August 1, 2016 (Vol. 36, No. 14)
Kate Marusina Ph.D.
Clinical-Style Validation, Regulation, and Quality Control Are Spreading To NGS-Based Tests
Drug development in oncology continues to experience high failure rate. We now understand that tumors are heterogeneous and dynamic, and we are seeing the importance of stratifying patients as well as continuously monitoring for resistant mutations. These measures may improve response rates to targeted therapies and their survival benefit.
Next-generation sequencing (NGS) is emerging as a powerful clinical diagnostic tool expediting molecular stratification, and it is enhancing our knowledge of tumor clonal changes. This article presents feasibility of NGS in the context of targeted therapeutic approaches to cancer.
“We are entering a promising time in cancer treatment, when the accumulated experience in cancer genome sequencing can be put to practical use in clinical practice,” says Shashikant Kulkarni, Ph.D., a professor of molecular and human genetics at Baylor College of Medicine, and a senior officer at Baylor Miraca Genetics Laboratories. “In just five years, NGS went from an experimental laboratory technology to clinically validated diagnostic tool.”
Dr. Kulkarni points to the recent guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing (Nex-StoCT) workgroup. Combined with more recent guidelines for bioinformatics, these important principles ensure that results from tests based on NGS are reliable and useful for clinical decisions.
The workgroup was convened by the Centers for Disease Control (CDC), which expanded its genetic reference testing program to include reference standards for NGS characterization. Complementary efforts are being pursued by the National Institutes of Health and the National Institute for Standards and Technology (NIST).
For example, NIST’s Genome in the Bottle team recently released the first DNA reference materials to gauge the performance of whole-genome and targeted panel sequencing tests. The development of these reference materials assists the FDA’s efforts to design a regulatory framework for NGS-based tests.
The Nex-StoCT workgroup has proposed a three-stage validation process: platform, test-specific, and informatics pipeline validation. Such a process, notes Dr. Kulkarni, could increase confidence in the “critical knowledge gained via NGS.”
Shaping Cancer Clonal Evolution
Dr. Kulkarni’s research is dedicated to the study of cancer clonal evolution and the analysis of the genetic changes that accumulate in the founder and side line clones. Dr. Kulkarni underscores critical knowledge gained through understanding of clonal selection and progression. Clonal evolution often leads to relapse of cancers such as acute myeloid leukemia (AML).
NGS data suggests that initial clones escape chemotherapy and acquire additional DNA transversions likely due to DNA damage cause by chemotherapy itself. “If clonal evolution is shaped by chemotherapy,” emphasizes Dr. Kulkarni, “we should be focusing our efforts to enlist targeted therapy whenever possible.”
Progress in personalized therapy, however, is still stymied by several major challenges. Correlation between mutations and phenotype is still poorly understood. ClinGen, a National Institutes of Health (NIH)-funded resource, is currently building an authoritative genomic knowledge database that defines the clinical relevance of genes and variants for use in cancer precision medicine, but these efforts are still in the early stages.
Other challenges include lack of insurance reimbursement for NGS diagnostics, low awareness among clinicians, and a slow process of bringing targeted therapies to the market. “Change is slow,” complains Dr. Kulkarni. “But I am fortunate to be involved in changing the cancer treatment paradigm, one DNA base at a time. My dream is to democratize access to precision medicine to all cancer patients.”
Targeting Actionable Mutations
The Center for Personalized Diagnostics (CPD) at the University of Pennsylvania aims to uncover actionable genetic mutations in individual cancers to allow more targeted and personalized treatment strategies. The Center’s core technology is NGS. The Center’s clinical director, Jennifer Morrissette, Ph.D., notes that the volume of analyses steadily grew from 50 cases per month at inception to reach the current volume of 250 sam>ples per month. The team zeroed in on a select panel of genes to create custom hematologic (68 genes) and solid tumor (47 genes) panels. “While our panels are not extensive, they are focused on targetable or prognostic genes,” adds Dr. Morrissette.
Two case studies highlight the power of personalized therapies enabled by NGS. Both concern intrahepatic cholangiocarcinoma (ICC), which is known to be highly genetically diverse, with multiple translocations and mutations affecting over half of the genome.
A stage IV patient of Arturo Loaiza-Bonilla, M.D., an assistant professor of clinical medicine at Penn, had undergone multiple rounds of standard-of-care therapies, only to develop new active metastasis. Genomic analysis reported a high-frequency mutation in the BRAF gene (pV600E), and the patient was deemed eligible for targeted BRAF/MEK inhibition therapy.
