DNA Molecular Testing and Personalized Medicine – Part 1

Over the last several years technological advancements in the molecular underpinnings of cancer have led to a revolution of cancer management by DNA molecular testing. By utilizing cancer genetics to guide the specific and customized treatment for each patient, the concept of personalized medicine has emerged. Melanoma has been one of the cancers extensively studied from a genetic and molecular aspect. This first of two articles explains genetic testing and molecular diagnostics in cancer medicine by highlighting several technologies employed in assessing tumors: cytogenetics, fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and microarray technology.

The predecessor to CGH and microarray analysis has been conventional cytogenetics where changes in chromosomes are visualized with staining under a microscope – karyotyping. This technology has been used since the 1950’s with few changes. Consider the fact that human genome has 3.2 billion base pairs. Karyotyping can easily visualize large (>10 mb, 10 megabase, 10 million base pairs or nucleotides) changes in chromosomes, such as whole chromosome duplication or deletion, but has the drawback of requiring dividing cells. Thus, it cannot be used on tissue that will not grow or has been fixed in formalin and paraffin embedded. Furthermore, genetic changes important in cancer are often below the resolution of karyotyping, for instance the amplification of Her-2/neu (ErbB2) commonly seen in breast cancer.

For these reasons, a higher resolution analysis was developed to detect submicroscopic changes in chromosomes, termed fluorescence in situ hybridization (FISH). With FISH analysis, fluorescently labeled DNA probes locate the positions of specific DNA sequences on chromosomes by hybridizing with the DNA being studied. This allows for the detection at higher resolution of mutations, deletions, and other genetic changes in cancer cells. Unfortunately, FISH is limited by the number of probes available across the genome, and the number of probes that can be used in each assay. Because of the cost, FISH is not a technology that can probe the entire genome on a routine basis.

The development of comparative genomic hybridization (CGH) provided the ability for genome-wide screening for chromosomal abnormalities (or more specifically copy number variants) at much higher resolution. CGH uses two genomes, a test and a control, which are differentially labeled and competitively hybridized to specific chromosome sequences across the genome in a microarray.

Watch these YouTube video to understand how microarrays are made and how microarrays function.

This video is a good overview of microarray testing options with CGH microarray demonstration starting at 1:40.

The fluorescent signal intensity of the labeled patient or tumor DNA relative to that of the reference DNA can then be analyzed, allowing the identification of DNA abnormalities (copy number changes). Copy number variation is one of the cornerstones of genetic variability and cancer development. The “copy” refers to a segment of DNA from one kilobase to several megabases in size. These DNA segments can be deleted, duplicated, or amplified during cell division causing new traits, disease, or cancer.

Current microarray technology utilizes several hundred thousand to several million DNA probes on each microarray, allowing the detection of aneuploidies (abnormal number of whole chromosomes, e.g. trisomy 21) and copy number changes of any evaluated DNA locus down to resolution of ~100,000 base pairs (100 kb). Thus, array CGH technique can achieve amazing efficiency of DNA analysis, with one assay equivalent to thousands of FISH experiments.

Another critical concept is the number of genes active in cancer growth. Of the 25,000 distinct genes in the human genome, it is estimated that only 200 to as many as 1,500 genes are important to oncology. At present, researchers understand only about 50 of the 200 + genes responsible for cancer behavior. It can be estimated that a single tumor type may have as few as 20 genes responsible for its behavior, but that number may be as high as 200 genes. Many clinical applications in oncology have emerged where whole panels of these gene expression markers can stratify patients into risk categories. For example, breast cancer management is aided by microarray technology such as MammaPrint where 70 genes that correlate with metastases are analyzed. CombiMatrix of Irvine uses DNA microarrays with up to 20,000 probes designed to target 500 genes and cancer specific loci within various cancers. Based upon changes in DNA copy number, these predictors can guide more aggressive or less aggressive clinical management. For instance, in Acute Myeloid Leukemia (AML), genetic markers help with decisions whether to treat with traditional chemotherapy or proceed straight to bone marrow transplantation. Melanoma risk analysis is also utilizing DNA microarray testing. Encouraging data suggests that MelTUMPS may be soon classified into melanomas or benign nevi based on analysis of DNA copy number variation. Thus microarray technology allows risk stratification of cancers or helps define tumors as benign or malignant thus guiding clinical management. This is primarily accomplished by evaluating copy number variations of tumor DNA. The ease of this analysis and its dropping cost usher in a renaissance in cancer management.

In our next article we will discuss reverse transcriptase polymerase chain reaction (RT-PCR) and gene expression microarrays and their effect on cancer treatment.