Microbiology Journal Club
9:10am alternating Mondays
Trailer D, next to Flex Labs
No topic!
www.mbio.ncsu.edu/MJC
James_Brown@ncsu.edu
Bring coffee!
Devil Facial Tumor Disease
Devil Facial Tumor Disease
First seen in 1996
Devil Facial Tumor Disease
First seen in 1996
Endemic in 65% of Tasmania
Devil Facial Tumor Disease
First seen in 1996
Endemic in 65% of Tasmania
Has reduced devil
population by 1/2, to ~75K
Devil Facial Tumor Disease
First seen in 1996
Endemic in 65% of Tasmania
Has reduced devil
population by 1/2, to ~75K
Rarely metastatic - death is by
starvation
Devil Facial Tumor Disease
First seen in 1996
Endemic in 65% of Tasmania
Has reduced devil
population by 1/2, to ~75K
Rarely metastatic - death is by
starvation
Spread by aggressive facial biting
Devil Facial Tumor Disease
First seen in 1996
Endemic in 65% of Tasmania
Has reduced devil
population by 1/2, to ~75K
Rarely metastatic - death is by
starvation
Spread by aggressive facial biting
Agent is the cancer cell itself!
Devil Facial Tumor Disease
Extreme inbreeding has resulted in genetic
homogeneity, and so the cancer “allograph” is
not rejected.
Devil Facial Tumor Disease
Extreme inbreeding has resulted in genetic
homogeneity, and so the cancer “allograph” is
not rejected.
All sarcoma cells
have the same odd
karyotype
Devil Facial Tumor Disease
Extreme inbreeding has resulted in genetic
homogeneity, and so the cancer “allograph” is
not rejected.
All sarcoma cells
have the same odd
karyotype
Probably originated
in ca. 1990
Devil Facial Tumor Disease
Extreme inbreeding has resulted in genetic
homogeneity, and so the cancer “allograph” is
not rejected.
All sarcoma cells
have the same odd
karyotype
Probably originated
in ca. 1990
Is there anything
else like it?
Canine Transmissible Venereal Tumor
Canine Transmissible Venereal Tumor
a.k.a. Stickers tumor
Canine Transmissible Venereal Tumor
a.k.a. Stickers tumor
primarily an STD of dogs
Canine Transmissible Venereal Tumor
a.k.a. Stickers tumor
primarily an STD of dogs
neoplasm of the genitalia,
& sometimes other sites
Canine Transmissible Venereal Tumor
a.k.a. Stickers tumor
primarily an STD of dogs
neoplasm of the genitalia,
& sometimes other sites
rarely metastatic, although
sometimes self-transmitted
Canine Transmissible Venereal Tumor
a.k.a. Stickers tumor
primarily an STD of dogs
neoplasm of the genitalia,
& sometimes other sites
rarely metastatic, although
sometimes self-transmitted
usually self-regressing,
resulting in immunity
Canine Transmissible Venereal Tumor
a.k.a. Stickers tumor
primarily an STD of dogs
neoplasm of the genitalia,
& sometimes other sites
rarely metastatic, although
sometimes self-transmitted
usually self-regressing,
resulting in immunity
easily treated by chemotherapy
Canine Transmissible Venereal Tumor
known since the early 1880’s
Canine Transmissible Venereal Tumor
known since the early 1880’s
experimentally transmitted
in 1876 by a Russian Vet
Canine Transmissible Venereal Tumor
known since the early 1880’s
experimentally transmitted
in 1876 by a Russian Vet
common worldwide,
especially in tropical and
subtropical rural areas,
where it is the most common
tumor found in dogs
Canine Transmissible Venereal Tumor
known since the early 1880’s
experimentally transmitted
in 1876 by a Russian Vet
common worldwide,
especially in tropical and
subtropical rural areas,
where it is the most common
tumor found in dogs
CTVT and devil facial tumor
are the only known directly
transmissible cancers
Is CTVT really an contagious cancer?
Is CTVT really an contagious cancer?
Only transmissible by live cells, not cell
extracts, filtrates, or killed cells.
Is CTVT really an contagious cancer?
