Case 1 -

FIP1L1-PDGFRA Positive Myeloproliferative Neoplasm James R. Cook, MD, PhD Cleveland Clinic

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Clinical Summary: A previously healthy 26 year old man first came to clinical attention for a thyroid nodule. FNA of the thyroid mass was positive for papillary carcinoma, and a total thyroidectomy was scheduled. A pre-operative CBC and peripheral smear review (Figure 1) demonstrated the following results: CBC 20.65 x103/ µl Abs Neut 2.68 x103/µl RBC 4.87 x106/ µl Abs Lymph 4.34 x103/µl Hgb 15.2 g/dL Abs Mono 0.21 x103/µl HCT 43.7 % Abs Eo 13.42 x103/µl MCV 89.7 fL Abs Baso 0.00 x103/µl PLT 183 x103/ µl To evaluate the patient's marked eosinophilia, a bone marrow aspirate and biopsy were performed. The bone marrow aspirate (Figures 2, 3 and 4) differential count revealed: 2% Blasts; 3% Promyelos; 45% Myelos/Metas/Bands/Segs; 13% Eos; 0% Basos; 0% Monos; 14% Erythroids; 22% Lymphs; 1% Plasma cells The bone marrow core biopsy demonstrated a 70% cellular bone marrow with trilineage hematopoiesis and eosinophilia (Figures 5, 6, and 7). No focal lesions were identified in routine sections. Immunohistochemical stains demonstrated mildly increased, scattered tryptase-positive mast cells that appeared spindled with coexpression of CD25 (Figure 8). Case 1 - Slide 1 Click to view with ImageScope Click to view with a Web-Based Viewer Case 1 - Figure 1 Peripheral blood smear displaying marked absolute eosinophilia. Case 1 - Figure 2 Bone marrow aspirate smear demonstrating eosinophilia. Eosinophil precursors frequently display coarse basophilic granules. Case 1 - Figure 3 Bone marrow aspirate smear demonstrating eosinophilia. Eosinophil precursors frequently display coarse basophilic granules. Case 1 - Figure 4 Bone marrow aspirate smear demonstrating eosinophilia. Eosinophil precursors frequently display coarse basophilic granules. Case 1 - Figure 5 Low power bone marrow core biopsy. The bone marrow is approximately 70% cellular with trilineage hematopoiesis. Focal lesions are not present. Case 1 - Figure 6 High power bone marrow core biopsy. There is trilineage hematopoiesis with focal areas showing marked eosinophilia. Case 1 - Figure 7 High power bone marrow core biopsy. There is trilineage hematopoiesis with focal areas showing marked eosinophilia. Case 1 - Figure 8 Bone marrow core biopsy. An immunohistochemical stain for mast cell tryptase demonstrates increased, scattered mast cells without aggregates. As shown in the inset, the mast cells appear spindled and appear to coexpress CD25. Molecular studies were performed on the bone marrow sample. FISH studies were negative for a BCR/ABL translocation, and PCR studies were negative for the KIT D816V mutation associated with systemic mastocytosis. However, FISH and RT-PCR studies were positive for a FIP1L1-PDGFRA fusion. Final Diagnosis: FIP1L1-PDGFRA positive myeloproliferative neoplasm Discussion: This case is an example of the recently described FIP1L1-PDGFRA-positive myeloproliferative neoplasm. In 2003, Cools et al [2] described an 800kb deletion on chromosome 4q12 which leads to the creation of a FIP1L1-PDGFRA fusion gene containing the 5' portion of FIP1L1 and the 3' portion of PDGFRA. This fusion tyrosine kinase receptor is constitutively active, and has been shown to induce self-phosphorylation and phosphorylation of the STAT5 pathway. [5] In the initial description of this abnormality, a FIP1L1-PDGFRA fusion was detected in 9 of 16 patients (56%) with hypereosinophilic syndrome. [2] More recently, however, a larger analysis by Pardanani et al [8] identified FIP1L1-PDGFRA fusion in 11 of 89 consecutive patients evaluated for moderate to severe eosinophilia, suggesting a lower incidence in this population (12%). Recognition of this entity is of great clinical importance as the FIP1L1-PDGFRA fusion protein is potently inhibited by imatinib. [5] Low dose (100 mg/day) imatinib therapy in FIP1L1-PDGFRA+ patients leads to rapid normalization of eosinophil counts, offering the potential to limit the end organ damage (such as endocardial fibrosis) observed in patients with chronic hypereosinophilia. Several series of FIP1L1-PDGFRA+ myeloproliferative neoplasms have been reported in the literature to date. [2, 6, 8, 9, 12] Each of these reports has demonstrated a striking male predominance (M:F approximately 16:1 overall). In the largest series to date to report detailed clinicopathologic findings, [6] the patients presented with a median age of 35 (range 17-77 years). Frequent clinical findings include fatigue, splenomegaly, anemia, pruritus, and cardiopulmonary symptoms. Peak absolute eosinophil counts ranged from 5.4 to 71.7x103/ µl (median 12.5x103/ µl). The serum tryptase at diagnosis was noted to range from 6.5-45 ng/mL (median 24 