FIP1L1-PDGFRA fusion: prevalence and clinicopathologic correlates in 89 consecutive patients with moderate to severe eosinophilia.

A novel oncogenic mutation (FIP1L1-PDGFRA), which results in a constitutively activated platelet-derived growth factor receptor-alpha (PDGFRA), has been invariably associated with a primary eosinophilic disorder. The current study examines both the prevalence and the associated clinicopathologic features of this mutation in a cohort of 89 adult patients presenting with an absolute eosinophil count (AEC) of higher than 1.5 x 10(9)/L. A fluorescence in situ hybridization (FISH)-based strategy was used to detect FIP1L1-PDGFRA in bone marrow cells. None of 8 patients with reactive eosinophilia displayed the abnormality, whereas the incidence of FIP1L1-PDGFRA in the remaining 81 patients with primary eosinophilia was 14% (11 patients). None (0%) of 57 patients with the hypereosinophilic syndrome (HES) but 10 (56%) of 19 patients with systemic mast cell disease associated with eosinophilia (SMCD-eos) carried the specific mutation. The bone marrow mast cell infiltration pattern in FIP1L1-PDGFRA(+) SMCD-eos was distinctly diffuse with loose tumoral aggregates. Treatment with low-dose imatinib (100 mg/d) produced complete and durable responses in all 8 FIP1L1-PDGFRA(+) cases treated. In contrast, only 40% partial response rate was seen in 10 HES cases. FIP1L1-PDGFRA is a relatively infrequent but treatment-relevant mutation in primary eosinophilia that is indicative of an underlying systemic mastocytosis.

FIP1L1-PDGFRA and c-kit D816V mutation-based clonality studies in systemic mast cell disease associated with eosinophilia.

Laboratory methods to detect both FIP1L1-PDGFRA and c-kit D816V mutations were combined with immunomagnetic cell separation to study the extent of clonal involvement by both myeloid and lymphoid cells in 3 patients with systemic mastocytosis associated with eosinophilia. The results suggested an early stem cell origin for the FIP1L1-PDGFRA mutation.

A novel tricolor, dual-fusion fluorescence in situ hybridization method to detect BCR/ABL fusion in cells with t(9;22)(q34;q11.2) associated with deletion of DNA on the derivative chromosome 9 in chronic myelocytic leukemia.

Dual-color, dual-fusion fluorescence in situ hybridization (D-FISH) can accurately detect and quantify cells with BCR/ABL fusion in <1% of 500 nuclei in 80% of patients with chronic myelocytic leukemia (CML) and t(9;22)(q34;q11.2). The remaining patients have one of three forms of atypical D-FISH patterns; these patterns have different sensitivities to detect disease. Neoplastic cells with one ABL, one BCR, and one BCR/ABL fusion are particularly problematic, because normal cells with coincidental overlap have the same pattern. For these patients, the normal cutoff for D-FISH is >23%. We tested a new method that incorporates an aqua-labeled probe for the argininosuccinate synthetase (ASS) gene into the conventional BCR/ABL D-FISH probe set. This tricolor D-FISH (TD-FISH) method takes advantage of the aqua-labeled ASS probe to distinguish between neoplastic and normal cells. We used TD-FISH to study 20 normal specimens and 35 specimens from 20 patients with known loss of both BCR and ABL from the derivative chromosome 9. The results show that TD-FISH effectively discriminates between cells with overlapping BCR and ABL signals from cells with true BCR/ABL fusion and improves the ability to quantify minimal residual disease from >23% to >1% of 500 interphase nuclei.

Comparison of interphase FISH and metaphase cytogenetics to study myelodysplastic syndrome: an Eastern Cooperative Oncology Group (ECOG) study.

Cytogenetic analysis can be important in determining the prognosis and diagnosis of a number of hematological disorders, including myelodysplastic syndromes (MDS). Here, we compared metaphase chromosomal analyses on bone marrow aspirates from MDS patients with interphase fluorescence in situ hybridization (FISH) using probes specific for chromosomes nos. 5, 7, 8, 11, 13 and 20. Forty-three patients enrolled in ECOG protocol E1996 for low risk MDS and five patients enrolled in ECOG protocol E3996 for high risk MDS were studied by both metaphase chromosomal analysis and interphase FISH. Excluding those with a clonal loss of the Y chromosome, an abnormal clone was detected by cytogenetic analysis in 18 of 48 samples (37.5%). In comparison, our FISH panel detected an abnormal clone in 17 of 48 samples (35.4%). Twenty-nine of 30 samples with apparently normal karyotypes, including those with a missing Y chromosome, were also normal by our FISH panel. One patient had an occult deletion of chromosome 11 that was detected by FISH. These results indicate that around 60% of patients with MDS do not have abnormalities that are detectable by either chromosomal or FISH studies. In addition, it appears that interphase FISH studies are nearly as sensitive as cytogenetic analyses and can be a useful tool in studying bone marrow aspirates where cytogenetic analysis is not possible.

New highly sensitive fluorescence in situ hybridization method to detect PML/RARA fusion in acute promyelocytic leukemia.

