Page 169 - Withrow and MacEwen's Small Animal Clinical Oncology, 6th Edition

148 PART II Diagnostic Procedures for the Cancer Patient

DNA product can be analyzed in a number of ways for mutations Detection of Fusion Gene Products by PCR
and to quantify the product for measurement of gene expression; One mechanism by which chromosomal translocation causes
malignant transformation of cells is to create novel proteins with
or, in the case of lymphoid malignancy, the DNA can be separated
VetBooks.ir by size to look for clonal populations of B and T cells. The same altered function. The best studied of these fusion genes, the Phila-
delphia chromosome, is the bcr-abl fusion gene found in greater
methods can be applied to the analysis of ribonucleic acid (RNA)
once the RNA has been transcribed into DNA (reverse transcrip- than 90% of all human chronic myelogenous leukemias (CMLs)
tase PCR [RT-PCR]). and occasionally acute lymphoblastic leukemia (ALL) and acute
For primer synthesis the sequence of the target gene typically myelogenous leukemia (AML). ABL is a tyrosine kinase that has
21
12
must be known. The publication of good quality canine and myriad activities in cell growth and differentiation. It is encoded
13
feline genomes has been invaluable in this regard—it is now pos- on human chromosome 9, and in CML is translocated to chro-
sible simply to use the known sequences, rather than hope for mosome 22. The site of the translocation varies within the bcr
sequence similarities with mice and humans. (breakpoint cluster region) gene, so that a new fusion gene, bcr-
abl, is formed. The new fusion protein allows for the constitutive
Detection of Genetic Insertions and Deletions activation of the ABL tyrosine kinase, which in turn promotes the
PCR-based assays commonly are used in human oncology to development of CML. This novel protein is the product of a novel
detect insertions or deletions in genes relevant to the prognosis RNA transcript, which can be readily detected by RT-PCR. This
or treatment of a neoplasm. In veterinary medicine detection of assay can detect as few as 1:10 tumor cells and therefore can
4
3
internal tandem duplications in the c-kit gene in canine mast cell be used both for diagnosis of CML and for quantifying residual
tumors is now a routine part of the diagnosis for the purpose disease after treatment.
of obtaining prognostic information. The primary mutations Assays for a large number of translocations in human cancers
described are internal tandem duplications (ITDs) in two dif- have been developed over the past 10 years. These assays are now
22
ferent exons, exon 8 and exon 11. The mutations involve the routinely available for characterization of human tumors, particu-
14
duplication of a small segment of DNA so that it is repeated, larly leukemia and sarcoma. The finding that canine leukemia and
resulting in a larger gene (Fig. 8.2). Approximately 14% to 20% lymphoma can exhibit the same translocations as their human
of canine mast cell tumors have a duplication in either exon 8 or counterparts 7,23 suggests that detection of novel fusion genes
14
exon 11. Dogs with tumors that carry the ITDs consistently will provide inexpensive and sensitive diagnostic testing both for
have been shown to have a worse outcome than dogs with a wild- detecting cancer and for monitoring disease in the near future.
type c-kit gene. 15,16
Detection of this type is fairly simple, because the presence of Assessment of Clonality in Lymphoma and Leukemia
a larger (or smaller, in the case of deletions) PCR product is deter- A clonality assay demonstrates that a group of cells is derived from
mined by size separation. As more genes are identified as targets of a single clone. The term usually is used to refer to detection of
therapy, such assays likely will become more frequent. Suter et al the unique genes found in each individual B or T cell—immu-
identified an internal duplication in the flt3 gene using the same noglobulin genes in B cells and T-cell receptor (TCR) genes in T
17
methods and provided preliminary evidence that the response cells. The portion of these genes that encodes the antigen binding
to a small molecule inhibitor is predicted by the presence of this region is the portion that varies between cells, both in size and
mutation in cell lines. Thus we are likely to see routine use of sequence. Once a B or T cell is mature and divides in response
mutation detection in the near future. to antigenic stimulation, the immunoglobulin and T-cell receptor
genes are passed on to the daughter cells. 24,25
Detection of Single-Base Mutations In the course of a normal immune response to a pathogen,
Some cancers will have predictable, single-base mutations that B and T cells are activated, expand, and eventually die, leaving
can be detected by standard sequencing. However, sequencing can behind a small number of residual memory cells. On the other
be insensitive; therefore a variety of PCR-based assays have been hand, when a cell becomes neoplastic, it no longer responds to
developed for mutation analysis. The best example of this type of growth controls and undergoes unlimited expansion. Therefore if
assay currently in use is detection of a single nucleotide change it can be established that most of the cells in a particular collection
18
found in the BRAF gene in 80% of cases of canine TCC. The of lymphocytes have the same immunoglobulin or T-cell receptor
mutation found in the canine gene is equivalent to a BRAF muta- gene, these cells most likely are neoplastic rather than reactive. 26
tion common in a variety of human cancers (V600E), and it causes When immunoglobulin and T-cell receptor genes rearrange
constitutive activation of the BRAF protein. BRAF is a serine/ during the course of B-cell and T-cell development, respectively,
threonine kinase, which activates a series of downstream signaling the length and sequence of the resultant gene differs from cell
pathways to drive cellular metabolism and proliferation. 19 to cell. This happens for many reasons; for example, nucleotides
Breen et al developed a PCR-based assay to detect this muta- are added between V, D, and J segments as they rearrange into
20
tion (in dogs the mutation is called V595E). The purpose of the a contiguous formation. The clonality assay takes advantage of
assay is to diagnose TCC in urine samples with suspicious cells, this development. A sample consisting of many different lympho-
which can often be difficult by cytology alone. The method used cytes, as in a reactive process (e.g., the lymph nodes of a dog with
for detection is a technique called droplet digital PCR (ddPCR). chronic pyoderma or poor dental hygiene), will have multiple,
ddPCR can detect the V595E mutation when it is present in different-sized, T-cell receptor and immunoglobulin genes. On
as little as .01% of the DNA. This method was considerably the other hand, in a sample consisting of neoplastic lymphocytes,
20
more sensitive than standard sequencing, which could not detect the immunoglobulin gene or the T-cell receptor gene (depending
the mutation when it was present in less than 10% of the DNA. on whether it is a B-cell or a T-cell lymphoma) will be a single
ddPCR probably will be used more commonly in the future, size (Fig. 8.3). (All methods used in veterinary medicine to detect
because it provides a way of quantifying mutations and DNA clonally rearranged T-cell receptor genes target the TCR gamma
copy number changes with high precision. gene.)