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A 54-year-old male patient presented in December 2008 with raised eosinophils on a routine full blood count. The white cell count was 10.3 × 109/L with hemoglobin 13.6 g/dL, platelets 154 × 109/L, and eosinophils 1.4 × 109/L. He was otherwise asymptomatic. BCR-ABL1 and FIPIL1-PDGFRA were both negative and his cytogenetics was reported as del(5)(q22q31) in 10 of 30 metaphases. Based on the above findings, he was formally diagnosed with myeloproliferative neoplasms, unclassified (MPN-U) with chromosome 5 abnormalities and eosinophilia. He was initially treated with low-dose dasatinib (the physician's choice), with a good hematological response and rapid resolution of the peripheral blood eosinophilia, but the treatment was discontinued after 10 months due to recurrent, symptomatic pleural effusions.

In 2013, nearly 5 years after the initial diagnosis, the patient continued to have persistent but asymptomatic mild eosinophilia off-therapy with otherwise normal blood counts. Cytogenetics was repeated and showed a 46,XY,ins(8;5)(p23;q33q35) in all 30 metaphases analyzed. Retrospective analysis of the original diagnostic sample revealed the same abnormality, which had originally been misinterpreted due to poor-quality metaphases. Fluorescence in situ hybridization (FISH) analysis using PDGFRB breakapart probes (Cytocell, Cambridge, UK) confirmed disruption of PDGFRB in both the original and follow-up samples. Based on this new finding and the reported efficacy of imatinib in PDGFRB-rearranged MPN [1][2], the patient was treated with low-dose imatinib (200 mg daily) in July 2013. This was well tolerated and within 3 months of therapy, a repeat bone marrow assessment showed complete morphological and cytogenetic remission including FISH negativity for the PDGFRBrearrangement. Ongoing assessments indicated continuing morphological and cytogenetic remission whilst being maintained on low-dose imatinib, with FISH negativity in January 2015 and July 2016.

In order to have a marker for more sensitive minimal residual disease analysis, we retrospectively sought to identify the putative PDGFRB fusion partner using archived frozen cells taken at the time of initial diagnosis. Despite only being able to extract poor-quality RNA from the sample (RNA integrity number <5), we performed RNA-seq analysis. PolyA+ RNA extraction, library preparation, and 100-bp paired-end sequencing was performed with multiplexing for a minimum of 75 million reads per sample using an Illumina HiSeq 2000. Bowtie, TopHat, and TopHat-Fusion were used to align reads, resolve splice junctions, and identify and filter potential tyrosine kinase fusions, as previously described [3]. On initial analysis no PDGFRBabnormalities were identified, but on relaxation of the filtering parameters, two breakpoint-spanning reads were identified with PCM1 fused to PDGFRB. Although this fusion has not been reported before, PCM1 was a plausible candidate since it is located at 8p22 and fuses to the tyrosine kinase JAK2in MPN with eosinophilia as a consequence of a recurrent t(8;9)(p22;p24) [4]. The PCM1-PDGFRB fusion was confirmed by RT-PCR and Sanger sequencing analysis, with PCM1 exon 27 joined to PDGFRB exon 11, and thus preserving the reading frame (Fig. 1a, b). The fusion protein is predicted to retain the bulk of PCM1, including several coiled-coil domains and the entire transmembrane and cytoplasmic domains of PDGFRβ (Fig. 2).

Fig. 1
PCM1-PDGFRB fusion. a Fusion junction of the chimeric PCM1-PDGFRB gene. b RT-PCR analysis: PCM1-PDGFRB was amplified from patient cDNA (E3419) but not negative control cases (E1180, E1181) using the primers PCM-AF (5′-ACACAGACTACTTGAGACAGAGGGC-3′) in PCM1 exon 25 and PDGFRB-AR (5′-GCTCCGACATAAGGGCTTGCTTC-3′) in PDGFRB exon 14. a, c MRD analysis. PCM1-PDGFRB cDNA was amplified by nested PCR for 30 cycles, each round using the primers PCM-AF plus PDGFRB-AR in the first step, followed by reamplification with the primers PCM-DF (5′-TGCTGATAATGCTAGTGTCCTGTCT) in PCM1exon 27 and PDGFRB-DR (5′-TGATAAGGGAGATGATGGTGAGCAC-3′) in PDGFRB exon 11 for the second step. c The resulting product of 134 bp was seen in dilutions of the ins(8;5) patient's presentation cDNA down to 10-3, but not in control samples or in blood or bone marrow samples taken 3 years after starting imatinib. b Confirmation of sample quality by amplification of normal ABL1 cDNA using primers ABL 123 F (5′- TGGAGATAACACCTTAAGCATTAACTAAAGGT-3′) and ABL R (5′-GATGTAGTTGGTTGGGACCCA-3′).
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PCM1-PDGFRB fusion. a Fusion junction of the chimeric PCM1-PDGFRB gene. b RT-PCR analysis: PCM1-PDGFRB was amplified from patient cDNA (E3419) but not negative control cases (E1180, E1181) using the primers PCM-AF (5′-ACACAGACTACTTGAGACAGAGGGC-3′) in PCM1 exon 25 and PDGFRB-AR (5′-GCTCCGACATAAGGGCTTGCTTC-3′) in PDGFRB exon 14. a, c MRD analysis. PCM1-PDGFRB cDNA was amplified by nested PCR for 30 cycles, each round using the primers PCM-AF plus PDGFRB-AR in the first step, followed by reamplification with the primers PCM-DF (5′-TGCTGATAATGCTAGTGTCCTGTCT) in PCM1exon 27 and PDGFRB-DR (5′-TGATAAGGGAGATGATGGTGAGCAC-3′) in PDGFRB exon 11 for the second step. c The resulting product of 134 bp was seen in dilutions of the ins(8;5) patient's presentation cDNA down to 10-3, but not in control samples or in blood or bone marrow samples taken 3 years after starting imatinib. b Confirmation of sample quality by amplification of normal ABL1 cDNA using primers ABL 123 F (5′- TGGAGATAACACCTTAAGCATTAACTAAAGGT-3′) and ABL R (5′-GATGTAGTTGGTTGGGACCCA-3′).

