant neoplasms grow faster than benign (oversimplified)Growth rate is not constant

HormonesAdequacy of blood supplyOther factors

Anticancer agents tend to work on fast-growing tumorsCells in proliferative phaseIf a low percentage (~5%) of the cells are in the proliferative phase = slow-growing tumor that is refractory to treatmentDebulking tumor with surgery surviving cells enter the cell cycle (leave G0) and become susceptible to anticancer agent treatment

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

Hyperplasia

• -plasia = formation• Neoplasia - new formation• Hyperplasia – enhanced formation• Metaplasia – changed formation• Anaplasia – backward formation• Dysplasia – abnormal formation

SUMMARY

LexiconOf

Carcinogenesis

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

Basics of Carcinogenesis• Molecular factors• Morphologic factors• Modulators and modifiers

From Robbins and Cotran Pathologic BasisOf Disease, 7th Edition, 2004.


Growth Factors

• Normal development– Embryogenesis

• Normal cell function– Locomotion, contractility

• Regeneration– E.g., hepatectomy

• Repair– Wound healing– Scar tissue formation


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

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




Proto-oncogene ActivationGrowth Factor (Proto-oncogene) [Mode of Action]

PDGF-β (SIS) [overexpression]FGF (HST-1; INT-2) [overexpression; amplification]TGTFa (TGFα) [overexpression]HGF (HGF) [overexpression]

Growth Factor Receptor (Proto-oncogene) [Mode of Action]EGF receptors (ERB-B1; ERB-B2) [overexpression; amplification]CSF-1 receptor (FMS) [point mutation]PDGF receptor (PDGF-R) [overexpression]Receptors for neurotrophic factors (KIT) [point mutation]

Proto-oncogene ActivationSignal Transduction (Proto-oncogene) [Mode of Action]

GTP-binding (K-RAS; H-RAS; N-RAS) [point mutation]Nonreceptor tyrosine kinase (ABL) [translocation]RAS signal transduction (BRAF) [point mutation]WNT signal transduction (b-catenin) [point mutation; overexpression]

Nuclear Regulatory Proteins (Proto-oncogene) [Mode of Action]Transcriptional activators (C-MYC; N-MYC; L-MYC)

[translocation; amplification]

Cell Cycle RegulatorsCyclins (CYCLIN D) [translocation; amplification] (CYCLIN E) [overexpression]Cyclin-dependent kinase (CDK4) [amplification; point mutation]

Human and Animal Neoplasms Associated with Activated Oncogenes

Multistage Hepatocarcinogenesis

normalfocus of altered

hepatocytes

hepatocellularadenoma

hepatocellularcarcinoma

H-rasactivation

altered Brca1

altered TGFa

CathepsinsOsteopontin

GoliathMIG

MHC class II

B-catenin

apoptosis c-foscyr61


NORMAL

PATHOLOGICAL HYPERPLASIAAND PRENEOPLASIA

ADENOMA

CARCINOMA

Thyroid hypertrophy, hyperplasia and adenoma secondary to liver enzyme induction

Normal thyroid Follicular cell hyperplasiaand hypertrophy

Follicular cell adenoma

Hepatic Foci of Cellular Alteration

Eosinophilic Focus Clear Cell Focus

Basophilic Focus Mixed Cell Focus

Hepatocellular Adenoma



Hepatocellular Carcinoma



Carcinoma arising in Adenoma



Hepatoblastoma arising in adenoma

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

Progression of Proliferative Liver Lesions

Basophilic Focus Hepatocellular adenoma

Metastatic carcinoma Hepatocellular carcinoma

Progression of Proliferative Forestomach Lesions




Modifying Factors

• Cell proliferation & apoptosis• Enzyme induction• Methylation & imprinting• Oncogenes & tumor suppressor genes• Hormones• Diet & body weight• Intercellular communication

promotioninitiation progression

Outline

• Overview of carcinogenesis• Lexicon of neoplasia (speaking the language)• Basics of carcinogenesis• Identifying & predicting potential carcinogens• Interpreting tumor bioassay data

Identifying potential carcinogens

Genotoxic vs non-genotoxic agentsRodent bioassays

History & evolutionPathology evaluation of

bioassayPeer review (previous Hardisty

presentation)Predicting carcinogenic hazard

Using toxicity study data

Carcinogenic agentsChemical carcinogensRadiant energy

UV, ionizing radiationOncogenic DNA viruses

PapillomavirusEpstein-Barr virusHepatitis B virus

Oncogenic RNA virusesHuman T-cell leukemia virus

Type 1

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

1700’s 1950 1960 1970 1980 1990 2000 2010

NCI NTP

CANCER BIOASSAY TIMELINE

1700’s 1950 1960 1970 1980 1990 2000 2010

NCI NTP

FDA

OECD

IARC

EPA

ICH

VICH

EUROTOX

IUTOX

SOT

DST

BTS

STP

BSTPASIATOX

ESTPCANCER BIOASSAY TIMELINE

1700’s 1950 1960 1970 1980 1990 2000 2010

NCI NTP


50 Male and 50 female F344 rats & B6C3F1 MiceMaximum tolerated dose & lower dosesRoutes: feed, gavage, drinking water,

inhalation, dermalTest duration of 2 yearsDiet: NIH-07 and NTP-2000Extensive histopathology & peer review

“Current” Testing Paradigm

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

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

Search for alternatives

1700’s 1950 1960 1970 1980 1990 2000 2010

NCI NTP


• 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

Model! Model! Who’s Got the Model?

