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Dr.
Breen's Berner-U Presentation
Shared
with permission from The Alpenhorn; published June 2012
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Berner-U Presentation May 8th
2012
by Dr. Matthew Breen
(Summarized by Pat Long, and edited by Dr.
Breen)
Human and Canine genetic researchers have been operating
independently, but the advantages of collaboration are becoming
more apparent. The sequencing of the Human Genome took 15 years
and cost between 3 and 5 billion dollars. The mouse genome took
5 years and cost $100M. Conversely, the canine genome took about
one year and cost $47M, and finally the horse genome took about
6 months and cost $15M. The research increases in speed each
year with better tools. The canine genome, however, is most
useful in cancer research. Unlike the mouse, canines and humans
both get spontaneous cancers.
In any discussion of genetic research, it is necessary to
understand the vocabulary. Genome is the sum total of all the
genetic material for an organism. Chromosomes are the structures
that house the DNA, a human cell has 23 pairs of chromosomes,
one set of those 23 chromosomes comes from each parent. The
canine cell has 38 pairs of chromosomes. The DNA in those
chromosomes are made up of the double helix, a twisted ladder,
with the rungs constituting the base pairs, those letters we
always see to depict the nucleotides A, C, G, and T. In the
canine genome, it is estimated that there are approximately 2.4
billion base pairs, and about 19,000 genes.
Humans in general have more genetic diversity than purebred
dogs. When the canine genome project was initiated, the decision
was made to use a breed with as little genetic variation as
possible. Berners were one of the handful of breeds considered,
but it was ultimately decided to use a Boxer for the genetic
mapping project, a female Boxer named Tasha. Of all the AKC
registered purebred dogs, 75% of them are made up of only 20
breeds. The top five breeds in 2009 were: Labrador Retrievers,
Yorkshire Terriers, German Shepherd Dogs, Golden Retrievers, and
Beagles. Through selection, many breeds now have a limited level
of genetic variation.
Quite simply, the lack of genetic variation within a
purebred population helps to simplify the study of genetics.
There are more than 300 recognized breeds, and there are more
than 360 inherited diseases, more than any mammal except humans.
Most of those diseases are recessive, and about 50% of those
genetic diseases that we know about occur predominately in one
or a few closely related breeds. A pure bred population is
strongly enriched for 'risk' alleles. Because there are large
families with multiple generations, it helps increase the
ability to map genes. A small number of founders, popular sires,
genetic bottlenecks - the very things that help set breed type
and consistency reduces genetic variation.
Dogs are plagued by the greatest number of documented,
naturally occurring genetic disorders of any non-human species.
Current estimates suggest that up to 25% of the purebred dogs
living in US households my have or carry a serious genetic
disease. Compare that to human health where a 1% disease rate is
considered high risk. Inherited canine disease affects almost
every body system: blood, cardiovascular, endocrine, eye,
gastrointestinal, immune, musculoskeletal, nervous system,
respiratory, skin, reproductive, and CANCER.
When the AKC CHF asks breed clubs to compile a list of the
top ten diseases, cancer is always at the top of the list. It is
the leading cause of death in pet dogs; about 25% will develop
cancer, and about 50% of dogs over the age of 10 will die of
cancer. Of the 42,000,000 vet visits per year in the US, about
10% of them result in a diagnosis of cancer. The presentation,
histology and biology of a number of spontaneous canine cancers
closely parallel those of human malignancies. Dogs and humans
share similar genomes, and are exposed to the same environment.
A number of canine cancers have been hypothesized to be
appropriate models for human disease: osteosarcoma,
non-Hodgkin's lymphoma, glial tumors, oral melanoma, mammary
carcinoma, and prostate cancer.
There are breed predispositions for cancer and some breeds
develop numerous cancers. The important associations in Dr.
Breen's work are the Golden Retriever (lymphoma, osteosarcoma,
soft tissue tumors, hemangiosarcoma, and histiocytic
malignancies), and the Berner (osteosarcoma, soft tissue tumors,
hemangiosarcoma, and histiocytic malignancies).
The Bernese Mountain Dog was created in the early 1900's
from a small number of "pure" individuals. In the USA,
the breed has increased 108 fold in the past 46 years (31 new
AKC registrations in 1966 compared to 3,338 in 2008). The breed
has a small breeding population, and thus a relatively small
gene pool. Most breeding is line breeding with very little out
crossing. Most BMDs are related to each other through a small
number of common ancestors. While this gives greater consistency
in the breed and more competitiveness at the show level, it
means that disease genes may be highly prevalent in the
population.