Fig. 1
PCM1-PDGFRB fusion. a Fusion junction of the chimeric PCM1-PDGFRB gene. b RT-PCR analysis: PCM1-PDGFRB was amplified from patient cDNA (E3419) but not negative control cases (E1180, E1181) using the primers PCM-AF (5′-ACACAGACTACTTGAGACAGAGGGC-3′) in PCM1 exon 25 and PDGFRB-AR (5′-GCTCCGACATAAGGGCTTGCTTC-3′) in PDGFRB exon 14. a, c MRD analysis. PCM1-PDGFRB cDNA was amplified by nested PCR for 30 cycles, each round using the primers PCM-AF plus PDGFRB-AR in the first step, followed by reamplification with the primers PCM-DF (5′-TGCTGATAATGCTAGTGTCCTGTCT) in PCM1exon 27 and PDGFRB-DR (5′-TGATAAGGGAGATGATGGTGAGCAC-3′) in PDGFRB exon 11 for the second step. c The resulting product of 134 bp was seen in dilutions of the ins(8;5) patient's presentation cDNA down to 10-3, but not in control samples or in blood or bone marrow samples taken 3 years after starting imatinib. b Confirmation of sample quality by amplification of normal ABL1 cDNA using primers ABL 123 F (5′- TGGAGATAACACCTTAAGCATTAACTAAAGGT-3′) and ABL R (5′-GATGTAGTTGGTTGGGACCCA-3′).
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PCM1-PDGFRB fusion. a Fusion junction of the chimeric PCM1-PDGFRB gene. b RT-PCR analysis: PCM1-PDGFRB was amplified from patient cDNA (E3419) but not negative control cases (E1180, E1181) using the primers PCM-AF (5′-ACACAGACTACTTGAGACAGAGGGC-3′) in PCM1 exon 25 and PDGFRB-AR (5′-GCTCCGACATAAGGGCTTGCTTC-3′) in PDGFRB exon 14. a, c MRD analysis. PCM1-PDGFRB cDNA was amplified by nested PCR for 30 cycles, each round using the primers PCM-AF plus PDGFRB-AR in the first step, followed by reamplification with the primers PCM-DF (5′-TGCTGATAATGCTAGTGTCCTGTCT) in PCM1exon 27 and PDGFRB-DR (5′-TGATAAGGGAGATGATGGTGAGCAC-3′) in PDGFRB exon 11 for the second step. c The resulting product of 134 bp was seen in dilutions of the ins(8;5) patient's presentation cDNA down to 10-3, but not in control samples or in blood or bone marrow samples taken 3 years after starting imatinib. b Confirmation of sample quality by amplification of normal ABL1 cDNA using primers ABL 123 F (5′- TGGAGATAACACCTTAAGCATTAACTAAAGGT-3′) and ABL R (5′-GATGTAGTTGGTTGGGACCCA-3′).

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Fig. 2
Predicted structure of the PCM1-PDGFRβ fusion protein. TM, transmembrane domain; TK, tyrosine kinase.
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Predicted structure of the PCM1-PDGFRβ fusion protein. TM, transmembrane domain; TK, tyrosine kinase.

Fig. 2
Predicted structure of the PCM1-PDGFRβ fusion protein. TM, transmembrane domain; TK, tyrosine kinase.
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Predicted structure of the PCM1-PDGFRβ fusion protein. TM, transmembrane domain; TK, tyrosine kinase.

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Having identified and confirmed a PCM1-PDGFRB fusion in this case, we designed nested PCR primers for sensitive detection of minimal residual disease after therapy. Using serial dilutions of the (poor-quality presentation) cDNA in water, we found that the nested PCR was able to amplify the fusion down to a 1:1,000 dilution. We then tested blood and bone marrow samples taken from the patient 3 years after starting imatinib; residual PCM1-PDGFRB mRNA was not detected in either sample (Fig. 1c).

It is known that signaling through PDGFRB plays an important role in mitogenesis, cytoskeletal rearrangements, and chemotaxis [5][6]. Disruption of PDGFRB, normally located on chromosome 5q33, was first described as the consequence of the t(5;12), in which the 5′ end of ETV6 is juxtaposed to the 3′ end of PDGFRB[7]. Subsequently, many more translocations involving 5q31-33 have been identified, resulting in the identification of >30 distinct PDGFRB fusion genes [8].

Pericentriolar material 1 (PCM1) encodes a component of centriolar satellites and is essential for the correct localization of several centrosomal proteins, and for anchoring microtubules to the centrosomes [9]. PCM1-PDGFRBand PCM1-JAK2 are thus further examples of fusions involving centrosome components, with potentially important functional consequences [10].

Many patients with PDGFRB fusions achieve long-term molecular remission on imatinib therapy [2], and it appears that our case is no exception. Whether, as in chronic myeloid leukemia, some of these patients may be functionally cured and can safely stop treatment [11] remains to be seen. In the meantime, PCM1-PDGFRB should be added to the list of abnormalities associated with the WHO-defined subtype “myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB, or FGFR1, or with PCM1-JAK2” [12].

N.C.P.C. and H.d.L. have received honoraria from Novartis, Incyte, and Pfizer for advisory boards and speaking engagements. N.C.P.C. received research support from Novartis and H.d.L. received research support from Incyte.

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