High frequency of IKZF1 deletions in Chinese adult patients with acute lymphoblastic leukemia detected by multiplex real- time quantitative PCR

Objectives: To examine the incidence and clinical dynamics of IKZF1 alterations in Chinese adult patients with ALL. Methods: Samples were studied from 328 newly diagnosed adult ALL patients at Peking University People’s Hospital from 2007 to 2012 by multiplex real-time quantitative PCR, multiplex fluorescent PCR and sequence analysis for four types of IKZF1 deletions. Albumin (ALB) was the plasmid standard. Results: All correlation coefficients for amplified ALB(albumin) and IKZF1 Δ4-7 deletion plasmids were above 0.99, and the sensitivity of detection was at least ten copies. About 36.3% (95% [CI], 31.1-41.7%) of 328 untreated cases showed IKZF1-deletion. The median IKZF1-deletion copies/ALB copy was 85.8% (0.1%697.9%). 27 IKZF1-deletion-positive patients (219 samples in total) were followed up after treatment, among them, 18 patients in hematologic remission continued to be tested negative within 8-66 weeks. In 5 relapsed cases, elevated levels of IKZF1 deletion were detected when relapse occurred. In 4 non responders, IKZF1 deletion maintained at high level. Patient with B-cell ALL subtype had much higher IKZF1-deletion rate (40.4%) than those with T-cell ALL subtype (2.8%, P<0.01). In particular, it was higher in common B-cell ALL group than those with other subtypes. Conclusion: This assay is reliable and sensitive. It is useful in diagnosis and monitoring minimal residual disease in adult ALL. High frequency of IKZF1 deletions occur in adult patients with common B-cell ALL. Correspondence to: Guo-Rui Ruan, Peking University People’s Hospital and Institute of Hematology 11 Xi-Zhi-Men, South Street, 100044 Beijing, China, Tel: 8610-88324672, Fax: 8610-88324672 # Li-Xin Wu and Jiao Zhou contributed equally to the work.

These techniques estimate the copy-number and therefore have limited sensitivity and they cannot be applied for the detection of minimal residual disease (MRD) in ALL. The deletion breakpoints for these IKZF1Δ4-7 alterations are usually located within a few nucleotides, suggested the feasibility of designing MRD (minimal residual disease) assays based on real-time quantitative polymerase chain reaction (RQ-PCR) in the same way as Ig/TCR-based minimal residual disease (MRD) tests [18]. In this study, in order to detect the levels of IKZF1Δ4-7 alterations, the albumin (ALB) gene was first amplified as the internal control, DNA from the bone marrow (BM) of untreated adult ALL patients from the Chinese mainland were examined by using the TaqMan® probe-based RQ-PCR, multiplex fluorescent PCR and sequence analysis. Simultaneously, the detections of IKZF1Δ4-8, IKZF1Δ2-7 and IKZF1Δ2-8 alterations were also performed.

Preparation of plasmid standards
The ALB plasmid standard was obtained as described earlier [24]. IKZF1Δ4-7 was amplified using the DNA isolated from the patients with the IKZF1Δ4-7. The 50-μl PCR mixture contained 25μl of 2× Universal PCR Master mix (TianGen Biotech, Beijing, China), 400 nM primers, and 300 ng DNA. The PCR protocol was as follows: pre-denaturing at 95°C for 5 min, followed by 36 cycles at 95°C for 40 s, 58°C for 40 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR products were purified and subcloned into the pMD18-T vector (Takara, Dalian, China). Transformation, screening, and sequencing were then performed to obtain IKZF1Δ4-7 plasmid standard. The copy number was calculated based on the optical density (OD) value, and the standards were subjected to a tenfold dilution (10 7 ,10 6 ,10 5 ,10 4 ,10 3 ,10 2 copies) in order to plot a standard curve. For each measurement, the threshold of amplification for the curve was set at 0.08, and it included a positive control (samples from the patients with the IKZF1Δ4-7), blank control (without template), and negative control (samples from patients without IKZF1Δ4-7).

Calculation of the IKZF1 deletion level
The copy numbers of ALB and IKZF1 deletions were calculated using the Ct value and standard curve. Our study revealed similar efficacies for the amplification of ALB and IKZF1Δ4-7 deletions genes (the slope of standard curve was -3.36 ± 0.15 and -3.46 ± 0.19, respectively). In order to decrease the differences in plasmid quantification, only one ALB standard curve was plotted. The IKZF1 deletions copy number in 100 ALB copies was used as the IKZF1 deletions gene content. If the ALB copy number was ≥3×10 4 , the samples were considered for quantitative detection. The results were analyzed based on the guidelines proposed by the European Study Group for the RQ-PCR detection of MRD [25].

Breakpoint-specific multiplex fluorescent PCR
Multiplex fluorescent PCR was completed as previously described [18]. Primers were designed that they could be combined in a single multiplex PCR and that the amplicons length and fluorescent labelling allowed direct identification of each type of deletion. DNA were amplified in a final volume of 25μl containing 2× Universal PCR Master mix (TianGen Biotech, Beijing, China), 400 nM of each primer, and 150-500 ng DNA. The PCR protocol was as follows: predenaturing at 95°C for 5 min, followed by 30 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. PCR products were run on an ABI 3500 analyser using a fragment size analysis program and analyses were performed using GeneMapper software (Applied Biosystems). The purified PCR products were subjected to direct sequence analysis with an ABI3700 DNA sequencer as described earlier. The screened complicated mutants were subcloned into pMD18-T vectors, followed by transformation, screening and further sequencing.