• 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?







Pathology Evaluation

An iterative process for identificationof subtle differences among groups

of experimental animals

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 benignNORMAL

PATHOLOGICAL HYPERPLASIAAND PRENEOPLASIA

ADENOMA

CARCINOMA

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

Drift Over Time

• Professional drift – changing criteria for a given lesion

• Personal drift – Increased familiarity with a given lesion with greater exposure

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







Rodent Liver Toxicity• Cytomegaly• Hypertrophy• Necrosis• Bile duct hyperplasia• Hepatocellular degeneration (rats)

• Liver weight

Cytomegaly Hypertrophy

Necrosis Bile duct hyperplasia

DegenerationToxicologic Pathology 39: 393-401 (2004)

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

Toxicological Sciences80: 225-229 (2004)

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, metabolism data, 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 results of 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.

SOT Annual MeetingSalt Lake City, UTMarch 9, 2010

Preneoplastic lesions not predictive

A completely negative12-month rat toxicity study don’t need to do a carcinogenicity study

Liver response appears generically predictive even for other target tissues

• 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

ANCHORING

• Biologically plausible

• Computational/Informatics– SAR & other alerts– Artificial intelligence– Modeling, including PBPK– Database mining– Focused epidemiology

C A N C E R

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

Interpreting Tumor Bioassay Data

Purpose of interpreting bioassay = detect differences that may be directly or indirectly related to exposure to the test agent

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

Modifying Factors

• Diet & body weight• Cell proliferation & apoptosis• Enzyme induction• Methylation & imprinting• Oncogenes & tumor suppressor genes• Hormones• Intercellular communication

Dose and Dose Relationships

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




Progression of Proliferative Liver Lesions

Basophilic Focus Hepatocellular adenoma

Metastatic carcinoma Hepatocellular carcinoma

Progression of Proliferative Forestomach Lesions

Considerations• Species/strain susceptibility

– Gallbladder adenoma/carcinoma– Hepatoblastoma– Stellate cell tumor

Hepatoblastoma Stellate cell tumor

GallbladderAdenoma

LungColon

LiverSkin

1.0

0.8

0.6

0.4

0.2

0

Relative Susceptibility of Inbred Mouse Strains toChemically Induced Carcinogenesis

A/J

AKR

BALB

/c

C3H

C57B

L/6

DBA/

2

P/J

SWR

Drinkwater & Bennett 1991

Male Mouse Liver Tumors(Spontaneous Frequency)

King-Herbert & Thayer - 2006

Relative susceptibilities of selected strains to liver tumor induction

Historical Control Incidences

Considerations

• Neoplasia• Benign vs. malignant• Latency• Multiplicity




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

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

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


N = 50

Lung - A/B adenoma 0 0 0 3Lung - A/B carcinoma 0 1 1 1Combined 0 1 1 4

• Equivocal evidence (EE) - a marginal increase of neoplasms that may be chemically related

Molybdenum Trioxide - EE in male rats


N = 50

Spectrum of Esophageal Lesions

Normal mucosa

Hyperplasia

PapillomaSquamous cell carcinoma

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.

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.

Evidence of carcinogenic activity (n=290) Liver 57 % Lung 22 % Kidney 22 % Mammary gland 14 % Hematopoeitic 13 % Forestomach 12 % Thyroid 10 % Vascular System 9 %

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

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

What might explain the lack of a clear tumor responsein the high dose group?

Liver tumor response in a 2-year rodent carcinogenicity study

What might explain the lack of a clear tumor responsein the high dose group?



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?


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?

Other types of liver tumorsHemangioma/hemangiosarcomaHistiocytic sarcomaKupffer cell sarcomaStellate cell tumorCholangiomaCholangiocarcinoma

Hemangiosarcoma

Cholangiocarcinoma Histiocytic sarcoma Stellate cell tumor

Liver tumor response in a 2-year rat carcinogenicity studyN = 50

What diagnostic entities are legitimate to combine?

Would you classify this as a positive carcinogenic response?

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




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

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

Case 6 – Uterine tumors in female Wistar rats

C LD MD HDNumber examined 40 49 50 50Fibromatous Polyp 7 11 12 10Multiple Fibrous Polyps 1 1 0 2

Adenocarcinoma 6 4 5 7Papilloma 0 0 1 0Carcinoma in situ 1 0 0 1Stromal Sarcoma 0 0 0 2Poorly Diff. Sarcoma 0 0 0 1Unclassified Sarcoma 0 0 0 1

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

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

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

Case 2 – Male B6C3F1 miceHistorical Control Data

All Sites Rate (%) Range (%)

Hemangioma 0.5 0-4

Hemangiosarcoma 5.4 0-12

Case 2 – Male B6C3F1 miceHistorical 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