DELFI approach as ‘pretest’ in early cancer detection

Amy Carpenter Aquino

December 2021—A cost-effective liquid biopsy focused on analyzing genomewide fragmentation profiles in cell-free DNA has been shown in proof-of-concept studies to detect early-stage lung and other cancers. And the goal is to move the needle for widespread adoption and accessibility, says Victor E. Velculescu, MD, PhD, co-director of cancer genetics and epigenetics and associate director for precision medicine, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine.

In a prospective study of 365 individuals at high risk for lung cancer, Dr. Velculescu and colleagues used a machine-learning model to detect tumor-derived cfDNA through genomewide analyses of cfDNA fragmentation patterns. The noninvasive DELFI (DNA evaluation of fragments for early interception) cancer detection model was validated with an independent cohort of 385 non-cancer individuals and 46 lung cancer patients (Mathios D, et al. Nat Commun. 2021;12[1]:5060).

“Between the discovery and validation cohorts, we found that the approach can detect lung cancer through a simple blood test,” Dr. Velculescu, who is also professor of oncology, pathology, and medicine at Johns Hopkins, said in a recent CAP TODAY interview. “It detects it in a high-performance way—in this initial proof-of-concept study—across all stages and subtypes.”

Dr. Velculescu describes it as a simple test: “We take the blood, we purify the DNA from it, we add on adaptors and sequence it. It’s three steps, as opposed to many types of liquid biopsy approaches that have a dozen, two dozen, steps.” He sees it as scalable and able to be performed at labs worldwide and affordable from a public health perspective.

Dr. Velculescu and colleagues examined patient blood samples obtained from 365 individuals at Bispebjerg Hospital in Copenhagen (LUCAS cohort) from September 2012 to March 2013. The majority of the subjects in the cohort were symptomatic individuals at high risk for lung cancer (age 50–80 and smoking history >20 pack–years). The cohort included 323 subjects (90 percent) with pulmonary, non-pulmonary, or constitutional symptoms, with the majority having smoking-related symptoms. The remainder were asymptomatic at enrollment, with an incidental chest image finding by X-ray or CT that was suspicious for lung malignancy.

The study’s authors isolated 2–4 mL of plasma from each patient in the LUCAS cohort and examined the extracted DNA using the DELFI approach. “Overall we detected more than 90 percent of these lung cancers across these different stages and subtypes, which is very encouraging for a test at this stage of development,” Dr. Velculescu says.

He described the DELFI approach and its use for cancer detection across seven cancer types in a 2020 AMP annual meeting virtual session, shortly after its publication in Nature (Cristiano S, et al. Nature. 2019;​570​[7761]:385–389). “This is a methodology that you can utilize broadly in both a high-risk and general population,” he says. The goal is to not only detect the cancer, he says, but also to identify the tissue of origin so individuals “don’t have to go through a complicated diagnostic odyssey.”

Using blood-based fragmentation profiles, the DELFI score—the probability that a person analyzed in this way has cancer—can more accurately identify those likely to have cancer. “The approach should be thought of as a pretest,” he says, used to send someone for imaging or a diagnostic follow-up test to identify the cancer, find out where it is, and determine the next intervention.

In his AMP presentation last year, Dr. Velculescu discussed what he called an earlier “monumental effort” within Johns Hopkins and internationally that led to the discovery that among individuals with breast, ovarian, lung, and colorectal cancers, a majority could be identified as having detectable alterations in the blood (Phallen J, et al. Sci Transl Med. 2017;9[403]:eaan2415).

In the study, the plasma of 44 healthy individuals and of 194 individuals with breast, colorectal, lung, or ovarian cancer provided “the first systematic analysis of sequence alterations in cell-free DNA for direct detection of early-stage tumors,” Dr. Velculescu said. The alterations were detectable at different stages, and the levels of circulating tumor DNA typically increased across different stages, with lower levels at earlier stages and higher levels at later stages.

Even multiple mutations in the blood could potentially be identified when looking at a targeted panel, sequencing deeply, and trying to identify cancer-associated alterations in the circulation, he said. “Although this study served as a precursor for our early detection efforts and was the first one to do it in this way, it had a number of weaknesses and highlights the kind of difficulties one has in utilizing mutations for early detection.”