Only transmissible by live cells, not cell
extracts, filtrates, or killed cells.
All CTVT cells worldwide have the same
chromosomal rearrangements & aneuploidies
Is CTVT really an contagious cancer?
Only transmissible by live cells, not cell
extracts, filtrates, or killed cells.
All CTVT cells worldwide have the same
chromosomal rearrangements & aneuploidies
All have a LINE-1 (Long Interspersed Nuclear
Element) near the c-myc gene
Is CTVT really an contagious cancer?
Only transmissible by live cells, not cell
extracts, filtrates, or killed cells.
All CTVT cells worldwide have the same
chromosomal rearrangements & aneuploidies
All have a LINE-1 (Long Interspersed Nuclear
Element) near the c-myc gene
BUT there have been repeated reports of
virus-like particles associated with the tumors.
Many originally thought Kaposi’s sarcoma was
a contagious cancer for similar reasons!
Clonal Origin and Evolution
of a Transmissible Cancer
Claudio Murgia,1,4 Jonathan K. Pritchard,2 Su Yeon Kim,3 Ariberto Fassati,1 and Robin A. Weiss1,*
1
MRC/UCL Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London,
46 Cleveland Street, London W1T 4JF, UK
2
Department of Human Genetics
3
Department of Statistics
University of Chicago, CLSC–507, 920 East 58th Street, Chicago, IL 60637, USA
4
Present address: Institute of Comparative Medicine, University of Glasgow Veterinary School, 464 Bearsden Road,
Glasgow G61 1QH, UK.
*Contact: r.weiss@ucl.ac.uk
DOI 10.1016/j.cell.2006.05.051
SUMMARY
The transmissible agent causing canine transmissible venereal tumor (CTVT) is thought to
be the tumor cell itself. To test this hypothesis,
we analyzed genetic markers including major
histocompatibility (MHC) genes, microsatellites,
and mitochondrial DNA (mtDNA) in naturally
occurring tumors and matched blood samples.
In each case, the tumor is genetically distinct
from its host. Moreover, tumors collected from
40 dogs in 5 continents are derived from a
single neoplastic clone that has diverged into
two subclades. Phylogenetic analyses indicate
that CTVT most likely originated from a wolf or
an East Asian breed of dog between 200 and
2500 years ago. Although CTVT is highly aneuploid, it has a remarkably stable genotype. During progressive growth, CTVT downmodulates
MHC antigen expression. Our findings have implications for understanding genome instability
in cancer, natural transplantation of allografts,
and the capacity of a somatic cell to evolve
into a transmissible parasite.
INTRODUCTION
CTVT, also known as Sticker’s sarcoma, is a histiocytic tumor that is usually transmitted among dogs through coitus
but may also spread through licking, biting, and sniffing
tumor-affected areas (Cohen, 1985; Das and Das, 2000).
First characterized 130 years ago (Novinski, 1876), CTVT
tumor cells, and not by killed cells or cell filtrates (Cohen,
1985). Second, the tumor karyotype is aneuploid but has
characteristic marker chromosomes in tumors collected
in different geographic regions (Murray et al., 1969; Oshimura et al., 1973; Weber et al., 1965). Third, a long interspersed nuclear element (LINE-1) insertion near c-myc
(Katzir et al., 1985) has been found in all tumors examined
so far (Katzir et al., 1987) and can be used as a diagnostic
marker to confirm that a tumor is CTVT (Liao et al., 2003).
In two animals that had been experimentally inoculated
with CTVT, the resulting tumors contained the LINE-1/
c-myc insertion, whereas the normal tissues did not (Katzir
et al., 1987; Liao et al., 2003). However, in natural transmission, inheritance of a LINE-1 insertion near c-myc in
the germline might represent a predisposition to develop
CTVT after exposure to an oncogenic agent, similar to
the Mendelian LINE-1 insertion in the factor IX gene, which
causes mild hemophilia B in dogs (Brooks et al., 2003).
