Dr. Breen - Oct, = 2006

From: "Gemt af Windows Internet Explorer 7" Subject: Dr. Breen - Oct, 2006 Date: Mon, 26 May 2008 08:31:37 +0200 MIME-Version: 1.0 Content-Type: multipart/related; type="text/html"; boundary="----=_NextPart_000_0012_01C8BF0A.E9EB28A0" X-MimeOLE: Produced By Microsoft MimeOLE V6.0.6000.16545 This is a multi-part message in MIME format. ------=_NextPart_000_0012_01C8BF0A.E9EB28A0 Content-Type: text/html; charset="utf-8" Content-Transfer-Encoding: quoted-printable Content-Location: http://www.bernergarde.org/Articles/html/DrBreen_Oct2006.htm =EF=BB=BF Dr. Breen - Oct, = 2006

Chromosomes, Genes and Cancer
A = molecular=20 cytogenetic approach to the study of malignant histiocytosis in = the=20 Bernese Mountain Dog

Dr. = Matthew Breen=20 & Tessa Breen
October 7, 2006
BMD Health Symposium,=20 SIBB
Como, Italy

The Canine Genome Project was a combined effort of numerous = people and=20 was led by Dr. Kerstin Lindblad-Toh at the Broad Institute. The = genome was=20 sequenced and assembled at the Broad and was anchored by a = combination of=20 approaches including an assessment of the chromosomes, which was = performed=20 in the lab of Dr. Matthew Breen at North Carolina State = University. The=20 scientific article detailing this work was formally published in = the=20 science journal =E2=80=98Nature=E2=80=99 on Dec 8th 2005. The = project cost approximately=20 $40 million and involved 35 million reads of genetic material over = seven=20 months, followed by a detailed assembly process t put all the = pieces into=20 their correct places. The cost was met by the National Human = Genetics=20 Research Institute, in recognition of the benefits that the = sequence would=20 offer towards the development of a greater understanding of = numerous human=20 genetic diseases, including cancers.

Dr. Breen=E2=80=99s research involves looking at genetic = changes that are=20 associated with cancers. In particular his laboratory looks at how = the=20 genome is =E2=80=98shuffled=E2=80=99 during the cancer process. = The genome is divided into=20 chromosomes (DR. Breen refers to these as nature=E2=80=99s = biological filing=20 cabinets) and each gene has a precise location on one of these=20 chromosomes. As cells replicate, chromosomes sometimes rearrange; = this is=20 particularly true with cancer. Chromosomes can be lost, they can = move, or=20 they can duplicate. These chromosomal aberrations are hallmarks of = gene=20 deregulation and genomic instability. As chromosomal material is = lost,=20 duplicated or relocated elsewhere in the genome, the genes within = such=20 regions are also affected accordingly. When genes move = neighborhood they=20 may not interact well with their new neighbors, if genes are lost, = their=20 function is absent from the cell, if genes are amplified their = function=20 may alter. All these features may lead to deregulation of the = cell=E2=80=99s=20 normal function and the development a cancer.

Many chromosome changes (aberrations) are so specific to = certain types=20 of cancers that they are regarded as diagnostic of that cancer. = Further,=20 many of these changes have been shown to correlate with response = to=20 treatment and so in addition to being diagnostic these chromosome=20 aberrations may also provide prognostic information and so = ultimately=20 assist in making treatment decisions.

Humans have 46 = chromosomes,=20 dogs have 78. Dog chromosomes all look very similar =E2=80=93 = except for the X and=20 Y chromosomes. This is problematic for researchers in that = chromosome=20 pairs are difficult to identify unequivocally. Dr. Breen has = developed a=20 series of molecular reagents which can be used to color code = chromosomes,=20 either a single locus or gene pair, or a full chromosome pair. = This=20 technique relies on a microscopic techniques referred to = fluorescence in=20 situ hybridization, or FISH.