The first is that these mutations typically make up only a small fraction of the circulating cell-free tumor DNA that’s present, Dr. Velculescu said. “In part, that’s because not all molecules have mutations and not all molecules that are tumor derived are even assessed. And these are orders of magnitude different in the circulation” (Cristiano S, et al. Nature. 2019;570​[7761]:385–389).

With a small fraction of molecules evaluated, only a few mutations will be detected in a panel of any reasonable size, “whether it’s a few genes that are highly mutated in cancer or even when looking at the top 50 or 100 genes that are mutated in cancer. One typically just gets a handful of mutations.”

The effect is that the limit of detection, simply because of the number of cfDNA molecules present, will be limited by the number of types of observations that can be obtained. “That may be inadequate if you have a low number of mutations,” he said, adding that the obvious goal would be to increase the number of potentially observed changes to increase the limit of detection.

There’s also a confounding difficulty in which mutations occur not only in cfDNA but also in white blood cells. An analysis of samples from patients in the CRITICS trial showed that in patients with gastric cancer, mutations identified in cfDNA came from both white blood cells and cancer. “Interestingly, those that were present in both typically were at similar levels,” Dr. Velculescu said, suggesting there is a population of WBCs that is rapidly turning over and releasing these types of alterations. However, “there’s also a population in the cell-free DNA that is not at all present in the white blood cells and more likely the tumor-derived alterations” (Leal A, et al. Nat Commun. 2020;11[1]:525).

“One can see from this type of effort that mutations in cell-free DNA can be truly confounded by clonal hematopoiesis, by the kinds of changes that occur in white blood cells,” Dr. Velculescu said. At first glance, then, it may be difficult to discern whether mutations are tumor or WBC derived.

Leal, et al., identified 21 alterations in p53, for example, and all but six alterations occurred in the white blood cells, Dr. Velculescu said. “Many of these occurred in hotspots in p53 and other pathogenically predicted changes, making it almost impossible to distinguish a priori which would have been tumor derived and which would have been derived from white blood cells.”

For these reasons, he said, the Johns Hopkins group and others began considering other types of alterations to evaluate early detection changes in individuals who may have cancer. One possibility is tumor fragmentation.

A number of observers noted cfDNA size in the blood is typically small, Dr. Velculescu said. A natural fragmentation occurs in the process of generating cfDNA and leaves only the protected DNA—around 167 bases—to be ultimately detectable in the blood (Cristiano S, et al. Nature. 2019;570[7761]:385–389).

“It turns out that individuals with cancer have slight shifts in that cell-free DNA,” Dr. Velculescu said. Although the overall difference is not significant enough to help identify individuals with cancer, “it got us thinking as to whether these fragmentation changes could perhaps be useful in another way.”

In looking through a targeted approach at the mutations they observed as altered in human cancer in the blood, they were able to see that in some cases the mutated DNA was smaller while in other cases it was larger, Dr. Velculescu said. “For example, in the case with a PIK3CA mutation, the DNA that includes the mutant molecules—those that are tumor derived—are smaller than those that are wild type.” However, the cfDNA derived from tumors with a CDKN2A mutation were larger than those that were wild type, “suggesting that different regions of the genome might be fragmented in different ways. And that may be a way to take advantage of this difference to identify those individuals with cancer.”

Though mutations can be confounded by clonal hematopoiesis, when they went back and looked at the cfDNA fragments with alterations in p53, all those that were tumor derived were shifted in size, while those that were mutant derived but from white blood cells were unchanged in size. This demonstrated that “fragmentation changes are likely to be tumor specific as opposed to a result of changes occurring during clonal hematopoiesis or other changes in white blood cells,” he said.

In looking at random variants occurring in the genome, whether germline or from other sources but not tumor derived, there is little difference between the mutated and wild-type fragments. “This concept of using fragment sizes, but thinking about them throughout the genome as a profile,” ultimately led to the study published in 2019 of genomewide cfDNA fragmentation in patients with cancer, by Cristiano, et al., and to the development of the DELFI approach, he said.