The recent emergence of a tumor transmitted by biting
in the endangered marsupial species the Tasmanian devil
(Sarcophilus harrisii) (Owen and Pemberton, 2006) has
attracted renewed interest in the concept of cellular transmission, for which CTVT is cited as a precedent (Pearse
and Swift, 2006). However, authors of reports describing
virus-like particles in CTVT (Ajello and Gimbo, 1965; Battistacci and Morriconi, 1974; Lombard and Cabanie,
1967) considered that an oncogenic virus might play a
role in tumorigenesis. Although most specialists in the field
accept the cellular transmission of CTVT, definitive data
that this is the case have been lacking, and the concept
of a contagious cancer cell has tended to be greeted
with skepticism by many oncologists and immunologists.
Molecular genetic markers have not previously been
used to resolve the issue of natural transmission, the breed
of origin, or the age of the canine tumor. Here, we compare
Clonal Origin and Evolution
of a Transmissible Cancer
Claudio Murgia,1,4 Jonathan K. Pritchard,2 Su Yeon Kim,3 Ariberto Fassati,1 and Robin A. Weiss1,*
1
MRC/UCL Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London,
46 Cleveland Street, London W1T 4JF, UK
2
Department of Human Genetics
3
Department of Statistics
University of Chicago, CLSC–507, 920 East 58th Street, Chicago, IL 60637, USA
4
Present address: Institute of Comparative Medicine, University of Glasgow Veterinary School, 464 Bearsden Road,
Glasgow G61 1QH, UK.
*Contact: r.weiss@ucl.ac.uk
DOI 10.1016/j.cell.2006.05.051
SUMMARY
The transmissible agent causing canine transmissible venereal tumor (CTVT) is thought to
be the tumor cell itself. To test this hypothesis,
we analyzed genetic markers including major
histocompatibility (MHC) genes, microsatellites,
and mitochondrial DNA (mtDNA) in naturally
occurring tumors and matched blood samples.
In each case, the tumor is genetically distinct
from its host. Moreover, tumors collected from
40 dogs in 5 continents are derived from a
single neoplastic clone that has diverged into
two subclades. Phylogenetic analyses indicate
that CTVT most likely originated from a wolf or
an East Asian breed of dog between 200 and
2500 years ago. Although CTVT is highly aneuploid, it has a remarkably stable genotype. During progressive growth, CTVT downmodulates
MHC antigen expression. Our findings have implications for understanding genome instability
in cancer, natural transplantation of allografts,
and the capacity of a somatic cell to evolve
into a transmissible parasite.
INTRODUCTION
CTVT, also known as Sticker’s sarcoma, is a histiocytic tumor that is usually transmitted among dogs through coitus
but may also spread through licking, biting, and sniffing
tumor-affected areas (Cohen, 1985; Das and Das, 2000).
First characterized 130 years ago (Novinski, 1876), CTVT
tumor cells, and not by killed cells or cell filtrates (Cohen,
1985). Second, the tumor karyotype is aneuploid but has
characteristic marker chromosomes in tumors collected
in different geographic regions (Murray et al., 1969; Oshimura et al., 1973; Weber et al., 1965). Third, a long interspersed nuclear element (LINE-1) insertion near c-myc
(Katzir et al., 1985) has been found in all tumors examined
so far (Katzir et al., 1987) and can be used as a diagnostic
marker to confirm that a tumor is CTVT (Liao et al., 2003).
In two animals that had been experimentally inoculated
with CTVT, the resulting tumors contained the LINE-1/
c-myc insertion, whereas the normal tissues did not (Katzir
et al., 1987; Liao et al., 2003). However, in natural transmission, inheritance of a LINE-1 insertion near c-myc in
the germline might represent a predisposition to develop
CTVT after exposure to an oncogenic agent, similar to
the Mendelian LINE-1 insertion in the factor IX gene, which
causes mild hemophilia B in dogs (Brooks et al., 2003).
The recent emergence of a tumor transmitted by biting
in the endangered marsupial species the Tasmanian devil
(Sarcophilus harrisii) (Owen and Pemberton, 2006) has
attracted renewed interest in the concept of cellular transmission, for which CTVT is cited as a precedent (Pearse
and Swift, 2006). However, authors of reports describing
virus-like particles in CTVT (Ajello and Gimbo, 1965; Battistacci and Morriconi, 1974; Lombard and Cabanie,
1967) considered that an oncogenic virus might play a
role in tumorigenesis. Although most specialists in the field
accept the cellular transmission of CTVT, definitive data
that this is the case have been lacking, and the concept
of a contagious cancer cell has tended to be greeted
with skepticism by many oncologists and immunologists.