We investigated a new fluorescence in situ hybridization (FISH) method to detect PML/RARA fusion and/or anomalies of the RARA gene (alias RARalpha) in interphase nuclei from patients with acute promyelocytic leukemia (APL). This method uses a commercially available product with two different colored fluorescent probes to detect both PML/RARA gene fusion products (double fusion signal or dual-color fluorescence in situ hybridization [D-FISH]). A total of 82 bone marrow specimens were studied, including 30 from normal bone marrow transplant donors, 33 from patients with untreated APL, 14 from patients with treated APL, and 5 from APL patients with known translocation variants or alternate translocations. The signal patterns and percentage of abnormal nuclei were determined in a blinded study on 500 interphase nuclei for each specimen. Based on 25 normal specimens, the normal cutoff was >0.6% and >1.6% for t(15;17) and t(17;var), respectively. The clinical sensitivity for this series of patients was 98% and the clinical specificity was 100%. The results suggest that the new D-FISH probe set can detect all t(15;17)(q22;q21) and all variant forms of this translocation associated with PML and RARA. In addition, this FISH method can detect all alternate translocations involving RARA and not PML. This FISH method can be used both for the accurate diagnosis of APL and to monitor low levels of disease in treated patients.

CHIC2 deletion, a surrogate for FIP1L1-PDGFRA fusion, occurs in systemic mastocytosis associated with eosinophilia and predicts response to imatinib mesylate therapy.

Imatinib mesylate is effective in the treatment of hematologic malignancies that are characterized by either abl- or PDGFR beta- activating mutations. The drug is also active in a subset of patients with eosinophilic disorders and systemic mast cell disease (SMCD). Recently, a novel tyrosine kinase that is generated from fusion of the Fip1-like 1 (FIP1L1) and PDGFR alpha (PDGFRA) genes has been identified as a therapeutic target for imatinib mesylate in hypereosinophilic syndrome (HES). We used fluorescence in situ hybridization (FISH) to detect deletion of the CHIC2 locus at 4q12 as a surrogate for the FIP1L1-PDGFRA fusion. CHIC2 deletion was observed in bone marrow cells for 3 of 5 patients with SMCD associated with eosinophilia. Deletion of this locus and expression of the FIP1L1-platelet-derived growth factor receptor alpha (PDGFRA) fusion was also documented in enriched eosinophils, neutrophils, or mononuclear cells by both FISH and reverse transcriptase-polymerase chain reaction (RT-PCR) for one patient. While all 3 patients with the FIP1L1-PDGFRA rearrangement achieved a sustained complete response with imatinib mesylate therapy, the other two, both carrying the c-kit Asp816 to Val (Asp816Val) mutation, did not. These observations suggest that the FIP1L1-PDGFRA rearrangement occurs in an early hematopoietic progenitor and suggests that the molecular pathogenesis for a subset of SMCD patients is similar to that of HES. Screening for the FIP1L1-PDGFRA rearrangement and Asp816Val mutation will advance rational therapy decisions in SMCD.

Chromosome anomalies detected by interphase fluorescence in situ hybridization: correlation with significant biological features of B-cell chronic lymphocytic leukaemia.

Fluorescence in situ hybridization (FISH) was used to detect 6q-, 11q-, +12, 13q-, 17p- and translocations involving 14q32 in interphase nuclei from blood and/or bone marrow from 113 patients with B-cell chronic lymphocytic leukaemia (B-CLL). A total of 87 patients (77%) had a FISH anomaly: 13q- x 1 was most frequent (64%) followed by 13q- x 2 (28%), +12 (25%), 11q- (15%), 17p- (8%) and 6q- (0%). FISH results for blood and bone marrow cells in 38 patients were similar. Purified CD5+/CD19+ cells from blood were studied in eight patients and results indicate that in some patients not all B cells have FISH anomalies. We used a defined set of hierarchical FISH risk categories to compare FISH results by stable versus progressive disease, age, sex, Rai stage, CD38+ expression and IgVH mutational status. Significant differences in FISH risk distributions were associated with Rai stage, disease status and CD38+, but not by age, sex or IgVH mutational status. To look for baseline factors associated with high-risk disease, multivariate analysis of age, sex, Rai stage, CD38+ and disease status versus FISH risk category was performed. Importantly, only CD38+ was significantly associated with high-risk FISH categories (+12, 11q- and 17p-) after adjustment for the effects of other variables.

A new method to extract nuclei from paraffin-embedded tissue to study lymphomas using interphase fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is difficult to accomplish using thin-sections of paraffin-embedded lymphoid tissue because of the high cellularity and truncated cells that interfere with accurate scoring of individual nuclei. We modified and tested a new technique to isolate individual nuclei from tissue cores of paraffin-embedded tissue processed with xylene, proteinase K, citric acid, and pepsin. The efficacy of this method to study paraffin-embedded tissue was investigated in six normal lymph nodes or tonsils and 32 malignant lymphomas including five mantle cell, five follicular, five Burkitt, five extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue, five anaplastic large-cell, and seven diffuse large B-cell. Fusion of CCND1 and IgH, BCL2 and IgH, c-myc and IgH, and MALT1 and API2 were detected using probes with a dual-fusion FISH strategy. Anomalies involving ALK and BCL6 were detected using break-apart FISH probes. FISH studies were successful for each of the 38 specimens. Chromosome anomalies were detected in each malignant specimen, but not in the normal lymphoid tissue. The correct chromosome anomaly was detected in 22 of 22 specimens with genetic abnormalities that were established by other genetic techniques. This FISH technique is useful to detect chromosome anomalies with high sensitivity and specificity in paraffin-embedded tissue and may provide important diagnostic and prognostic genetic information.