The top cancers in the breed in order based on the two most
recent health surveys are:
|
|
2000 |
2005 |
|
histiocytic malignancies |
54% |
47% |
|
lymphoma |
24% |
29% |
|
hemangiosarcoma |
9% |
8% |
|
osteosarcoma |
7% |
10% |
|
mast cell |
6% |
6% |
About 75% of all Berner cancer deaths are a result of
histiocytic malignancies or lymphomas.
In Bernese Mountain Dogs recruited into Dr. Breen's study
the common presentation of patients with HM (histiocytic
malignancies) are:
|
Age of diagnosis
|
Youngest = 18 months |
|
Oldest = 13 years |
|
Average is about 6 years |
|
|
Clinical signs
|
Lethargy, anorexia, weight loss |
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Enlargement of the spleen and/or liver
and/or lymph nodes |
|
|
Common organ involvement
|
Spleen, liver, lung |
|
Kidney, lymph node, bone marrow, brain
|
|
|
Survival
|
Less than one hour up to a couple of
months |
|
Usually with a primary tumor in the
spleen and removed early |
|
[Pat Long note: I've heard of it as young as 7 months. We
have a number of cases that have been detected early with a
tumor in the lung, found on x-ray in preparation for a surgery
in most cases. Lomustine (CCNU) has been the treatment of
choice, and anecdotal evidence has seen survival of up to 18
months. Survival without any treatment has been as long as 18
months. I do know, however, that dogs that I would have only
given days or weeks based on my experience have lasted several
months with chemotherapy.]
Several of the dogs that were recruited for Dr. Breen''s
study as unaffected siblings of an affected Berner have
subsequently been diagnosed with HM within a year. The most
common misdiagnosis of HM in Berners is either lymphoma or
hemangiosarcoma.
HM is the 'Honey Badger' of tumors; the Honey Badger is a
weasel like animal known for its aggressive fighting spirit -
once it latches on, it doesn't let go. HM is aggressive and not
typically responsive to treatment. The vocabulary of HM has
varied over the years, and there is still a wide variety of
terms used by pathologists. Dr. Breen uses the following
terminology:
|
- Histiocytic sarcoma consists of an
initial single tumor, perhaps present on a limb. It is
diagnosed more in Flat Coated Retrievers (FCR) than in
Berners. It presents as either a mass on a limb (which has a
better prognosis) or as a tumor on a single internal organ
(which has a poor prognosis). |
|
- Malignant histiocytosis consists of
tumors that arise in multiple tissues/organs simultaneously,
not metastasis. It usually involves internal organs such as
the lung, spleen, liver. It is diagnosed more in Berners
than in FCR. Diagnosis is more likely if an internal mass is
observed.
Note: it can only be diagnosed if a pathologist is
given tumor tissue from multiple organs. This is the only
way that a pathologist can determine if the tumors arose
from the organ or were metastasized from a single organ. |
|
- NOTE: Even if a dog has multiple
histiocytic malignancies throughout its body, if only one
mass is evaluated the pathology diagnosis will be a HS.
Disseminated disease can be confirmed only if multiple
tissues are evaluated. |
Lymphoma is the most widely studied cancer across all dog
breeds. Consequently researchers have made more headway with
gene discovery. It can also be treatable and is often responsive
to chemotherapy.
Hemangiosarcoma is a silent killer. There are often no
signs of problems until the highly vascularized tumor ruptures
and the dog bleeds out.
Cancer is a genetic disease, it is caused by mutations in
the DNA at the cell level. The DNA is made up of the four
component parts termed A, C, G, and T. One A looks just like all
the other A's, so finding the specific genes responsible for
disease is very difficult. Having a detailed road map of those
genes is the first step.
The canine genome was sequenced and assembled at the Broad
Institute at MIT. It was a collaborative effort, and Dr. Breen's
lab contributed significantly with their development of
cytogenetic tools to help anchor the genome. The sequencing took
about 35 million reads of genetic material over a period of 7
months. The genome is approximately 2,400,000,000 base pairs of
DNA, and it is estimated that there are about 19,000 genes.
The dog has 78 chromosomes, and during the cell division
process each one is replicated, not always exactly. With cancer,
those replication errors are of key importance. If we think of a
chromosome as a filing cabinet, and the genes in that chromosome
as the folders, sometimes those folders get duplicated, or lost
(each of these are examples of unbalanced changes), or just
rearranged during the replication process (a balanced change).