The patient showed an extraordinary response, resolving all previous liver metastases. Dr. Morrissette mentions that the same mutation was since found in several other ICC patients, suggesting that it may be worth paying particular attention to V600E mutation in ICC. Of all cancers, ICC is especially genetically variable, and finding a targetable, if rare, mutation offers a promise to those patients who have it.
In the second case, a multidisciplinary approach was particularly effective in determining the course of personalized therapy. The Abramson Cancer Center tumor board at Penn uses the genotype-to-phenotype concept to select targeted therapies or recommend a pertinent clinical trial to the patients. Martin Carroll’s group, using information from the CPD has been validating an Integrated Genetic Prognostic (IGP) model that combines cytogenetics with mutations in nine genes to predict a treatment course for younger patients with acute myeloid leukemia.
“Being able to predict the course of disease informs clinicians about urgency of actions, for instance, to use targeted therapy, seek an immediate transplant, or to pursue palliative care,” explains Dr. Morrissette. “Our goal is to validate this model in a larger patient cohort.” The Center pursues other synergistic approaches, such as a combination of NGS and targeted single-gene testing to create an optimized and robust analysis workflow.
Finding Genomic Rearrangements
“Genomic rearrangements represent a significant proportion of genetic abnormalities in cancer cells,” says George Vasmatzis, Ph.D., director of the Biomarker Discovery Program, Mayo Clinic. “Our program focused on this space early on and achieved significant advances in both sample preparation and informatics analysis.”
Dr. Vasmatzis brought together a unique combination of technologies to identify large genomic rearrangements and translocations in liquid and solid tumors. The process begins with laser capture microdissection (LCM) to efficiently and accurately extract pure populations of tumor cells. The cells are applied directly into whole-genome amplification protocol, followed by mate-pair (MP) sequencing on Illumina instrumentation.
In MP sequencing, the library preparation yields short inserts containing two adjacent fragments that were previously separated by 2–5 kB from each other in the genome. Combining data generated from MP library sequencing with that from short-insert paired-end reads provides a powerful ability to identify complex genomic rearrangements.
“The MP approach is a rapid and economic way of sequencing the whole genome for such abnormalities,” asserts Dr. Vasmatzis. “Our Center has already completed over 1,500 tumor genomes.”
Later this year, the Mayo Clinic plans to offer this test at their CLIA-certified lab. At first, it will support constitutional genetic analysis. Eventually, it will substitute for fluorescence in situ hybridization (FISH) and cytogenetic tests in the analysis of hematologic malignancies and solid tumors.
Dr. Vasmatzis describes a typical clinical scenario where this technology may provide valuable information for clinicians: “It is critical to determine whether two distant nodules are genetically related. If they are related, it may indicate an aggressive late-stage tumor, but if they are not related, the disease may still be at stage I, making patient eligible for a potentially curable surgery.”
Using just a few cells from the fine needle biopsy aspirations, the team is able to detect somatic translocations that are unique for each tumor clone. An added benefit is potential discovery of targetable rearrangements.
This protocol may find a wider application in a nationwide screening of individuals with increased cancer risk, such as smokers. “It is still somewhat unclear how the trough of information that our test generates could be presented to physicians,” allows Dr. Vasmatzis. “We have been working on visualization strategies to make this complex information more readily understood and actionable.”
Fluid Cancer Monitoring
Most patients with metastatic prostate cancer respond well to androgen deprivation therapy. However, many relapse and develop castration-resistant prostate cancer (CRPC).
“Novel therapies targeting the androgen receptor (AR) demonstrate significant survival benefit,” says Delila Gasi-Tandefelt, Ph.D., Marie Curie Research Fellow, the Institute of Cancer Research (London). “However, 30% of patients do not respond at all, and among those who respond to therapy, invariably all develop resistance.”
The Institute’s laboratory studies the origins and progression of treatment resistance. Molecular characterization of tumors taking serial biopsies could be impractical considering that most of CRPC metastasis develops in bones. In addition, heterogeneity of tumors renders comprehensive sampling rather challenging.
Liquid biopsies present a minimally invasive way for longitudinal tracking of tumor molecular makeup. As part of the Institute’s treatment resistance team, Dr. Gasi-Tandefelt evaluated the liquid biopsy approach by conducting a comprehensive comparison of cancer tissues and circulating cell-free DNA from the same patients. She showed that tumor DNA found in blood is representative of the entire tumor burden.
