The most common method for large-scale analysis of RNA expression in use in research laboratories has been based on DNA microarrays, but newer methods involving RNA sequencing that offer a more comprehensive and quantitative assessment of RNA expression are beginning to supplant older methods. However, RNA is prone to degradation and is a more difficult analyte to work with than DNA in clinical practice. Furthermore, DNA sequencing is technically simpler than RNA sequencing, permitting the development of methods that rely on massively parallel sequencing (so-called “next-generation [NextGen] sequencing”) that can be readily performed on virtually any tissue specimen (Chapter 7). The increases in DNA sequencing capacity and speed that such methods have enabled over the past decade have been breathtaking, and are matched by an equally remarkable decrease in cost. The first reasonably complete draft of the sequence of the human genome, released in 2003, took 12 years of work and cost about $2,700,000,000. The cost of sequencing the whole genome has now decreased to less than $5000 in some commercial laboratories, making the test readily affordable. At present, using NextGen sequencing, the process of whole-genome of individual tumors can be completed in as little as a few weeks, which includes the time required for the extraordinarily complex task of assembling and analyzing the sequencing data.
These advances have enabled the systematic sequencing and cataloging of genomic alterations in various human cancers, an effort sponsored by the National Cancer Institute called The Cancer Genome Atlas (TCGA). The main impact of cancer genome sequencing to date has been in the area of research: identification of new mutations that underlie various cancers; description of the full panoply of genetic lesions that are found in individual cancers; and a greater appreciation of the genetic heterogeneity that exists in individual cancers from area to area. While whole-genome sequencing to manage patients can be performed, most efforts in the clinical realm are focused on developing sequencing methods that permit identification of therapeutically “actionable” genetic lesions in a timely fashion at a reasonable cost. Such approaches seem particularly applicable to tumors, such as lung carcinomas, that are genetically diverse and require a “personalized” approach if targeted therapy is to succeed (
Fig. 6.37 ). Thus, the current trend in molecular diagnostic laboratories is to develop methods that permit several hundred exons of key genes to be sequenced simultaneously at sufficient “depth” (fold coverage of the sequence in question) to reliably detect any mutations that might be present in as few as 5% of tumor cells. A second method that is moving into clinical practice involves the use of DNA arrays to identify changes in DNA copy number, such as amplifications and deletions. Arrays containing probes that span the entire genome at some standard spacing can detect all but the smallest copy number aberrations, providing information that is complementary to that obtained from focused DNA sequencing. Other “omics,” such as proteomics and epigenomics, are currently being used mainly in the realm of clinical research, but with many drugs that target the cancer epigenome moving into the clinic, it can be anticipated that tests directed at assessing the state of the epigenome that predict response to such agents are soon to follow.
The excitement created by the development of new techniques for the global molecular analysis of tumors has led some scientists to predict that the end of histopathology is in sight. However, histopathologic inspection of tumors provides information about important characteristics of cancers, such as anaplasia, invasiveness, and tumor heterogeneity, that cannot be gleaned from DNA sequences. Histopathology coupled with in situ biomarker tests performed http://ebooksmedicine.net
From anatomy... ...to molecular target
Fig. 6.37 Genetic analysis of cancers is being utilized to identify mutations that can be targeted by drugs.