Diverse B-cell tumors associated with t(14;19)(q32;q13)/IGH::BCL3 identified by G-banding and fluorescence in situ hybridization

Abstract

We characterized 5 B-cell tumors carrying t(14;19)(q32;q13) that creates the IGH::BCL3 fusion gene. The patients’ ages ranged between 55 and 88 years. Two patients presented with progression or recurrence of B-cell chronic lymphocytic leukemia (B-CLL)/small lymphocytic lymphoma (SLL), two with diffuse large B-cell lymphoma (DLBCL) of non-germinal center B-like phenotype, and the remaining one with composite angioimmunoblastic T-cell lymphoma and Epstein-Barr virus-positive DLBCL. The presence of t(14;19)(q32;q13) was confirmed by fluorescence in situ hybridization (FISH), showing colocalization of 3′ IGH and 3′ BCL3 probes on der(14)t(14;19) and 5′ BCL3 and 5′ IGH probes on der(19)t(14;19). One B-CLL case had t(2;14)(p13;q32)/IGH::BCL11A, and 2 DLBCL cases had t(8;14)(q24;q32) or t(8;11;14)(q24;q11;q32), both of which generated IGH::MYC by FISH, and showed nuclear expression of MYC and BCL3 by immunohistochemistry. The IGH::BCL3 fusion gene was amplified by long-distance polymerase chain reaction in 2 B-CLL/SLL cases and the breakpoints occurred immediately 5′ of BCL3 exon 1 and within the switch region associated with IGHA1. The 5 cases shared IGHV preferentially used in B-CLL cells, but the genes were unmutated in 2 B-CLL/SLL cases and significantly mutated in the remaining 3. B-cell tumors with t(14;19)(q32;q13) can be divided into B-CLL/SLL and DLBCL groups, and the anatomy of IGH::BCL3 in the latter may be different from that of the former.

INTRODUCTION

t(14;19)(q32;q13) was first described in 3 out of 30 patients with B-cell chronic lymphocytic leukemia (B-CLL),¹ and the translocation was found to create a fusion gene between the IGH gene at the 14q32 chromosomal band and BCL3 gene at 19q13.^2–5 BCL3 contains 7 tandem copies of the ankyrin repeat in the central domain and is included in the NF-κB inhibitor IκB family of proteins.⁴ Unlike prototypical IκB proteins, BCL3 is a nuclear protein containing a transactivation domain and interacts specifically with p50 and p52 homodimers.^6,7 Transgenic mice that carry the BCL3 transgene under the control of the IGH-Eμ enhancer, mimicking t(14;19)(q32;q13), show splenomegaly and an accumulation of mature B cells in lymph nodes, bone marrow, and the peritoneal cavity.⁸

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.⁹ A large proportion of patients with t(14;19)(q32;q13) are aged <40 years, and the median age is significantly lower compared with that of t(14;19)(q32;q13)-negative patients.^9–12 t(14;19)(q32;q13) is associated with atypical morphological and immunophenotypical characteristics and patients show an aggressive clinical course and poor overall survival.^9–12 Thus, some investigators proposed the existence of a t(14;19)(q32;q13)-positive small B-cell leukemia/neoplasm that is independent of B-CLL/small lymphocytic lymphoma (SLL).¹¹ On the other hand, lymphoid tumors carrying t(14;19)(q32;q13) or its light chain variants exhibit heterogeneous presentation, including not only many types of B-cell tumors other than B-CLL/SLL,^2,13–20 but also Hodgkin lymphoma and peripheral T-cell lymphoma.⁷ The t(14;19)(q32;q13) in these tumors is identical to that in B-CLL/SLL at the cytogenetic level, but not necessarily at the DNA level.¹⁹ In this study, we present 5 B-cell tumors with t(14;19)(q32;q13) characterized in our laboratory and describe their clinical, histopathologic, and cytogenetic features in detail.

PATIENTS AND METHODS

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.²¹ Their clinical features, laboratory data, and treatment outcomes were obtained from the clinical records.