By following mutations in anchor genes, the team characterized the clonality of metastatic disease. The analysis suggested emergence of distinct mechanisms of resistance in different clones that arose or disappeared under treatment selection pressure. Sequential monitoring by liquid biopsies may ensure early discontinuation of therapies that drive resistance.
“Our future research will further focus on evaluation of clonal response to targeted drug therapies,” informs Dr. Gasi-Tandefelt. “Tumors are very dynamic, especially under selective pressure. Thus, stratifying patients on the basis of past tumor biopsies is problematic.”
NGS analysis of genetic AR aberrations in CRPC patients, before and after the start of abiraterone treatment, suggested the important role of somatic mutations and AR gain in resistance. The point mutations could be observed months before any clinical manifestation. This data is at the heart of a prospective clinical trial led by Gerhardt Attard, M.D., Ph.D., a senior researcher at the institute.
At present, it appears that NGS could inform on the choice of treatment based on the type of the genomic aberration. The next frontier, Dr. Gasi-Tandefelt predicts, will be expansion from the targeted gene panels to whole-genome/whole-exome sequencing, garnering even more information for intelligent cancer treatments and single-cell analysis to enable detection of rare resistant clones.
Strengthening Quality Control
“NGS is revolutionizing our approach to cancer therapeutics,” says Francine de Abreu, Ph.D., a genomic analyst at Dartmouth College. “It is essential for CLIA-certified laboratories to implement quality control (QC) programs to ensure accuracy and reproducibility of sequencing results.
“At present, every CLIA laboratory has its own QC practices. We have established a comprehensive six-step QC process that ensures accurate sequencing results using formalin-fixed paraffin embedded (FFPE) tissues.”
The workflow is based on the College of American Pathologists (CAP) framework developed over the past few years. It conceptualizes the overall NGS test process as composed of two major analytical components: a “wet bench” component and a “dry bench,” or bioinformatics component.
The wet bench includes pre- and post-analytical checkpoints: DNA extraction, DNA quality, library preparation, and quantification. The dry bench includes bioinformatics steps: post-sequencing run, sample, and variant metrics. For each run, the metrics verified are as follows: chip loading, usable sequences, polyclonality, and low-quality reads. Additional values that may be assessed for each individual sample include coverage uniformity and on-target reads.
The complete NGS process can be monitored step by step. Dr. de Abreu explains that in addition to the mandatory routine testing of blinded CAP samples, many CLIA laboratories also agree to participate in additional proficiency testing through inter-laboratory sample exchanges. For its part, the Dartmouth-Hitchcock Medical Center has organized a Next-Generation Sequencing Project Team. It reviews and updates the accreditation checklist requirements specific to NGS to adapt them to meet the rapid evolution of NGS and its translation to clinical diagnostic testing.
The Center’s laboratory for Clinical Genomics and Advanced Technology (CGAT) analyzed over 1,700 FFPE tumor tissues composed of multiple tumor types, with about 80% passing the “wet bench” QC checkpoints. A large percentage of identified somatic mutations were actionable.
The Dartmouth-Hitchcock Medical Center established the Multidisciplinary Molecular Tumor Board to evaluate potential treatments for the actionable cases identified by the sequencing laboratory. In over 50% cases, the board was able to recommend therapy with a targeted agent. In a few patients treated with the recommended therapy, disease outcomes were positive, suggesting that increasing the awareness among patients and clinicians of the benefits of molecular testing could improve patient care.
Quantifying Cancer Biomarkers
The inherent sensitivity, rapid time to results, and cost effectiveness of droplet digital PCR (ddPCR) in the detection and quantification of DNA biomarkers identified by next-generation sequencing (NGS) makes ddPCR an attractive complement to NGS in the study
and monitoring of cancer.
One example of NGS and ddPCR working in tandem comes out of the laboratory of Lao Saal, M.D., Ph.D., assistant professor and head of the Translational Oncogenomics Unit at Lund University in Sweden. His team is studying whether levels of cell-free circulating tumor DNA, serially monitored from patients’ blood, can predict metastasis in early-stage cancer. His use of a liquid biopsy is less invasive than traditional, solid tumor biopsies, facilitating periodic measurement.
Using low-coverage whole-genome NGS, Dr. Saal’s approach starts with identifying chromosomal rearrangements in patients’ primary tumors, which occur early in tumor development and are often shared among the tumor’s subclones. Although these rearrangements generally do not drive the cancer’s growth, they do serve as biomarkers for measuring tumor burden and are widely applicable to different cancer types.
