Demystifying Liquid Biopsies

Tumor biopsies have long been the standard method for diagnosing, monitoring, and treating cancer and, in the era of precision medicine, molecular analyses of tissue samples can reveal information that helps guide treatment strategies. However, tissue biopsies can be invasive and, depending on the site of the tumors, difficult or impossible to perform. Additionally, repeated surgical biopsies are often an impractical method to track changes in a patient’s disease over time.

Enter circulating tumor DNA (ctDNA) assays: blood-based tests that cancer researchers say can provide a more comprehensive view of a patient’s cancer than a single tissue biopsy. These so-called “liquid biopsies” can allow clinicians to detect tumors early and non-invasively track tumor changes in real time. Non-invasive ctDNA tests are still years away from routine clinical use, though.

The goal of ctDNA research “is to understand whether these liquid biopsies are a suitable proxy for conventional biopsies,” Viktor Adalsteinsson, PhD, a group leader of the Blood Biopsy Team at the Broad Institute in Boston, who is working on blood tests using ctDNA to profile cancer genomes, told ASH Clinical News.

What makes ctDNA a promising biomarker for cancer detection and monitoring, and will “liquid biopsies” be replacing tissue biopsies in the near future? ASH Clinical News spoke with researchers developing and testing ctDNA assays for answers.

Liquid Biopsies 101

Liquid biopsies take advantage of a concept scientists observed more than 100 years ago: Cells in the body turn over and, as they do, their DNA is shed into the bloodstream. This is known as cell-free DNA. Tumor cells also turn over and fragments of their tumor DNA, known as ctDNA, end up in the bloodstream.1 Analyses of ctDNA can reveal much of the same information as tumor biopsies, and collecting a blood sample to measure ctDNA is less invasive and more easily repeatable.

Investigators are now working to understand the biology and refine the measurement of ctDNA, including developing methods to distinguish the small proportion of ctDNA in the blood from the more abundant DNA from non-malignant cells that also circulates in the blood. For the most part, these methods home in on the somatic DNA mutations unique to tumor cells.

“It’s amazing how quickly this field has progressed in the last five years,” said Maximilian Diehn, MD, PhD, an associate professor at Stanford University and a radiation oncologist whose lab is developing ctDNA detection assays for clinical applications. “[We went] from just a few researchers discussing development methods to [having scientific] meetings focused solely on ctDNA and the start of clinical trials to test these assays.”

In the Clinic

In June 2016, the U.S. Food and Drug Administration (FDA) approved the first DNA-based liquid biopsy: the cobas® EGFR Mutation Test v2. The assay is a polymerase chain reaction–based test that detects and sequences the epidermal growth factor receptor (EGFR) gene in patients with metastatic non-small cell lung cancer.2 The test was approved as a companion diagnostic along with two EGFR-targeting therapies (erlotinib and osimertinib).

Liquid biopsies can allow clinicians to detect tumors early and non-invasively track tumor changes in real time.

The FDA has also permitted several other commercial ctDNA tests (developed in Clinical Laboratory Improvement Amendments–certified labs) to accept human samples for diagnostic testing, but, according to the National Cancer Institute (NCI), these tests still need to be rigorously studied to understand limitations, sensitivity, and whether the results can inform clinical decision-making and improve patient care.1

Andrew Spencer, MD, a myeloma researcher and clinician who leads the Malignant Haematology and Stem Cell Transplantation Service at The Alfred Hospital in Melbourne, Australia, began exploring the potential of ctDNA to monitor tumor burden and genetic abnormalities in patients with myeloma after realizing the limits of what a single bone marrow (BM) biopsy could tell him about his patients’ disease.

“All the genetic information of a patient’s myeloma was based on this single biopsy of the BM, even though a scan would show … lesions all over their body,” said Dr. Spencer, who also is a professor of hematology at Monash University in Melbourne. “We hypothesized that the disease wouldn’t be the same in the different myeloma foci throughout the body.”

