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DOI: 10.1177/0192623311436181

published online 9 February 2012Toxicol PatholPrashant R. Nambiar, Susan E. Turnquist and Daniel Morton

Spontaneous Tumor Incidence in RasH2 Mice: Review of Internal Data and Published Literature

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Spontaneous Tumor Incidence in rasH2 Mice:Review of Internal Data and Published Literature

PRASHANT R. NAMBIAR1, SUSAN E. TURNQUIST

2, AND DANIEL MORTON3

1Drug Safety Research and Development, Pfizer Inc., Groton, Connecticut, USA2University of Georgia, Tifton, Georgia, USA

3Drug Safety Research and Development, Pfizer Inc., Cambridge, Massachuesettes, USA

ABSTRACT

Alternate transgenic mouse models are accepted as replacements for the standard carcinogenicity mouse bioassay by regulatory agencies with

a companion 2-year rat bioassay. The slower rate of industry acceptance of these shorter transgenic mouse cancer bioassays has been due to lack of

historical data and diagnostic criteria, and the use of nonstandardized terminologies in published data. To address these issues, especially that

of generating a large historical database, a retrospective analysis of the spontaneous tumor incidences in rasH2 mice from internally sponsored

6-month carcinogenicity studies was compared to the published literature. Incidences of common spontaneous tumors (incidences > 1%) observed

in these studies were lung bronchiolo-alveolar adenomas (mean 3.9–9.9%; range 0–18%), lung bronchiolo-alveolar adenocarcinomas (mean

1.4–2.4%; range 0–5%), splenic hemangiosarcomas (mean 3.0–3.9%; range 0–17%), cutaneous squamous cell papillomas (mean 1.1–1.2%; range

0–4%), Harderian gland adenoma (mean 0.8–1.2%; range 0–4%), and hepatocellular adenomas (mean 1.8%; 0–9% in males only). The remarkable

similarity in the tumor incidences in multiple rasH2 studies over a decade and the observed stability of the inserted human gene are important

indicators of the minimal drift in this model. Overall, the historical control data for spontaneous neoplasms should assist in the interpretation

of future rasH2 mouse studies.

Keywords: rasH2; ras; background; historical control; mice; carcinogenicity.

INTRODUCTION

For cancer hazard identification of pharmaceuticals,

alternate short- or medium-term mouse models including the

p53þ/– mouse and rasH2 mouse are generally accepted as pos-

sible replacements of the standard lifetime carcinogenicity

mouse bioassay by the International Conference on Harmoni-

zation (ICH) and the U.S. Food and Drug Administration

(FDA) (Anonymous 1998). The use of these alternate mouse

models can provide significant advantages with similar predic-

tivity for human carcinogens, fewer irrelevant positive results,

more flexible resourcing, decreased animal use, shorter testing

duration and reduced costs (Alden, Smith, and Morton 2002).

Among the alternate shorter-term mouse models, p53þ/– and

rasH2 models appear to be the most commonly used (Long

et al. 2010). p53þ/– mice are generally accepted only for geno-

toxic pharmaceuticals, while rasH2 mice are recommended for

both non-genotoxic and genotoxic pharmaceuticals (Long et al.

2010; MacDonald et al. 2004). The transgenic rasH2 mouse

model was initially developed in Japan to evaluate the role of

ras over expression in tumorigenesis (Saitoh et al. 1990). The

rasH2 mouse, officially known as CByB6F1-Tg(HRAS)2Jic,

is a hemizygous transgenic mouse carrying the human c-Ha-

ras gene with its own promoter/enhancer elements and harbor-

ing a single point mutation in the 30-end (in the last intron),

which induces high enhancer activity (Morton et al. 2002;

Suemizu et al. 2002; Tamaoki 2001). This prototypic human

transgene, which encodes for the p21ras protein, was noted to

be inserted tandemly (3–6 copies) in the murine chromosome

15E3 with approximately 2- to 3-fold the levels found in

wild-type mice. The hemizygous transgenic mice are produced

by breeding transgenic male C57BL/6 J mice with wild type

BALB/CByJ female mice.

Major gaps identified in the use of these alternate mouse

models (as gauged by a recently published survey of the

Society of Toxicologic Pathology membership) include issues

with study interpretation primarily due to lack of historical

background data and issues with tumor identification and

characterization of pre-neoplastic lesions (Long et al. 2010).

Although there are several reports of control data from rasH2

mice, they have typically been conducted in multiple labora-

tories, are several years old, have generally lacked details on

study-design parameters such as age of animals, study duration,

dosing procedures, laboratory practices, and pathology diag-

nostic criteria, and often have interpathologist variability.

