Page 172 - Withrow and MacEwen's Small Animal Clinical Oncology, 6th Edition

CHAPTER 8 Molecular Diagnostics 151

inflammatory bowel disease from gastrointestinal (GI) lymphoma inactivated in all the cells in a particular sample. Because not all
in dogs 30,31 and cats 32,33 and to aid in the diagnosis of nodular animals will be heterozygous, the assay is useful only in a subset of
female animals—68% of cats and 50% of dogs. The assay has
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splenic lymphoma, which can be difficult to distinguish from
VetBooks.ir nodular hyperplasia. 34 been used for canine patients with possible chronic myelogenous
Clonality assays sometimes can be useful for establishing the
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leukemia and for cats with possible myelodysplastic syndrome.
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lineage (B cell vs. T cell) in cytologically unambiguous lympho- In dogs this assay has been named the canine androgen receptor
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mas if additional case material is not obtainable from the patient. assay (CANARA assay).
However, flow cytometry or immunohistochemistry generally is
preferable for this purpose, for several reasons: first, aberrant rear- Whole Genome and Whole Exome Sequencing—
rangements occasionally can be seen in nonlymphoid tumors, such
as myelogenous leukemias. Second, simply knowing whether a Next Generation Sequencing
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neoplasm is derived from a B cell or a T cell may not be clinically Next generation sequencing (also known as massively parallel
useful. Both lineages can give rise to markedly different tumor sequencing) is a technology that allows investigators to sequence the
types with significantly different outcomes. For example, the entire genome of an individual (or an individual’s tumor). Sequenc-
two most common forms of T-cell lymphoma (peripheral T-cell ing an entire genome was considered an enormous and expensive
lymphoma and T-zone lymphoma) both have clonally rearranged endeavor not many years ago, but it is now a routine part of inves-
T-cell receptor genes. Peripheral T-cell lymphoma, however, has a tigating the biology of cancer. Thousands of human cancers have
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median survival time of 150 days, whereas T-zone lymphoma is been fully sequenced, and the cost and efficiency of this process
indolent and may not require treatment at all. 37 means that individual tumors can be sequenced to aid clinical deci-
The principle of the clonality assay also can be used to quan- sion making. The main types of information that can be obtained
tify tumor cells in blood, bone marrow, or node and to monitor from whole genome sequencing of a tumor include (1) mutations
minimum residual disease. For this type of analysis PCR primers in potential oncogenes or tumor suppressor genes, including single
specific for the immunoglobulin or T-cell receptor gene carried by nucleotide variants (SNVs) and insertions and deletions (indels);
the tumor are used instead of the broadly reactive primers used (2) structural changes in chromosomes, including fustion genes
to screen samples. In this way the investigator is certain that only and copy number variations (CNVs), in which whole genetic
tumor DNA is amplified, and not nonneoplastic lymphocytes. regions may be duplicated; and (3) the mutational load of a par-
The specificity of this reaction permits determination of the num- ticular tumor, which may predict responsiveness to checkpoint
ber of tumor cells in a sample of blood, even when those cells are inhibitor therapy and also identify potential neoantigens that may
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as rare as 1:10 cells. Yamazaki et al demonstrated that with cur- be targeted in immunotherapy strategies. The principles of next
rent chemotherapy protocols, all seven dogs they examined had at generation sequencing are described in the next section.
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least 1:10 cells in their peripheral blood, even though the dogs
achieved clinical remission. Although this kind of analysis may Quantifying Genes and Gene Expression
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not be practical for routine diagnostics, it is a powerful research
tool to compare the efficacy of novel chemotherapy regimens, and The complete genetic code, or DNA sequence, is present within
it also can be used to refine current protocols. Indeed, this method every cell in the body. The effective genetic information that uniquely
subsequently was used by Sato et al to evaluate the responsive- defines each cell type within the body is defined by the genes
ness of B-cell lymphoma to individual components of the cyclo- expressed (transcribed) as messenger RNA (mRNA). The complete
phosphamide, hydroxydaunorubicin, Oncovin and prednisone or profile of mRNA expression is more responsible for the phenotype
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prednisolone (CHOP) chemotherapy protocol. of a cancer than the individual genes, although mutations in a gene
often are individual drivers of a cancer phenotype. To assess the level
Assessment of Clonality in Nonlymphoid Neoplasms of expression of one or a few genes, three methods could be used:
Nonlymphoid cells do not have unique DNA sequences that dif- (1) real-time quantitative PCR (RT-qPCR), which has been used
fer from cell to cell, so other methods must be used for clonality for many years; (2) ddPCR, which is a more recent development
assessment. The question of whether a collection of cells is neo- that has many advantages over RT-qPCR and which probably will
plastic arises in a variety of circumstances—eosinophilia in the replace RT-qPCR for most applications; and (3) non-PCR–based
cat, for example, or pleural effusion in a dog, which is dominated gene counting methods, such as Nanostring.
by cells that could be either reactive or neoplastic mesothelium. If the goal is to measure expression of all the genes in a tumor,
One way to determine whether the cells in these cases are derived microarrays and, more recently, RNA sequencing (RNA seq)
from the same clone is to examine the pattern of X chromosome methods have been used. Assessment of the global level of gene
inactivation. One copy of the X chromosome in the cells of female expression is called gene expression profiling.
animals is inactivated by DNA methylation. The purpose of the
inactivation is to ensure that both male and female animals have Real-Time PCR and Droplet Digital PCR
the same “dosage” of genes found on the X chromosome. Inacti-
vation is thought to be random, so in a nonneoplastic collection RT-qPCR refers to the quantitative (Q) measurement of
of cells, any given cell would have an equal chance of either the DNA—either genes or, more commonly, RNA that has be
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maternal or paternal X chromosome being inactivated. If the cells reversed transcribed to cDNA. The principle of RT-qPCR
are all derived from the same clone, however, the same X chromo- is that DNA is amplified using primers, just as in a routine
some would be inactivated in each cell. PCR reaction, but at each round of amplification, the amount
In female animals, X chromosome inactivation can be mea- of PCR product is quantified. Unlike endpoint PCR, in which
sured using the androgen receptor gene. This gene contains the reaction runs to completion and the product is separated by
repeated DNA elements (CAG repeats), the number of which size, real-time PCR is quantitative and relies on the change in
varies. If an animal is heterozygous for the number of repeats, the amount of PCR product, as measured by fluorescence, as
it is possible to determine whether the same X chromosome is the reaction progresses.