Real-Time Quantitation of Three CBFB-MYH11 Fusion Transcripts in ...

A novel method for inv(16);CBFβ-MYH11 fusion gene quantitation by real-time PCR using single-concentration calibrator plasmids N. Scott Reading1*, Jonathan A. Schumacher1*, Todd W. Kelley1,2 1ARUP

Institute for Clinical and Experimental Pathology, Salt Lake City, UT, and 2Department of Pathology, University of Utah, Salt Lake City, UT. *Co-first authors

BACKGROUND Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disease of the bone marrow characterized by the accumulation of acquired somatic aberrations in hematopoietic progenitor cells that alter normal cellular mechanisms. These aberrations, which range from single nucleotide changes to chromosomal rearrangements, often occur in genes encoding transcription factors or disrupt elements of the transcription pathway that regulate growth rate, survival, differentiation and/or maturation. In AML cases with normal karyotypes, five genes (NPM1, FLT3, CEPBA, MLL, NRAS), among a variety of other genes, most often mutated.1,2 Chromosomal rearrangements frequently observed in AML cases include t(8;21); RUNX1RUNX1T1, t(15;17);PML-RARA, inv(16);CBFβ-MYH11 and others. Additionally, chromosome loss or gain is common, frequently involving chromosomes 7, 8, 21, X and Y. These varied and diverse genetic aberrations provide the basis for the wide array of phenotypic expression and prognosis seen within this disorder. The inv(16);CBFβ-MYH11 abnormality is one of the more frequent chromosomal rearrangements found in de novo AML. The rearrangement generates a core binding factor β (CBFβ)-myosin 11 (MYH11) chimeric fusion protein.3 The CBFβ-MYH11 chimeric protein blocks differentiation of myeloid leukemic cells. Over 24 different breakpoints have been identified for these gene rearrangements. All CBFβ breakpoints occur in a single intron resulting in a mRNA transcript fusion of exon 5 with the MYH11 partner. The majority of MYH11 breakpoints (21/24) occur within a single intron. The remaining three breakpoints occur upstream. Approximately 98% of all breakpoints generate three different fusion gene products, types A, D, and E (Figure 1).4 CBFβ-MYH11 type A is the most common fusion product, found in 88% of inv(16)-positive AML. Clinically, CBFβ-MYH11 is associated with the French-American-British (FAB) M4E0 subtype. Patients with CBFβ-MYH11 generally have good long-term prognosis and response rates to treatment, however relapses occur in 30-35% of cases.5,6 Testing for minimal residual disease using a sensitive quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) test provides a clinical benefit.7 We have previously described quantitative PCR assays that utilize a known ratio calibrator plasmid containing the targeted mutation and its wild-type form.8 We have applied the principles used in quantitative allele-specific PCR assays to the detection and quantitation of chromosomal translocations. By combining the translocation target and reference gene at a fixed ratio into a single plasmid we can normalize quantitation between sample testing and maximize patient testing to reduce costs while maintaining a highly specific and accurate assay. Here we describe a qRT-PCR method that does not require standard curves to calculate CBFβ-MYH11 fusion transcripts. The assay is specific to detect each of the CBFβ-MYH11 types A, D, and E and sensitive to allow for monitoring patient transcript levels over time. Relative quantitation using a single heterozygous calibration standard for qRT-PCR assay provides a sensitive, accurate and cost effective quantitation strategy that maximizes patient sample testing over absolute quantitative strategies that rely on internal standard curves. Here we describe a novel approach to quantitate CBFβ-MYH11 fusion transcripts using plasmid calibrators specific for the A, D, and E types in a multiplex PCR format.

RESULTS

MATERIALS AND METHODS

 Quantitation is linear in multiplex reaction • Dilution series of CBFβ-MYH11 FusionQuant fusion gene standard in singlet (A) and multiplex reaction (B) • PCR efficiency consistent in multiplex PCR • Linear response over 5-log dilution (A) Standard alone

Figure 1 - Assay design: Primers span CBFβ and MYH11 exon junctions for type specific amplification of CBFβ/MYH11 fusion transcript with MYH11 exon specific 6-FAM-labeled hydrolysis probe for realtime detection. Reaction is multiplexed with ABL1 primers/Cy5-probe for internal standardization.