The major objectives of this study were to generate a control

database of spontaneous tumors in rasH2 mice using consistent

diagnostic criteria/nomenclature and to compare it with previ-

ous rasH2 literature. In this report compiled from eleven carci-

nogenicity studies, retrospective analysis of the background

tumor prevalence in untreated rasH2 hemizygous transgenic

mice was performed by an individual veterinary pathologist.

The author(s) declared no potential conflicts of interests with respect to the

authorship and/or publication of this article. The author(s) received no financial

support for the research and/or authorship of this article.

Address correspondence to: Prashant R. Nambiar, Drug Safety and

Research Development, Pfizer Inc, Eastern Point Road, Groton, CT 06340,

USA; e-mail: [email protected].

1

Toxicologic Pathology, 00: 1-10, 2012

Copyright # 2012 by The Author(s)

ISSN: 0192-6233 print / 1533-1601 online

DOI: 10.1177/0192623311436181


Furthermore, the background tumor incidences in these rasH2

mice were compared to the incidences published in the literature.

Comparison between Pfizer studies and published studies also

includedMNU-treated tumor incidences. Thus, this is a first col-

lation of data from a large number of rasH2 mice (N¼ 333–363)

from studies conducted with pharmaceuticals or studies designed

to generate historical control data. Some of the key highlights of

this retrospective analysis are adherence to the best practice

guidelines for historical control data such as detailed information

of study design parameters, relatively shorter time interval for

data collection (4–5 years), and peer review of the data by an

individual pathologist to ensure consistent application of diag-

nostic criteria and nomenclature (Keenan et al. 2009).

MATERIALS AND METHODS

Study Design

Data reported in this article are from control/vehicle groups

from 10 carcinogenicity studies for males and 11 studies for

females of 6-month dosing duration conducted between March

2004 and July 2009 under good laboratory practice (GLP) con-

ditions. All studies and animal procedures were conducted in

accordance with animal use protocols approved by the animal

care and use committee in compliance with The Guide for Care

and Use of Laboratory Animals and all applicable regulations.

CB6F1/Jic-Tg (rasH2-Tg) male and female mice were pur-

chased from Taconic (Germantown, NY) at 6 weeks of age and

housed individually in suspended polycarbonate or stainless

steel wire cages in an environmentally controlled room (tem-

perature 18–26�C, relative humidity 30%–70%, controlled

12-hour light cycle) and provided ad libitum access to water

and a certified rodent diet. Each mouse was genotyped before

leaving the vendor’s facility. The mice were typically allowed

to acclimate for 2 weeks before study start, and weighed between

17.6 to 25.4 g at study initiation. The total number of control

males and control females evaluated in these carcinogenicity

studies were 333 and 363, respectively. The number of control

animals in each study ranged from 25 to 90/sex. These control

mice received either certified rodent diet (n¼ 4 diet admix stud-

ies) alone or a vehicle such as 0.5%methylcellulose+ Tween or

sterile water via oral gavage (n ¼ 7 studies). The alkylating car-

cinogen, N-methyl-N-nitrosourea (MNU), was used as a positive

control in 8 out of 11 studies. A single dose of MNU pH 4.0 in

citrate buffer was given at 75 mg/kg IP (n¼ 7 studies) and at 58

mg/kg IP (n ¼ 1 study). Only 6 of these 8 studies were used for

MNU-related incidence reporting, as only these 6 included eva-

luation of a full tissue list. The numbers of rasH2 mice that were

evaluated from the MNU-treated groups were 87/sex.

Pathology Evaluation

All surviving animals were humanely euthanized at the end

of each 6-month study. A complete necropsy was performed

with fixation of most tissues in 10% neutral buffered formalin

followed by embedding in paraffin, sectioning at 3–5 �m and

staining with hematoxylin and eosin (H&E) using standard

procedures. Tissues such as eyes and testes/epididymides were

fixed in 3% glutaraldehyde or Davidson’s solution, respectively.

The H&E stained tissue sections from the control animals were

retrospectively evaluated by a board-certified veterinary pathol-

ogist using published nomenclature and criteria (Mohr 2001).

The standard tissues collected and evaluated in these studies are

included in Table 1. Individual control mice that were eutha-

nized moribund or found dead prior to scheduled necropsy were

also included in the data.

To compare the internally generated tumor incidencedatawith

previous reports in rasH2mice, tumor incidence from control and

MNU-treated rasH2 mice from 6-month carcinogenicity studies

were also collated from literature published from 2001–2011

(Table 2) (Cho et al. 2010; Jin et al. 2007; Machida et al. 2008;

Okamura et al. 2006, 2007; Palazzi and Kergozien-Framery

2009; Usui et al. 2001). It is important to note that MNU-

related tumor incidences in the Pfizer studies were obtained

from study reports and not by retrospective analysis of slides.