Some chromosomal aberrations = are=20 balanced aberrations in which there is no net gain or loss DNA in = the=20 cell- examples include inversions of gene groupings and reciprocal = translocations;. Other aberrations are imbalanced, in which DNA is = either=20 lost or duplication, i.e. the gene copy number changes =E2=80=93 = examples include=20 deletions and insertions. In order to look at these aberrations = Dr. Breen=20 needs viable cells, or fresh tumor tissue shipped quickly. In = order to get=20 this, it requires owner and veterinary awareness and willingness. = Dr.=20 Breen=E2=80=99s lab thus requests specific samples to be sent as = listed=20 below:

The samples are used in the following manner:

Formalin fixed biopsy =E2=80=93 this us used to obtain (usually = to confirm) a=20 pathology diagnosis, and may also be used in FISH

Blood = sample=20 (EDTA) =E2=80=93 this is used to isolate constitutional DNA and is = shared with Dr.=20 Ostrander at the NHGRI

Sterile tumor biopsy =E2=80=93 this = is use as=20 source of tumor cell DNA and also as source of viable cells to = make=20 chromosome preparations that are evaluated using FISH with Dr. = Breen=E2=80=99s=20 panel of chromosome specific reagents.

Of the tumors that = have=20 been submitted to Dr. Breen=E2=80=99s lab for Bernese Mountain = Dogs (from USA dogs=20 over approx 2 years), the breakdown is as follows:

	Malignant = histiocytosis/Histiocytic=20 Sarcoma	58 (70% of total affected samples)
	Hemangiosarcoma	6
	Lymphoma	5
	Other	15
	Relatives	27

The sterile tissue is placed in a culture to grow. The cells = are then=20 fixed at metaphase. There is a very different look to the = chromosomes in=20 normal cells versus tumor cells. Dr. Breen cannot use tumor tissue = that=20 has been subjected to chemotherapy. Tumor tissue that has been = preserved=20 in wax blocks can (sometimes) be used for his research.

Dr. = Breen=20 uses a variety of molecular techniques to determine which = chromosomes are=20 involved in the cancers. Chromosome painting is an approach that = literally=20 paints the length of a chromosome. This approach is very well = suited to=20 looking for chromosome translocations but is not able to identify = changes=20 within individual chromosomes. Chromosome tiling is a technique = that=20 generates a multicolored bar code of each chromosomes and so a = change in=20 the order of the colors along the length of a chromosome = identifies=20 regions that have been changed. This process thus allows Dr. Breen = to see=20 insertions and deletions. Dr. Breen has also shown that chromosome = changes=20 seen in dog cancers may be closely related to those seen in the=20 corresponding human cancer. This may have important consequences = for the=20 development of new therapies for both dogs and people.

In = Malignant=20 histiocytosis/Histiocytic Sarcoma there are a number of = chromosomal=20 abnormalities that can be seen. The findings so far:

		a tendency toward = extremely=20 chaotic genome reorganization
		chromosomal gains and losses = are=20 widespread
		translocation events are = apparent with a=20 reduction in total chromosome number
		some things are shared between = the Flat=20 Coat Retriever and the BMD
		others exist only within the = breed
		MH is=20 NOT the same = cancer=20 between FCRs and BMDs at the genetic=20 level

There are key regions of interest that are now being evaluated = in more=20 detail.

Dr. Ostrander uses blood and has received samples = from 49=20 affected Berners, and 59 controls. Her control samples come from = Berners=20 who have reached the age of 10 years without having had any = cancer. Her=20 study has identified regions of the genome that may correlate = highly with=20 MH. These are found in normal cells that may indicate a genetic=20 predisposition for the disease.

The studies of both Drs. Breen and Ostrander are making = exciting=20 progress. To continue making this progress requires many more = samples to=20 help refine their data and Dr. Breen urged the audience to remain = very=20 active in submitting samples for these research projects.=20

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