Submitting Author: Jamali, Mina, MD MSc FRCPC
Institution: Sunnybrook Health Sciences Centre, The University of Toronto
Additional authors:Huburt Tsui, Marciano D. Reis
Session: Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1

The patient is a 54-year-old man with a medical history significant for a massive thromboembolic stroke following a 2-week history of diarrhea and resulting in a dense hemiplegia. No cause for his stroke was found upon multiple investigations at an outside hospital; however, he was found to have a prothrombin G20210A mutation and peripheral eosinophilia. He presented to our hospital a year later clinically stable but with a persistently elevated eosinophil count and spleen measuring 16cm on ultrasound. Given the suspicion of a hematologic stem cell process, several investigations were undertaken including a bone marrow study (BMS).

Laboratory values from the same day as the BMS:

WBC 19x10^9/L, Hb 123g/L, Platelets 328x10^9/L

Absolute WBC differential count (x10^9/L): promyelocytes 0.2, myelocytes 1.5, metamyelocytes 0.8, neutrophils 7.8, basophils 0.6, eosinophils 7.0, lymphocytes 1.0, monocytes 0.2

HCT 0.37L/L, RBC 3.88x10^12/L, MCV 95.3fL, RDW 13.6%, MCH 31.6pg, MCHC 332g/L

LDH 293IU/L, liver function tests and creatinine were within normal limits

Serum tryptase and vitamin B12 levels were not done

Peripheral blood smear:

The peripheral blood smear shows a leukocytosis due to a mild neutrophilia and eosinophilia (7.0x10^9/L). Rare circulating blasts and occasional immature granulocytes are identified which, together, account for 13% of total leukocytes. A small number of segmented neutrophils show hypolobated nuclei and hypogranular cytoplasm. Eosinophils are mostly mature with rare eosinophilic myelocytes. Many are sparsely granulated. A slight absolute basophilia (3% of leukocytes) is seen but there is no monocytosis. Red cells show mild normocytic normochromic anemia with occasional teardrop cells and stomatocytes. Platelets are adequate in number by visual estimate but with anisocytosis and poorly granulated forms. Rare bare megakaryocyte nucleus is noted.

Bone marrow biopsy:

Left posterior iliac crest, B.Plus fixative, RDO Rapid (HCl-based) decalcifier.

The bone marrow biopsy consists of 2 cores measuring 5mm each revealing a cellularity close to 90%, which is markedly hypercellular for age. The increased cellularity is due to a granulocytic hyperplasia comprised of predominantly eosinophils and neutrophilic granulocytes in all stages of maturation. There is no overt increase or clustering of blasts on the H&E-stained sections alone although distinction between nucleolated precursors is somewhat difficult. Erythropoiesis progresses to maturity but is slightly reduced and present as scattered late precursor forms. Megakaryocytes are moderately increased in number with no appreciable clustering. Many are small and hypo/monolobated with some showing features similar to ‘dwarf’ megakaryocytes and micromegakaryocytes. Bare nuclei are also noted.

Bone marrow aspirate:

The bone marrow aspirate smears show numerous hypercellular spicules with cellularity in keeping with that seen in the biopsy.

A 500 cell count differential yields:

Blasts 0%

Promyelocytes 3%

Myelocytes 5%

Metamyelocytes 8%

Bands 14%

Seg 16%

Eos 31%

Baso1%

Lymph 2%

Plasma cell 0%

Mono 1%

Erythroid precursors 19%

M:E = 4:1

Blasts are not increased on the differential cell count and the M:E ratio is appropriate. There is maturing trilineage hematopoiesis. Eosinophils and eosinophilic myelocytes are increased. Some of the segmented neutrophils are abnormally lobated with hypercondensed chromatin. Occasional immature granulocyte shows cytoplasmic vacuoles. Significant dyserythropoiesis is not appreciated. Megakaryocytes are markedly increased and are predominantly small, hypolobated forms. A number of megakaryocytes resemble ‘dwarf’ megakaryocytes. Mast cells are not conspicuous. Lymphoid and plasma cells are not increased and are morphologically unremarkable. Hemophagocytic activity is noted.

A Prussian blue iron stain performed on the aspirate smear shows no iron stores and no evidence of ring sideroblasts.

Immunohistochemical and special stain analysis:

A CD34 stain shows positive expression in rare mononuclear cell. CD34+ cytoplasmic granular staining is also seen in rare mature megakaryocyte. CD117 and mast cell tryptase highlight rare to occasional scattered round and spindled mast cells. Factor VIII reveals the striking megakaryocytic hyperplasia with numerous smaller, hypolobated forms.