RESULTS

Body Weight and Survival Rate

The terminal body weight of male and female rasH2 mice in

the Pfizer studies ranged from 28.4–34.5 and 22.9–25.1 g,

respectively (Table 3). Over a period of 6 months, the body

weight gain, as expected, was slightly higher in the males

(4.6–9.0 g) than females (4.1–6.2 g). The survival rate in these

mice at the end of 6 months was high (>95% average) with a

TABLE 1.—General list of tissues examined.

Adrenal (2) Ovary (2)

Aorta Oviduct

Brain Pancreas

Cecum Pituitary

Cervix Preputial glanda

Clitoral glanda Prostate

Colon Rectuma

Duodenum Salivary gland [mandibular (2)]

Epididymis (2) Sciatic nerve

Esophagus Seminal vesicle

Eye (2) Skeletal muscle (thigh)

Femur with bone marrow Nasal cavitya

GALT (Peyer’s patch) Optic nerve

Harderian gland Skin/adnexa

Heart Spinal cord

Ileum Spleen

Jejunum Sternum with bone marrow

Kidney (2) Stomach

Larynxa Testis (2)

Lesions Thymus

Liver Thyroid (2 lobes) with parathyroid

Lung with large bronchi Tongue

Lymph node (mesenteric) Trachea

Lymph node (inguinofemoral) Ureter

Mammary gland (females) Urinary bladder

Uterus

aTissues collected but not examined microscopically unless examined because of a

gross lesion.

2 NAMBIAR ET AL. TOXICOLOGIC PATHOLOGY


range between 92–100%. Cause of death or early euthanasia was

due to hemangiosarcomas, pulmonary bronchiolo-alveolar

tumors, squamous cell carcinoma, urinary tract obstruction, pre-

putial abscess, and so forth (Table 3).

Spontaneous Tumor Incidence in rasH2 Mice

Spontaneous tumor incidences in control rasH2 mice were

evaluated from the Pfizer 6-month carcinogenicity studies. The

common tumors, defined as tumors with incidence � 1% over

all studies, are listed in Table 4. A comprehensive list of the

tumor incidences in these mice is provided in the supplemen-

tary table (S1). The mean incidence of common tumors

observed in these studies were lung bronchiolo-alveolar adeno-

mas / adenocarcinomas (1.4–9.9%), splenic hemangiosarcomas

(3.0–3.9%), cutaneous squamous cell papillomas (1.1–1.2%),

Harderian gland adenoma (0.8–1.2%), and hepatocellular

adenomas (1.8% in males only). Isolated occurrences of

hemangiosarcomas in individual organs (such as testes, skin,

kidney, bone marrow, etc.) were observed in individual ani-

mals across studies. When combined (as they were mutually

exclusive), the incidences of hemangiosarcomas in all organs

in both males and females were 5.1% and 6.1%, respectively.

The tumor incidences were similar between males and females

except for pulmonary bronchiolo-alveolar adenoma incidence,

which had a higher incidence in males (9.9%) compared to

females (3.9%), and liver adenomas which had a higher inci-

dence in males (1.8%) compared to females (0%). The histolo-

gic phenotype of these tumors was based on previously

reported criteria and was similar to those described in rasH2

mice previously (Mitsumori et al. 1998; Mohr 2001).

To compare the spontaneous tumor incidence in the Pfizer

studies with those reported previously, we reviewed the litera-

ture for rasH2 6-month studies spanning from 2000 to 2011

(Table 2). Overall, the spontaneous tumor incidence in Pfizer

studies was generally comparable to those reported previously

(Table 5). For example, the mean incidences of the most com-

mon tumors for both sexes reported in the literature include

pulmonary bronchiolo-alveolar adenomas (5.3–5.4%), splenic

hemangiosarcomas (3.0–3.7%), cutaneous squamous cell

papillomas (1.3–1.8%), lymphomas (0.3–2.4%), and gastric

squamous cell papillomas (0.6–1.3%). The primary differences

noted in the Pfizer data and the published literature include the

incidence of lung bronchiolo-alveolar adenomas/adenocarcino-

mas in both sexes, which were slightly higher (5.3–12.3%) in

the Pfizer studies compared to the literature (5.9–6.4%;

Table 5). Furthermore, the incidence of lymphomas in females

was relatively low (0.3%) in the Pfizer studies compared to pre-

vious reports (2.4%; Tables 4, 5).

Histologic Phenotype

A brief discussion of the key diagnostic features of the com-

mon tumors in rasH2 mice based on previously published cri-

teria (Mohr 2001; Renne et al. 2009) are presented below.

Pulmonary bronchiolo-alveolar adenomas were typically

small in size (< 3–4 mm diameter), discrete without encapsula-

tion, and often located on the peripheral border of the lung

lobes. The tumor cells were arranged in glandular or papillary

patterns with frequent compression of the adjacent normal

alveoli. The tumor cells were uniformly cuboidal to columnar

with basophilic cytoplasm and supported by a thin fibrovascu-

lar stroma. Mitotic figures were rare or absent (Figure 1).

