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Proof-of-Principle Study Points to Possibility of Monitoring Pediatric ALL With Plasma Samples

NEW YORK – Blood plasma sequencing appears to offer a noninvasive method for tracking disease clearance or persistence in children with acute lymphoblastic leukemia (ALL), along with the presence or absence of potentially infectious microbes, new research published in Science Advances on Wednesday suggests.

"We believe that, with additional work, we will identify the mutations and microbe dynamics that predict treatment success, as well as morbidity as a result of a given treatment strategy," senior and corresponding author Charles Gawad, a pediatrics, oncology, and computational biology researcher affiliated with Stanford University, St. Jude Children's Research Hospital, and the Chan Zuckerberg Biohub, said in an email

"With this deeper understanding of patient response that can be monitored by clinicians," Gawad added, "we anticipate that this approach will complement existing methods to inform more dynamic, precise, and efficacious treatment strategies."

Using a custom circulating tumor DNA capture and next-generation sequencing panel, the researchers profiled 168 blood plasma and bone marrow samples collected over six weeks from 20 children going through inductive chemotherapy treatment for ALL through the Total Therapy XVII clinical trial. Along with circulating tumor DNA (ctDNA)-based detection of disease persistence or ALL relapse, they showed that such sequencing approaches can be used to pick up microbes that may spur infection in the immune-suppressed pediatric patients.

"[B]eing able to monitor both the disease and patient-infectome interface provides a new vantage point from which we can monitor our patients," Gawad explained, noting that "infectious complications are the leading cause of early death and treatment changes in children with leukemia."

With their custom capture sequencing panel, the investigators identified at least one ctDNA variant in all but three of the pediatric ALL patients profiled at the time of diagnosis. Most of the variants also turned up in corresponding bone marrow samples, though distinct alterations were also detected in the blood and the bone marrow.

By the end of induction therapy treatment, four of the patients had minimal residual disease detectable by conventional flow cytometry-based measurements of persistent cancer cells, the team reported, while other patients appeared MRD-free but continued to carry disease-related variants in the blood.

With their microbe capture, sequencing, and detection pipeline, meanwhile, the investigators searched for potentially risky bacterial, and fungal species within cells and in cell-free DNA, comparing the microbial cell-free DNA profiles with those found in five healthy control individuals and five individuals being treated for acute myeloid leukemia.

Among other microbial patterns, the team found that cell-free microbial DNA in blood plasma samples from the pediatric ALL cases fluctuated over the course of treatment. Even so, many of the same bacterial culprits tended to turn up in the same individuals over time, including dominant microbial species not found in cancer-free control individuals. On the other hand, viral species appeared more prone to change as children experienced immune suppression related to the chemotherapy treatment.

"We have demonstrated that a customized hybrid capture [next-generation sequencing] panel can noninvasively measure the two leading causes of mortality in pediatric patients with ALL: leukemic disease burden and the invasion of infectious microbes," the authors wrote. "This proof of concept helps establish a technical and conceptual framework that we anticipate will be expanded and applied to different populations of patients with leukemias, as well as additional cancers."

The researchers expect to profile more pediatric patients with ALL to more fully characterize the tumor mutation profiles and microbial dynamic features that may track with clinical outcomes, Gawad explained, noting that the panels used to capture tumor and microbial DNA will likely continue to expand as the sequencing costs decline further. In addition, he and his coauthors pointed to the possibility of bringing in additional RNA sequencing data to detect informative structural variants for guiding treatment or management of some cases.

"Molecular monitoring can help accelerate the attainment of insights into the temporal biology of host-microbe-leukemia interactions, including how these changes correlate with and alter anticancer therapy efficacy," the authors reported. "We also anticipate that fewer invasive bone marrow examinations will be required, as these methods improve with standardization and are validated for clinical use."