Molecular genetic markers have not previously been
used to resolve the issue of natural transmission, the breed
of origin, or the age of the canine tumor. Here, we compare
Goal:
To show once and
for all whether or
not CTVT is a
transmissible
cancer.
Clonal Origin and Evolution
of a Transmissible Cancer
Claudio Murgia,1,4 Jonathan K. Pritchard,2 Su Yeon Kim,3 Ariberto Fassati,1 and Robin A. Weiss1,*
1
MRC/UCL Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London,
46 Cleveland Street, London W1T 4JF, UK
2
Department of Human Genetics
3
Department of Statistics
University of Chicago, CLSC–507, 920 East 58th Street, Chicago, IL 60637, USA
4
Present address: Institute of Comparative Medicine, University of Glasgow Veterinary School, 464 Bearsden Road,
Glasgow G61 1QH, UK.
*Contact: r.weiss@ucl.ac.uk
DOI 10.1016/j.cell.2006.05.051
SUMMARY
The transmissible agent causing canine transmissible venereal tumor (CTVT) is thought to
be the tumor cell itself. To test this hypothesis,
we analyzed genetic markers including major
histocompatibility (MHC) genes, microsatellites,
and mitochondrial DNA (mtDNA) in naturally
occurring tumors and matched blood samples.
In each case, the tumor is genetically distinct
from its host. Moreover, tumors collected from
40 dogs in 5 continents are derived from a
single neoplastic clone that has diverged into
two subclades. Phylogenetic analyses indicate
that CTVT most likely originated from a wolf or
an East Asian breed of dog between 200 and
2500 years ago. Although CTVT is highly aneuploid, it has a remarkably stable genotype. During progressive growth, CTVT downmodulates
MHC antigen expression. Our findings have implications for understanding genome instability
in cancer, natural transplantation of allografts,
and the capacity of a somatic cell to evolve
into a transmissible parasite.
INTRODUCTION
CTVT, also known as Sticker’s sarcoma, is a histiocytic tumor that is usually transmitted among dogs through coitus
but may also spread through licking, biting, and sniffing
tumor-affected areas (Cohen, 1985; Das and Das, 2000).
First characterized 130 years ago (Novinski, 1876), CTVT
tumor cells, and not by killed cells or cell filtrates (Cohen,
1985). Second, the tumor karyotype is aneuploid but has
characteristic marker chromosomes in tumors collected
in different geographic regions (Murray et al., 1969; Oshimura et al., 1973; Weber et al., 1965). Third, a long interspersed nuclear element (LINE-1) insertion near c-myc
(Katzir et al., 1985) has been found in all tumors examined
so far (Katzir et al., 1987) and can be used as a diagnostic
marker to confirm that a tumor is CTVT (Liao et al., 2003).
In two animals that had been experimentally inoculated
with CTVT, the resulting tumors contained the LINE-1/
c-myc insertion, whereas the normal tissues did not (Katzir
et al., 1987; Liao et al., 2003). However, in natural transmission, inheritance of a LINE-1 insertion near c-myc in
the germline might represent a predisposition to develop
CTVT after exposure to an oncogenic agent, similar to
the Mendelian LINE-1 insertion in the factor IX gene, which
causes mild hemophilia B in dogs (Brooks et al., 2003).
The recent emergence of a tumor transmitted by biting
in the endangered marsupial species the Tasmanian devil
(Sarcophilus harrisii) (Owen and Pemberton, 2006) has
attracted renewed interest in the concept of cellular transmission, for which CTVT is cited as a precedent (Pearse
and Swift, 2006). However, authors of reports describing
virus-like particles in CTVT (Ajello and Gimbo, 1965; Battistacci and Morriconi, 1974; Lombard and Cabanie,
1967) considered that an oncogenic virus might play a
role in tumorigenesis. Although most specialists in the field
accept the cellular transmission of CTVT, definitive data
that this is the case have been lacking, and the concept
of a contagious cancer cell has tended to be greeted
with skepticism by many oncologists and immunologists.