Figure 3. The percentage mutant allele for 4 HCL samples was calculated from the pBRAFHET calibrator plasmid run at different concentrations ranging over 3 logs. (C) Relative concentration determination of standard in cDNA

CONCLUSIONS Figure 3: Linear regression plot of crossing threshold (Ct) vs log concentration (CN) for (A) CBFβMYH11 fusion gene standards (A, D, E) and (B) standards spiked into cDNA simulating patient sample multiplex reaction detecting ABL1 transcript. (C) Relative concentration (ratio) determination of CBFβMYH11D standard spiked into cDNA normalized to calibrator pCBFβD/MYH11. Target/reference ratio (cyan column) and normalized ratio (red column). Table 1 lists the calculated and observed relative ratios determined for the standards in a multiplex reaction with ABL1 transcript containing cDNA. Similar results were obtained for A and E standards (data not shown).

 Sensitivity of detection of AML inv(16) type A & E patient samples • Diluted in cDNA 5 logs (1/10, 1/10, 1/10, 1/5, 1/5, 1/2, 1/2, 1/2, 1/2, 1/2) • Linear correlation of quantitation values between copy number and relative ratio values • LOD approaches 1 copy/reaction (A)

 Quantification methodology: • Calibrator plasmid (pCBFβ-MYH11/ABL1, type A,D,E) • Single concentration • Quantification equation: [1/(E∆Cp+1)] – E = PCR efficiency – ∆Cp = (Cpinv16-Cpabl1)sample-(Cpinv16-Cpabl1)calibrator  PCR efficiency: • 5-log dilution series of CBFβ-MYH11 FusionQuant fusion gene standards (A,D,E) • 6-log dilution series of calibrator plasmid (A,D,E) • E = 10-1/slope (plot of Cp values vs. log plasmid concentration) • Eavg = 1.97 [Etype A = 1.98; Etype D = 1.97; Etype E = 1.96]

F

ABL1

R

F

pCBFβ-MYH11/ABL1 (3.5 kb)

R

Figure 2: Calibrator plasmid design. The fusion sequences of CBFβ-MYH11 were placed in tandem with ABL1 exon 2-3 sequence to create calibrator plasmids for each of the inv(16); CBFβ/MYH11 fusion partners (types A, D, and E).

 We describe a sensitive, quantitative RT-PCR assay for detection and quantitation of inv(16);CBFβ-MYH11 fusion transcripts.  The assay uses a calibrator plasmid containing inv(16) and ABL1 (pCBFβ-MYH11/ABL1) at a defined ratio.  Calibrator is run at a single concentration.  Distinguishes and quantitates inv(16);CBFβ-MYH11 types A, D, E.  Single point calibrator quantitation yields similar values as data calculated using conventional standard curves.  Does not require the generation or purchase of standards of known copy number.

REFERENCES

(B)

(C)

(D)

Inv16 (A,D,E)

Figure 5: Comparison of CBFβMYH11:ABL1 ratios obtained from Ipsogen CBFβA-MYH11 Fusion Quant Assay and ARUP qRT-PCR for the quantitation of CBFβAMYH11 fusion transcripts in clinical samples.

(B) Standard in cDNA

 Sample Type: • Total RNA isolated from peripheral whole blood • Acute Myeloid Leukemia inv(16) type A, E patient cDNA • CBFβ-MYH11 FusionQuant Fusion Gene Standards (Ipsogen) • University of Utah IRB# 7275  PCR methodology: • Reverse Transcription kit (Ipsogen) • qPCR forward primers and reverse primers • Dual-label hydrolysis probes (IDT) 6-FAM/CY5 reporter dyes Type A: 5’/TGG AGA CCC AGA TGG AGG AGA TGA A/3’ Type D: 5’/TGA AGT TGA GAG CGT CAC AGG GAT /3’ Type E: 5’/ACG CTG GAG AAA GAG AAC GCA GAC /3’ ABL1: 5’/ACA CTG CTT CTG ATG GCA AGC TCT /3' • QuanTitect Multiplex PCR Kit (Qiagen) • LightCycler 480 instrument (Roche)

 Quantitation comparison of qRT-PCR assay results with CBFβMYH11A FusionQuant Assay (Ipsogen) • 18 AML [inv(16) type A] patient cDNA samples • Linear correlation of quantitation results

Figure 4. Linear regression plots of crossing threshold (Ct) vs log concentration (CN) for Patient cDNA with (A) CBFβA-MYH11 and (B) CBFβE-MYH11 fusion transcript diluted serially in cDNA. Relative concentration (ratio) determination of Patient cDNA with (C) CBFβA-MYH11 and (D) CBFβE-MYH11 transcript serial dilutions normalized to calibrator pCBFβA,E/MYH11. inv(16)A,E/ABL1 ratio (cyan column) and normalized ratio (red column). Tables lists the calculated and observed relative ratios based on the initial ratio value of the undiluted sample in a multiplex reaction with ABL1 transcript. Copy number for the samples were determined using standard curves of CBFβA,E-MYH11 fusion gene standards.

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