ng/mL). Morphologically, bone marrow aspirates will demonstrate numerous eosinophils and eosinophilic precursors. [5, 6] In one study, the median bone marrow eosinophil count was reported to be 40%. [12] In routine H&E stained sections, bone marrow core biopsies will demonstrate eosinophilia without obvious mast cell aggregates (at least in most cases). With immunohistochemical staining for tryptase, however, increased, generally scattered mast cells will be identified with a characteristic abnormal spindled appearance. In some cases, mast cells are so numerous as to form loose aggregates. However, the dense, compact aggregates of spindled mast cells typically associated with systemic mastocytosis appear to be rare in FIP1L1-PDGFRA+ cases. Using immunohistochemistry or flow cytometry, the mast cells are shown to be positive for both CD117 and CD25. CD2 expression, also frequently seen in the mast cells of systemic mastocytosis, has not been described in the few cases examined to date. Common Features of FIP1L1-PDGFRA - associated myeloproliferative neoplasm Clinical Marked male predominance Adult presentation (median 4th-5th decade) Peripheral blood/bone marrow Moderate to severe peripheral eosinophilia Mildly to moderately elevated serum tryptase (usually <50ng/mL) Marrow eosinophilia Increased mast cells - scattered or in loose aggregates Spindled mast cell morphology CD25 expression in mast cells Genotypic Findings FIP1L1-PDGFRA demonstrated by FISH or RT-PCR Negative for KIT codon 816 mutations Normal karyotype The 800kb deletion on chromosome 4q12 in FIP1L1-PDGFRA+ myeloproliferative neoplasms is too small to be detected by banded cytogenetics, and most cases display a normal karyotype. However, the FIP1L1-PDGFRA fusion may be detected by either RT-PCR or FISH studies. [7, 8, 9, 12] Because the fusion breakpoints in the FIP1L1 gene are variable, the size of the product amplified by RT-PCR may vary between patients, and primers must be constructed to account for all known translocation breakpoints. Because the normal FIP1L1 and PDGFRA genes are closely located on chromosome 4q12, the common "fusion probe" strategy employed to detect balanced translocations cannot be used for detection of this abnormality by FISH. Rather, a tri-color FISH strategy may be utilized, with one probe located in the 800kb region deleted by the FIP1L1-PDGFRA fusion (including the CHIC2 gene) and two probes flanking the fusion breakpoints. Deletion of the intervening sequence with retention of the probes telomeric and centromeric to the breakpoint serves as a surrogate marker for the FIP1L1-PDGFRA fusion gene. The limited data in the literature to date indicates that both techniques are similarly effective in identifying the FIP1L1-PDGFRA fusion. In theory, FISH assays may be preferred at initial diagnosis. Rare cases of imatinib-responsive eosinophilic myeloproliferative disorders with balanced translocations of the PDGFRA gene with several other translocation partner genes have also been described, [3, 11] and tri-color FISH assays offer the potential to identify similar translocations which would be missed by RT-PCR studies. For follow-up studies or detection of minimal residual disease, RT-PCR studies would be expected to have a greater diagnostic yield due to a higher sensitivity. The precise classification of FIP1L1-PDGFRA+ myeloproliferative neoplasms has been controversial. Given that identification of a FIP1L1-PDGFRA fusion gene provides evidence for a clonal population of eosinophils, some investigators have classified these cases as chronic eosinophilic leukemia. Other investigators, however, have classified such cases as systemic mastocytosis with eosinophilia. While compact mast cell aggregates appear to be uncommon in FIP1L1-PDGFRA+ patients, the finding of spindled, CD25+ mast cells and elevated tryptase levels characteristic of FIP1L1-PDGFRA+ cases would meet 3 minor criteria for systemic mastocytosis, thus allowing for this diagnosis by current WHO criteria. WHO (2001) Criteria for Systemic Mastocytosis Major Criteria: 2 or more dense mast cell aggregates (>15 mast cells/aggregate) Minor Criteria: >25% spindled, immature, or atypical mast cells KIT point mutation at codon 816 detected Aberrant expression of CD2 and/or CD25 on mast cells Serum tryptase >20 ng/mL Diagnosis requires 1 major and 1 minor, or 3 minor criteria WHO (2001) Criteria for Hypereosinophilic Syndrome / Chronic Eosinophilic Leukemia HES: Persistent eosinophilia (>1.5x103/µL) <20% blasts PB or BM Exclude reactive conditions Exclude neoplasms with secondary reactive eosinophilia Exclude other myeloid neoplasms Exclude abnormal T-cell proliferation CEL: All of the above, plus EITHER: Blasts >2% in PB, or >5% and <19% in BM, OR Evidence of clonality As highlighted in