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Single-Cell Analysis, Immunosequencing ID Multiple Clonotypes in CLL Patients

NEW YORK (GenomeWeb) – Researchers from the University of Alberta and Cross Cancer Institute in Edmonton, Canada have used single-cell PCR analysis and next-generation immunosequencing to characterize clonal diversity in patients with chronic lymphocytic leukemia.

Publishing their results this week in PLOS One, the researchers reported that around 13 percent of CLL patients analyzed had more than one immunoglobulin heavy chain (IGH) gene rearrangement, which is typically a "unique molecular signature."

Typically, CLL patients are stratified based on their unique IGH gene rearrangements and the mutational status of their immunoglobulin heavy variable (IGHV) gene. Patients with mutations in IGHV, dubbed M-CLL, tend to have a better prognosis than those without, U-CLL.

In the PLOS One study, the researchers looked in more detail at the IGH clones of 198 CLL patients and found that 26 patients had more than one IGH rearrangement, and that the vast majority of those patients (20) were those with U-CLL. For the majority of patients with more than one rearrangement, one clone was dominant, and the patient's disease was classified based on that dominant clone. But, in five patients at least one of the so-called "partner" clones was at a high enough frequency to be considered a second CLL.

Although multiple IGH rearrangements have previously been reported in CLL patients, the authors wrote that it was "unclear whether these derived from distinct/unrelated clones or if two productive rearrangements [arose] in a single B-CLL cell."

In the study, the authors set out to determine the incidence and clonal origin of multiple rearrangements, as well as their persistence throughout the course of disease.

They analyzed 198 patients, including 193 with CLL, four with small lymphocytic leukemia, and one with monoclonal B-cell lymphocytosis. Seventy-nine patients had an unmutated IGHV gene and 119 patients' IGHV gene was mutated.

The team first determined the IGH clones using a real-time PCR technique. Next, for samples that were determined to have more than one IGH clone, they analyzed between 96 and 110 single cells using nested PCR to determine clonal frequency. For samples with just one clone, PCR analysis was done in 16 to 24 cells.

The team then used Adpative Biotechnologies' next-generation ImmunoSeq assay — an NGS assay that amplifies and sequences the CDR3 region of the IGH — to validate the clonal analyses and to further screen for multiple clones. The team ran the assay on 13 patients, seven with more than one clone and six monoclonal patients. The ImmunoSeq assay confirmed the original findings and also found several additional clones.

The researchers evaluated the patients over time and found that "partner clones were consistently detectable for many years, at relatively constant ratios with the primary CLL clone," the authors wrote.

"The relatively frequent presence of partner clones suggests that evaluation of clonal heterogeneity and clonal evolution in CLL would benefit from inclusion of molecular analysis for IGHV [and immunoglobulin heavy diversity and joining gene] signatures to distinguish between intra-clonal and inter-clonal diversity."

In addition, in five patients with at least one partner clone, the partner clone was "sufficiently frequent at some points in time for designation as a second CLL," they wrote. Although the clinical significance of these partner clones is still unknown, the authors wrote that their presence warrants further study. In particular, it may be important to analyze the partner clones for additional driver mutations that could impact disease progression or prognosis.