These aberrations are hallmarks of gene deregulation and genome
instability. Some aberrations have been shown to be present only
in specific types of cancer, making them valuable diagnostic
tools. In addition, the presence of some aberrations are tightly
associated with response to certain therapies and so offer
prognostic value and guide the type of treatment most likely to
combat the cancer.
In looking at a grouping of the chromosomes in a canine
cell, it is difficult for even a trained eye to group all of the
chromosomes into their pairs (see
http://www.breenlab.org/karotype.html).
Determining whether material on those chromosomes has been lost,
duplicated, or rearranged is quite impossible without some sort
of assistance. Reagents have been developed in human gene
studies to assist with this research. The reagents available for
canine genomic study are much more sparse. Fluorescence in situ
hybridization, or FISH, was used at North Carolina State
University in Dr. Breen's lab to assist with canine genomic
research. FISH uses fluorescently labeled/detected DNA fragments
to mark pieces of the genome ranging from a few thousand base
pairs to an entire chromosome. Think of a yellow colored tab on
one file folder in that filing cabinet, a quick glance will show
if the new cabinet has that same yellow tab, or more than one,
or none at all. With the use of the chromosome identification
tools developed by Dr. Breen's lab, detecting aberrations
becomes much more simple (see
http://www.breenlab.org/cytogenetics.html)
Another technique used is called array comparative genomic
hybridization (aCGH). This uses whole genome fluorescent
labeling of tumor and non-tumor samples to compare directly the
relative amount of DNA in each sample at intervals throughout
the genome – the smaller the intervals the
higher the resolution of the array. If there are not differences
in the amount of DNA in a tumor and a non-tumor sample, the
amount of fluorescence from each is the same. Where the tumor
sample has missing, or extra DNA, the arrays reveal the genome
location of this aberrations. (see http://www.breenlab.org/array.html).
Technology continues to advance; in 2005 there would have
been one observation every 10,000,000 base pairs, but now the
lab is evaluating genome staus with an observation every 2,500
base pairs. It becomes much easier to see the specific part of
the chromosomes that have the aberrations. We see the same
increase in clarity with a digital image when we increase the
number of pixels.
To look for new structural chromosome changes in canine
tumors requires viable cells. The submissions have to be fresh
tumor biopsies, collected using sterile techniques. They need to
be shipped and processed quickly. This is not always possible,
the dog must come first. But if participation is possible,
having a vet willing to follow the simple procedures carefully
is key. Once the samples are received, the sterile biopsy cells
are put into a culture and live on for a short while. Dr.
Breen's lab is well aware of the fact that our dogs live on in
their study, as evidenced by his request for photos that are
displayed on the lab wall.
Research of cancer in humans is marked by the wide
variation of genetic material among humans. Canine cancer
research, however, is marked by very little variation, making it
much easier to detect the specific genetic aberrations
associated with cancers.
As a good example, meningiomas are marked by either a
partial or total deletion of chromosome 22 in humans, a piece of
the genome that contains over 550 genes. This human chromosome
shares DNA sequence with regions of three dog chromosomes,
numbers 10, 26 and 27. In the dog we don't see deletion of
chromosomes 10 and 26, but we see that most cases are missing a
copy of chromosome 27. The actual size of the genome sequence
shared between human 22 and dog 27 is only very small and
contains fewer than a dozen genes. This tells us that if human
and dog meningioma are caused by the same genes (which we
believe they are) the hunt for the key genes in both species
should concentrate on just those 12 shared genes, not the other
500+ genes that are not involved with dog meningioma. The
findings in the canine research enabled human researchers to
focus on the equivalent areas on the human genome. Areas that
they had not planned to study for a number of years and a great
many research dollars later.
By studying cancers in dogs and people, we are able to
focus on what they share and this accelerates the process of
gene discovery. Cancer has subtypes, caused by aberrations of
different genes.
These subtypes are often associated with vastly different
responses to therapies, and with different prognoses.