The pulmonary bronchiolo-alveolar carcinomas were

mostly poorly demarcated neoplasms with diameter often >

TABLE 2.—List of published studies that were mined for common and/or N-methyl-N-nitrosourea (MNU)-related tumors in rasH2 transgenic used

in 6-month carcinogenicity studies.

Chemical Sponsor

Route and

frequency Male Female Reference

Di-isodecyl phathalate University of Edinburgh PO diet 15 15 Cho et al. (2010)

HDPE s/c implants Biomatech-Namsa S/C 15–20 15–20 Palazzi and Kergozien-Framery (2009)

Citrate buffer CIEA IP 15 15 Machida et al. (2008)

Troglitazone Tokyo University of Agriculture and Technology PO diet 15 15 Jin et al. (2007)

2-amino-3-methylimidazo

[4,5-f] quinoline

Tokyo University of Agriculture and Technology PO diet 15 15 Okamura et al. (2006)

Multiple sources Multiple Different routes 505 502 Usui et al. (2001)

TABLE 3.—Body weight and survival for untreated/vehicle rasH2 mice

at end of 6-month study (Pfizer Studies).

Male Female

Number of studies examined 10 11

No. of animals 333 363

Initial body weight mean range (g) 21.4–25.4 17.6–19.8

Final body weight mean range (g) 28.4–34.5 22.9–25.1

Body weight gain range (g) 4.6–9.0 4.1–6.2

Survival range (%) a 92–100 88–100

Mortality (No. of animals)b 11 17

Cause of death

Neoplasms 5c 8d

Non-neoplasms 5e 1f

Undetermined 1 8

aDoes not include mortality associated with unequivocal gavage trauma.bTotal number of animals from these studies that were euthanized or found dead prior

to scheduled necropsy.cHemangiosarcoma.dHemangiosarcoma, lung bronchiolo-alveolar carcinoma, stomach squamous cell

carcinoma, vertebral osteosarcoma, lymphoreticular neoplasm.eUrinary tract obstruction, preputial abscess, renal disease, polyarteritis nodosa.fThoracic hemorrhage.

Vol. 00, No. 0, 2012 SPONTANEOUS TUMOR INCIDENCE IN RASH2 MICE 3


3–4 mm and occupying an entire lobe with distortion of and

invasion into underlying architecture. The tumor cells were

cuboidal to columnar with anisocytosis and anisokaryosis and

often arranged in solid glandular/tubular and papillary patterns.

Occasional infiltration into bronchioles and lymphatics were

also noted. Increased inflammatory cells (macrophages) and

TABLE 5.—Percentage incidences of the common and N–methyl–N–nitrosourea (MNU)–related tumors (> 1% incidence) in rasH2 transgenic mice

used in 6–month carcinogenicity studies (from literature).

Untreated MNU-treated

Males Females Males Females

No. of Studies 24 24 11 11

No. of Animals 623 597 373 375

Organ Tumor Mean % (Range %) Mean % (Range %) Mean % (Range %) Mean % (Range %)

Lung Adenoma 5.3 (0–20) 5.4 (0–6.7) 9.4 (0–33.3) 12.8 (26.7)

Adenocarcinoma 0.6 (0–6.7) 1.0 (0–6.7) 0.3 (0–0.3) 0.5 (0–0.6)

Spleen Hemangiosarcoma 3.7 (0–6.7) 3.0 (0–26.7) 3.8 (0–3.8) 2.1 (0–2.5)

Hemangioma 0.8 (0–6.7) 1.0 (0–1.2) 1.1 (0–6.7) 1.9 (0–13.3)

Liver Hepatocellular adenoma 1.4 (0–7) 0.3 (0–0.4) 0 0

Lymphoreticulara Lymphoma 0.3 (0–6.7) 2.4 (0–20) 44.0 (36–93.3) 43.2 (35.9–100)

Skin Papilloma 1.8 (0–20) 1.3 (0–1.6) 28.4 (33.9–73.3) 34.9 (30.2–93.3)

Keratoacanthoma 0 0 1.9 (0–33.3) 2.9 (0–40)

Squamous cell carcinoma 0 0 1.1 (0–1.3) 3.2 (0–6.7)

Stomach Squamous cell Papilloma 0.6 (0–0.8) 1.3 (0–6.7) 50.9 (44.4–100) 52.3 (46.7–93.3)

Squamous cell carcinoma 0.2 (0–0.2) 0.2 (0–0.2) 7.8 (0–13.3) 7.2 (0–26.7)

Harderian Gland Adenoma 0.2 (0–6.7) 1.2 (0–6.7) 1.6 (0–1.9) 2.4 (0–2.9)

Adenocarcinoma 0 0 0 1.1 (0–1.3)

