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HERCULES, Calif.—As the industry searches for a faster way to diagnose and treat blood disorders, life-sciences company Bio-Rad Laboratories Inc. has announced that its droplet digital PCR (ddPCR) technology fits the bill by providing a more sensitive, precise and rapid method of diagnosing and monitoring leukemia than any counterpart.
Scientists attended the American Society of Hematology (ASH) conference in Orlando in December, presenting more than 40 abstracts highlighting research, driven in part Bio-Rad’s ddPCR technology. Some of the work illustrated how a simple blood test, using ddPCR, can predict treatment outcomes for leukemia patients, leading to improving patients' lives by helping their physicians determine the right treatment at the right time and enabling clinicians to monitor biomarkers of malignant and nonmalignant blood disorders cost-effectively and at scale.
Other conference posters demonstrated how ddPCR has been used to monitor patients with hairy cell leukemia (HCL) and chronic myeloid leukemia (CML), particularly in cases when testing must identify low levels of the biomarkers to provide an early indication of whether or not a patient is responding to treatment.
Two researchers from the University of Bologna, Italy—Dr. Pier Luigi Zinzani, head of the Lymphoma Unit and a professor of hematology, and Alessandro Broccoli, research fellow in the Department of Experimental, Diagnostic and Specialty Medicine—had searched for a more sensitive test to determine if patients with HCL were in complete response after cladribine (2CdA) treatment. But after using ddPCR, the Italian researchers demonstrated how patients with active disease display a higher fractional abundance of BRAF V600E ctDNA than patients in complete response.
With ddPCR, Broccoli was able to measure the ctDNA and find residual HCL DNA in the blood following treatment with cladribine (CdbA), indicating the cancer would likely return earlier than expected.
“The study we presented last December at ASH, in fact, indicates that some patients with a long-lasting (> 5 years) complete response after one course of cladribine display no evidence of the BRAF V600E mutation, regarded as the key molecular event in the pathogenesis of the disease in peripheral blood,” Broccoli explains. “On the contrary, patients with active disease (i.e. disease at onset or relapse, meeting the criteria for the initiation of an effective treatment) display a positive assay, and in any case with a fractional abundance of the mutated allele (always higher than 1 percent).”
“Given that HCL displays only a few leukemic circulating cells, even when the disease is active, a very sensitive assay is required to detect even a small fraction of affected (mutated) cells—and ddPCR shows more sensitivity than other quantitative PCR methods,” he adds.
Broccoli argues that with ddPCR, researchers must take steps “to assess comparability between peripheral blood and bone marrow samples in defining a molecular negativity/positivity of the assay (depending on different disease time-points: onset, relapse, first response, long-lasting first response, further complete response), to define a new category of ‘molecular responders’ that may better categorize patients in terms of long-term prognosis and tendency to relapse over time.” They must also “define a threshold for fractional abundance to discriminate between negative results, minimal residual disease with no evidence of active disease and active disease.”
With the goal of evaluating the potential diagnostic value of ddPCR in monitoring tyrosine kinase (TKI)-treated patients with CML, Carmen Fava, assistant professor at the University of Turin, Italy, and her colleagues recently conducted a multi-centric study comparing ddPCR with the standard method of reverse transcription quantitative PCR (RT-qPCR).
Fava found that ddPCR is just as effective as RT-PCR at detecting minimal residual disease in CML under TKI treatment, and that ddPCR is more precise than RT-PCR at low levels of residual disease.
“[The] ddPCR kit’s reproducibility, ability to express results in IS and promising findings in several trials and reports, suggest ddPCR’s potential extends to routine use in clinical settings to determine when treatment can be discontinued,” Fava says.
For TKI-resistant patients with CML, ddPCR enables more timely changes in treatment than NGS Sanger sequencing, a common method used to detect BCR-ABL1 mutations which causes TKI treatment resistance among patients with CML who don't respond well to therapy, Fava explains.
Simona Soverini, assistant professor of the Department of Experimental, Diagnostic and Specialty Medicine of the University of Bologna, and other researchers recently tested next-generation sequencing (NGS) and a novel ddPCR based multiplex assay as potential alternatives to Sanger sequencing for screening a panel of 13 BCR-ABL1 mutations relevant for TKI selection. She found that she could use ddPCR to detect resistance mutations in TKI-resistant CML patients faster than NGS. Within the first 24 hours, ddPCR showed its superiority by yielding its findings in just one day, compared to 15 working days for NGS.