Molecular genetic markers have not previously been
used to resolve the issue of natural transmission, the breed
of origin, or the age of the canine tumor. Here, we compare
Approach:
To test whether
cancer cells are
genetically
related to the
host, or represent
a genetically
independent
population.
Table S1. Specimens
Table 1. Sources of CTVT Samples
Fresh Tumors with Matching Blood Sample
Place
Number
Catania, Italy
5
Messina, Italy
5
Kolkata, India
4
Nairobi, Kenya
2
Paraffin-Embedded Archival Tumors
Country
Number
Brazil
4
Italy
5
Spain
4
Turkey
9
USA
2
Total
40
Details of age, sex, breed of dog, and site of tumor are in Table S1.
widely dispersed across different chromosomes in the
normal karyotype (Parker et al., 2004). These were chosen
to compare tumor and normal DNA in 11 of the dogs from
which both types of tissue were available. A neighbor-joining tree was constructed using chord distance (Figure 2),
which showed that the tumors and the hosts were genetically separate, with all tumors clustered together. A
neighbor-joining tree based on the proportion of alleles
shared between pairs of samples gave a similar result (Figure S2). None of the host dogs showed close relatedness
to any of the others, consistent with the fact that they
came from three locations in Europe, Asia, and Africa
Data sources
Dogs Providing Fresh Tumor and Blood Samples
Sample
Date Breed
Sex
Age (Yrs)
A
2001 mixed
male
2
B
2001 mixed
male
1
C
2001 mixed
male
10
Tumor Location
penis
penis
penis, eye, skin,
lymph nodes
D
2001 mixed
male
2
penis
E
2001 mixed
female 5
vagina
F
2003 mixed
male
3
penis
G
2003 mixed
male
2
penis
H
2003 mixed
female 2
vagina
I
2003 mixed
female 3
vagina
L
2003 mixed
male
nd
penis
M
2003 mixed
male
nd
penis
N
2005 mixed
female 7
vagina
P
2005 mixed
female 8
vulva
Q
2005 mixed
female 2
vulva
R
2005 mixed
female 3
vagina
S
2005 mixed
female 2
vagina
Paraffin-Embedded Samples Used for Microdissection of Tumors
Sample
Date Breed
Sex
Age (Yrs)
Tumor Location
2
nd
nd
nd
nd
nd
3
nd
nd
nd
nd
nd
4
nd
nd
nd
nd
nd
5
nd
nd
nd
nd
nd
6
nd
nd
nd
nd
nd
8
nd
nd
nd
nd
nd
9
nd
nd
nd
nd
nd
11
nd
nd
nd
nd
nd
12
nd
nd
nd
nd
nd
14
1985 mixed
female 3
vagina
17
1985 mixed
female 2
vagina
18
1986 mixed
female 2
vagina
19
1986 mixed
female 3
vagina
20
1999 mixed
male
1
penis
21
1999 beagle
male
2
penis
25
1999 husky
female 4
vulva
27
1997 husky
male
1
penis
29
1999 mixed
male
4
penis/skin
30
1995 mixed
female nd
cervix
32
1995 maremmano male
2
penis
33
1995 mixed
male
nd
penis
35
1992 mixed
female nd
vagina
36
1983 mixed
female nd
vagina
37
1976 mixed
female nd
vagina
Country
Italy (Catania)
Italy (Catania)
Italy (Catania)
Italy (Catania)
Italy (Catania)
India (Kolkata)
India (Kolkata)
India (Kolkata)
India (Kolkata)
Kenya (Nairobi)
Kenya (Nairobi)
Italy (Messina)
Italy (Messina)
Italy (Messina)
Italy (Messina)
Italy (Messina)
Country
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Spain
Spain
Spain
Spain
USA (Georgia)
USA (Georgia)
Brazil
Brazil
Brazil
Brazil
Italy (Sardinia)
Italy (Sardinia)
Italy (Sicily)
Italy (Sicily)
Italy (Sicily)
Tumor and normal (blood) tissues from Sicily were brought to the UK with permission of the Department of
Environment, Foods and Rural Affairs and were tested for the absence of rabies by RT-PCR at the Veterinary
Laboratory Agency (Weybridge, UK) prior to use. DNA was extracted from tumours and blood samples of Indian
and Kenyan specimens on site. The Messina samples of matched tumor and normal tissues were analyzed for the
LINE-1/c-myc insertion only. nd = not determined.