Small Intestine Adenoma 0 0 1.9 (0–2.2) 4.8 (0–15.2)

Adenocarcinoma 0 0 1.9 (0–6.7) 4.8 (0–13.3)

Oral Cavity Papilloma 0 0 2.7 (0–33.3) 0

Tongue Papilloma 0 0 1.9 (0–6.7) 1.3 (0–13.3)

Urethra Transitional cell papilloma 0 0 0.5 (0–6.3) 4.0 (0–4.8)

Uterus Polyp NA 0 NA 3.5 (0–4.1)

Endometrial adenoma NA 0 NA 2.1 (0–40)

Hemangioma NA 0 NA 1.6 (0–20)

Vagina Papilloma NA 0 NA 3.5 (0–13.3)

NA ¼ not applicable.aInclude tumors that were classified as lymphomas arising in the hematopoietic system (thymus, spleen, lymph nodes).

TABLE 4.—Background incidences of common tumors in rasH2 transgenic mice (from Pfizer Studies).

Control MNUa

Male Female Male Female

No. of studies 10 11 6 6

No. of animals examined 333 363 87 87

Organ Tumors Mean % (Range %) Mean % (Range %) Mean % (Range %) Mean % (Range %)

Lung Adenoma 9.9 (0–18) 3.9 (0–16) 8.0 (0–20) 18.4 (0–27)

Adenocarcinoma 2.4 (0–5) 1.4 (0–4) 2.3 (0–7) 2.3 (0–7)

Spleen Hemangiosarcoma 3.0 (0–8) 3.9 (0–17) 3.4 (0–20) 4.6 (0–13)

Liver Hepatocellular adenoma 1.8 (0–9) 0 0 0

Stomach Squamous cell papilloma 0.3 (0–4) 0.3 (0–4) 71.3 (47–83) 63.2 (33–87)

Squamous cell carcinoma 0.3 (0–4) 0 34.5 (7–60) 29.9 (20–40)

Lymphoreticular Lymphoma 0 0.3 (0–1) 65.5 (47–87) 66.7 (47–80)

Skin Squamous cell papilloma 1.2 (0–4) 1.1 (0–4) 29.9 (7–60) 46.0 (20–53)

Squamous cell carcinoma 0 0.3 (0–0.1) 6.9 (0–33) 10.3 (0–27)

Harderian gland Adenoma 1.2 (0–4) 0.8 (0–4) 3.4 (0–7) 6.9 (0–13)

Adenocarcinoma 0.6 (0–4) 0.8 (0–4) 0 4.6 (0–13)

Small Intestine Adenoma 0 0.3 (0–4) 4.6 (0–13) 2.3 (0–7)

Adenocarcinoma 0 0 8.0 (0–27) 11.5 (0–25)

aMNU–related tumors from these studies were obtained from the study reports and not prospectively evaluated.



FIGURE 1.—Representative photomicrographs of spontaneous neoplasms noted in rasH2 mice in 6-month carcinogenicity studies. These include

pulmonary bronchiolo-alveolar adenoma (A) and adenocarcinomas (B), cutaneous squamous cell papilloma (C), gastric squamous cell carcinoma

(D), Harderian gland adenoma (E), and adenocarcinoma (F). Insets represent low-magnification images of the neoplasm while the boxed area is

the magnified portion of the region. H&E stain.



loosely attached neoplastic cells were noted within the alveoli

adjacent to the tumors (Figure 1).

Splenic hemangiosarcomas were often expansile infiltrative

masses with effacement of the underlying normal structures.

These tumors were hypercellular and composed of elongate to

plump atypical cells with large anisokaryotic nuclei forming vas-

cular channels separated by varying amount of connective tissue

as well as solid areas with little evidence of vascular channels

and composed of atypical elongate to plump neoplastic cells

arranged in a storiform pattern. Mitotic figures were frequent

(Figure 2). Metastases of these tumors were uncommon.

Hepatocellular adenomas were well demarcated and

slightly compressed the adjacent hepatic parenchyma. The

cells within the neoplasm were uniform, well differentiated

with uniform nuclei forming regular hepatocyte plates, and

lacking lobular architecture (i.e., portal triads, central veins,

etc.). The hepatocyte cytoplasm ranged from eosinophilic, baso-

philic, and clear to vacuolated.

Cutaneous papillomas were solitary, mostly exophytic,

well-circumscribed neoplasms with single or multiple finger-

like projections and a fibrovascular connective tissue core. The

overlying epidermis was composed of hyperplastic stratified

squamous epithelium and there was no evidence of dermal

invasion (Figure 1).

Gastric squamous cell papillomas were usually solitary,

exophytic masses arising within the squamous/non-glandular

stomach and composed of a thin fibrovascular connective tis-

sue core. The epithelium was frequently hyperplastic with nor-

mal maturation and no evidence of proprial invasion.