A reticulin stain shows 1+ to focal 2+/3+ reticulin fibrosis.

Flow cytometric analysis (peripheral blood):

CD45 vs side scatter analysis reveals a population of cells in the lymphoid region accounting for 10% of total events, comprised of predominantly unremarkable CD3+ T-cells (T4:T8 of 1.6:1) and small number of polyclonal CD19+ B-cells.

Flow cytometric analysis (bone marrow aspirate):

CD45 vs side scatter analysis reveals a small population of cells with a myeloid profile in the blast region accounting for only 2% of total events.

Cytogenetic/FISH studies (bone marrow aspirate):

Karyotype:

Results by G-banded karyotype revealed an abnormal clonal proliferation with t(9;12)(q34;p13) and a 13q deletion: 46,XY,t(9;12)(q34;p12~13),del(13)(q12~14q22~34)[9]/46,XY[2]

Nine of eleven cells analyzed show a deletion of the long arm of chromosome 13 with breakpoints estimated between q12~q14 and q22~q34. In addition, these abnormal cells show a balanced translocation between the long arm of chromosome 9 and the short arm of chromosome 12 with breakpoints estimated at 9q34 and 12p12 or 12p13. The remaining two cells show a normal male karyotype.

FISH:

ish t(9;12)(q34;p12~13)(ABL1+;ABL1+)[3]

Results by metaphase FISH are positive for ABL1 rearrangement, with a partner gene on the short arm of chromosome 12. This confirms the presence of a t(9;12)(q34;p12~13) involving a rearrangement of the ABL1 gene, as suspected by G-banding.

Molecular studies (peripheral blood):

BCR-ABL1 fusion transcripts were not detected by real-time quantitative PCR (RQ-PCR) consistent with the absence of Philadelphia chromosome positive cells in the peripheral blood.

There was also no evidence of FIP1L1-PDGFRA fusion transcripts by RQ-PCR.

Molecular studies (bone marrow aspirate):

The JAK2 V617F mutation was NOT detected using the PCR ARMS assay.

Course:

A diagnosis of ETV6-ABL1+ myeloproliferative neoplasm with eosinophilia was made and the patient was started on imatinib mesylate, up to 400mg daily. In five months, the patient has shown no adverse side effects and complete hematologic response. He is to be seen in 6 weeks for a repeat bone marrow study to assess for cytogenetic and molecular response.

Interesting aspects of case:

This case illustrates a myeloid neoplasm carrying the rare rearrangement resulting in a chimeric protein containing the helix-loop-helix domain of ETV6 and the tyrosine kinase domain of ABL1. A number of translocations involving ETV6 have been reported since the identification of ETV6-PDGFRB in patients with chronic myelomonocytic leukemia (CMML) and t(5;12)(q33;p13). In many of these variants, the common theme is fusion of the homodimerization domain of ETV6 to a tyrosine kinase domain, resulting in a constitutively active kinase. Despite their rarity, these translocations are important to detect as they are likely to respond to small molecule inhibitors such as imatinib. Our patient has responded well to imatinib therapy thus far and will soon be assessed for cytogenetic/molecular remission.

Reports on the hematologic features of ETV6-ABL1 positive cases emphasize an abnormal eosinophilic proliferation, as is the hallmark of the WHO 2008 category ‘Myeloid and lymphoid neoplasms with eosinophila and abnormalities of PDGFRA, PDGFRB or FGFR1’. As in that category, the clinical and hematologic features may be influenced by the partner gene involved. Morphologic findings do not support a diagnosis of chronic myelogenous leukemia (CML), atypical CML or CMML. Although features of the case and clinical history best classify the disorder morphologically as chronic eosinophilic leukemia (CEL), the degree of dysplasia noted in the granulocytic and megakaryocytic lineages, rarity of myeloblasts and increased presence of neutrophilic precursors and basophils in the peripheral blood possibly warrant a more nuanced category. It must be noted that our patient also carried an additional cytogenetic abnormality, del13q, which has been reported in myeloid neoplasms, especially in CML.

Myeloproliferative neoplasm (MPN) with eosinophilia associated with ETV6-ABL1

Myeloproliferative neoplasm with eosinophilia with t(9;12)(q34;p13); ETV6-ABL1

Case 237