LINE-1
c-myc
Figure 1. Specific LINE-1/c-myc and DLA Haplotype Genetic Markers for CTVT Detected by Specific PCR Amplification
(A) For each of 11 dogs (A–M), fresh normal and tumor samples are indicated as N and T, respectively. The panel is assembled from three separate
gels visualized by ethidium bromide. The invariant DLA-88 intron sequence serves as a positive control for each of the 22 specimens.
(B) PCR amplification of DNA using Cy5-labeled forward primers from 21 microdissected tumor cells from paraffin-embedded specimens. The panel
is assembled from four separate gels.
exon
intron
exon
T-specific primers
Figure 1. Specific LINE-1/c-myc and DLA Haplotype Genetic Markers for CTVT Detected by Specific PCR Amplification
(A) For each of 11 dogs (A–M), fresh normal and tumor samples are indicated as N and T, respectively. The panel is assembled from three separate
gels visualized by ethidium bromide. The invariant DLA-88 intron sequence serves as a positive control for each of the 22 specimens.
(B) PCR amplification of DNA using Cy5-labeled forward primers from 21 microdissected tumor cells from paraffin-embedded specimens. The panel
is assembled from four separate gels.
Microsatellite genotyping
simple sequence repeats
additional Structure analyses (Figure
wolves and the subset of breeds that
To determine the specific origin of CTVT, we perf
larity to CTVT in the initial analysis. T
additional Structure analyses (Figure 4A), focus
lowed the CTVT samples to have m
wolves and the subset of breeds that showed som
that if the progenitor animal was a mo
larity to CTVT in the initial analysis. The model us
spread
a
lowed theCTVT
CTVT ancestry
samples should
to have be
mixed
ances
breeds. Inanimal
this analysis,
the tumor
that if the progenitor
was a mongrel
dog, th
most strongly
wolves.
A second
CTVT ancestry
should bewith
spread
across
two or
a nonparametric
clusteringclut
breeds. Inapplied
this analysis,
the tumor samples
bor-joining
tree A
based
onmethod
pairwise
most strongly
with wolves.
second
of al
a
applied a genotypes)
nonparametric
technique
(a
andclustering
again indicated
simila
bor-joiningand
treethe
based
pairwise(Figure
allele sharing
a
wolfon
samples
4B). Thu
genotypes)based
and again
indicated
similarity
between
method
and the
neighbor-joinin
and the wolf
samples
4B). Thus,
bothDLA
the am
satellites
are(Figure
consistent
with our
based method
themay
neighbor-joining
method
for
that and
CTVT
have originated
in wolv
satellites are consistent with our DLA analysis and in
caution is required due to the small sa
that CTVT may have originated in wolves. However
breed and the fact that the available do
caution is required due to the small sample sizes o
to pedigree breeds (Parker et al., 20
breed and the fact that the available dog data were
domestic
dogs isetnot
to pedigree
breeds (Parker
al.,excluded.
2004), so an or
domestic dogs is not excluded.