To test this hypothesis, Dr. Spencer and colleagues conducted a pairwise comparison in 33 patients with relapsed and/or refractory disease and 15 patients with newly diagnosed myeloma.3 They sampled patients’ blood, analyzed their ctDNA, and compared the mutations in four genes known to be mutated in myeloma with those detected in a single tumor BM biopsy. Mutations occurred at a higher frequency in relapsed and/or refractory patients, compared with newly diagnosed patients, suggesting that tumors grow in size and number in advanced disease. As they progress, the tumors also become more spatially and genetically complex.

Reviewing ctDNA samples from seven patients collected throughout the course of their disease, the researchers also found that the mutations changed in type, demonstrating that liquid biopsies can capture the evolution of the patients’ myeloma.3

The Monash University team is now working on a series of hypothesis-generating clinical trials in Australia and New Zealand to analyze ctDNA and mutational changes related to therapy and tumor progression in patients with myeloma. Dr. Spencer hopes the results will inform how to best use this technology in the clinic to help care for these patients.

Dr. Spencer also predicts that a ctDNA detection assay could assess if there are any remaining myeloma cells after treatment, or minimal residual disease (MRD), which is typically measured by quantifying certain serum biomarkers or taking an additional, invasive BM biopsy. However, neither technique is highly sensitive and each can produce false-negative results.4

Tracking Resistance

ctDNA-based liquid biopsies could be useful for monitoring patients’ response to therapy during and after treatment and could allow clinicians to adjust treatment strategy in real time.

In solid tumors, researchers have previously shown that tracking blood-borne tumor DNA during treatment can detect whether a patient’s cancer is responding to a specific drug and whether tumor clones have developed resistance mutations to targeted agents.5,6 Investigators are now attempting to extend the use of ctDNA-based blood assays to hematologic malignancies.7-9

Dr. Diehn and Ash Alizadeh, MD, PhD, an associate professor at the Stanford Comprehensive Cancer Center, evaluated CAPP-Seq (Cancer Personalized Profiling by Deep Sequencing), a liquid biopsy technique that sequences for more than 200 lymphoma-relevant genes, in 92 patients with diffuse large B-cell lymphoma (DLBCL).10

According to their findings, CAPP-Seq independently predicted patient outcomes, allowed researchers to classify tumor subtypes, and identified treatment-related emergence of resistance mutations. In addition, “by simultaneously tracking multiple somatic mutations in ctDNA, our approach outperformed immunoglobulin sequencing and radiographic imaging for the detection of MRD,” the authors reported. When they validated the CAPP-Seq method in a first-in-human pilot study, the researchers also showed that they could detect resistance mutations in the blood of patients treated with the Bruton tyrosine kinase inhibitor ibrutinib before patients displayed clinical signs of resistance.

“DLBCL has a high level of ctDNA compared with the average carcinoma, so it is ideally suited for ctDNA studies,” Dr. Diehn told ASH Clinical News. The researchers also found that unlike their prior analysis of ctDNA in lung cancer, CAPP-Seq for DLBCL could track approximately 10 times more mutations, boosting the assay’s sensitivity 10-fold.

Since 2016, the team has followed up their study with international collaborations that show CAPP-Seq could be used to risk-stratify patients with DLBCL and to detect, after a single cycle of therapy, whether a patient is responding well to a curative-intent therapy.11,12

Other scientists, including Dr. Adalsteinsson, are taking a more holistic approach by sequencing all of the ctDNA rather than focusing on specific genes.13 While whole-exome sequencing of ctDNA is too costly for clinical applications, it can define every alteration present in ctDNA – offering the opportunity to identify and discover new targetable mutations.

Too Early for Early Detection?

The holy grail of liquid biopsy research is developing a test that can detect tumors before they cause symptoms. With this type of test, people could be routinely screened to identify their risks of developing cancer.

Another team of researchers at Johns Hopkins University, led by Nickolas Papadopoulos, PhD, appears to have made progress in this area, according to a study published in February 2018 in Science.14 The study examined 1,005 patients whose tumors had not yet metastasized. Focusing on mutations within just 16 genes that are often mutated in cancer – and adding an analysis of eight protein biomarkers known to be associated with specific types of cancer – the test detected 33 to 98 percent of cases, depending on the tumor type. The test also appeared to provide reliable results, with sensitivity levels of 69 to 98 percent for tumors for which there is no approved screening test, including ovarian, liver, and pancreatic cancers.