Harderian gland adenomas were usually isolated, variable

sized, well-demarcated tumors composed of acinar glands lined

by a single layer of finely vacuolated/foamy cells with projec-

tion into the lumen (Figure 1). Harderian gland adenocarcino-

mas were expansile infiltrative tumors, poorly demarcated with

disruption of normal architecture. The neoplastic cells formed

solid glandular structures often piling up to form multiple

layers, with a variable degree of cytoplasmic vacuolation, and

prominent oval to round nuclei. The mitotic figures were

increased and occasional necrotic foci were noted within the

neoplasm (Figure 1).

The intestinal neoplasms noted in these rasH2 mice were

adenomas (Figure 3).

Tumor Incidence in the Commonly Used Positive

Control, MNU

MNU, a known alkylating genotoxic carcinogen, is often

used as the positive control to confirm the sensitivity of the

rasH2 mouse model to chemical carcinogens. We therefore

evaluated the tumor incidence in MNU-treated (positive

control) rasH2 mice and compared our data to the published

literature. The target organs, with respect to the observed

MNU-related tumors, were comparable in the Pfizer studies and

previous reports. In the Pfizer studies, the most common MNU-

related tumors in both sexes were gastric papillomas (63.2–

71.3%), lymphomas (65.5–66.7%), cutaneous papillomas

(29.9–46.0%), gastric squamous cell carcinomas (29.9–34.5%),

pulmonary bronchiolo-alveolar adenomas (8.0–18.4%), small

intestinal adenocarcinomas (8.0–11.5%), cutaneous squa-

mous cell carcinomas (6.9–10.3%), harderian gland adenomas

(3.4–6.9%), and small intestine adenomas (2.3–4.6%;

Table 4). MNU-related increases in incidence of squamous

cell papillomas, squamous cell carcinomas, hemangiosarco-

mas, and so forth were also sporadically noted in the female

reproductive tract including vagina, cervix, and uterus (data

not shown) in both Pfizer studies and previous reports.

Some of the unique MNU-related features in the Pfizer

studies relative to previous reports include higher incidence

of the more common MNU-related tumors such as lymphomas,

gastric papillomas, and cutaneous papillomas. Tumors such as

gastric squamous cell carcinoma, cutaneous squamous cell car-

cinomas, and small intestinal adenocarcinomas were also noted

at higher incidences (Tables 4, 5). Similar to previous reports,

MNU treatment in the Pfizer studies failed to increase the inci-

dence of splenic hemangiosarcomas, the second most common

spontaneous tumor in rasH2 mice.

The most common and consistent MNU-related neoplasm in

the Pfizer studies and published literature were gastric papillo-

mas and lymphomas. The incidences across studies ranged from

33–87% and 47–87% for gastric papillomas and lymphomas,

respectively. Overall, MNU was a robust positive control elicit-

ing a tumorigenic response comparable to previous reports.

DISCUSSION

The limitations of lifetime rodent bioassays include sev-

eral factors such as protracted study duration (2–3 years),

study size (> 600 animals), cost (*1 MM USD), high mor-

bidity and mortality rates during study, lack of clarity on

mechanism of tumorigenesis, and questionable relevance

to human risk assessment (Alden et al. 2009). The accep-

tance of short-term (i.e., 6-month) alternate mouse trans-

genic models such as rasH2 and p53þ/– mice for cancer

biohazard testing of pharmaceuticals by the FDA and ICH

has been an important milestone from drug development

and human risk assessment perspectives (Friedrich and Olej-

niczak 2011).

In line with regulatory acceptance of the transgenic mouse

models, recent trends in the use of these models in carcinogeni-

city testing have been encouraging. In the last decade (2002–

2011), 135 out of 506 mouse carcinogenicity protocols sub-

mitted to the FDA were transgenic mouse protocols (ranging

from 11–52% per year). Of these 135 transgenic mouse proto-

cols, 78 (57.8%; range 9.1–92.9% per year) were from studies

using rasH2 mice. Interestingly, the trends in the use of p53þ/–

mice and rasH2 mice have reversed in the past 5 years, with an

increase in rasH2 mouse protocol submission to the FDA (50–

85% per year) relative to p53þ/– mice (0–31.3% per year; per-

sonal communication). The trend toward greater use of the

rasH2 model likely reflects the FDA’s view that the p53þ/–

mouse is suitable only for pharmaceuticals that are positive

in at least one genotoxicity assay.



FIGURE 2.—Representative photomicrographs of hemangiosarcomas noted in rasH2 mice in 6-month carcinogenicity studies. These tumors were

most common in the spleen (A, B), while isolated occurrences were noted in skin (C), testis (D), kidney (E), bone marrow (F), and occasionally

other organs, independent of the spleen. Note the two distinct patterns of splenic hemangiosarcomas–neoplastic cells forming vascular channels

(A) and the more solid fibrous component (B). In other organs, the hemangiosarcomas typically formed vascular channels. Insets represent low

magnification images of the neoplasm, while the boxed area is the magnified portion of the region. H&E stain.



