Ponatinib as targeted therapy for FGFR1 fusions associated with the 8p11 myeloproliferative syndrome

The 8p11 myeloproliferative syndrome is a rare, aggressive myeloproliferative neoplasm characterised by constitutively active FGFR1 fusion proteins that arise from specificchromosomal translocations and which drive aberrant proliferation. Although FGFR1 inhibitors have shown in vitro activity against FGFR1 fusions, none are in use clinically and there is a need to assess additional compounds as potential therapy. Here we use cell linesand primary cells to investigate ponatinib (AP24534). Ponatinib-treated Ba/F3 cells transformed by ZMYM2-FGFR1 and BCR-FGFR1 and the FGFR1OP2-FGFR1 positiveKG1A cell line showed reduced proliferation and decreased survival when compared to control cells. Inhibition induced apoptosis and reduced phosphorylation of the FGFR1 fusionproteins and substrates. Ponatinib-treated cells from five 8p11 myeloproliferative syndrome patients showed reduced colony growth compared to controls. In one evaluable patient, ponatinib specifically reduced numbers of FGFR1-fusion gene positive colonies. Ponatinib therefore shows considerable promise for the treatment of patients with 8p11myeloproliferative syndrome

Megalencephaly syndromes: exome pipeline strategies for detecting low-level mosaic mutations

Two megalencephaly (MEG) syndromes, megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyriapolydactyly?-hydrocephalus(MPPH), have recently been defined on the basis of physical and neuroimaging features. Subsequently, exome sequencing of ten MEG cases identified de-novo postzygotic mutations in PIK3CA which cause MCAP and de-novo mutations in AKT and PIK3R2 which cause MPPH. Here we present findings from exome sequencing three unrelated megalencephaly patients which identified a causal PIK3CA mutation in two cases and a causal PIK3R2 mutation in the third case. However, our patient with the PIK3R2 mutation which is considered to cause MPPH has a marked bifrontal band heterotopia which is a feature of MCAP. Furthermore, one of our patients with a PIK3CA mutation lacks syndactyly/polydactyly which is a characteristic of MCAP. These findings suggest that the overlap between MCAP and MPPH may be greater than the available studies suggest. In addition, the PIK3CA mutation in one of our patients could not be detected using standard exome analysis because the mutation was observed at a low frequency consistent with somatic mosaicism. We have therefore investigated several alternative methods of exome analysis and demonstrate that alteration of the initial allele frequency spectrum (AFS), used as a prior for variant calling in samtools, had the greatest power to detect variants with low mutant allele frequencies in our 3 MEG exomes and in simulated data. We therefore recommend non-default settings of the AFS in combination with stringent quality control when searching for causal mutation(s) that could have low levels of mutant reads due to post-zygotic mutatio

How cured are CML patients in complete molecular remission (CMR) after stem cell transplantation or imatinib?