Flow cytometry (FCM)

Mononuclear cells were separated from peripheral blood (PB) or bone marrow (BM) aspirates using Lymphocyte Separation Solution (Nacalai Tesque, Kyoto, Japan). 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 FCM. Fluorescence was captured using a NAVIOS 3L flow cytometer and analyzed by Kaluza Flow Cytometry Analysis Software (Beckman Coulter, Brea, CA, USA).

Histopathological examination

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 (PS1; Nichirei Biosciences), CD5 (4C7; Leica Biosystems, Newcastle upon Tyne, UK), CD10 (56C6; Leica Biosystems), CD20 (L26; Leica Biosystems), CD30 (Ber-H2; DAKO, Glostrup, Denmark), CD79a (JCB117; DAKO), BCL2 (124; DAKO), BCL6 (LN22; Nichirei Biosciences), MUM1 (NCL-L-MUM1; Leica Biosystems), Ki-67 (MIB-1; DAKO), MYC (Y69; Abcam PLC, Cambridge, UK), BCL3 (clone 1E8; Novocastra, Newcastle upon Tyne, UK), and PD-1 (NAT105; Abcam). In situ hybridization (ISH) for Epstein-Barr virus (EBV)-encoded RNA (EBER) was performed using EBER Probe (Leica Biosystems) according to the manufacturer’s instructions.

Cytogenetic study

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 CO_² 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 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 2020.²²

Fluorescence in situ hybridization (FISH)

Cytogenetic preparations were hybridized with fluorophore-labeled probes. The following FISH probes were used: the Vysis LSI IGH break-apart (BA) probe (Abbott Laboratories, Abbott Park, IL, USA), Vysis LSI BCL6 BA probe (Abbott Laboratories), Vysis LSI MYC BA probe (Abbott Laboratories), and XL BCL3 BA probe (MetaSystems GmbH, Altlussheim, Germany). The B-CLL FISH panel was provided by Cytocell Ltd. (Cambridge, UK). Denaturing of the chromosome/probe, hybridization, and washing conditions followed the manufacturer’s recommendations. FISH results were analyzed using fluorescence microscopes (Nikon Corporation, Tokyo, Japan; Carl Zeiss, Oberkochen, Germany) equipped with DAPI, fluorescein isothiocyanate (FITC), and tetramethylrhodamine B isothiocyanate (TRITC) fluorescence filters as well as a DAPI/FITC/TRITC triple band-pass filter.

Polymerase chain reaction (PCR) and nucleotide sequencing

Genomic DNA was isolated from specimens using proteinase K and phenol/chloroform. BIOMED-2 multiplex PCR to detect rearrangements of antigen receptor genes was performed as previously described.²³ The sequences of primers for long distance (LD)-PCR to relevant regions of IGH and BCL3 are shown in Supplementary Table S1.^24,25 Allele-specific (AS) PCR for detecting the G17V mutation in the RHOA gene was as described previously.²⁶ All PCR procedures were conducted using a Veriti 96 Well Thermal Cycler (Applied Biosystems, Inc., Forester city, CA, USA). PCR products were visualized by ethidium bromide (EtBr)-stained agarose gel electrophoresis, purified using a MinElute^® PCR Purification Kit (QIAGEN, Hilden, Germany), subjected to the cycle sequencing reaction (BigDye^TM Terminator v3.1 Cycle Sequencing Kit; Thermo Fisher Scientific), and then sequenced using a SeqStudio^TM Genetic Analyzer (Thermo Fisher Scientific). The data obtained were applied to BLASTn and IgBLAST programs to identify closely related sequences.