Estimating the Age of CTVT
We
investigated
whether CTVT repres
Estimating
the
Age of CTVT
a recently
emerged
or whether
i
We investigated
whether
CTVT tumor
represents
an epide
Figure 2. Microsatellite DNA Analysis of 11 Fresh Tumors and
Figure
S2. Comparison of 21 Microsatellites
Matched
Tumor
and
a recently emerged
tumor
whether
it hasparasite
a moreNa
origin in
and
is in or
effect
a stable
Host Samples
FigureMatched
2. Microsatellite
DNA Analysis of 11 Fresh Tumors and
origin and The
is in effect
a stable
parasite
of would
dog popul
Novinski
(1876)
report
arg
Matched
Host Samples
Unrooted
neighbor-joining tree based on chord distance compiled
reportold,
would
argue that
Unrooted
treeloci.
based
on chord
distance compiled
least(1876)
130 years
assuming
that CTV
the
fromneighbor-joining
21 microsatellite
A similar
neighbor-joining
tree basedThe
on Novinski
Neighbor-joining
tree
based
on
proportional
shared
alleles.
least 130 years
assuming
that the tumors
ob
from 21
microsatellite
A similar
neighbor-joining
tree based on
allele
sharing is loci.
provided
in Figure
S2.
wereold,
clonal
with modern
tumors.heThe
257bp sequence of mtDNA control region
Figure 3. Analysis of mtDNA in CTVT
Tumor haplotypes from fresh and paraffin-embedded tissues showing two main clusters of mtDNA. Diameter of each circle is proportional to the
number of tumor samples. Each branch represents one base pair change, with black dots representing intermediates not found in the tumor samples
analyzed. The outlined boxes indicate that tumor samples with homozygous (diploid) and hemizygous (haploid) DQA1 genes coincide with mtDNA
clusters, except for tumor 9, which is diploid.
257bp sequence of mtDNA control region
Figure S4. Maximum-Likelihood Tree of mtDNA Sequences
The tree was constructed from 21 CTVT samples and 45 additional dogs, wolves and a coyote
outgroup, without assuming that the tumor sequences are monophyletic. Given the short (257bp)
amplified region from archival samples, there may be uncertainty about the true position on the
tree of the two sequences that group quite separately from the main tumor clusters; moreover
they may represent contaminating host mtDNA.
Figure S6. CTVT in Relation to Wolves and 85 Dog Breeds
Data are based on 73 microsatellite loci. Each panel shows the results from a model-based clustering algorithm, Structure, that assigns
sampled individuals, based on their genotypes, to a prespecified number, K, of clusters. Each tumor sample, or individual dog, is
represented by a vertical bar, with colored segments indicating the proportion of that individual's membership in each cluster. At K=2,
the tumors cluster clearly with wolves and 'old' dog breeds; for larger, more stringent K values, the tumors form a distinct group,
indicating a common origin.
Figure 4. Relationship of CTVT to Wolves and Dog Breeds
(A) Results of a Structure analysis of the canids that appeared most closely related to CTVT (yellow at K = 2 in Figure S6). The clustering was based on
the nontumor samples only, and the three tumor samples with nearly complete data were then assigned to the appropriate clusters.
(B) Neighbor-joining tree based on pairwise differences among the same set of individuals as in (A). The relationship between wolves and CTVT is
similar when the tree is constructed using all dogs (Figure S6).
Based on 73 microsatellites - just ‘old’ breeds
Figure
4. Relationship
of of
CTVT
totoWolves
Figure
4. Relationship
CTVT
Wolvesand
andDog
Dog Breeds
Breeds
(A) Results
of a of
Structure
analysis
of of
the
canids
closelyrelated
relatedtotoCTVT
CTVT
(yellow
at=K2=in2Figure
in Figure
The clustering
was based
on
(A) Results
a Structure
analysis
the
canidsthat
thatappeared
appeared most
most closely
(yellow
at K
S6).S6).
The clustering
was based
on
the nontumor
samples
only,
and
thethe
three
completedata
datawere
were
then
assigned
to the
appropriate
clusters.
the nontumor
samples
only,
and
threetumor
tumorsamples
samples with
with nearly
nearly complete
then
assigned
to the
appropriate
clusters.
(B) Neighbor-joining
based
pairwisedifferences
differences among
among the
asas
in (A).
TheThe
relationship
between
wolves
and CTVT
is
(B) Neighbor-joining
treetree
based
onon
pairwise
the same
sameset
setofofindividuals
individuals
in (A).
relationship
between
wolves
and CTVT
is
similar
when
the
tree
is
constructed
using
all
dogs
(Figure
S6).
similar when the tree is constructed using all dogs (Figure S6).
