Next generation sequencing of myeloid neoplasms with eosinophilia harboring the<i>FIP1L1-PDGFRA</i>mutation
Authors: Animesh Pardanani, Terra Lasho, Daniela Barraco, Mrinal Patnaik, Yoseph Elala, Ayalew Tefferi
Journal: American Journal of Hematology (2015)

To the Editor: A subset of patients with peripheral blood and bone marrow eosinophilia harbor the FIP1L1-PDGFRA mutation; this fusion gene results from a cryptic interstitial chromosomal deletion at 4q12 that leads to constitutive activation of PDGFRA tyrosine kinase activity 1. While FIP1L1-PDGFRA targets a pluripotent hematopoietic stem cell, it appears to impose a predominant commitment toward eosinophilic lineage differentiation, possibly in cooperation with interleukin-5 signaling. Patients harboring this mutation are predominantly male and present with a multisystem disease characterized by tissue invasion with eosinophils. A unique morphologic characteristic seen in conjunction with eosinophilia is bone marrow proliferation of loose/non-cohesive clusters of spindled CD25-positive mast cells associated with elevated serum tryptase levels. Patients with a FIP1L1-PDGFRA-positive chronic myeloid disease are uniquely sensitive to treatment with low doses of imatinib mesylate, with virtually all patients achieving a complete hematological/molecular remission that is durable; there are reports of some patients in unmaintained remission even after discontinuing imatinib therapy 2. While FIP1L1-PDGFRA is the candidate driver mutation, the objective of the current study was to identify and characterize other genetic variants that may contribute to disease pathogenesis, phenotype, or clinical outcome by screening a set of myeloid disease-relevant genes using next generation sequencing. The current study was approved by the Mayo Clinic institutional review board. Consecutive patients with FIP1L1-PDGFRA mutation for whom archived bone marrow aspirate samples were available for DNA extraction were studied. Capture libraries were assembled according to the Nimblegen standard library protocol (Roche Nimblegen, Inc, Basel, Switzerland). A list of 27 heme-related genes was selected for custom target capture using Agilent SureSelect Target Enrichment Kit (Agilent Technologies Inc, Santa Clara, CA): IKZF1, PTPN11, SH2B3, SUZ12, ZRSR2, ASXL1, CALR, CBL, CEBPA, CSF3R, DNMT3A, EZH2, FLT3, IDH-1/2, JAK2, KIT, MPL, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, and U2AF1. Single nucleotide variants and small insertion/deletions were called using the Genome Analysis Toolkit (GATK-Broad Institute, Cambridge, MA). Alamut®Visual mutation analysis software (Interactive Biosoftware, Rouen, France) was used to filter variations through public genomic databases. Variants were not further analyzed if the sequencing read depth was <50 and/or were present with ≥1% minor allele frequency (MAF) in the National Center for Biotechnology Information Single Nucleotide Polymorphism public archive database (dbSNP). Finally, every genomic variant was manually curated before classification as confirmed somatic mutation or single nucleotide polymorphism or unknown variant. The latter were classified as potentially pathogenic as per American College of Medical Genetics/Association of Molecular Pathology criteria. Patient information was updated through November 2015 through review of patient histories and correspondence, social security death index, or a telephone call to the patient or their local physician. A total of 10 patients with a chronic myeloid neoplasm with eosinophilia and FIP1L1-PDGFRA mutation were evaluated; Table 1 outlines their clinical and laboratory details at presentation. All patients were male with involvement of peripheral blood and bone marrow. Eight patients received imatinib treatment; one patient died of disease-related complications before imatinib treatment could be initiated (vide infra) and treatment details for another patient were not available. All 8 imatinib-treated patients achieved complete hematological remission which was maintained at last contact (median follow-up =141 months; range 104–218). Next generation sequencing using a customized panel that targeted regions of interest of 27 myeloid-related genes was conducted (note: FIP1L1 and PDGRA were not part of the panel). A single genetic variant per case was identified in four patients: CSF3RM696T (read depth = 2,045, alternate allele fraction = 46%), KITP155S (read depth = 4,680, alternate allele fraction = 49%), ASXL1A1312V (read depth = 7,337, alternate allele fraction = 49%), and CEBPAH195_P196insPH (read depth = 427, alternate allele fraction = 29%). Of these, two genetic variants (20%) were adjudicated as being pathogenic: CSF3RM696T, previously shown to be somatic mutation in chronic myelomonocytic leukemia 3, and KITP155S, a potentially novel variant in the Ig-like C2-type 2 motif in the extracellular domain predicted to be pathogenic by in silico analysis. In contrast, CEBPAH195_P196insPH is a known SNP (MAF 5%) and ASXL1A1312V has been previously reported as a germline variant 4. There were no clinical or laboratory characteristics that distinguished the two patients with CSF3R/KIT mutation versus those without additional mutations (P > 0.05 for all comparisons). There was one death in the entire cohort, namely the patient with the CSF3RM696T mutation. The patient presented with a subacute multisystem illness characterized by constitutional symptoms, weight loss, neurological deficits, renal insufficiency, leukocytosis, and eosinophilia. There was marginal improvement with corticosteroid therapy, however, the patient succumbed to sepsis from ascending cholangitis/gallstone pancreatitis before imatinib treatment could be initiated. While the quality of treatment response to imatinib, which potently inhibits PDGFRA tyrosine kinase activity, strongly supports the notion that FIP1L1-PDGFRA is the driver mutation, other data have pointed to possible additional oncogenic influences in this disease such as co-occurrence of KITD816V mutation or presence of clonal T-lymphocytes. Further, a very small subset of FIP1L1-PDGFRA harboring patients exhibit leukemic transformation that also points to additional oncogenic insults. Our study clarifies these questions by demonstrating that mutations in other myeloid disease-relevant genes are relatively rare. The genetic variants in CSF3R and KIT in two patients likely represent passenger mutations given the absence of phenotype-modifying effects although the sample size is too small to reach definitive conclusions. Further, unlike other chronic myeloid diseases characterized by driver mutations such as chronic neutrophilic leukemia (CSF3RT618I) or systemic mastocytosis (KITD816V) 5, 6, FIP1L1-PDFRA driven disease may be unique in that clinically detrimental mutations, in particular those targeting ASXL1, have not yet been demonstrated yet, which is consistent with the generally favorable prognosis of this disease entity. Animesh Pardanani*, Terra Lasho, Daniela Barraco, Mrinal Patnaik, Yoseph Elala, Ayalew Tefferi Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, Minnesota