RESULTS

Clinical characteristics

The patients’ ages ranged between 55 and 88 years and male/female ratio was 4/1 (Table 1). Two patients presented with progression or recurrence of B-CLL/SLL 6 (case 1) and 5 (case 2) years after their initial presentation, while the remaining 3 (cases 3, 4, and 5) had de novo diseases of advanced-stage aggressive non-Hodgkin lymphoma. All patients showed significant lymphadenopathy, and the enlarged spleen in 3 patients was diffusely stained by ¹⁸F-fluorodeoxyglucose-positron emission tomography combined with computed tomography. The lactate dehydrogenase level increased in 3 patients, and soluble interleukin 2 receptor and β2 microglobulin levels were elevated in all. Two patients responded to treatments, while two died of disease progression.

Table 1. Clinical features of 5 B-cell tumors associated with t(14;19)(q32;q13)/IGH::BCL3.

Case no.	Case 1	Case 2	Case 3*	Case 4	Case 5
Age/Sex	64/male	55/male	68/male	77/male	88/female
Disease at presentation	Progression of B-CLL	Recurrence of B-CLL/SLL	DLBCL (non-GCB)	DLBCL (non-GCB)	EBV+ DLBCL composite with AITL
Risk category	Rai: high	Rai: intermediate	IPI: high	IPI: low-intermediate	IPI: high
“B” symptoms	+	−	+	−	+
Lymphadenopathy	+	+	+	+	+
Splenomegaly	+	+ (FDG-avid)	+ (FDG-avid)	−	+ (FDG-avid)
BM involvement	+	+	+	−	−
Hemoglobin (g/dL)	12.9	14.8	12.8	12.1	11.2
White cell count (×103/μL)	289.3 (lymphocytes, 95.0%)	4.88	6.10 (lymphoma cells, 1%)	5.86	7.02
Platelet count (×103/μL)	88	266	185	230	201
LDH (U/L)	4,433	150	402	174	374
sIL-2R (U/mL)	9,880	985	5,190	1,197	6,095
β2 Microglobulin (μg/mL)	10.6	2.18	7.82	2.12	4.98
Treatment	Ibrutinib	Acalabrutinib	R-CHOP, DA-EPOCH-R	R-CHOP	Best supportive care
Outcome	Died of Richter transformation	PR	Died of progression	CR	Lost to follow-up

*Case 3 was described previously.²¹

B-CLL, B-cell chronic lymphocytic leukemia; SLL, small lymphocytic lymphoma; DLBCL, diffuse large B-cell lymphoma; EBV, Epstein-Barr virus; AITL, angioimmunoblastic T-cell lymphoma; Rai, Rai staging system for CLL; IPI, international prognostic index for aggressive non-Hodgkin lymphoma; FDG, ¹⁸F-fluorodeoxyglucose; BM, bone marrow; LDH, lactate dehydrogenase; sIL-2R, soluble interleukin-2 receptor; R-CHOP, rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone; DA-EPOCH-R, dose-adjusted etoposide, prednisolone, vincristine, cyclophosphamide, doxorubicin, and rituximab; PR, partial response; CR, complete response.

G-banding and FISH

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 (Table 2, Figures 1, 2, and 3). In case 5, both diploid- and tetraploid-range karyotypes had a single copy of t(14;19)(q32;q13) (Figure 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 (Table 2; Figures 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 (Table 2; Figures 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 (Table 3; Figure 2).

Table 2. Cytogenetic findings in 5 B-cell tumors with t(14;19)(q32;q13)/IGH::BCL3.

