Tissue and Blood Based Tests for Cancer Diagnosis


Conventional Cancer Diagnostic Tests

The diagnosis of cancer is a complex procedure, which may range from the initial physical examination to modern molecular diagnostic tests. Cancer itself is a heterogeneous disease that may involve a range of histopathologic and genetic changes.

The biopsy sample evaluation remains the gold standard in the diagnosis of most common cancer types. Depending upon the site, size, location, and type of cancer different diagnostic techniques are generally employed to establish the diagnosis of particular cancer type/specific defect.

The following is a list of various conventional diagnostic tests that are generally carried out for the diagnosis of most common cancer types:

Histopathologic Evaluation

In this technique, biopsy sample obtained from the cancer tissue is first fixed on a glass slide and then is observed under the microscope by a histopathologist who is expert in diagnosing morphological cancer changes in the cells/tissue.

Histopathological studies help in determining the severity of cancerous changes involved (grade of cancer) in a biopsy sample by estimating the concentration of cancerous cells and the extent of cancerous changes involved. These tests are required to be performed by an expert pathologist who can swiftly and precisely identify cancerous cells and distinguish them from the normal ones in the biopsy sample.

Traditionally various dyes, such as, hematoxylin and eosin were used to detect some color changes that indicate some characteristics of cancer cells. However, these color changes were not very specific and are now replaced with more advanced immunohistochemical stains.

Immunohistochemical (IHC) analysis

This technique involves the use of antibodies that can bind to certain specific proteins/antigens in a sample of cancer tissue. The antibodies are modified in such a way to possess fluorescence or any other microscopically observable property upon binding to the antigen. Thus, a number of characteristic proteins on the surface of cancer cells can be detected using IHC analysis in a quick and sensitive manner.

This technique has been successfully utilized to detect the following surface proteins on breast cancer cells: estrogen receptors (ERs), progesterone receptors (PRs), and human epidermal growth factor receptor 2 (HER2). Similarly, for various cancer types, a range of characteristic surface proteins can be identified by this technique which can be targeted with a targeted therapy.

Flow cytometry

This cytogenetic technique enables the identification and quantification of hematologic cancer cells based on the characteristic surface protein. The blood sample is first treated with some fluorescent antibodies that get attached to certain specific proteins/antigens on the surface of the cancer cells.

The treated sample is then analyzed using a laser beam and a detector attached to a computer. This test can establish the diagnosis of hematologic malignancies through detection of different types of cells (with specific cell surface proteins) in the blood sample along with the quantification of each type of cells. Presence of certain characteristic cluster of differentiation (CD) cell surface proteins usually establishes the diagnosis of a particular type of hematologic malignancy.

Further, the restricted expression of either kappa or lambda immunoglobulin light chains on the cell surface membrane is utilized for establishing the clonality of hematologic cells. This technique can also be utilized for human leukocyte antigen (HLA) testing that enables the identification of an appropriate donor for patients who are the good candidate for allogeneic stem cell transplant (SCT).

Fluorescence in situ hybridization (FISH): This is a cytogenetic analysis tool that plays a crucial role in the diagnosis of hematogenous malignancies. FISH allows detection of chromosomal abnormalities like translocations, inversions, addition, or deletion. A classic example of FISH utility is the detection of Philadelphia chromosome (Ph), the most common genetic abnormalities in the CML cells.

The sample cells are first grown into the culture medium and are observed under a microscope after adding certain reagents that bind only to a specific portion of a chromosome. Different types of probes, such as fluorescent RNA probes, DNA probs, and others are used, which bind to a specific portion of a chromosome in the sample cells.

Then, the sample can be examined under a fluorescence microscope to determine the presence of certain chromosomal abnormalities targeted by the RNA probe. One of the major advantages of FISH is the detection of multiple chromosomal abnormalities in a single run simultaneously using probes with different targets and colors.

This technique is very sensitive, fast, and accurate. This technique is preferably used for detecting common genetic abnormalities in hematologic malignancies and soft tissue sarcomas.

Sanger Sequencing

It has been the gold standard for DNA sequence analysis for many years and is also referred to as dideoxy sequencing or chain termination. DNA sequencing reveals precise organization of nucleotides in a DNA segment.

This analysis is based on the use of normal DNA nucleotides and dideoxynucleotides, the modified nucleotides that prevent the addition of further nucleotides when integrated into a DNA strand. Radiolabeled or fluorescent complimentary DNA primers are first attached to the DNA fragment to be analyzed. Then, deoxynucleosides triphosphates are sequentially added to the primer by DNA polymerase.

Dideoxynucleotides terminate the elongating chains at specific sites. Many oligonucleotides are generated with different length and sequence complementary to the parent DNA strand. Gel electrophoresis is generally utilized to segregate and analyze the mixture of newly synthesized DNA strands, which further elucidate the sequence of template DNA strand. Any mutation in the template DNA sequence can be identified by comparing the tumor DNA sequence and normal DNA sequence.

Although a small amount of starting DNA sample is required for this technique due to amplification by polymerase chain reaction (PCR), analytical sensitivity is limited requiring 50% tumor cell concentration in the sample. With advancement in diagnostic technology and emerging new diagnostic techniques, this technique has been replaced with more advanced diagnostic methods.

Polymerase chain reaction (PCR)

This technique was first invented by Kerry Mullis in 1985. This technique makes use of DNA polymerase that amplifies a target segments of DNA using two types of primers, a forward and a reverse primer. These primers are highly selective of the target DNA region and provide the starting point for nucleotides to get added sequentially.

Any mutation in the template DNA strand leads to a mismatch at elongating (3-prime) end and thus lead to the failure of DNA amplification. Alternatively, mutation-specific fluorophores are first added in the sample and then DNA amplification is done to displace the fluorophores from their binding sites.

Displaced fluorophores are estimated using a real-time PCR instrument that reveals the extent of a specific mutation. The limitation of this technique is that only a single mutation can be studied in one reaction. Although a very small amount of sample DNA is required for single analysis, the amount may escalate for multiple mutation analysis.

Thus, this technique is suitable for analyzing a small number of specific mutations. This is a very sensitive diagnostic tool which can detect a very small number of cancer cells with a specific genetic change, for example, Immunoglobulin heavy-chain variable (IGHV) region gene mutation can be identified with high sensitivity and specificity. Quantitative PCR (qPCR) technique is generally used to diagnose minimum residual disease (MRD) in patients after treatment.

Reverse-transcriptase polymerase chain reaction (RT-PCR)

This technique is a variation of the PCR technique. In this technique, messenger RNA (mRNA) is generally used as a template to produce complimentary cyclic DNA (cDNA) using reverse transcriptase. Then the cyclic DNA is amplified using PCR.

Amplified DNA can be identified using different real-time DNA identification tools. This technique enables the quantification of mRNA, which in turn help in the estimation of gene expression. This is the most sensitive diagnostic tool available today, which can detect a very small number of cancer cells with a specific genetic change (e.g. BCR-ABL1 gene) in blood or bone marrow sample.

This technique can be used either as a qualitative tool to establish the diagnosis of a particular cancer type or as a quantitative tool after treatment to assess the number of cancer cells in the blood or bone marrow, and thus, the efficacy of treatment or the minimum residual disease (MRD) can be assessed.


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