B-Cell Tumors With t(14;19)(q32;q13)/IGH::BCL3 Identified by G-Banding and FISH

Journal of Clinical and Experimental Hematopathology. 2024; 64 (1): 21-31.

Authors: Ohno H, Maekawa F, Hayashida M, Nakagawa M, Fukutsuka K, Matsumura M, Takeoka K, Maruyama W, Ukyo N, Sumiyoshi S, Tanaka Y, Haga H.

t(14;19)(q32;q13) was first described in 3 out of 30 patients with B-cell chronic lymphocytic leukemia (B-CLL), and the translocation was found to create a fusion gene between the IGH gene at the 14q32 chromosomal band and BCL3 gene at 19q13. In a large series of B-CLL patients tested by fluorescence in situ hybridization (FISH) using relevant probes, t(14;19)(q32;q13)/IGH::BCL3 was found in 16 (0.9%) out of a total of 1,683 patients.

We searched the database of cytogenetic analysis in our institution between 2010 and 2023 and selected 5 patients who carried t(14;19)(q32;q13), including one patient described previously.

• Mononuclear cells were separated from peripheral blood (PB) or bone marrow (BM) aspirates. Lymph node (LN) biopsy specimens were aseptically minced to prepare a cell suspension. Cells were resuspended in phosphate-buffered saline and aliquots were subjected to flow cytometry (FCM).
• Pathological specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, and then subjected to histopathological examination. The monoclonal antibodies used for immunohistochemistry (IHC) were: anti-CD3, CD5, CD10, CD20, CD 30, CD79a, BCL2, MYC, BCL3, and PD-1.
• Tumor cells prepared from PB/BM/LN were incubated in RPMI 1640 medium supplemented with 15% heat-inactivated fetal bovine serum at 37°C under a CO₂ concentration of 5%. Cells were then cultured in the presence of 0.1 μg/mL colcemid for 2 hr. After harvesting, the cells were treated with the hypotonic solution and fixed in methanol: acetic acid (3:1). Chromosomes were banded by trypsin-Giemsa and the results of chromosome analysis are presented according to ISCN 2020.
• Cytogenetic preparations were hybridized with fluorophore-labeled probes. The following FISH probes were used: the Vysis LSI IGH break-apart (BA) probe, BCL6 BA probe, MYC BA probe, and XL BCL3 BA probe. FISH results were analyzed using fluorescence microscopes equipped with DAPI, fluorescein isothiocyanate (FITC), and tetramethylrhodamine B isothiocyanate (TRITC) fluorescence filters as well as a DAPI/FITC/TRITC triple band-pass filter.
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• G-banding of metaphase spreads obtained from PB, BM, or LN revealed t(14;19)(q32;q13), resulting in the characteristic 14q+ and 19q− morphology, in all 5 cases. (Fig.1, 2, and 3). In case 5, both diploid- and tetraploid-range karyotypes had a single copy of t(14;19)(q32;q13) (Fig. 3). The translocation was confirmed by FISH in all cases, demonstrating colocalization of red-colored centromeric 3' IGH and green-colored telomeric 3' BCL3 probes at q32 of der(14)t(14;19), corresponding to the IGH::BCL3 fusion gene, and red-colored centromeric 5' BCL3 and green-colored telomeric 5' IGH probes at q13 of der(19)t(14;19), corresponding to the reciprocal gene fusion (Fig. 1, 2, and 3). Other 14q32/IGH translocations included t(2;14)(p13;q32) in case 1, t(8;14)(q24;q32) in case 3, and t(8;11;14)(q24;q11;q32) in case 4 (Fig. 1, 2, and 3). In the latter 2 translocations, FISH revealed colocalization of red-colored centromeric 3' IGH and green-colored 3' MYC at q32 of der(14)t(8;14) and der(14)t(8;11;14), respectively, proving the generation of the IGH::MYC fusion gene (Fig. 2). Additional abnormalities recognized by G-banding and FISH were trisomy 12 in cases 1, 2, and 3, 3 copies of MYC in cases 1 and 3, and 4 copies of BCL6 in association with deletion of a red-colored telomeric 5' BCL6 probe in case 4 (Fig. 1 and 2). A CLL FISH panel test to detect del(6q), del(11q), del(13q), and del(17p) was negative in all cases tested (Fig. 1B).

Fig. 1 Cytogenetic findings in case 1.

Fig. 2 Cytogenetic findings in case 4.

Fig. 3 Cytogenetic findings in case 5.

• Histopathological examination of the LN biopsy in case 2 revealed diffuse proliferation of small cells admixed with larger prolymphocytes/para immunoblasts. The cells were positive for CD20 and BCL2 and negative for CD5 by IHC. The Ki67 index was 20%. In cases 3 and 4, on the other hand, tumor cells were large and had round, vesicular nuclei with prominent nucleoli; the proportion of tumor cells was lower in the latter case compared with the former (Fig. 4). The cells were negative for CD10 and positive for CD20, MUM1, and BCL2. A fraction of large cells was positive for CD5 in case 3. BCL6 was negative in case 3 and positive in case 4. Thus, cases 3 and 4 were classified into the non-germinal center B-like (non-GCB) category of diffuse large B-cell lymphoma (DLBCL). The tumor cell nuclei of both cases were stained positive by anti-MYC and anti-BCL3 antibodies (Fig. 4). The Ki67 index was >90% in case 3 and 40% in case 4. In case 5, the LN structure was effaced with infiltration of CD3-positive small to medium-sized cells, CD20-positive large B cells, and CD30-positive cells in the setting of proliferation of high-endothelial venules and polymorphic cellular infiltrates composed of histiocytes, plasma cells, and eosinophils (Fig. 5). Some T cells were positive for PD-1 and clusters of BCL6-positive cells were found. CD10 was negative. EBER ISH revealed many EBV-positive cells, most likely corresponding to large B cells (Fig. 5).

Fig. 4 Histopathology of cases 3 (A to C) and 4 (D to I).

Fig. 5 Histopathology of case 5.

We characterized 5 B-cell tumors carrying t(14;19)(q32;q13) that creates the IGH::BCL3 fusion gene. The presence of t(14;19)(q32;q13) was confirmed by FISH, showing colocalization of 3' IGH and 3' BCL3 probes on der(14)t(14;19) and 5' BCL3 and 5' IGHprobes on der(19)t(14;19).

Karyotyping (G-Banded) Service

Fluorescent In Situ Hybridization (FISH) Service

Reference

1. Ohno H, et al. (2024). "Diverse B-cell tumors associated with t(14;19)(q32;q13)/IGH::BCL3 identified by G-banding and fluorescence in situ hybridization." J Clin Exp Hematop. 64 (1): 21-31.

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