Breakpoint-specific multiplex polymerase chain reaction allows the detection of IKZF1 intragenic deletions and minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia

Abstract

Deletion of the Ikaros (IKZF1) gene is an oncogenic lesion frequently associated with BCR-ABL1-positive acute lymphoblastic leukemias. It is also found in a fraction of BCR-ABL1-negative B-cell precursor acute lymphoblastic leukemias, and early studies showed it was associated with a higher risk of relapse. Therefore, screening tools are needed for evaluation in treatment protocols and possible inclusion in risk stratification. Besides monosomy 7 and large 7p abnormalities encompassing IKZF1, most IKZF1 alterations are short, intragenic deletions. Based on cohorts of patients, we mapped the microdeletion breakpoints and developed a breakpoint-specific fluorescent multiplex polymerase chain reaction that allows detection of recurrent intragenic deletions. This sensitive test could also detect IKZF1 subclonal deletions, whose prognostic significance should be evaluated. Moreover, we show that consensus breakpoint sequences can be used as clonal markers to monitor minimal residual disease. This paper could be useful for translational studies and in clinical management of BCP-ALL.

Figure 1.

(A) Array-CGH plots showing an IKZF1 intragenic Δ4-8 deletion (case CGH #1). (B) Schematic representation of IKZF1 gene and recurrent intragenic deletions with primer sequences for breakpoint-specific PCR. Boxes represent exons, with the non-coding exon 1 in dark gray. Blue and green arrows represent forward fluorescent-labeled primers, black arrows represent non-labeled reverse primers. GL, germline, designates the primer for control amplification of a non-rearranged IKZF1 sequence. (C) Representative Genescan pattern of each type of IKZF1 rearrangement amplified by breakpoint-specific fluorescent multiplex PCR. Amplicon size ranges are indicated. Dotted lines indicate amplicons size ranges for variant breakpoints. GL, germline, indicates the 760 bp control amplicon.

Figure 2.

(A) and (C) Numbers of IKZF1 intragenic deletions detected by either MLPA or PCR, in BCR-ABL1-positive ALL and BCR-ABL1-negative BCP-ALL cases, respectively. (B) and (D) Pie charts representing the frequency of IKZF1 deletions in patients with BCR-ABL1-positive ALL and BCR-ABL1-negative BCP-ALL, respectively. *include 2 cases with biallelic intragenic deletions (one case with Δ4-7 + Δ4-8 and one case with Δ4-7 + Δ2-8) and 3 cases with a clonal rearrangement associated to a subclonal rearrangement (one case with Δ4-7 + minor Δ2-7, one case with Δ2-7 + minor Δ4-7 and one case with 2 Δ4-7). ^†include one case with biallelic intragenic deletions and 2 cases with a clonal rearrangement associated to a subclonal rearrangement. ^‡include one case with biallellic broad deletion and 2 cases with broad deletion and a subclonal intragenic deletion.

Figure 3.

(A) Schematic representation of IKZF1 gene with primers and probes used for Q-PCR for MRD monitoring. Δ2c primer and probes are used for variant breakpoints. Arrows represent primers; solid lines with lozenges represent Taqman probes. (B) Concordance of 54 paired MRD results obtained by Ig/TCR and IKZF1 Q-PCR. Detectable but not quantifiable MRD under the 10^-4 threshold is termed “pos <10^-4” and the opposite “neg <10^-4” for undetectable MRD measurement. R2 is the Pearson's correlation coefficient of the 11 MRD values positive ≥ 10^-4 measured by both methods. As Ig/TCR MRD analysis was performed with 2 markers for most patients, the marker with the best sensitivity and quantitative range was retained for non-quantifiable results, and the mean result of the 2 markers for quantifiable results.