Hairy cell leukemia‐variant without typical morphology and with near‐tetraploid DNA content

A 75-year-old male was referred to our medical center due to abdominal pain. Clinical record showed splenomegaly 4 years prior to his visit and a mild microcytic anemia during the last 2 years. Physical examination showed splenomegaly without lymphadenopathy. Complete blood count showed anemia (RBC 4.21 3 10/mL, Hg 10.3 g/DL, MCV: 76.5), normal leukocyte count 4.93 3 10/mL (with normal differential), and thrombocytopenia (107 3 10/mL). Bone marrow (BM) aspirate that was sent for flow cytometry showed lymphocyte clusters that accounted for 30% of the total nucleated cell component and Kappa restricted B cells that accounted for 30% of the total lymphocytes. DNA analysis that we performed showed near tetraploid DNA content (DI 5 1.95) coexpressing the B cell marker CD20. Further immuno-phenotyping of the B cells showed bright expression of CD45, positive expression of FMC7, CD22, CD23, CD79b, and CD200 and negative expression of CD5, CD10, and CD43. Detection of HCL-relevant antigens showed positive expression of CD11c and CD123, dim CD103 expression and negative expression of CD25. Staining with LAIR1(CD305) showed bright expression. BM biopsy was mild hyper-cellular with increased small to intermediate size lymphocytes showing diffuse interstitial pattern and minimal nodular pattern. CD45 was positive in 30% of cells, part of which was positive to CD20 and CD79a and part to CD3 and CD5. The percentage of leukemic cell out of the total hematopoietic cellularity in the bone marrow biopsy was 10%. TRAP immunoreactivity was inconclusive. Flow cytometry analysis of the peripheral blood (PB) showed the malignant B cells with incidence of 42.1% among the total lymphocyte component (901 leukemic cells/mL) and identical immuno-phenotype and aneuploidy as observed in the BM aspirate (Fig. 1a–d). A search for pathological cells in the blood smear showed medium to large size cells with prominent nucleolus and wide cytoplasm that lack visible hairy branches (Fig. 1g). Due to uncertain diagnosis, the extremely enlarged spleen (2.48 kg, 28 cm long as determined by ultrasound 2 month before splenectomy) was removed from the patient. Flow cytometry analysis of the spleen biopsy showed lymphocyte cluster that accounted for 85% of the total nucleated cell component and Kappa restricted B cells that accounted for 45% of the total lymphocytes. The B cells in the spleen showed immunophenotype and aneuploidy similar to the BM aspirate and the peripheral blood (PB) (Fig. 1e,f). Histological assessment of the spleen showed little and condensed white pulp with massive lymphocyte infiltration in the red pulp. The lymphocytes were medium size with irregular cell nuclei, partly folded with clumping chromatin; some of them with prominent nucleoli. The lymphocytes were positively stained with CD20, CD79a, PAX-5, CD23, and TRAP (Fig. 1h). Immunoreactivity to Annexin-1A, DAB44, BCL-6, MUM-1, CD10, CD5, and P53 were negative. Ki67 was positive in 30% of the lymphocytes and the larger atypical cells represented the greater fraction of the proliferating cells. Immunoreactivity to cMYC and BCL-1 were positive only in a few cells. Molecular analysis of the spleen biopsy showed negative t(11:14) translocation, wild type BRAF (exon 15 V600E) and MEK1/MAP2K1 were determined to be without mutation. An unbiased search for the clonal immunoglobulin heavy chain (IGH) gene rearrangement harbored by the tumor was performed. This analysis revealed the tumor IGH gene rearrangement to contain a 100% unmutated VH4-34 variable gene. Flow cytometry analysis of BM aspirate that was taken from the patient one month after splenectomy showed lymphocyte clusters that accounted for 26% of the total nucleated cell component and Kappa restricted B cells that accounted for 36% of the total lymphocytes. The malignant B cells showed similar immuno-phenotype to the cells at diagnosis and they were clearly recognized by their near-tetraploid peak. BM sample that was sent for cytogenetics showed no abnormality in the 19 cells in metaphase that were screened. Nevertheless, BM sample that was sent for FISH analysis detected cells with nuclear atypia that showed four signals for centromeres 2, 4, 8, 10, 15, and 17 as well as four MLL and four IGH signals. Analysis of centromere 9 showed three signals. By contrast, p53 was found to be with only 2 signals, suggesting a deletion of p53 in the atypical cells with the neartetraploid DNA. Sequencing of TP53 exons 4–9 showed no mutations. The immuno-phenotype of the cells by flow cytometry in our case fits with those of HCL-variant as determined by absence of CD25 (1). As was previously shown (2,3), the wild-type BRAF gene that we found in our case gives another molecular support to the diagnosis of HCL-variant. HCL-variant is difficult to differentiate from splenic diffuse red pulp small B-cell lymphoma. However, in our case we have several evidences that are in favor of HCL-variant and against splenic diffuse red pulp small B-cell lymphoma. First, the cells in our case are medium size whereas in splenic diffuse red pulp small B-cell lymphoma cell size should be small monomorphous and with common villous cytology. Second, TRAP staining was determined to be positive in the spleen in our case and according to WHO classification usually not present in splenic diffuse red pulp small B-cell lymphoma. To the best of our knowledge, our case represents the first documentation of HCL-variant with near-tetraploid DNA content in vivo. We found this case important to be reported as this neartetraploid DNA content was associated with atypical morphological appearance of the cells as shown by lack of typical Cytometry Part B (Clinical Cytometry) 94B:169–171 (2018)