Despite the positive signs, the adoption of these alternate

transgenic mouse models by the pharmaceutical industry

has been slow (Friedrich and Olejniczak 2011). Some of the

factors contributing to limited use of these models by the

biopharmaceutical industry have been concerns regarding

lack of historical data, application of consistent diagnostic

criteria, use of nonstandardized terminologies in published

data, and lack of comfort using a novel model (Long et al.

2010). To address these issues especially that of generating

a large historical database, we conducted comprehensive

retrospective analysis of the spontaneous tumor incidence

in rasH2 mice from internally sponsored 6-month carcino-

genicity studies.

Overall, the incidences of tumors in the Pfizer internal

rasH2 studies were similar to the compiled incidence from all

the published rasH2 studies of similar duration (i.e., 6-month

studies). Despite variables such as the wide range of the study

conduct dates (i.e., over 10 years), geographical locations, and

sponsoring institutions, the tumor spectrum in the published lit-

erature was remarkably similar to the Pfizer studies. For exam-

ple, the common spontaneous tumors noted in rasH2 mice in

the Pfizer studies were pulmonary adenomas/carcinomas, sple-

nic hemangiosarcomas, cutaneous squamous cell papillomas,

hepatocellular adenomas, and harderian gland adenomas. A

few exceptions noted in the Pfizer studies relative to the liter-

ature were the low incidence of lymphomas and gastric

FIGURE 3.—Representative photomicrographs of rare neoplasms (<1% incidence) noted in rasH2 mice in 6-month carcinogenicity studies. Ade-

nocarcinoma within the Brunner’s gland in the duodenum (A, B), small intestinal adenoma (C), and a mesenteric leiomyoma (D) are shown. Insets

represent low-magnification images of the neoplasm. H&E stain.



papillomas and slightly higher incidence of pulmonary tumors.

The stability of the inserted human transgene in the mouse gen-

ome over several generations (Suemizu et al. 2002; Tamaoki

2001) and the remarkable similarity in the tumor incidences

in multiple rasH2 studies over a long interval of time (i.e.,

decade) demonstrate the robustness and minimal drift in this

animal model.

Although the inserted human c-ha-ras transgene is

expressed in most tissues of rasH2 mice, tumors develop only

in select organs without any direct correlation between trans-

gene expression and tumorigenesis. The mechanistic basis for

the higher predilection for pulmonary tumors, hemangiosarco-

mas, and epithelial tumors in rasH2 mice is unclear. Although

protein levels of the human transgene product (p21) and acti-

vating mutation of the human transgene play important roles

in tumorigenesis, tumorigenesis in rasH2 mice can be indepen-

dent of mutational activation (Mitsumori 2002). Higher tumor

incidence in specific organs or cell types can be a function of

the genetic background of the parental strains. The background

strains of rasH2 mice are BALB/c and C57BL/6. The sponta-

neous tumor spectrum in rasH2 mice was roughly in accor-

dance with the background strains of BALB/c and C57BL/6

(Mitsumori 2002; Tamaoki 2001). In fact, the reported inci-

dences of lung adenomas and gastric papillomas in the wild

type littermates of the rasH2 mice around 7–9 months of age

were 1.7% and 0.6%, respectively (Mitsumori 2002; Tamaoki

2001). Thus, the low spontaneous tumor incidence in untreated

rasH2 mice and greater susceptibility to chemical carcinogens

highlights the model’s sensitivity to chemical carcinogens.

MNU, an alkylating carcinogen, is the commonly used

positive control to demonstrate the enhanced sensitivity of the

rasH2 mouse model. To evaluate if MNU causes an increase in

the background tumor incidence or causes induction of tumors

in organ systems distinct from those in which background

tumors are seen, we surveyed the literature for tumor inci-

dences in MNU-treated (single dose) 6-month rasH2 studies.

These data were then compared with the data generated in the

MNU positive control groups in our 6-month carcinogenicity

studies. The most common tumors induced by MNU (gastric

papillomas, lymphomas, cutaneous papillomas, and gastric car-

cinomas) were similar between the two data sets. The inci-

dences of these common MNU-related tumors were slightly

higher in Pfizer studies compared to previous reports. These

differences in MNU sensitivity may be a function of the

preparation and immediate administration of MNU in the

laboratory setting. MNU is a highly volatile agent with a short

half-life; therefore, careful preparation and immediate adminis-

tration are critical elements in eliciting a positive tumor

response. Overall, MNU was a robust positive control with con-

sistent induction of tumors such as lymphoma (47–87% range)

and gastric papilloma (33–87% range), as previously reported

(Usui et al. 2001). This high positive tumor rate permits the use

of only 10–15 animals/sex in MNU-positive control groups

(Morton et al. 2008). For the positive control cohorts, histologic

evaluation of thymus alone is recommended while collecting

only stomach and tissues with macroscopic findings for further

evaluation if needed. The FDA has endorsed this approach for

at least 2 pharmaceutical candidates.

