Screening for diverse PDGFRA or PDGFRB fusion genes is facilitated by generic quantitative reverse transcriptase polymerase chain reaction analysis

Abstract

Background: Rapid identification of diverse fusion genes with involvement of PDGFRA or PDGFRB in eosinophilia-associated myeloproliferative neoplasms is essential for adequate clinical management but is complicated by the multitude and heterogeneity of partner genes and breakpoints.

Design and methods: We established a generic quantitative reverse transcriptase polymerase chain reaction to detect overexpression of the 3'-regions of PDGFRA or PDGFRB as a possible indicator of an underlying fusion.

Results: At diagnosis, all patients with known fusion genes involving PDGFRA (n=5; 51 patients) or PDGFRB (n=5; 7 patients) showed significantly increased normalized expression levels compared to 191 patients with fusion gene-negative eosinophilia or healthy individuals (PDGFRA/ABL: 0.73 versus 0.0066 versus 0.0064, P<0.0001; PDGFRB/ABL: 196 versus 3.8 versus 5.85, P<0.0001). The sensitivity and specificity of the activation screening test were, respectively, 100% and 88.4% for PDGFRA and 100% and 94% for PDGFRB. Furthermore, significant overexpression of PDGFRB was found in a patient with an eosinophilia-associated myeloproliferative neoplasm with uninformative cytogenetics and an excellent response to imatinib. Subsequently, a new SART3-PDGFRB fusion gene was identified by 5'-rapid amplification of cDNA ends polymerase chain reaction (5'-RACE-PCR).

Conclusions: Quantitative reverse transcriptase polymerase chain reaction analysis is a simple and useful adjunct to standard diagnostic assays to detect clinically significant overexpression of PDGFRA and PDGFRB in eosinophilia-associated myeloproliferative neoplasms or related disorders.

Figure 1.

(A) Patients with PDGFRA fusion genes showed significantly increased PDGFRA expression levels (PDGFRA/ABL) as compared to patients with non-reactive eosinophilia without known molecular aberrations (n=191) and healthy controls (n=35). The expression level was not different between patients with FIP1L1-PDGFRA (n=45) and alternative PDGFRA fusion genes (X-PDGFRA: BCR-PDGFRA, CDK5RAP2-PDGFRA, ETV6-PDGFRA, KIF5B-PDGFRA). The cut-off point for overexpression of PDGFRA was determined for PDGFRA at a ratio of 0.030 PDGFRA/ABL (35 healthy controls, mean + 3 SD, dotted line). PDGFRA expression levels were not different between patients with non-reactive eosinophilia and healthy controls. (B) Patients with different PDGFRB fusion genes (X-PDGFRB [n=7]: ETV6-PDGFRB, CCDC6-PDGFRB, GIT2-PDGFRB, GPIAP1-PDGFRB and MYO18A-PDGFRB showed significantly increased PDGFRB expression levels compared to patients with non-reactive eosinophilia without known molecular aberrations and healthy controls. In the screening group, 13 patients showed significant overexpression of PDGFRB. In one of these patients, with uninformative cytogenetics and an excellent response to imatinib a new SART3-PDGFRB fusion gene was identified by 5′-RACE-PCR. No increased PDGFRB expression was found in five patients with chromosomal aberrations and involvement of chromosome bands 5q31–32. In four of these cases, alternative fusion genes with involvement of ETV6, NPM1, MLF1 and ACSL6 could be confirmed by RT-PCR. The cut-off point for overexpression was determined at a ratio of 23 PDGFRB/ABL (35 healthy controls, mean+ 3 SD, dotted line). PDGFRB expression levels were not different between patients with non-reactive eosinophilia and healthy controls.

Figure 2.

At diagnosis and during the first 12 months on imatinib the normalized PDGFRA ratio was not statistically different in patients in chronic phase (n=37) compared to patients in blast phase or with secondary AML (n=8).

Figure 3.

(A) Junction sequence and corresponding amino acids of the SART3-PDGFRB fusion protein. SART3-PDGFRB splice variants contain intron-derived sequences which are spliced in-frame between SART3 exon 14 and PDGFRB exon 10. (B) Structure of SART3, PDGFRB and the predicted fusion protein. TM: transmembrane domain, WW: WW-like domain, TK: tyrosine kinase domain.