Still, further research and clinical trials are needed to determine whether performing a blood test in healthy individuals for some or all types of cancer is practical, and whether early intervention changes the natural history of the disease.

The Limitations of Liquid Biopsies

According to scientists who spoke with ASH Clinical News, there are as many limitations to ctDNA-based liquid biopsies as there are potential clinical applications. For example, most cancer types lack well-established biomarkers for use in these types of assays, and specific DNA mutations can vary among patients with the same disease.

“The question for ctDNA assays is whether an early detection would be sensitive enough and clinically relevant,” said Lynn Sorbara, PhD, a pathologist at NCI’s Division of Cancer Prevention. The NCI is supporting liquid biopsies by partnering with the National Institute of Standards and Technology to develop standardization and quality-control tools that research labs can use when assaying ctDNA, according to Sudhir Srivastava, PhD, MPH, chief of the NCI’s Cancer Biomarkers Research Group.

He added, though, that two major, partially unanswered questions about ctDNA remain: Is a comprehensive capture of ctDNA representative of the heterogeneity of all the patient’s tumors? And are there factors that can change the ctDNA after it is shed from tumor cells?

Researchers are optimistic that prospective clinical trials will soon answer these questions, including how ctDNA testing can change diagnosis and treatment.

“We envision the ctDNA tools being developed now will be applied to every aspect of cancer patient care, from early detection to care of patients with advanced-stage disease,” said Dr. Diehn.

Dr. Alizadeh agreed, adding, “studying and treating lymphoma for many years, I think ctDNA will be a transformative tool. For hematologists treating hematologic malignancies, it’s a particularly unique opportunity because the blood is our analyte.”—By Anna Azvolinsky


References

  1. National Cancer Institute. “Liquid Biopsy: Using DNA in Blood to Detect, Track, and Treat Cancer.” Accessed March 15, 2018, from https://www.cancer.gov/news-events/cancer-currents-blog/2017/liquid-biopsy-detects-treats-cancer.
  2. S. Food and Drug Administration. “cobas EGFR Mutation Test v2.” Accessed March 15, 2018, from https://www.fda.gov/drugs/informationondrugs/approveddrugs/ucm504540.htm.
  3. Mithraprabhu S, Khong T, Ramachandran M et al. Circulating tumour DNA analysis demonstrates spatial mutational heterogeneity that coincides with disease relapse in myeloma. Leukemia. 2017;31:1695-705.
  4. Flores-Montero J, Sanoja-Flores L, Paiva B, et al. Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma. Leukemia. 2017;31:2094-103.
  5. Diehl F, Li M, Dressman D, et al. Detection and quantification of mutations in the plasma of patients with colorectal tumors. Proc Natl Acad Sci. 2005;102:16368-73.
  6. Dawson SJ, Tsui DW, Murtaza M, et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med. 2013;368:1199-209.
  7. Diaz LA, Williams RT, Kinde I, et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature. 2012;486:537-40.
  8. Russo M, Siravegna G, Blaszkowsky LS, et al. Tumor heterogeneity and lesion-specific response to targeted therapy in colorectal cancer. Cancer Discov. 2016;6:147-53.
  9. Murtaza M, Dawson SJ, Tsui DW, et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature. 2013;497:108-12.
  10. Scherer F, Kurtz DM, Newman AM, et al. Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA. Sci Trans Med. 2016;8:364ra155.
  11. Kurtz D, Jin M, Soo J, et al. Circulating tumor DNA is a reliable measure of tumor burden at diagnosis of diffuse large B cell lymphoma: an international reproducibility study. Abstract #310. Presented at the 2017 ASH Annual Meeting, December 10, 2017; Atlanta, Georgia.
  12. Kurtz D, Scherer F, Jin M, et al. Development of a dynamic model for personalized risk assessment in large B-cell lymphoma. Abstract #826. Presented at the 2017 ASH Annual Meeting, December 11, 2017; Atlanta, Georgia.
  13. Adalsteinsson V, Ha G, Freeman SS, et al. Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors. Nat Commun. 2017;8:1324.
  14. Cohen JD, Li L, Thoburn C, et al. Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science. 2018;359:926-30.

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