Case no.	G-banding and FISH (ISCN)22
Case 1	47,XY,t(2;14)(p13;q32),der(4)t(4;8)(q35;q22),+12,t(14;19)(q32;q13).ish t(2;14)(p13;q32)(5′IGH+;3′IGH+),der(4)t(4;8)(5′MYC+,3′MYC+), t(14;19)(q32;q13)(3′IGH+,3′BCL3+;5′BCL3+,5′IGH+)
Case 2	47,XY,del(1)(p34),add(6)(p21),+12,t(14;19)(q32;q13).ish t(14;19)(q32;q13)(3′IGH+,3′BCL3+;5′BCL3+,5′IGH+)
Case 3*	51,XY,−2,+3,del(6)(q21),t(8;14)(q24;q32),+der(12)t(8;12)(q13;q22),t(14;19)(q32;q13),+18,+18,+2mar.ish t(8;14)(q24;q32)(5′MYC+,5′IGH+;3′IGH+3′MYC+),der(12)t(8;12)(5′MYC+,3′MYC+),t(14;19)(q32;q13)(3′IGH+,3′BCL3+;5′BCL3+,5′IGH+)
Case 4	52,XY,+X,der(1)t(1;2)(p36;p13)del(1)(q32),der(2)t(1;2)(p36;p13),+der(3)dup(3)(q26q28)?del(3)(q27),+del(7)(q32),t(8;11;14)(q24;q11;q32),+11, t(14;19)(q32;q13),del(15)(q11q21),der(21)t(1;21)(q21;p11)del(1)(q32),der(22)t(1;22)(q11;p11),+2mar.ish der(3)dup(3)(3′BCL6+,5′BCL6+)?del(3)(3′BCL6+,5′BCL6−),t(8;11;14)(q24;q11;q32)(5′MYC+;5′IGH+;3′IGH+,3′MYC+), t(14;19)(q32;q13)(3′IGH+,3′BCL3+;5′BCL3+,5′IGH+)
Case 5	48,XX,inv(1)(q25q42),+3,t(14;19)(q32;q13),+15,add(15)(p13)×2.ish t(14;19)(q32;q13)(3′IGH+,3′BCL3+;5′BCL3+,5′IGH+)/94<4n>,XXXX,inv(1)(q25q42),+3,t(14;19)(q32;q13),+15,add(15)(p13)×2.ish t(14;19)(q32;q13)(3′IGH+,3′BCL3+;5′BCL3+,5′IGH+)

*Case 3 was described previously.²¹

Fig. 1.

Cytogenetic findings in case 1. (A) G-banding karyotype. t(14;19)(q32;q13) and t(2;14)(p13;q32) are indicated by open arrowheads and arrows, respectively. The closed asterisk indicates +12 and the open asterisk indicates der(4)t(4;8)(q35;q22). (B) Interphase FISH using the CLL FISH panel. The top left picture shows trisomy 12. (C) Interphase FISH using the BCL3 BA probe. Green, red, and yellow signals are indicated by arrowheads with their respective colors. (D) Metaphase FISH using the IGH BA probe (left), BCL3 BA probe (middle), and MYC BA probe (right). Pictures of G-banding and FISH through the triple band pass filter are aligned side by side. On the left, t(14;19)(q32;q13) and t(2;14)(p13;q32) are indicated by arrowheads and arrows with respective colors. In the middle, normal chromosome 19 is labeled with a yellow signal, der(14) with the telomeric 3′ BCL3 green signal, and der(19) with the centromeric 5′ BCL3 red signal. On the right, the ends of the long arms of chromosome 8s and der(4)t(4;8) are labeled with yellow signals. Diagrams of the probes provided by the manufacturers are shown at the bottom for reference.

Fig. 2.

Cytogenetic findings in case 4. (A) G-banding karyotype. t(14;19)(q32;q13) and t(8;11;14)(q24;q11;q32) are indicated by open arrowheads and arrows, respectively. Additional abnormalities are shown by closed asterisks for numerical abnormalities and open asterisks for structural abnormalities. (B) Metaphase FISH using the BCL3 BA probe. G-banding and FISH pictures are aligned side by side. Normal chromosome 19 is labeled with a yellow signal, der(14) with a green signal, and der(19) with a red signal. (C) Interphase FISH using the BCL3 BA probe. A break-apart signal is observed in the nucleus of the large cell at the bottom. (D) Metaphase FISH using the IGH BA probe (left), MYC BA probe (middle), and BCL6 BA probe (right). Pictures of G-banding and FISH through the triple band pass filter are aligned side by side. On the left, the centromeric 3′ IGH red signals are located on the two der(14) and the telomeric 5′ IGH green signals are located on der(11)t(8;11;14) and der(19)t(14;19). In the middle, the centromeric 5′ MYC red signal is located on der(8)t(8;11;14) and the telomeric 3′ MYC green signal is located on der(14). On the right, der(3)dup(3)?del(3) is labeled with one yellow signal and one green signal indicative of deletion of 5′ BCL6. A diagram of the probe provided by the manufacturer is shown at the bottom for reference.

