Detection and molecular monitoring of FIP1L1-PDGFRA-positive disease by analysis of patient-specific genomic DNA fusion junctions

Abstract

To evaluate current detection methods for FIP1L1-PDGFRA in hypereosinophilic syndrome (HES), we developed a means to rapidly amplify genomic break points. We screened 202 cases and detected genomic junctions in all samples previously identified as RT-PCR positive (n ¼ 43). Genomic fusions were amplified by single step PCR in all cases whereas only 22 (51%) were single step RT-PCR positive. Importantly, FIP1L1-PDGFRA was detected in two cases that initially tested negative by RT-PCR or fluorescence in situ hybridization. Absolute quantitation of the fusion by real-time PCR from genomic DNA (gDNA) using patient-specific primer/probe combinations at presentation (n ¼ 13) revealed a 40-fold variation between patients (range, 0.027-1.1 FIP1L1-PDGFRA copies/haploid genome). In follow up samples, quantitative analysis of gDNA gave 1-2 log greater sensitivity than RQ-PCR of cDNA. Minimal residual disease assessment using gDNA showed that 11 of 13 patients achieved complete molecular response to imatinib within a median of 9 months (range, 3-17) of starting treatment, with a sensitivity of detection of up to 1 in 10 5 . One case relapsed with an acquired D842V mutation. We conclude that detection of FIP1L1-PDGFRA from gDNA is a useful adjunct to standard diagnostic procedures and enables more sensitive follow up of positive cases after treatment.

The low frequency of clinical resistance to PDGFR inhibitors in myeloid neoplasms with abnormalities of PDGFRA might be related to the limited repertoire of possible PDGFRA kinase domain mutations in vitro

“…This is similar to CML, where imatinib resistance occurs more frequently and is more often associated with kinase domain mutations in advanced phase than in chronic phase of the disease (Baccarani et al, 2009). In accordance with our results, in six out of seven cases either FP/T674I or FP/D842V were identified (Cools et al, 2003a;von Bubnoff et al, 2005a;Ohnishi et al, 2006;Gotlib and Cools, 2008;Lierman et al, 2009;Score et al, 2009), including one patient relapsing with FP/D842V and receiving treatment with sorafenib (Lierman et al, 2009). The remaining patient was found with a S601P þ L629P double mutation (Simon et al, 2008), which we did not identify.…”

“…Both act as activating mutations, at the same time giving rise to imatinib resistance (Ma et al, 2002;Hirota et al, 2003), probably by destabilizing the inactive conformational state of the kinase to which imatinib binds. FP/ D842V recently has been identified in two cases of FPpositive myeloproliferation and acquired resistance to imatinib (Score et al, 2009) and sorafenib (Lierman et al, 2009). We recovered FP/D842V with nilotinib and sorafenib, but missed this exchange with imatinib, although three other exchanges at the same position emerged with imatinib (D842E/G/H).…”

“…In the meantime, more than 70 different BCR-ABL exchanges that confer clinical imatinib resistance have been identified in patients (Apperley, 2007). In contrast, only seven cases of FP-positive myeloproliferation with acquired imatinib resistance due to a point mutation in the PDGFRA kinase domain have been reported so far (Cools et al, 2003a;von Bubnoff et al, 2005a;Ohnishi et al, 2006;Gotlib and Cools, 2008;Simon et al, 2008;Lierman et al, 2009;Score et al, 2009). Strikingly, in six cases, either a FP/T674I (corresponding to T315I in cABL) or a D842V exchange of the FP kinase domain was identified, whereas in one case, a S601P þ L629P double mutation was detected (Simon et al, 2008).…”

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

“…3 Although these abnormalities are very uncommon, they are associated with excellent responses to imatinib and thus their detection is critical for optimal management of patients. [7][8][9][10][11][12] Accurate detection is, however, complicated by several factors: (i) bone marrow cytogenetic assessment, which is critical to the detection of 4q (PDGFRA) or 5q (PDGFRB) rearrangements for all fusions apart from FIP1L1-PDGFRA, may fail to yield adequate metaphases; (ii) the size of the clone in peripheral blood may be very small and the abnormality thus escapes detection by cytogenetics; (iii) split apart fluorescence in situ hybridization (FISH) may fail to detect small clones or cases with complex rearrangements; 13,14 (iv) the heterogeneity of fusion partners and breakpoints makes it difficult and expensive to develop comprehensive and specific reverse transcriptase polymerase chain reaction (RT-PCR) assays. 15 Although some clinicians consider that a short trial of imatinib might be the best way to identify sensitive cases, this is simply not possible in many countries due to budgetary and prescribing restrictions.…”

Acute myeloid leukaemia with associated eosinophilia: justification for FIP1L1‐PDGFRA screening in cases lacking the CBFB‐MYH11 fusion gene

“…The 100-day posttransplant BM was in complete morphological remission, 97% donor chimerism and negative for FIP1L1-PDGFRA by nested RT-PCR, but positive by a DNA based real time PCR assay (Case E2245; Score et al, 2009). The 6-month BM was also in morphological remission, but weakly positive for FIP1L1-PDGFRA by RT-PCR and imatinib was recommenced at 400 mg daily.…”