Using microfluidics to isolate circulating leukemia cells

Amy Carpenter Aquino

February 2020—Microfluidic assays are being used to isolate circulating leukemia cells and manage minimal residual disease in some patients with acute myeloid leukemia and B-cell/T-cell acute lymphoblastic leukemia.

“There is a lot of popularity in liquid biopsies, but there’s still a lot of work to do,” said Steven A. Soper, PhD, foundation distinguished professor of chemistry, mechanical engineering, and bioengineering at the University of Kansas. Dr. Soper, who also holds a teaching appointment at Ulsan National Institute of Science and Technology in Ulsan, South Korea, was a co-presenter with Sunitha Nagrath, PhD (see story), at the 2019 AMP annual meeting.

Most molecular analyses that depend on liquid biopsy markers will fail if the input into that assay is of low quality, Dr. Soper said, which is why “the isolation is extremely important.” Isolation removes potentially interfering blood cells and at the same time enriches the target cells such as the circulating leukemia cells (CLCs) and even circulating tumor cells (CTCs) for solid tumors.

Microfluidic platforms provide the capability to perform assays or use processing strategies that cannot be performed on the benchtop “and can provide some very elegant results,” Dr. Soper said. “I say this with a heavy ‘but be careful,’” he added. While there are several “elegant” technologies in the microfluidics community for the isolation of CTCs or CLCs, or both, “many of them don’t translate well into the clinic.”

Dr. Soper presented the results of studies using his laboratory’s microfluidic device to detect minimal residual disease in hematologic cancers by isolating CLCs. CLCs differ from CTCs morphologically and biologically, he said, most notably in size. CLCs are comparable in size to WBCs, so filtration-based isolation techniques won’t work.

Another challenge with CLCs, Dr. Soper said, is that the antigens targeted for enriching CLCs can be expressed by normal blood cells. “When we use anti-EpCAM [anti-epithelial cell adhesion molecule] antibodies to isolate CTCs, the purity is high, and the amount of interfering cells is low because of the simple fact that WBCs for the most part don’t express EpCAM.”

In the case of B-cell acute lymphoblastic leukemia, for example, CD19 is used as the enrichment antigen. However, many B cells express CD19 but are not leukemia cells. The inferior biological purity of CLCs compared with CTCs “creates a bit of a conundrum,” Dr. Soper said.

Acute myeloid leukemia (AML) is a heterogeneous disease. “There’s no common marker that’s ubiquitous across all patients in this particular disease state. For example, personalized, patient-specific PCR tests can be used for each particular type of patient to monitor prognostic indicators,” Dr. Soper noted. “Because there is not a common marker expressed across all CLCs in AML, it makes it very difficult to enrich CLCs from patients with AML.”

With AML, “we don’t have one antigen that we can target to cover a large cohort of patients characterized with AML. We have to use a combination.” Dr. Soper’s laboratory’s microfluidic assay targets CD33, CD34, and CD117 antigens and in so doing, “we’re covering about 70 to 90 percent of the population with AML. However, in the case of ALL, CD19 is used exclusively to cover most patients with this disease.”

Since some non-leukemic cells will express CD34, for example, “we need to use aberrant markers to identify the leukemia cells from the normal hematopoietic cells that also express CD34.”

The ability to detect minimal residual disease is analytical method dependent, Dr. Soper said. “For example, multiparameter flow cytometry is typically used to monitor MRD. That’s typically done from bone marrow biopsies or aspirates because the leukemia cell content in the bone marrow is about 100- to 1,000-fold higher than it is in circulation. You need sensitive techniques to enrich those leukemia cells out of blood, and use them as an indicator of relapse from MRD.” And the need for bone marrow aspirates limits the frequency of sampling that can be done. “As such, a patient may relapse much sooner than detected via flow cytometry analysis of a bone marrow sample and, as such, the prognosis for that patient may be poor,” he said.

Dr. Soper shared results of a study in which his laboratory’s microfluidic assay was used to detect MRD in AML patients who had undergone hematopoietic stem cell transplantation (Jackson JM, et al. Analyst. 2016;141[2]:640–651).

The microfluidic assay can be performed on a sample of three milliliters of peripheral blood, Dr. Soper said, and is equipped with three different microchips surface-decorated with antibodies targeting CD33, CD34, or CD117 antigens. “We phenotypically sort these cells out because unique genetic alterations can occur in each one of these phenotypes.” Aberrant markers specific to the patients—CD7 and CD56—are used to identify the CLCs from the isolated fraction of cells.

The biological purity and analytical abilities of the microfluidic assay allow for the capture of sufficient numbers of cells to do molecular profiling of those cells, he said. “We don’t need to do single-cell analysis. We can elute off the chip using a photocleavable linker and then do any molecular analysis on them directly.”

The study results showed that “we were able to detect relapse two months prior to the clinically accepted standard of care,” which was PCR or flow cytometry analysis from a bone marrow biopsy, Dr. Soper said.

“PCR tests fail for 50 percent of AML patients,” he added.

Following treatment, relapse occurs in 15 to 20 percent of children with B-cell acute lymphoblastic leukemia (B-ALL), Dr. Soper said, and only half of those children who relapse are expected to be cured. MRD testing is unsuccessful in about 10 percent of children, Dr. Soper said. “And PCR for MRD is 20 percent unsuccessful” (Conter V, et al. Blood. 2010;115[16]:3206–3214).

In a pilot clinical study of 20 B-ALL patients at Children’s Mercy Hospital, Kansas City, Mo., Dr. Soper and collaborators Keith August, MD, and Maggie Witek, PhD, used the same microfluidic assay but with CD19 as the selection antigen. “It’s the same chip,” Dr. Soper said, retooled with a different antibody to target a different type of cell.

“To identify the leukemia cells from the normal B cells that express CD19, we used a panel of markers, in particular terminal deoxynucleotidyl transferase [TdT], which does untemplated extension of nucleic acids with deoxynucleotides,” Dr. Soper said. “We also used CD34, which is a stem cell marker, and CD10, which is a membrane metalloendopeptidase.”

“In these cases, the leukemia cells are nucleated, but they express TdT, a kind of ubiquitous marker that’s found in leukemia cells from B-ALL patients,” he said. Since TdT is present in the nucleus and cannot be used as an enrichment marker, “we used CD19. Almost all B cells express some amount of CD19.”

Dr. Soper and his collaborators tracked patients during therapy and up to 85 days after the period of consolidation therapy. By looking at the phenotypic distribution of cells for expression of TdT, CD34, or CD10, “we could see phenotypic changes that occurred as a result of chemotherapy.”