Statistical analysis
Statistical analysis was performed using SPSS software version 13.0 (Chicago, IL, USA). Independence of categorized parameters was calculated using Chi-square test (or Fisher Exact test). Distribution of continuous variables was calculated using Wilcoxon two sample tests. Values of P<0.05 were considered to be significant.
MRD analysis was subsequently performed on 48 paired samples from 8 patients and compared with MRD results obtained using IgH markers ( Figure 4). The analyses use the log10MRD of the dilution of diagnosis sample giving the same amplification as each sample. A very good correlation (R 2 = 0.986, P <0.01) was observed for the quantifiable paired samples. R 2 is the Pearson correlation coefficient of the 29 MRD values positive ≥ 10 -4 measured by both methods. The results, as previously shown by others for IKZF1 Δ4-7, [17][18] confirm the good accuracy of MRD quantification using IKZF1 markers.

Multiplex fluorescent PCR and sequencing results
Multiplex fluorescent PCR and sequence analysis revealed that the 119 positive cases included 80 (67.2%) cases with type Δ4-7, 29 (24.4%) cases with type Δ2-7, seven (5.9%) cases with type Δ4-8, three (2.5%) cases with type Δ2-8, and six cases with deletion of both alleles. In the 119 positive cases, one case with type Δ2-7 and five cases with type Δ4-7 were detected by the multiplex fluorescent PCR and multiplex RQ-PCR but not direct sequencing possibly due to low level of the deletion. The four kinds of IKZF1 deletions with type Δ4-7, Δ2-7, Δ4-8, and Δ2-8 were shown with arrows indicating the region in which the breakpoints occur ( Figure 5). Genomic breakpoint sequences of the IKZF1 deletion in the 113 ALL cases were described in the supplementary appendix.

Discussion
RQ-PCR shows the IKZF1-deletion rate was as high as 36.3% in the untreated 328 ALL patients and the combination rate of BCR/ABL was more frequent than not(68.8% vs.22.4%, P<0.01), as previously published data shown [11,15,[26][27][28]. Patients with B-ALL had a higher IKZF1-deletion rate than those with T-ALL, especially, our result showed that IKZF1-deletion rate in common B-cell ALL was the highest among all B-ALL subtype. Sequence analysis and multiplex fluorescent PCR revealed that 125 IKZF1 intragenic deletions were  MRD analysis was subsequently performed on 54 paired samples and compared with MRD results obtained using IgH markers ( Figure  2). A very good correlation (R2 = 0.986, P<0.01) was observed for the quantifiable paired samples. The results, as previously shown by others for IKZF1 Δ4-7 [18][19], confirm the good accuracy of MRD quantification using IKZF1 markers. They examined reproducibility measurement of MRD using markers based on IKZF1 deletions compared with Ig/TCR rearrangements and found that the IKZF1 results were as close to the Ig/TCR results. It is therefore likely that this IKZF1 marker will be at least as stable as Ig/TCR rearrangements, although this will need to be confirmed in more extensive studies. Two DNA MRD markers with high sensitivity (at least 10 -4 ) are generally required in MRD intervention clinical trials [29][30], and in a large cohort of 2854 pediatric precursor B-cell ALL patients, 20% of patients had only one sensitive marker and 8% had none [30]. At present the concept of using disease-related markers for MRD testing has been already established for fusion transcripts such as BCR-ABL and for gene rearrangements such as for SIL-TAL1 in T-ALL and for MLL rearrangements in infant ALLs [31]. The use of oncogenic lesions for MRD monitoring has been limited by the fact that recurrent chromosomal translocations are not found in all ALL and are usually studied at the RNA level, so that IKZF1 gene deletions will provide a useful addition to the repertoire of MRD markers currently available for monitoring MRD in ALL and inclusion of this marker in standard screening for MRD targets would be an easy way to provide more patients with two sensitive markers.
By using ALB as the internal control, we found that in 119 IKZF1deletion positive patients, the average IKZF1-deletion copies/ALB copies was 138.5% (0.1%-697.9%) at diagnosis. Of 96(80.7%) patients hold higher levels of IKZF1-deletion (12.5%-697.9%), which might be a helpful condition for MRD assay based the IKZF1-deletion.27 IKZF1-deletion-positive patients (219 samples in total) were followed up after treatment, among them, 18 patients in complete hematologic remission maintained in negativity for IKZF1-deletion within 8-66 weeks. In five relapsed cases, elevated levels of IKZF1 deletion were detected when relapse occurred. In the four non responders, IKZF1 deletion maintained at high level. Kuiper et al. [32] in an analysis of paired diagnosis and relapse samples from 34 patients found IKZF1 deletions at diagnosis and showed that all were conserved at relapse, in contrast to other recurrent genetic lesions found at diagnosis such as PAX5, CDKN2A and EBF1. Therefore, to detect the levels of IKZF1deletion during the clinical course would be helpful for monitoring early relapse cases in ALL patients.
In summary, we found that RQ-PCR based on ALB as control gene was a reliable and sensitive method for detecting IKZF1 deletions. Consensus breakpoint sequences can be used as markers for MRD monitoring. This work should be useful for adjunctive diagnosis, risk assessment and studying the underlying pathogenesis of adult B-ALL, especially in common-B-cell ALL.

Declaration of interest
The authors report no declarations of interest.

Ethical approval
The study design adhered to the principles of the Helsinki Declaration and was approved by the ethics committees of our hospital.

Informed consent
Informed consent was obtained from all the patients prior to their enrollment in the study.