Polycythemia Vera

Myeloproliferative neoplasm

CD34+ cell

SETD1B is a SET-domain containing protein with histone H3-Lys4 methyltransferase activity, which is associated with active gene expression (Lee et al. 2007). SET-like enzyme genes include those implicated in various leukemias and cancers like MLL. SETD1B interacts with RBM15 through its LSD motif, creating a complex important for epigenetic regulation (Lee et al. 2002). RBM15 (RNA binding motif protein 15) utilizes the epigenetic mechanism to control alternative splicing of MPL (myeloproliferative leukemia virus oncogene), leading to the regulation of thrombopoietin response in hematopoietic stem cells (Xiao et al. 2014). In the SETD1B/GTF2H3 chimeric protein, SETD1B loses the LSD domain, possibly leading to an altered epigenetic regulation. GTF2H3 has been related to the nucleotide excision repair pathway, and resulted upregulated in HL-60 cell line resistant to ATRA (Liu et al. 2014).

The SETD1B/GTF2H3 rearrangement was found in only one out of 60 patients with myeloproliferative neoplasms evaluated. However, FISH analysis revealed a splitting signal in a very low percentage of nuclei (3/412, 0.7%) in one of 13 analyzed patients, suggesting a possible rearrangement of SETD1B with other partner genes (Storlazzi et al. 2014).

The SETD1B/GTF2H3 rearrangement has been found in a patient diagnosed with Polycythemia Vera that evolved in post-polycythemia vera myelofibrosis after two years.

Blood smear showed no leukoerythroblastic picture but only few dacrocytes.

The bone marrow biopsy showed the presence of hyperplasia of myeloid and erythroid lineages, increased scattered megakaryocytes without overt morphologic abnormalities and an increase in reticulin fibrosis compatible with grade 1 of 3 (european scale).

The subject was treated with phlebotomies and hydroxyurea according to the European Leukemia Net (ELN) recommendations (Barbui et al. 2011), but considered as refractory/resistant to hydroxyurea (Barosi et al. 2010). The patient was then enrolled in a clinical trial with the histone deacethylase inhibitor givinostat, obtaining prompt relief from pruritus and rapid reduction up to stop of phlebotomies (Finazzi et al. 2013). However, the treatment was interrupted after six months because of progression of a pre-existing mild renal insufficiency, and the subject was shifted again to low-dose hydroxyurea plus phlebotomies.

Two years after the detection of the SETD1B/GTF2H3 fusion gene, the patient was diagnosed with post polycythemia vera myelofibrosis according to the diagnostic criteria of the International Working Group for Myeloproliferative Disorders Research and Treatment (IWG-MRT) (Barosi et al, 2008). At that time, the gene fusion was still detectable in bone marrow sample.

The rearrangement leading to the genesis of this fusion gene (a cryptic deletion on chromosome 12) was not evident at cytogenetic level.

The only described PV patient harboring this novel fusion gene showed a complex karyotype.

The SETD1B/GTF2H3 fusion gene was identified by genomic massive sequencing. FISH analysis with an appropriate probe set for SETD1B disclosed the occurrence of a 110Kb interstitial deletion, confirming the gene rearrangement.

A ins(6;15) insertion accompanied by chromosome losses at all the breakpoint regions [6p22.1p22.3 (11,6 Mb), 15q21.3q22.2 (6 Mb), and 15q25.1q25.2 (3 Mb)]; a cryptic and inverted insertion of a short chromosome 15q25.1 fragment (chr15:79,476,511-79,486,994) into chromosome 18, accompanied by a 25.7 Kb heterozygous deletion

5 SETD1B/3 GTF2H3. SETD1B (accession no. NM_015048) at exon 11 was fused in frame with GTF2H3 (accession no. NM_001516) at exon 7.

Genomic paired-end massive sequencing. The fusion transcript was validated by RT-PCR and Sanger sequencing.

In silico translation of the fusion transcript, obtained by ORF finder, showed a putative protein maintaining the SETD1B RNA binding domain (RRM_S) at its N-terminus and a portion of GTF2H3 DNA binding domain (Tfb4) at its C-terminus