David J. Argyle, BVMS, PhD, DECVIM-CA (Oncology), MRCVS
This lecture will cover specific advances in the diagnosis of cancer. More specifically:
Advanced imaging technologies
The use of immunohistochemistry and immunophenotyping
PCR-based diagnostics in lymphoma
Transcriptomics to define molecular signatures
Proteomics for serum diagnosis and monitoring
The use of genomics to define at risk patients
Advanced Imaging Technologies and Their Indications in Oncology (Table 1)
Table 1. Advanced imaging in oncology.
Classically used for evaluation of the GI tract and the urinary tract. Now largely replaced by availability of endoscopy. Although can be used for myelography, MRI is safer and far superior.
Ultrasound has largely replaced abdominal radiography for the evaluation of the abdomen. Essential for:
--Evaluation of abdominal organs for evidence of primary or secondary disease (e.g., staging mast cell tumours).
--Aid in guided biopsy of deep tumours.
--Evaluation of internal lymph nodes (especially sub-lumbar in cases of low GI/perianal tumours.
--Evaluation of tumour invasion into vital structures (e.g., thyroid tumours).
Largely replaced contrast radiography for the evaluation of:
--Upper and lower GI tract
--This involves the administration of radiopharmaceuticals that localize to areas of tumour or inflammation.
--Most commonly used to detect bony metastasis (e.g., in osteosarcoma of dogs).
--Technetium-99M (99mTc) most commonly used because of short half-life, good imaging qualities, and can be bound easily to localizing pharmaceuticals (e.g., 99mTc-methylene diphosphonate for bone scans).
--Generally sensitive for disease lesions but non-specific for disease aetiology.
Computerized Tomography (CT)
--This is becoming more widely used in veterinary medicine with greater availability. It gets over the disadvantage of superimposition in conventional radiography by portraying images as computer generated slices.
--Produces imaged in the transverse plane in real time. Can reconstruct from transverse images to produce images in the sagittal and dorsal planes.
--Greater superiority over radiography for detection of pulmonary metastasis and evaluation of lymph nodes.
--Used in treatment planning for radiotherapy.
--Good for imaging lesions of the skull (e.g., oral tumour and nasal tumour staging.
--Used in combination with contrast agents (contrast enhanced CT), can be invaluable in determining the extent of tumour invasion.
Magnetic Resonance Imaging (MRI)
--Can produce images in the transverse, sagittal and dorsal planes in real time.
--Images generated through the properties of hydrogen atoms in the body when they are placed in a magnetic and radiofrequency field.
--Primary use in veterinary medicine is the evaluation of the central nervous system.
--Excellent evaluation of soft-tissue structures, less useful for cortical bone.
Positron Emission Tomography (PET)
--This is a sophisticated form of nuclear scintigraphy. Where CT and MRI are concerned with anatomy, PET is concerned with function.
--PET uses positron emitting radionuclides in association with a fixed or rotating gamma camera that reconstructs the images in cross section.
--Tumour cells tend to have increased glucose utilization through glycolysis. The increased energy demand in tumour cells is met through the up-regulation of the hexose monophosphate pathway, cell membrane glucose transporter proteins, and hexokinase. A glucose analogue, 18F-fluorodeoxyglucose (FDG) acts as a glucose molecule and is preferentially up taken by tumour cells.
--Particularly useful in detecting metastases. New machine combine CT and PET (PET-CT scanners) which give the optimum imaging modality (anatomy and function) for cancer patients.
Immunohistochemistry and Tumour-specific Histologic Dyes
Immunohistochemistry (IHC) involves the use of labelled antibodies as reagents to localize antigens and proteins in cells to help identify the cell type. The use of IHC or special histologic dyes is indicated when a diagnosis is difficult using standard H/E sections.
Typical routine stains include those shown in Table 2.
Table 2. Routine stains.
S100, Melan A
Mast cell tumour
Toluidine Blue*, Giemsa*
Using Immunohistochemistry to Diagnose Round Cell Tumours
Collectively, mast cell tumours, cutaneous lymphoma, cutaneous plasma cell tumours, transmissible venereal tumours (TVT), histiocytomas, and neuroendocrine (Merkel cell) tumours are referred to as round cell tumours due to the morphology of the cancer cells.
Often the clinician is faced with a histopathological diagnosis of round cell tumour, and will require special stains or immunohistochemistry to further identify the cell type and ascertain the appropriate diagnosis.
