IMPROVED LIQUID BIOPSIES continued
When initial ddPCR plasma genotyping does not identify common drivers, or for tumors with a large set of defining mutations, NGS is critical for charting a personalized treatment path. The technique provides a global view of driver mutations as well as additional genomic information about the patient. To confirm NGS results, a more targeted and sensitive assay like ddPCR is often used.
A key benefit for physicians is the serial use of digital PCR to monitor one or more driver mutations throughout treatment to determine response and recurrence since the level of mu- tated DNA found in liquid biopsies has been found to reflect the size of the tumor(s). It is also possible to use NGS to track progress; however, in these cases it is less practical due to cost and longer turnaround time.
In a clinical study conducted by Garcia-Murillas et al., early-stage breast cancer patients were initially genotyped by targeted NGS of DNA from tissue biopsy. Patient-specific ddPCR as- says then identified minimal residual disease and tracked disease progression by analyz- ing the presence of ctDNA in liquid biopsies after conventional treatment (Figure 1).4
The
likelihood of disease recurrence was pre- dicted almost eight months earlier than would have been possible using traditional clinical diagnostics.
Siravegna et al. examined the liquid biopsies of patients with colorectal cancers (CRCs) to deter- mine mutational dynamics of CRCs treated with a therapeutic antibody specific to the EGFR.5
The researchers used ddPCR to investigate the mutational status of seven genes critical in CRC growth and resistance. This study uncovered how the CRC genome adapts to intermittent drug schedules, allowing a drug to which the tumor had previously grown insensitive to again be effective. Such “real-time” monitoring of ctDNA levels for driver and resistance mu- tations will ultimately help point to the best course of treatment for CRC patients.
When resistance mutations cannot be pre- dicted, NGS is needed to give a global view of the genetic landscape and to identify the im- portant new mutations driving cancer growth. The zoomed-in view of ddPCR for detecting and tracking actionable mutations with very high sensitivity complements the zoomed-out view provided by NGS to assess larger numbers of possible driver and resistance mutations.
Monitoring chromosomal rearrangements in
heterogeneous tumors Due to the heterogeneous nature of tumors and their evolving subclones, it is important to find genetic markers present in all cells within the tumor. Lao Saal, Ph.D., assistant professor and head of the Translational Oncogenomics Unit in the Division of Oncology and Pathology at Lund University Cancer Center, describes why chromosomal rearrangements are key markers for monitoring cancer. “Chromosomal rearrangements are often early events in cancer development; thus they are shared across the subclones, and rearrangements are present
in essentially all cancers,” he said. “Therefore, chromosomal rearrangements can be used to monitor all patients, whereas the gene-based cell-free DNA tests won’t be informative in cases that don’t have the typical mutations.”
Chromosomal fusions (also called structural variants) found in tumors can serve as straight- forward biomarkers to identify and monitor cancer progression, whereas any given single nucleotide polymorphism (SNP) or insertion- deletion mutation might not be representative of the whole tumor. NGS enables researchers to cast a wide net to identify chromosomal rearrangements that mark—but do not neces- sarily drive the cancer’s growth—and allow monitoring of potentially all of a tumor’s cells. With tumor-specific rearrangements identified, Saal can convert these patient-specific fusion markers into highly sensitive ddPCR assays for a fast, cost-effective, real-time biomarker assay through liquid biopsy.
Olsson et al. studied early detection of metasta- sis in breast cancer. Metastasis caught too late cannot be effectively treated. By monitoring about a half dozen of these structural variants using liquid biopsies with ddPCR analysis, the researchers were able to detect metastasis as much as three years earlier (with an average of 11 months) than conventional techniques. They also demonstrated that the quantity of ctDNA was predictive of the likelihood of recurrence and death.6
This approach is also being studied by other laboratories and with other cancers. Reinert et al. analyzed ctDNA in patients with CRC follow- ing surgery.7
Colorectal cancers are difficult
to biopsy, which makes their liquid biopsy especially advantageous for serial monitor- ing. Reinert’s team first analyzed the tumor tissue with NGS to identify somatic structural variants specific to each patient. Similar to Olsson’s group, Reinert was able to detect cancer recurrence an average of 10 months in advance of traditional methods by converting these rearrangements into easily monitored ddPCR assays.
Clinical applications of liquid Figure 1 – Droplet Digital PCR enables liquid biopsy for monitoring cancer treatment. AMERICAN LABORATORY 18 MARCH 2016
biopsy and genomic biomarkers Liquid biopsy for solid cancer is beginning to gain traction outside of the research laboratory.
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