Figure 5. Average Pairwise Divergence
of Microsatellite DNA in Wolves, Dogs,
and CTVT
The expected heterozygosity at microsatellite
loci in different subsets of canids and CTVT
was plotted. Non-CTVT data were derived
from Parker et al. (2004). The Australian shepherd represents the most diverse defined
breed and the miniature bull terrier the least
diverse breed for microsatellite DNA.
CTVT are immune to tumor development upon reinoculation, naive dogs of many breeds are susceptible to tumor
growth (Cohen, 1985). A recent study indicated that secretion of tumor growth factor b (TGF-b1) may play a role in
local immune suppression during progressive growth but
that interleukin 6 secretion by tumor-infiltrating lymphocytes aids eventual immune destruction during tumor
were the majority population (!90%) in the microdissected tumor tissue (Figure 6B). If class I genes were
wholly unexpressed, NK cells might eliminate the tumor;
hence, our finding of low expression appears more plausible than the suggestion of defective b2-microglobulin (Cohen et al., 1984). A systematic and quantitative analysis
of several tumors during different phases of growth and
We therefore performed RT-PCR with tumor-specific
and host-specific primers within the tumor tissue of Sicilian dog C in order to investigate differential expression of
tumor and host DLA genes. Figure 6A shows that class I
expression was lower in tumor cells than in stromal cells
(which serve as a loading control) and that class II expression was absent. This result indicates significant downmodulation of DLA expression in the tumor cells because they
immune responses.
MHC expression is repressed in tumor cells
DISCUSSION
Our results, based on several independent genetic
markers in tumor-bearing dogs living on five continents,
show that CTVT arose from a common ancestral neoplastic cell. Early in its evolution, the clone diverged into two
Figure 6. MHC Expression in CTVT
(A) Expression of DLA class I (DLA-88) and
class II (DRB1) in the penile tumor of dog C
by RT-PCR using tumor-cell-specific primers
(T) and primers specific to the alleles of this animal for host stromal cells and infiltrating normal cells (N). M = marker lanes.
(B) Histopathology of a hematoxylin-andeosin-stained 4 mm section of the same tumor.
Scale bar = 30 mm. Cells with large round nuclei
are tumor cells, and mitoses are apparent. A
stromal cell is indicated with an arrow.
Cell 126, 477–487, August 11, 2006 ª2006 Elsevier Inc. 483
Figure S1. Gene Dosage of DLA Class II Alleles in Fresh and Microdissected Tumor Samples
The copy number of the target DLA genes was compared to the reference gene !-actin using
qPCR with SYBR-Green and the kinetic method (standard curve) so that the diploid !-actin gene
was calibrated as 1.0. !-actin was compared to glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) as a second standard for diploid gene dosage. Fresh tumors are denoted by letter and
paraffin extracted tumors by number; normal tissues from matched animals A, B, G and M are
shown as white bars. Ratios of 0.8 and above were scored as diploid and of 0.6 or below as
haploid.
tions spanning five
authors first establ
origin of the tum
contained the tumo
1 element, even sa
two decades apa
whereas DLA ana
large number of var
DNA, only two m
appeared in the tu
ing the existence o
subtypes. Results
by mtDNA analysis
tumors into two d
within a single clad
canine phylogeny. G
from the same do
uted among three c
al., 2006).
The authors then
origin of the tumors
morphism and mic
indicated that all
were closely relate
wolves and East As
providing convinci
a common origin (F
satellite analysis o
subgroups reveal
lent degree of var
a divergence even
tions spanning five
authors first establ
origin of the tum
contained the tumo
1 element, even sa
two decades apa
whereas DLA ana
large number of var
DNA, only two m
appeared in the tu
ing the existence o
subtypes. Results
by mtDNA analysis
tumors into two d
within a single clad
canine phylogeny. G
from the same do
uted among three c
al., 2006).
CTVT
(Canis parasiticus)
The authors then
origin of the tumors
morphism and mic
indicated that all
were closely relate
wolves and East As
providing convinci
a common origin (F
satellite analysis o
subgroups reveal
lent degree of var
a divergence even