It is unclear if CMR, i.e., absence of BCR-ABL mRNA, is synonymous with, or even required for, cure of chronic myeloid leukemia (CML). This is particularly relevant for management of the minority of patients who achieve CMR with imatinib (IM). Although most patients in long-term remission (LTR) post-stem cell transplantation (SCT) are considered "functionally cured", BCR-ABL mRNA is occasionally detected in their peripheral blood (PB), at a level similar to that detectable in the PB of healthy individuals. Most CML patients in CMR on IM relapse shortly after treatment discontinuation. We sought to elucidate the quality of the molecular response in these two groups of CML patients by using a genomic DNA (gDNA) real-time quantitative PCR (RQ-PCR) with patient-specific primers/probe combinations for detection of BCR-ABL genomic fusions (gBCR-ABL). gBCR-ABL - a molecular signature of each CML case - was sequenced from pre-SCT or pre-/early-IM therapy using inverse PCR (I-PCR) or long-range genomic PCR (LR-PCR). The I-PCR involved digestion of gDNA with RsaI, circularization of the fragments, and amplification with 2 sets of inverse primers located in the 5' end of the RsaI-fragments of the major breakpoint region of BCR for cloning and sequencing of the BCR-ABL band. The LR-PCR is a multiplex reaction with forward primers on BCR exons 13 or 14, and 20 reverse primers, spanning 150kb of the ABL breakpoint region. The patient-specific products are then directly sequenced. Knowledge of the sequence allowed us to design patient-specific primers/probe combinations that were then used to test gDNA from PB follow-up (FU) samples using novel single-step or nested RQ-PCR assays. When tested in serial dilutions of the sample from which the breakpoint sequence was obtained, both methods generated standard curves of similarly good quality; the nested approach did not improve the sensitivity of the assay, with both methods being capable of detecting one single target DNA molecule per reaction. The specifity of the assay was demonstrated using at least 2 different BCR-ABL-positive gDNAs and a no-gDNA negative controls, whereas the sensitivity was maximized by testing a minimum of 7.2µg gDNA in multiple reactions. From 6 patients in LTR post-SCT (median time post-SCT 186 months; range: 87 to 333) we tested 9 FU samples collected between 87 and 321 months post-SCT (median: 168). From 3 IM-treated patients we tested 5 samples in CMR collected between 36 and 75 months (median: 63) after the start of IM. Six of the 9 post-SCT samples had been classified as low-level positive for BCR-ABL transcripts (BCR-ABL/ABL 0.001 to 0.012%): only 1 was positive for gBCR-ABL (BCR-ABL/ABL from this sample - 0.003%). Of the 3 patients in CMR on IM, 1 had 1 sample negative for gBCR-ABL; 1 had 1 sample positive and 1 sample negative 37 months later; 1 had 2 positive samples separated by 36 months. The FU samples positive for gBCR-ABL were positive at a very low level, with only 1 or 2 positive reactions out of a minimum of 24 replicates, with Ct values very close to the threshold of detection of the standard curves. In conclusion, the results so far suggest that, in post-SCT patients in LTR, the original BCR-ABL positive clone is rarely detected, and in most instances may not be the cause of low-level positivity for BCR-ABL mRNA. The leukemic clone may be more frequently present in IM-treated patients in CMR, which suggests the need for continuing IM even after achievement of CMR

RecurrentCEP85L-PDGFRBfusion in a patient with a t(5;6) and an imatinib-responsive myeloproliferative neoplasm with eosinophilia

Fusion genes involving the catalytic domain of tyrosine kinases (TKs) play an important role in the pathogenesis of hematological malignancies and solid tumors. In BCR-ABL1-negative myeloproliferative neoplasms (MPNs) several different tyrosine kinase fusion events have been described, most commonly involving the genes encoding the platelet-derived growth factor receptor alpha (PDGFRA) or beta (PDGFRB). Since the introduction of small molecule kinase inhibitors, TK fusions have emerged as prime therapeutic targets. Here, we report a recurrent CEP85L-PDGFRB fusion in a patient with eosinophilia and an MPN. The fusion was confirmed by specific amplification of the genomic breakpoints and reverse transcription polymerase chain reaction (PCR).The patient was treated with imatinib and achieved hematologic and cytogenetic remission. Minimal residual disease screening over 3 years with nested PCR failed to detect CEP85L-PDGFRB mRNA or genomic DNA, confirming a long term molecular remission on imatinib<br/

Transcription factor mutations in myelodysplastic/myeloproliferative neoplasms

Background: aberrant activation of tyrosine kinases, caused either by mutation or gene fusion, is of major importance for the development of many hematological malignancies, particularly myeloproliferative neoplasms (MPN). We hypothesized that hitherto unrecognized, cytogenetically cryptic tyrosine kinase fusions may be common in non-classical or atypical MPN and related myelodysplastic/myeloproliferative neoplasms (MDS/MPN).Design and Methods: to detect genomic copy number changes associated with such fusions, we performed a systematic search in 68 patients using custom designed, targeted high-resolution arraycomparative genomic hybridisation (CGH). Arrays contained 44,000 oligonucleotide probes that targeted 500 genes including all 90 tyrosine kinases plus downstream tyrosine kinase signaling components, other translocation targets, transcription factors, and other factors known to be important for myelopoiesis.Results: no abnormalities involving tyrosine kinases were detected, however, nine cytogenetically cryptic copy number imbalances were detected in seven patients including hemizygous deletions of RUNX1 or CEBPA in two cases with atypical chronic myeloid leukemia. Mutation analysis of the remaining alleles revealed non-mutated RUNX1 and a frameshift insertion within CEBPA, respectively. A further mutation screen of 187 MDS/MPN patients identified RUNX1 mutations in 27 (14%) and CEBPA mutations in seven (4%) patients. Analysis of other transcription factors known to be frequently mutated in acute myeloid leukemia revealed NPM1 mutations in six (3%) and WT1 mutations in two (1%) MDS/MPN patients, respectively. Univariate analysis indicated that patients with mutations had a shorteroverall survival (28 vs. 44 months, P=0.019) compared with mutation negative cases, with the prognosis for cases with CEBPA, NPM1 or WT1 mutations being particularly poor.Conclusions: we conclude that mutations of transcription and other nuclear factors are frequent in MDS/MPN and are generally mutually exclusive. CEBPA, NPM1 or WT1 mutations may beassociated with a poor prognosis, an observation that will need to be confirmed by detailed prospective studie

