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

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 × 106/µL, Hg 10.3 g/DL, MCV: 76.5), normal leukocyte count 4.93 × 103/µL (with normal differential), and thrombocytopenia (107 × 103/µL). 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 = 1.95) co-expressing 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 LAIR-1(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/µL) 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). HCL-variant with tetraploidization. (a) DNA analysis of the peripheral blood showing cells with near-tetraploid DNA content. The picture on the right is representative of FISH analysis showing 4 signals for centromere 17 and only 2 signals for p53. (b) CD20 immunoreactivity in cells with near-tetraploid DNA content. (c) CD103 and CD25 expression on the B cells (green) as compared to non B cell lymphocytes (gray) in the peripheral blood. (d) LAIR-1 expression on the B cells (green) as compared to non B cell lymphocytes (red) in the peripheral blood. (e) DNA analysis of spleen biopsy showing cells with near-tetraploid DNA content. (f) CD103 and CD25 expression on the B cells (green) as compared to non B cell lymphocytes (gray) in the spleen biopsy. (g) Cell morphology in the peripheral blood. (h) H&E and TRAP staining of the spleen biopsy. [Color figure can be viewed at wileyonlinelibrary.com] 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 immuno-phenotype 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 near-tetraploid 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 near-tetraploid DNA content was associated with atypical morphological appearance of the cells as shown by lack of typical hairy branches. The near-tetraploid cells we observed can theoretically represent truly tetraploid cells. Alternatively, these near-tetraploid cells could represent diploid cells arrested in G2-M. The distance between the markers of the centromeres in our case would tend to make unlikely that our near-tetraploid DNA content cells are diploid cells arrested in G2 (In which each pair of centromeres should be attached together). Absent of overlap between the proportion of leukemic cells and the mitotic figures in the spleen biopsy as well as the lack of mitotic figure in the blood film would tend to make unlikely an arrest of diploid cells in M. On the other hand, the significant Ki-67 positivity of total spleen cells would tend to make unlikely an arrest of truly tetraploid cells in G0 (when Ki-67 is not expressed). As Ki67 was previously showed to stain non-proliferating cells in G1 4, it might be possible that our near-tetraploid DNA content cells represent true tetraploid cells arrested in G1. Indeed, the relatively lower proportion of cells in S-phase may be compatible with little proliferation and arrest. Chromosomal abnormalities were reported in 67% of HCL patients and they were mostly involving insertions and deletions in chromosomes 1, 2, 5, 6, 11, 19, and 20. Except trisomy 5 numerical abnormalities and translocations were rare 5. In a study of chromosomal abnormalities in HCL-variant, numerical chromosome changes included loss of chromosomes 2, 3, 4, 6, 10, 19, 21, and X 6. In another study, trisomy 12 was shown by FISH in 8.5% and 6–8% of cells in HCL-variant and classical HCL, respectively 7. The only record for tetraploidization in HCL in the literature was shown in a cell line named Hair-M that was originally established from an 86-year-old male patient 8. However, this tetraploidization was probably developed by continuous genetic instability in vitro as the karyotype of this cell line was initially described to be diploid. Furthermore, The Hair-M cell line lacks the BRAF-V600E mutation and HCL typical immuno-phenotype being therefore of questionable HCL origin 9. In contrast to FISH the tetraploidization detected by flow cytometry in our case was not detected by conventional cytogenetics. This discrepancy could be explained by the relatively lower incidences of leukemic cells in the BM specimen which may provide a few mitoses to be detected by the conventional cytogenetic method. Screening of normal dividing cells that were abundant in the sample could also explain the absence of corresponding cytogenetic abnormality. Genetic instability, abortive cell cycle, and cell fusion were suggested as candidate mechanism by which tetraploidization is developed 10. Interestingly, a remarked tendency to harbor chromosome numbers in the tetraploid range was previously reported in blastic variants of mantle cell lymphoma (MCL) 11. Due to its crucial role in cell cycle regulation, cyclinD1 (BCL-1) overexpression resulting from the t(11:14) translocation was postulated to contribute to this phenomenon in MCL. Although overexpression of cyclin D1 mRNA and protein previously was shown in HCL which lacks the t(11:14) translocation, this overexpression was much lower compared to that in MCL 12. Indeed the lack of t(11:14) translocation and the corresponding few BCL-1 positive cells that were detected in our case indicate that another mechanism may be involved with the tetraploidization we observed. Our case showed the deletion of p53 in two of the four chromosomes 17 in the tetraploid cells. This incidence emphasized that tetraploidization can obscure the presence of P53 deletion due to a duplication of the hybridization pattern. As p53 was deleted in two chromosomes and not in three chromosomes of the tetraploid cells, we assume that this deletion initially occurred in a diploid cell that later become tetraploid. P53 is a tumor suppressor gene, one of whose functions is the maintenance of genetic integrity 13. Correspondingly, it was previously shown that development of tetrploidization is interdependent with the inactivation of p53 14. HCL-v is known to frequently carry an unmutated or lowly mutated IgVH4-34 rearrangement and that this is usually associated to a worse prognosis (Refs. 3 and others), in contrast to the rather indolent course of the case described here. Apparent cell cycle arrest and little S-phase activity may be partly explaining the indolent disease of our patient. In this regard it is tempting to speculate that this cell cycle arrest and the corresponding indolent disease course resulted by re-acquisition of two functional copies of P53 upon tetraploidization. Although such a dynamics would be unusual and needs to be further approved. We wish to thank Dr. Robert J Kreitman for his tremendous help regarding this case. David Azoulay* Department of Hematology Galilee Medical Center Nahariya, Israel Vadim Sonkin Department of Pathology Galilee Medical Center Nahariya, Israel Luiza Akria Department of Hematology Galilee Medical Center Nahariya, Israel Ayala Rozano Gorelick Department of Hematology Galilee Medical Center Nahariya, Israel Luba Trakhtenbrot Department of Hematology Sheba Medical Center Tel Hashomer, Israel Dov Hershkovitz Department of Pathology Rambam Health Care Campus Haifa, Israel Ety Shaoul Department of Hematology Galilee Medical Center Nahariya, Israel Simona Rozen Department of Hematology Galilee Medical Center Nahariya, Israel Eugene Dementiev Department of Pathology Galilee Medical Center Nahariya, Israel Hector I Cohen Department of Pathology Galilee Medical Center Nahariya, Israel Celia Suriu Department of Hematology Galilee Medical Center Nahariya, Israel Andrei Braester Department of Hematology Galilee Medical Center Nahariya, Israel