Diagnosis and monitoring of residual disease by quantitative RT-PCR in eosinophilia-associated myeloproliferative disorders with rearrangements of PDGFRA and PDGFRB

Erben, Philipp, Mueller, Martin C., Metzgeroth, Georgia, Popp, Helena, Walz, Christoph, Schenk, Thomas, Lauber, Stephanie, Kruth, Jens, Cross, Nicholas C.P., Hehlmann, Rudiger, Hochhaus, Andreas and Reiter, Andreas (2006) Diagnosis and monitoring of residual disease by quantitative RT-PCR in eosinophilia-associated myeloproliferative disorders with rearrangements of PDGFRA and PDGFRB. Blood, 108 (11), 760A-761A.

Abstract

In eosinophilia-associated myeloproliferative disorders with rearrangements of PDGFRA or PDGFRB, molecular diagnosis of the respective fusion genes and monitoring of minimal residual disease (MRD) during treatment with imatinib are compromised by the heterogeneity of the fusion partners. We therefore sought to establish a rapid and reliable quantitative RT-PCR assay (RQ-PCR) using the LightCycler technology for the detection and quantification of PDGFR fusion transcripts by universal amplification of regions which are located downstream to known breakpoint cluster regions within PDGFRA exon 12 and between intron 9 and 11 of PDGFRB. Diagnostic analyses were performed in cDNAs derived from bone marrow or peripheral blood samples of 39 patients (pts) with FIP1L1-PDGFRA (n=31), BCR-PDGFRA (n=1), CDK5RAP2-PDGFRA (n=1), H4-PDGFRB (n=2), ETV6-PDGFRB (n=2), GIT2-PDGFRB (n=1) and GPIAP1-PDGFRB (n=1) fusion genes (36m, 3f, median age 56 ys, range 20 – 73). Except in FIP1L1-PDGFRA positive cases, all patients revealed involvement of chromosome bands 4q12 (PDGFRA) or 5q31–33 (PDGFRB) in chromosomal aberrations identified by conventional cytogenetics. As external standards for quantification, serial dilutions of plasmids containing normal PDGFRA and PDGFRB sequences were employed. ABL transcripts were quantified as internal control and results were expressed as ratios PDGFRA/ABL or PDGFRB/ABL. A cut-off point for overexpression of PDGFRA and PDGFRB (mean+/–2SD) was determined by analysis of a series of 30 healthy volunteers. In healthy individuals, PDGFRA is expressed at very low levels if at all, whereas PDGFRB is expressed at comparable levels to ABL. Serial dilutions of the FIP1L1-PDGFRA positive EOL1 cell line in HL60 cells and of mRNAs derived from patients with known fusion genes (FIP1L1-PDGFRA and H4-PDGFRB) in control mRNA revealed an assay sensitivity of up to 1:1,000 for both fusion genes which was two logs lower than the sensitivity of the specific nested RT-PCRs (1:100,000). At diagnosis, all pts with PDGFR fusion genes showed significantly increased transcript levels compared to healthy controls. The transcript levels ranged within 4 orders of magnitude for PDGFRA (ratio PDGFRA/ABL 0.03–51) and one order of magnitude for PDGFRB fusion genes (ratio PDGFRB/ABL 190–1350). Serial quantification for MRD monitoring during treatment with imatinib has been performed in 21 pts with PDGFRA (100–400 mg/d) and 5 pts with PDGFRB (400mg/d) fusion genes, respectively. In PDGFRA cases, RQ-PCR for PDGFRA transcripts became negative in 21 of 21 patients after a median of 13 weeks (range, 8–67) which was confirmed by fusion gene specific nested RT-PCR in 19 of 21 cases after a median of 21 weeks (range, 28–67). In PDGFRB cases, RQ-PCR for PDGFRB became negative in 4 of 5 patients after a median of 44 weeks (range 16–72 weeks) which was confirmed by fusion gene specific nested RT-PCR in 1 of 5 patients after 40 weeks. We conclude that the universal quantification of regions which are located downstream to known breakpoint cluster regions of PDGFRA and PDGFRB is a sensitive and reliable assay for the routine screening of constitutive activation of PDGFRA or PDGFRB and for monitoring of residual disease during treatment with tyrosine kinase inhibitors.

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Contributors

Author: Nicholas C.P. Cross

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