Inactivation of polycomb repressive complex 2 components in myeloproliferative and myelodysplastic/myeloproliferative neoplasms

The polycomb repressive complex 2 (PRC2) is a highly conserved histone H3 lysine 27 methyltransferase that regulates the expression of developmental genes. Inactivating mutations of the catalytic component of PRC2, EZH2, are seen in myeloid disorders. We reasoned that the other two core PRC2 components SUZ12 and EED may also bemutational targets in these diseases, as well as associated factors such as Jarid2. SUZ12 mutations were identified in 1 of 2 cases with myelodysplastic/myeloproliferative neoplasm (MDS/MPN) with 17q aUPD and 2 of 2 myelofibrosis cases with focal 17q11 deletions. All three were missense mutations affecting the highly conserved VEFS domain. Analysis of a further 146 MDS/MPN cases revealed an additional VEFS domain mutant, yielding a total mutation frequency of 2/148 (1.4%). We did not find mutations of Jarid2 or EED in association with aUPD for chromosome 6p or 11q, respectively, however screening unselected cases identified missense mutations in EED (1/148; 1%) and Jarid2 (3/148; 2%). All three SUZ12 mutations tested and the EED mutation reduced PRC2 histone methyltransferase activity in vitro, thus demonstrating that PRC2 function may be compromised in myeloid disorders by mutation of distinct genes

Screening for diverse PDGFRA or PDGFRB by fusion genes is facilitated by generic quantitative RT-PCR

Backround: rapid identification of diverse fusion genes with involvement of PDGFRA or PDGFRB in eosinophilia-associated myeloproliferative neoplasms (Eos-MPN) is essential for adequate clinical management but is complicated by the multitude and heterogeneity of partner genes and breakpoints. Design and Methods. We established generic quantitative RT-PCR assays (RQ-PCR) to detect overexpression of 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 vs. 0.0066 vs. 0.0064, p&lt;0.0001; PDGFRB/ABL: 196 vs. 3.8 vs. 5.85, p&lt;0.0001). Sensitivity and specificity of the activation screening test is 100% and 88.4% for PDGFRA and 100% and 94% for PDGFRB, respectively. Furthermore, significant overexpression of PDGFRB was found in an Eos-MPN patient with uninformative cytogenetics and excellent response to imatinib. Subsequently, a new SART3-PDGFRB fusion gene was identified by 5'-RACE-PCR.Conclusion: RQ-PCR is a simple and useful adjunct standard diagnostic assays to detect clinically significant overexpression of PDGFRA and PDGFRB in Eos-MPNs or related disorder

Limited duration of complete remission on ruxolitinib in myeloid neoplasms with PCM1-JAK2 and BCR-JAK2 fusion genes

Rearrangements of chromosome band 9p24 are known to be associated with JAK2 fusion genes, e.g., t(8;9)(p22;p24) with a PCM1-JAK2 and t(9;22)(p24;q11) with a BCR-JAK2 fusion gene, respectively. In association with myeloid neoplasms, the clinical course is aggressive, and in absence of effective conventional treatment options, long-term remission is usually only observed after allogeneic stem cell transplantation (ASCT). With the discovery of inhibitors of the JAK2 tyrosine kinase and based on encouraging in vitro and in vivo data, we treated two male patients with myeloid neoplasms and a PCM1-JAK2 or a BCR-JAK2 fusion gene, respectively, with the JAK1/JAK2 inhibitor ruxolitinib. After 12 months of treatment, both patients achieved a complete clinical, hematologic, and cytogenetic response. Non-hematologic toxicity was only grade 1 while no hematologic toxicity was observed. However, remission in both patients was only short-term, with relapse occurring after 18 and 24 months, respectively, making ASCT indispensable in both cases. This data highlight (1) the ongoing importance of cytogenetic analysis for the diagnostic work-up of myeloid neoplasms as it may guide targeted therapy and (2) remission under ruxolitinib may only be short-termed in JAK2 fusion genes but it may be an important bridging therapy prior to ASCT