The sensitivity and precision afforded by RNA-Seq makes it an ideal technology to characterize the gene-expression signatures of circulating tumor cells (CTCs). “CTCs are often used for early diagnosis and monitoring of responses to cancer therapies,” notes Abizar Lakdawalla, Ph.D., marketing manager, new technologies, Illumina.
“The gene-expression profile is usually unique to the cell’s lineage (where the CTC has originated). It clearly shows the level of heterogeneity in a patient if multiple CTCs are isolated and sequenced from that individual’s blood. Further, RNA sequencing shows changes in RNA structure (different isoforms formed by alternative splicing, novel RNAs produced by translocation or other structural changes in the genome) that often correlate with the origin and progression of a disease.”
Dr. Lakdawalla says that RNA sequencing of individual CTCs also can be extremely useful for detecting the generation of drug-resistant clones from blood for individuals undergoing therapy. “This allows for the optimization of the therapy regimen with the type of lesions prevalent in new CTC clones.”
In order to perform single-cell sequencing, a number of modifications in the procedure needed to be optimized. Dr. Lakdawalla notes, “RNA sequencing of individual cells is based on three steps. First, individual CTCs are isolated from blood by immunolabeling them with cell-specific markers and isolating them through magnetic affinity capture.
“The mRNA in the single cell is converted to cDNA from which a sequencing library is derived with a modified Illumina library prep method. This is quantitated and sequenced on a sequencer with paired 50 or 75 bp reads.”
There are a number of potential applications for RNA-Seq of individual cells, according to Dr. Lakdawalla. “The method is applicable to samples where there is a limited amount of biological materials. Molecular phenotyping of CTCs from blood, single-cell capture, and RNA sequencing is extremely useful for the isolation of CTCs or other cells from breast exudates (breast cancer), urine (kidney-related diseases), and feces (colorectal cancers), etc.
“The high sensitivity and specificity of this method expands the range of samples that can be analyzed. For example, cells isolated by laser capture from frozen or FFPE tissues sections also can be sequenced.”
Dr. Lakdawalla also reports that single-cell RNA sequencing is highly useful in developmental biology (to track spatial and temporal gene-expression changes in individual cells in a developing embryo), to create a three-dimensional gene-expression map of an organ at millimeter resolution, and for evaluating clonal heterogeneity in production cell lines, for example.
Translation to Clinic
Utilizing targeted sequencing to identify cancer biomarkers may soon translate into better clinical patient care. “For biomarker discovery, one wants the most comprehensive way to analyze the genome,” says Olivier Harismendy, Ph.D., assistant professor of pediatrics at the Moores Cancer Center, University of California, San Diego.
“The sequencing of all exons offers great opportunities to discover cancer somatic mutations and DNA-based biomarkers that can translate into new therapies. This is a very powerful way to discover new tumor suppressor genes and oncogenes.”
There are, however, significant drawbacks to such a broad approach, which hinders its wide adoption for clinical care. First, the clinical significance of the vast majority of identified mutations is still unclear. Then, the quality of the samples collected in the clinic is suboptimal due to cellular heterogeneity.
Heterogeneity arises in several ways: inclusion of normal cells during cancer resection or biopsy and also from the presence of several subclones in the tumors themselves. In both cases it impairs the ability to detect the somatic mutations. Dr. Harismendy and colleagues have developed a streamlined approach for massively parallel sequencing of cancer mutational hotspots in heterogeneous samples.
“We devised a novel ultra-deep targeted sequencing (UDT-Seq) assay that enhances laboratory workflow and mutation detection. The idea is that targeted sequencing assesses all clinically actionable genes and allows for high sequencing coverage depth. As a result there is a much more sensitive analysis of heterogeneous clinical samples, and that enhances its clinical utility.”
The UDT-Seq is a direct sequencing assay with ~200-nucleotide long PCR amplicons generated via multiplexing utilizing microdroplet PCR.
“We initially focused on 71 kilobases of mutational hotspots in 42 cancer genes. We use chimeric primer pairs with both locus-specific and adapter sequences to generate PCR amplicons. These were directly sequenced on the Illumina Genome Analyzer II platform. This process simplifies the workflow because it removes the time-consuming and error-prone step of sample fragmentation and library preparation.”
Dr. Harismendy and team are now engaged in a pilot clinical study that uses an updated version of this assay using a faster and more accurate DNA sequencer (MiSeq from Illumina), for evaluation of 47 genes in 40 patients with breast cancer.
“The goal of the clinical trial is to improve clinical care. Somatic mutations in the tumor DNA will inform us if particular patients are eligible for a targeted treatment. Pharmacogenomic markers in the patient’s germline DNA can help avoid adverse effects of some therapies.
“Finally, some inherited DNA variants will be helpful for prognosis but could also help the patients’ relatives that could be at increased risk for cancer. We will report the validated results back to the patient’s physician. As a result we hope that some patients may be eligible to enroll in the latest targeted therapies clinical trials for breast cancer.”