Novel NPM1 gene fusion transcripts. (A) NPM1/RPP30 rearrangement. In MLL patient 1, NPM1 was rearranged with the RPP30 gene at the end of exon 9 (breakpoint at position 772 of NPM1 complementary DNA [cDNA] based on transcript ENST00000296930), and RPP30 was rearranged with NPM1 at exon 11 (breakpoint at position 697 in RPP30 cDNA ENST00000371703.7). The encoded fusion was predicted to be out of frame. The new fusion protein is 269 aa long, with a predicted molecular weight (MW) of 29.6 kDa. The predicted protein sequence of the C-terminus of the new fusion protein (red) is shown as compared with the C-terminus of either NPM1 WT or mutant A. The newly acquired NES domain is underlined (LLLL). (B) NPM1/CCDC28A rearrangement. In MLL patient 3 and PG patient 4, NPM1 was rearranged with the CCDC28A gene at the end of exon 11 (breakpoint at position 846 of NPM1 cDNA based on transcript ENST00000296930), and CCDC28A was rearranged with NPM1 at the beginning of exon 2 (breakpoint at position 228 in CCDC28A cDNA ENST00000611852.4). The encoded fusion was predicted to be in frame. The new fusion protein is 480 aa long, with a predicted MW of 53.1 kDa. Two NES domains at the C-terminus of the new fusion protein (red) are underlined (LLLL, LILL). (A-B) Representative images of NIH-3T3 overexpressing the new GFP-NPM1 fusion protein NPM1/RPP30 (MLL patient 1) (A) and NPM1/CCDC28A (MLL patient 3 and PG patient 4) (B) showing the aberrant localization in the cytoplasm (right). Of note, the nucleoli are also positive because of the loss of NoLS. Images were acquired using a Zeiss LSM 800 confocal microscope (Carl Zeiss) with a 488-nm (for eGFP) laser line for excitation and 63×/1.4 oil Plan-Apochromat objective (×63 original magnification). (C) Left, WB analysis with either the anti-NPM1 antibody (clone 376) recognizing the N-terminus of NPM1 (upper left) or the anti-CCDC28A antibody raised against the C-terminal part of CCDC28A protein (upper right) on the total protein extract from MLL patient 3. The AML sample carrying the NPM1/CCDC28A fusion transcript shows a band recognized by both antibodies just above 50 kDa, corresponding to the predicted 53-kDa MW of the new fusion protein. OCI-AML3 protein extract was used as control for the clone 376 anti-NPM1 antibody (band at ∼37 kDa). Samples were loaded in duplicate. The white vertical line indicates where the membrane was cut. Blotting for histone H3 was used as control for protein loading (lower subpanel). Right, BM trephine from PG patient 4 carrying the NPM1/CCDC28A fusion transcript showing diffuse BM infiltration by leukemic blasts (hematoxylin eosin [HE]; left) with cytoplasmic NPM1 (inset for details; right). NPM1 staining: mouse monoclonal anti-NPM1 clone 376 by antialkaline phosphatase technique with hematoxylin counterstaining. Images were collected using an Olympus B61 microscope and a UPlanApo 40×/0.85 (×40 original magnification) and UPlan FI 100×/1.3 NA oil (×100 original magnification) objective for the inset, Camedia 4040 (Dp_soft version 3.2), and Adobe Photoshop CC 2019. (D) Table illustrating the most relevant characteristics of patients carrying the new NPM1 fusion transcripts. Dash indicates not applicable. ActD, actinomycin D at 12.5 mg/kg per day for 5 days²⁵; CR, complete remission; FU, follow-up; Hb, hemoglobin; IC, standard intensive chemotherapy; NA, not available; PLT, platelets; VAF, variant allele frequency; WBC, white blood cells.