Another facet of this animal model, noted in the Pfizer stud-

ies and in the literature, was the tumor spectrum (i.e., organ

susceptibility) after treatment with MNU. MNU-related tumor

spectrum was slightly discordant with the spontaneous/back-

ground tumor spectrum. For example, the most common spon-

taneous tumors in rasH2 mice were pulmonary adenomas,

hemangiosarcomas, hepatocellular adenomas, and cutaneous

papillomas. MNU administration did not result in a propor-

tional increase in the incidence of these background tumors.

For example, the incidence of hemangiosarcomas, the second

most common spontaneous tumor in rasH2 mice, did not

change after treatment with the alkylating carcinogen MNU.

The most common MNU-related tumors were gastric papillo-

mas, lymphomas, cutaneous papillomas, and gastric squamous

cell carcinomas. Similarly, urethane (another alkylating geno-

toxic carcinogen) treatment was associated with a tumor spec-

trum distinct from MNU, that is, high incidence of lung tumors

and splenic hemangiomas / hemangiosarcomas (Ozaki et al.

2005). Although the basis of these differences between sponta-

neous tumors and different genotoxic agents is unclear, earlier

reports have highlighted organ susceptibility and mutational

pattern as the two main differences between MNU-related

tumors and spontaneous tumors in rasH2 mice (Ando et al.

1992; Saitoh et al. 1990). Thus, it is important to keep in mind

that the organ susceptibility in rasH2 mice is dependent on the

type of the positive control used (MNU or urethane).

An ideal animal model, as defined by the National Research

Council, should be based on ‘‘appropriateness as an analog,

transferability of information, genetic uniformity, background

knowledge of biological properties, generalization of the

results, ease of experimental manipulation and ecological con-

sequences and ethical implications’’ (Wall and Shani 2008). The

rasH2 mouse model satisfies most of these requirements as they

have been shown to be as effective if not better than the model

they have replaced, that is, 2-year mouse bioassay; are robust

and reproducible with respect to the tumor incidence; have been

shown to be genetically stable over several generations; and

have low mortality rates and spontaneous tumor incidence,

which could compromise long-term studies. Despite the positive

attributes of rasH2 mouse carcinogenicity studies, it is important

to note that these studies in transgenic models are always a part

of an integrated, weight of evidence evaluation that considers

mechanisms of action, genotoxicity, structure activity relation-

ships, toxicokinetic information, and results from other in vitro

and in vivo bioassays (MacDonald et al. 2004).

In summary, a 6-month rasH2 mouse carcinogenicity study is

an accepted alternative to a 2-year rodent bioassay for cancer

hazard identification of genotoxic and non-genotoxic small phar-

maceutical molecules when combined with one 2-year rat carci-

nogenicity study. The rasH2 mouse expresses multiple copies of

the human c-ha-ras gene that is aberrantly expressed in many

human neoplasms. The rasH2 model predicts known human car-

cinogens with similar accuracy as 2-year rodent studies, but

demonstrates fewer positive results that are considered irrelevant



or of low relevance for human risk assessment. Other advantages

include reduced animal use, high survival at study termination,

greater flexibility in study scheduling, and overall resource sav-

ings. The rasH2 mouse model should be considered to support

registration of small molecule pharmaceuticals that require carci-

nogenicity assessment. The standard study design for a rasH2

mouse study includes 3 treatment groups and a vehicle control

group composed of 25 animals/sex/group. A positive control

group (usually MNU or urethane) composed of at least 10 ani-

mals/sex is required. It is acceptable to examine microscopically

only the thymus in the positive control (MNU-treated) cohort.

Limited historical control data and lack of confidence in interpre-

tation of positive results likely have slowed the use of the rasH2

mouse model by sponsors. The historical control data for sponta-

neous neoplasms and neoplasia induced by MNU should assist in

the interpretation of future rasH2 mouse studies. Overall, the

remarkable similarity in the tumor incidences in multiple rasH2

studies over a long interval of time (i.e., decade) further high-

lights the robustness and minimal drift in this animal model.

AUTHOR’S NOTE

Supplemental material located online at http://tpx.sage

pub.com/supplemental.

ACKNOWLEDGMENTS

The authors wish to express their sincere thanks to Drs. Roy

Kerlin and Christopher Houle and Ms. Kimberly Ebersole in

critically reviewing the article and Walter Bobrowski for help

with the images.

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