Table 3 is intended as a guide and an indication of the service that your pathologist should offer (i.e., a typical round cell immunohistochemical panel).
Table 3. Stains for round cell tumours.
Positive = mast cell tumour
--Positive = T cell lymphoma
--If CD18 and MHCII are also positive, the tumor may be of histiocytic origin
--Positive = B cell lymphoma OR plasmacytoma
--If CD18 and MHCII are also positive, the tumor may be of histiocytic origin
When other stains are negative:
--If CD18 positive and MHC II positive, the tumor may be of histiocytic origin
--CD18 positive and MCH II negative, the tumour may be a mast cell tumour
Immunophenotyping of canine lymphoma tissue is straightforward. Laboratories can classify tumours as T-cell (CD3) or B-cell (CD79); the latter being more common and the former associated with a poorer prognosis. In the clinic, the fact that a tumor may be T or B cell does not alter the type of conventional therapy that is offered. However, it may alter an owner's willingness to treat and can be offered as part of the diagnostic work-up. It is possible, however, as we progress to classification systems based upon molecular and immunological markers, that we may adopt different treatment protocol tailored to sub-classifications.
Immunoglobulin Rearrangements in Lymphoma
The monoclonality of a population of neoplastic lymphoid cells lends itself to providing supporting evidence for a diagnosis of lymphoma. Both B and T cells have cognate receptors which enable them to take part in the immune response. PCR amplification of either the T cell Receptor (TCR) or immunoglobulin chains on B cells will demonstrate either a mixed population (i.e., with a reactive lymphadenopathy) or a clonal population of cells (as with lymphoma)
Other diagnostic and prognostic markers have been suggested including serum levels of alpha 1-acid glycoprotein (AGP) and matrix metalloproteinases (MMP 2 and 9) as indicators of relapse. However, currently there are no commercially available tests for these markers. Additionally, validation in large scale clinical trials needs to be performed to give confidence to their clinical use. However, new markers and the use of sophisticated molecular techniques (such as micro and tissue arrays) are being developed to provide information for the clinician. It is possible that their widespread use in clinical practice will become evident over the next 10 years.
Tissue array (or tissue microarray) is a method of relocating multiple tissues from conventional histologic paraffin blocks so that tissues from multiple patients can be seen on a same slide. Tissue microarrays (also TMAs) consist of paraffin blocks in which up to 1000(1) separate tissue cores are assembled in array fashion to allow simultaneous histological analysis. In the tissue microarray technique, a hollow needle is used to remove tissue cores as small as 0.6 mm in diameter from regions of interest in paraffin embedded tissues such as clinical biopsies or tumor samples. These tissue cores are then inserted in a recipient paraffin block in a precisely spaced, array pattern. Sections from this block are cut using a microtome, mounted on a microscope slide and then analyzed by any method of standard histological analysis. Each microarray block can be cut into 100-500 sections, which can be subjected to independent tests. Tests commonly employed in tissue microarray include immunohistochemistry, and fluorescent in situ hybridization.
A DNA microarray (also commonly known as 'gene or genome chip', 'DNA chip', or 'gene array') is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to a chemical matrix. Since there can be tens of thousands of distinct probes on an array, each microarray experiment can potentially accomplish the equivalent number of genetic tests in parallel. Microarray-based gene expression profiling can be used to identify cancer disease genes by comparing gene expression in diseased and normal cells. Using this technology attempts have been made to identify specific genetic signatures of tumour types that predict response to therapy or prognosis.
The term 'proteome' was first coined in 1994, and refers to all the proteins in a cell, tissue, or organism. Proteomics refers to the study of the proteome. Because proteins are involved in almost all biological activities, the proteome is a rich source of biological information. The goal of clinical proteomics is to develop proteomics technology for the benefit of patient care. This new research technology is now being used in clinical research studies ranging from cancer to cardiovascular disease and organ transplants. Researchers are searching for proteins that can be used as early biomarkers of disease, or that may predict response to therapy or the likelihood of relapse after treatment in blood, urine, or diseased tissue
Genomics to Identify at Risk Patients
With the cloning of the canine genome, we now have the tools to try and identify specific genetic signatures associated with genetic predisposition to cancer. Researchers are particular concerned with identifying risk factors in such breeds as the flat-coated retriever that predispose them to certain mesenchymal malignancies.