one recent large study, [6] however, there are distinct clinical and biological differences between FIP1L1-PDGFRA+ cases and cases of systemic mastocytosis that lack the FIP1L1-PDGFRA fusion. In particular, FIP1L1-PDGFRA+ cases display a marked male predominance, with cardiac and pulmonary symptoms being more common and gastrointestinal symptoms being less common than in FIP1L1-PDGFRA-negative systemic mastocytosis, even if eosinophilia is also present. While tryptase levels are often elevated in FIP1L1-PDGFRA+ patients, the elevation is moderate (usually <50 ng/mL) in contrast to systemic mastocytosis which frequently displays tryptase levels >100 ng/mL. Finally, the KIT codon 816 mutations typical of systemic mastocysis are not identified in FIP1L1-PDGFRA positive cases. The demonstration of distinct clinical, morphologic and genetic features in FIP1L1-PDGFRA+ cases warrants their distinction from systemic mastocytosis. The current draft outline of the revised WHO classification (available online at www.socforheme.org/new.htm ) has therefore recognized FIP1L1-PDGFRA+ myeloproliferative neoplasm as a distinct clinicopathologic entity. While the diagnosis of FIP1L1-PDGFRA+ myeloproliferative neoplasm is defined largely on the basis of molecular testing, several entities must be considered in the differential diagnosis. In particular, FIP1L1-PDGFRA+ cases must be distinguished from other myeloproliferative disorders that may be associated with eosinophilia, from T-cell lymphoproliferative disorders that may have accompanying eosinophilia, and other forms of HES/CEL. Other myeloproliferative neoplasms may also demonstrate prominent eosinophilia. Chronic myeloid leukemia may present with an absolute eosinophilia, but this is usually less prominent than the neutrophilic leukocytosis with left shift and characteristically frequent circulating myelocytes. Other imatinib-responsive myeloid neoplasms, often meeting criteria for diagnosis as chronic myelomonocytic leukemia, may present with prominent eosinophilia and are associated with translocations involving the PDGFRB locus at chromosome 5q33. The best characterized of these abnormalities is the t(5;12)(q33;p13) involving PDGFRB and ETV6 (TEL). However, many other PDGFRB translocation partners have also been reported, many of which are also imatinib-responsive. [4] Molecular studies are critical to differentiate between these entities. FISH for BCR/ABL performed on peripheral blood is helpful to rule out CML, while metaphase cytogenetic studies are needed to assess for possible translocations involving 5q33. While a number of reference laboratories offer home-brew FISH assays to specifically detect PDGFRB/ETV6 translocations, break-apart FISH probes to detect any PDGFRB translocation (including non-ETV6 partner genes) are unfortunately not currently commercially available in the United States. Some cases of unexplained, persistent hypereosinophilia have been reported to be associated with abnormal lymphoid proliferations. In particular, studies of some patients with HES have identified either phenotypically abnormal T-lymphocytes by flow cytometry and/or clonal T-cell populations by PCR. [5, 10] In these cases, expansion of an abnormal proliferation of Th2 type cells, through production of IL-5 and perhaps other cytokines, is thought to contribute to the persistent eosinophilia. It is currently unclear whether these T cell proliferations represent unusual reactive processes, or are possibly early peripheral blood involvement by a subtle T-cell lymphoma or leukemia. Reactive eosinophilia also can be seen accompanying clear cut T-cell non-Hodgkin lymphomas and leukemias, however. Flow cytometric studies of the peripheral blood and/or PCR studies may therefore be helpful to assess for possible abnormal T-cell proliferations in this setting. Finally, there is a set of patients who, despite thorough investigation, will be found to have no known inciting reactive etiology and no known lymphoproliferative or myeloproliferative disorder. These patients should remain classified as HES/CEL using WHO criteria. It is worth noting that a subset of patients with HES/CEL who lack the FIP1L1-PDGFRA fusion gene nevertheless respond to imatinib therapy. [1, 5] Empiric trials of imatinib may therefore be warranted in this patient population. Those patients who do respond to imatinib presumably may possess other, not-yet-characterized tyrosine kinase abnormalities that drive the hypereosinophilia. The molecularly defined classification of eosinophilia-associated myeloproliferative disorders certainly remains a work in progress, and additional classification changes in the future can be anticipated. References: Baccarani M, Cilloni D, Rondoni M, et al. 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