Dr. Saal’s team then uses
Bio-Rad’s QX200 ddPCR system to monitor the quantity of the rearrangements in patients’ plasma samples using patient-specific ddPCR assays. With this method, the team was able to identify breast cancer recurrence in 86% of patients an average of 11 months (but as much as 3 years) prior to conventional clinical techniques.
Dr. Saal’s laboratory also uncovered that biomarker levels are a predictor of metastasis and poor survival. Other research laboratories using similar methods have also illustrated the utility of digital PCR in liquid biopsy for monitoring disease load and treatment response in colorectal, rectal, gynecologic, and lung cancers.
Dr. Saal is now commercializing this technique by forming SAGA Diagnostics, which offers liquid biopsy and companion diagnostic testing to healthcare organizations, biopharmaceutical companies, and academic institutions.
Detecting CNVs in Clinical Samples
Several techniques have been employed to determine copy number, including array comparative genomic hybridization (aCGH) and quantitative PCR (qPCR).
However, none of these methods are amenable to high-throughput, directed copy number variation (CNV) detection, according to a research team from the City of Hope National Medical Center and Archer DX. In a poster on detecting CNVs in clinical samples, Haimes et al. described the development of a directed next-generation sequencing (NGS)-based method to rapidly and quantitatively measure the copy number of tens and potentially hundreds of genes simultaneously.
“This complete workflow, found in the Archer™ Universal DNA Kit, is powered by Anchored Multiplex PCR (AMP™) chemistry and processes dozens of samples in about six hours,” explained Josh Stahl, CSO and general manager. “By ligating a molecular barcode to randomly fragmented input DNA and then using AMP to simultaneously enrich for several regions of each target gene, we can accurately measure the relative copy number of each target gene in test samples by counting unique molecular barcodes associated with each target region.”
The scientists validated their methodology with a 25-gene panel on a subset of NCI-60 cell lines by comparing their copy number measurements to those determined by both aCGH and qPCR. Results from both orthogonal methods strongly correlated with data from the team’s NGS-based method.
“We multiplexed hundreds of samples on a single MiSeq® run and detected CNVs, both amplifications and deletions, of 2× magnitudes (and often lower) at extremely high confidence, indicating that this panel is amenable to highly multiplexed screens of potentially hundreds of samples,” added Stahl.
“Furthermore, we demonstrated that our NGS-based CNV detection workflow and analysis is compatible with DNA extracted from formalin-fixed, paraffin-embedded (FFPE) samples, suggesting that this system could be adapted for use in clinical applications.”
Determining Allelic Frequencies of Human Cancers
Earlier this year, a team of scientists from Directed Genomics and New England Biolabs® (NEB®) presented a poster entitled “Application of target enrichment combined with unique molecular identifiers to determine allelic frequencies of human cancers” at the AACR conference in New Orleans. The study focused on the use of NEBNext Direct™, a target-enrichment technique developed for hybridization-based capture of genomic regions of interest.
When this method is used, target genomic DNA sequences are isolated and converted into an Illumina-compatible library within seven hours. Unlike some alternative approaches that convert the entire genome into a library and select the targets as a final step, NEBNext Direct target-enrichment enzymatically removes off-target sequences and converts only target regions of the native DNA molecules into sequencer-ready libraries, according to Andrew Barry, target enrichment product marketing manager, NEB.
Target-enrichment and library-preparation strategies for next-generation sequencing typically utilize PCR amplification steps that introduce substantial bias across amplicons. They can also lead to the creation of duplicate sequence reads, which in turn affects the quantification accuracy of somatic allele frequencies, an important factor in understanding tumor progression.
Unique molecular identifiers (UMIs) are molecular tags that label each molecule prior to library amplification with a 12-basepair randomized sequence incorporated into the the library adaptor. The UMI, notes Barry, allows sequence reads to be disambiguated as PCR duplicates, enabling an accurate assessment of variant allele frequencies and molecular copy number of the original sample.
“In this study, we enriched control samples of known variant allele frequencies representing challenging sample types with the NEBNext Direct Cancer HotSpot Panel, and we used the incorporated UMIs to determine allelic frequencies of select cancer targets,” explains Barry. “This novel approach to target enrichment in conjunction with library preparation and the inclusion of UMIs enables filtering of PCR duplicate reads, offering improved sensitivity and more accurate assessment of variant allele frequencies.”