Laboratory Assessment of Patients with Hypereosinophilic Syndrome

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Abstract

In FY 2022, we performed a study that revealed that false-negative testing for FIP1L1::PDGFRA gene fusion by fluorescence in situ hybridization (FISH) is a frequent cause of diagnostic delay in patients with the myeloproliferative variant of HES. The imatinib-sensitive fusion gene FIP1L1::PDGFRA is the most frequent molecular abnormality identified in patients with eosinophilic myeloid neoplasms. Rapid recognition of this mutation is essential given the poor prognosis of PDGFRA-associated myeloid neoplasms prior to the availability of imatinib therapy. We reported a case of a patient in whom delayed diagnosis resulted in cardiac transplantation for eosinophilic endomyocardial fibrosis. The delay in diagnosis was due, in part, to a false-negative result in fluorescence in situ hybridization (FISH) testing for FIP1L1::PDGFRA. To explore this further, we examined our cohort of patients presenting with confirmed or suspected eosinophilic myeloid neoplasms. Sixty-five patients with confirmed or suspected eosinophilic myeloid neoplasms were identified, of which 37 (57%) were positive for FIP1L1::PDGFRA by RT-PCR and/or FISH testing, 19 had other molecular abnormalities, and 9 had clinical and laboratory features of HESN with no mutation identified. Both RT-PCR and FISH testing for FIP1L1::PDGFRA were performed in 39 of the 65 patients with presumed or confirmed HESN. Twelve were positive for FIP1L1::PDGFRA in both tests, and 8 were positive only by RT-PCR (p = 0.008). The remaining 19 patients were negative in both tests. In 8 patients who showed false negative FISH results despite a positive reverse-transcriptase polymerase chain reaction test for FIP1L1::PDGFRA, false-negative FISH results delayed the median time to imatinib treatment by 257 days in these patients. Neither clinical nor demographic features were different between the patients who tested positive or false-negative by FISH. A need for higher percentage of cells expressing FIP1L1::PDGFRA has been suggested as a potential explanation for the relative insensitivity of FISH testing, and could have contributed to the false negative results in some of the patients in the current series. To explore this further, interrogation of whole-genome sequences from FISH-positive and negative HES cases for the variant allele fraction (VAF) of the PDGFRA-FIP1L1 fusion was performed. Whole-genome sequence was available for 15 of the 20 PDGFRA-FIP1L1-positive patients, including 11 of the patients who underwent both FISH and PCR testing. As previously reported, the results revealed variable breakpoints inside the FIP1L1 locus in different patients. Breakpoint data were available for 7 patients who underwent both FISH and PCR testing and showed no obvious differences between the two groups. However, FISH-positive cases (n = 6) had significantly higher fusion VAFs (median 0.36) compared to FISH-negative cases (n = 5, median 0). Although the sequencing depth limits the detection sensitivity of the fusion, this result suggests that samples with relatively low tumor cell content are more likely to lead to false-negative FISH results. These data emphasize the importance of adequate molecular testing in these patients, and the need for use of empiric imatinib therapy in patients with clinical features suggestive of PDGFRA-associated disease. Unbiased genomics of PDGFR-rearranged HES have not been comprehensively studied at genome scale. To better characterize genomics of PDGFR-rearranged hypereosinophilic syndrome, whole-genome sequencing (WGS) on purified eosinophils from 11 patients with chronic phase PDGFRrelated HESN was conducted. Paired normal samples were peripheral blood mononuclear cells collected in hematologic and molecular remission after imatinib treatment, in the setting of normal eosinophil counts and no detectable PDGFR rearrangement. The median age at tumor sampling was 43 years (range, 13-60); there were 10 males and 1 female. Nine patients had evidence of an FIP1L1::PDGFRA (F/P) fusion. One patient had a PDGFRB::ETV6 rearrangement, and 1 had a novel complex rearrangement involving IQGAP2 and PDGFRB on chromosome 5 and UVRAG on chromosome 11. This created an in-frame protein fusion between exon 10 of IQGAP2 and exon 12 of PDGFRB, with the other ends of these breakpoints connecting to the first intron of UVRAG. Single-nucleotide variants (SNVs) and insertions and deletions occurred at a median rate of 0.37 events per megabase (range, 0.23-0.58), comparable with that of other myeloproliferative neoplasms (0.38 mutations per megabase) and lower than that of other hematologic malignancies. Signature analysis revealed COSMIC SBS5 (a DNA mutation pattern enriched for C>T transitions) as the most frequent tumor-associated abnormality. SBS5 is found across all tumor types, including myeloid neoplasms, and exhibits clock-like behavior that correlates with age. No signature suggesting exposure to environmental or endogenous mutagens was detected. Next, clonal structure using single-sample inference was investigated. Most tumors exhibited linear phylogenies with 1 or 2 subclones. The PDGFR fusion occurred in the truncal clone in all cases. In total, 87 non-silent somatic mutations in protein-coding genes were identified in HESN tumors, with no recurrently mutated genes. With the caveat of small cohort size, this suggests that there are no obvious additional somatic driver mutations in HESN beyond the PDGFR rearrangement.