NEW YORK (GenomeWeb) – An incidental finding from a noninvasive prenatal test has prompted at team at the University of Leuven in Belgium to develop a blood test for Hodgkin lymphoma that analyzes circulating cell-free DNA (ccfDNA).
In a proof-of-concept study published in Lancet Haematology this week, the researchers demonstrated that they can identify genomic imbalances found in Hodgkin lymphoma cells in cell-free DNA from the blood of patients diagnosed with early and advanced disease, and monitor these profiles over the course of treatment.
The noninvasive assay, which relies on shotgun next-generation sequencing of ccfDNA, could be useful for identifying patients unlikely to respond to therapy and provide an alternative for monitoring therapy response.
The idea for the study was prompted by a NIPT assay performed at the University Hospitals Leuven that came back with genome-wide copy number abnormalities, a result that was confirmed in a second blood sample from the same woman and led to further testing. Eventually, she was diagnosed with early-stage Hodgkin lymphoma.
To confirm that the woman's abnormal genomic profile was related to the cancer, the researchers studied a biopsy of her tumor that contained malignant cells, so-called Hodgkin/Reed-Sternberg (HRS) cells, by fluorescent in situ hybridization and found that their DNA had some of the same abnormalities as the blood DNA.
This finding suggested that a fraction of the ccfDNA in the blood was derived from HRS cells, a somewhat surprising finding.
According to Peter Vandenberghe, a professor in the Department of Human Genetics at Leuven and the lead author of the study, only a small percentage of cells in a Hodgkin lymphoma biopsy, such as an enlarged lymph node, are HRS cells, which are surrounded by non-malignant cells.
"This poses a difficult technical obstacle when you want to analyze these HRS cells," he said. "They are mixed with other cells, and if you want to analyze what happened in the genome of HRS cells, you have to purify them, which is time-consuming, technically challenging, and difficult."
Because some of the same genomic features of HRS cells could be found in a sequencing-based blood test that is "demanding but not very difficult," he said, it could be a promising alternative to analyzing HRS cells.
In order to confirm their findings, the researchers sequenced ccfDNA from nine additional patients, most of whom were recently diagnosed with Hodgkin lymphoma at their hospital, using an Illumina HiSeq 2500 in fast run mode. Seven of the cases had early-stage disease and two had advanced disease.
In eight patients, they found abnormal DNA profiles. The only negative result came from a patient with early-stage disease, which might be related to the fact that the disease had not spread widely in this patient.
They validated their results on HRS cells from patient biopsies using FISH, looking at a number of genomic regions that were recurrently abnormal in the DNA profiles. They found that "there was a striking overall concordance between genomic imbalances in ccfDNA and altered copy numbers of the corresponding chromosomal regions in nearly all FISH experiments," according to the paper.
In addition, the researchers analyzed blood samples from the patients that were taken several weeks after they were started on chemo- and radiotherapy and found that in most cases, their ccfDNA profiles returned to normal very quickly.
According to Vandenberghe, a blood test for Hodgkin lymphoma-related genomic abnormalities has promise in several clinical areas, in particular for predicting therapy response and for monitoring disease after therapy.
Hodgkin lymphoma accounts for up to 30 percent of all lymphomas, and is the most common type of lymphoma in adolescents and young adults. The disease has a cure rate of around 90 percent with chemotherapy and radiotherapy, but 10 to 15 percent of patients fail first-line therapy. "It would be very helpful if we could identify such cases upfront, so we do not expose them to therapy that is not very likely to work," Vandenberghe said.
A similar fraction of patients is overtreated by first-line therapy, leading to toxicity effects later in life that can include other types of cancer. "If you want to reduce treatment, it's important that we can recognize those patients who will be equally well cured with less intensive therapy," Vandenberghe said. "This is now not possible upfront."
The fact that ccfDNA profiles returned to normal quickly after the onset of therapy suggests that the test could also be used to monitor response to therapy, which is currently done by PET/CT imaging. Imaging "is a very informative technique but it also has some risks in terms of radiation exposure," Vandenberghe said. "This novel approach of sampling blood could also have some potential in this setting."
However, like PET/CT scanning, the blood-based sequencing assay, which relies on counting sequencing reads in different regions of the genome and does not pick up point mutations, is unlikely to detect minimal residual disease because it is not sensitive enough, he said.
Other, more sensitive techniques, such as digital PCR, might be able to detect very small amounts of free DNA derived from HRS cells in the blood, and thus be better suited to look for minimal residual disease.
Going forward, Vandenberghe and his team plan to study their blood test in at least 100 Hodgkin lymphoma patients as part of a clinical trial. The goal is to collect patient blood samples at diagnosis, at the interim evaluation point, and at the end of treatment and to correlate their genomic profiles with their disease and outcome. "We will be able to establish … the sensitivity of the technique to detect the disease at an early or more advanced stage, and whether these profiles correlate to good response to therapy or whether some profiles correspond with less favorable responses to therapy. We also want to look at how rapidly the abnormal profiles disappear upon treatment, and how that is correlated with PET/CT," he said.
In addition, the researchers plan to explore techniques other than shotgun sequencing to analyze ccfDNA, including PCR, digital PCR, and targeted sequencing, which could allow them to look for recurrent Hodgkin lymphoma mutations and measure levels of minimal residual disease.
"One of the strong assets of this technique is that obtaining a blood sample is quite easy, much easier than going back to the lymph node biopsy and having to dissect individual HRS cells from these biopsies," Vandenberghe said. "We can do many cases, because the capacity is there and it's not technically too demanding or laborious, whereas the older procedures are only possible in a research context and will never be able to produce a result in 14 days, or even one week, as this test can do."