Platelet expression of the transcription factor ETV6 points toward ETV6-related thrombocytopenia and can be detected by immunofluorescence on the blood smear

Introduction Inherited platelet disorders (IPD) present with congenital reduced or dysfunctional blood platelets and sometimes with additional, acquired manifestations affecting the morbidity of the patients more than the thrombocytopenia itself. ETV6-related thrombocytopenia (ETV6-RT), as well as IPD due to mutations in RUNX1 and ANKRD26, causes mild-to-moderate thrombocytopenia with normal platelet size and makes the patients prone to develop throughout-life hematological malignancies [1] [2]. ETV6 protein is a transcriptional suppressor, which is usually located in the nucleus of megakaryocytes but redistributes into the cytoplasm when its DNA-binding site is hit by mutations. Recognizing ETV6-RT-patients is challenging. While the diagnostic confirmation could be relevant for the interpretation of their phenotype, it could raise at a time ethical issues because of the associated risk of leukemic transformation ([Fig. 1]).

Fig. 1 Platelet expression of ETV6; Platelet expression of ETV6 protein in platelets from an ETV6-RT patient with respect to a healthy control by immunofluorescence-microscopy. The platelets are counterstained for GPIbIX.

Method We analyzed blood smears by light- and immunofluorescence-microscopy upon staining with May-Grünwald-Giemsa technique and with a set of primary antibodies against 13 markers belonging to different platelet structures (granules, cytoskeleton, surface receptors) and ETV6 protein. Their expression was assessed after staining with fluorescence-labelled secondary antibodies [3] [4].

Results We investigated 11 patients belonging to 6 unrelated families with genetically confirmed ETV6-RT. In all but one patient, diverse abnormalities of dense granule markers LAMP1, LAMP2 and CD63 were reported (variable reduction of granule number; diffused distribution of the staining; granule structure presenting on the outer membrane instead inside the platelets). ETV6 protein was detectable in the cytoplasm of platelets of all patients, suggesting a pathological expression. In parallel, we assessed ETV6 expression in platelets from healthy blood donors (n=20) and from patients affected with other forms of IPD (n=20). We found no platelet expression of ETV6 in any of the investigated control individuals.

Conclusion We have established an immunofluorescence-based screening method for ETV6-RT on the blood smear. Typical features are: platelet expression of ETV6, normal platelet size, and evidence of dense granule abnormalities. Due to the ethical implications of this finding, i.e., the association of ETV6-RT with an increased risk for hematologic malignancies, patients have to be informed and should provide explicit consent prior to performing this investigation.

• References

• 1 Galera P, Dulau-Florea A, Calvo KR. Inherited thrombocytopenia and platelet disorders with germline predisposition to myeloid neoplasia. Int J Lab Hematol 2019; 41 (Suppl. 01) 131-141 PMID: 31069978
• 2 Di Paola J, Porter CC. ETV6-related thrombocytopenia and leukemia predisposition. Blood 2019; 134 (08) 663-667 Epub 2019 Jun 27. PMID: 31248877; PMCID: PMC6706811
• 3 Greinacher A, Pecci A, Kunishima S, Althaus K, Nurden P, Balduini CL, Bakchoul T. Diagnosis of inherited platelet disorders on a blood smear: a tool to facilitate worldwide diagnosis of platelet disorders. J Thromb Haemost 2017; 15 (07) 1511-1521 Epub 2017 Jun 4. PMID: 28457011
• 4 Zaninetti C, Greinacher A. Diagnosis of Inherited Platelet Disorders on a Blood Smear. J Clin Med 2020; 9 (02) 539 PMID: 32079152; PMCID: PMC7074415