The current NCSU BMD samples, as of March 31st, 2012
|
Total submissions |
231 |
% of
191 diagnosed cancers |
|
|
|
|
|
Confirmed MH/HS |
117 |
61% |
|
Hemangiosarcoma |
13 |
7% |
|
Lymphoma |
13 |
7% |
|
Genuine STS* |
40 |
21% |
|
Osteosarcoma |
8 |
4% |
|
Pending diagnosis (submitted as HS and HSA)
|
19 |
|
|
Unsuitable** |
21 |
|
|
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|
|
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* includes fibrosarcoma, hemangiopericytoma, carcinoma
|
|
|
|
** necrotic tissue, non-tumor tissue, bad
packaging |
|
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|
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And blood from 110 healthy relatives |
|
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The age of diagnosis of HM is different between Berners and
FCRs: it peaks at 7-8 years for Berners, and at 9-10 years for
FCRs. The anatomical location varies by breed. Out of 146 HM
samples, 68 US Berners, 33 French Berners, and 45 US FCRs:
Location
|
|
FCR |
BMD |
|
One internal organ |
23% |
38% |
|
Disseminated |
26% |
51% |
|
Skin |
8% |
3% |
|
Limb |
38% |
3% |
|
Lymph Node |
5% |
5% |
Genetically, the aberrations have some similarities and
some differences between the two breeds as well. The findings
summarized:
|
- chaotic genome |
|
- highest recurrence of loss located on
chromosomes 2, 11, 16, 22, and 31
- Chr16 highest overall, with two regions at ~86%
recurrence
- 47-53Mb (telomeric end)
- 41.8-44.2Mb
|
|
- Several genes of interest now being
investigated:
- PTEN
- CDKN2A/B (p15, p16, ARF)
- RB1
- FAT1
- MTAP
|
|
- Some noted differences between BMD/FCR
|
Looking at chromosome changes, hemangiosarcomas are much
less complex than histiocytic malignancies; it is marked by far
fewer DNA copy number aberrations. However, the genome-wide
differences between hemangiosarcoma in BMDs and Golden
retrievers show more complex changes in the Berners than in the
Goldens.
What's next? Dr. Breen's lab has developed an assay to look
at the function of genes using archival tissue samples. They
have identified a list of genes that their data suggest are
drivers of MH, but it's still quite a long list. That list needs
to be narrowed, and then they need to assess the role of those
genes in patients.
Dr. Breen has a unique opportunity to partner with a human
pediatric oncologist. Dr. Joshua Shiffman owned a Berner that
died of histio, and he works at the Huntsman Cancer Institute at
the University of Utah. Comparing genetic changes in Berners and
humans will assist in identifying what is shared. It will allow
the list of candidate genes to be narrowed.
Tests on the horizon
Dogs were recruited for a study of lymphoma treatments. The
genetics of the lymphoma subtypes were studied and compared to
the treatment outcomes. A genetic test has been developed that
identifies duration of the first remission of lymphoma patients
treated with chemotherapy. That test is anticipated to be
commercially available within a year.
Genetic comparison of HM, lymphoma and hemangiosarcoma is
also possible, and a commercial test is feasible within two
years. The real importance of this test is evidenced by the
number of presumed HM samples Dr. Breen's lab has received that
were determined to be lymphoma instead. Always ask your
veterinarian to send in tumor biopsies as coming from a
Yorkshire Terrier rather than from a Berner. Too many
pathologists see Berners and assume histiocytic disease. Yorkies
almost never get cancer, so it would enable a pathologist to
work without having any preconceived notions.
Acknowledgement
Dr Breen and his lab expressed their sincere thanks to all
the BMD owners, breeders and vets who have had the courage to
submit samples from their beloved companions to help their
research projects move forward. He also paid tribute to Joye
Neff's fundraising efforts, particularly the mountain of tickets
she got for the Visit to Dr. Breen's lab, won by Wendy Djang.
He will be donating that offer again, and Wendy has offered
her hospitality to future winners.
From Pat Long: I would like to give a very special thank
you to Tessa. She works with each person who submits a sample to
the research lab. She is dealing with the most emotionally
trying of circumstances, and she does it with grace and care.
Dr. Breen met with the people who had come for the Berner-L get
together Monday night in Gettysburg. He claimed that Tessa knew
the name of each person and each dog that has participated in
the research. Several of us raised eyebrows - we know there have
been hundreds of participants. He asked Lori Simidian the name
of her girl who had died in 2008 as an example. He called Tessa
and asked if she remembered Lori Simidian's girl. on speaker
phone, no less. Yes, her name was Mallomar, and she participated
in the study four years ago. To say that we were all blown away
is an understatement.
Dr. Breen may provide the brains of the lab, but Tessa
provides the heart. While HM is one of the worst aspects of our
breed, we are truly lucky to have the participation of
researchers like those at NCSU.
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