Fig. 3.

Cytogenetic findings in case 5. (A) G-banding of diploid-range (top) and tetraploid-range (bottom) karyotypes. t(14;19)(q32;q13) is indicated by open arrowheads, and additional numerical abnormalities are indicated by closed asterisks and additional structural abnormalities by open asterisks. The tetraploid-range karyotype is considered to be generated by cell fusion between a cell with the diploid-range karyotype and a normal diploid cell.³⁵ (B) Metaphase FISH using the BCL3 BA probe. Pictures of G-banding, FISH through the triple band filter, and FISH through the FITC filter are aligned to clearly show the telomeric 3′ BCL3 green signal on der(14). (C) FISH of a tetraploid-range karyotype using the IGH BA probe. Three chromosome 14s are labeled with yellow signals, one der(14) with the centromeric 3′ IGH red signal, and one der(19) with the telomeric 5′ IGH green signal.

Table 3. Immunophenotypes and genetic features of 5 B-cell tumors with t(14;19)(q32;q13)/IGH::BCL3.

Case no.		Case 1	Case 2	Case 3*	Case 4	Case 5
FCM	CD5	+	Dim or −**	Dim	Dim	ND
	CD10	−	−	−	−	ND
	CD20	+	+	+	+	ND
	CD23	−	−	−	−	ND
	CD38	+	+	+	+	ND
	SmIg	IgMDλ	IgGκ	IgMκ	IgMDκ	ND
IHC	CD5	NT	−	+ (partial)	−	ND
	BCL3	NT	NT	+	+	NT
+12		+	+	+	−	−
del(17p) (TP53)		−	−	−	−	NT
del(6q) (MYB)		−	−	−	ND	NT
del(11q) (ATM)		−	−	−	ND	NT
del(13q) (RB1)		−	−	−	ND	NT
Other additional abnormalities		IGH Sγ2::5′ BCL11A, 3 copies MYC	−	IGH::MYC, 3 copies MYC	IGH::MYC, 4 copies BCL6, del(5′ BCL6)	RHOA G17V
IGHV		IGHV1-69	IGHV4-34	IGHV3-7	IGHV4-34	IGHV4-34
IGHD		IGHD3-3	IGHD4-23/5-18/5-5	IGHD3-10	IGHD1-1/1-20	IGHD2-15
IGHJ		IGHJ5	IGHJ6	IGHJ4	IGHJ3	IGHJ6
IGHV identity		100%	99.7%	96.6%	84.8%	91.5%
CDR3 length		21	17	12	18	10
t(14;19) anatomy		IGH Sα1::5′ BCL3	IGH Sα1::5′ BCL3	Not amplified	Not amplified	Not amplified

*Case 3 was described previously.²¹ **See text.

FCM, flow cytometry; IHC, immunohistochemistry; SmIg, surface membrane immunoglobulin; ND, not determined; NT, not tested.

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 (Tables 2 and 3; Figures 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 (Table 3; Figure 1B).

Immunophenotype of tumor cells and histopathology based on biopsies

FCM of single-cell suspensions prepared from PB, BM, or LN isolated a clonal B-cell population in all but case 5 (Table 3). The cells in cases 1 through 4 expressed CD19, CD20, and monoclonal immunoglobulins on their cell surface. CD5 was positive in case 1 and dim in cases 3 and 4. In case 2, B-CLL cells at initial presentation dimly expressed CD5, but SLL cells at relapse lost the expression. CD10 and CD23 were negative and CD38 was positive. Other antigens studied included CD2^dim and FMC7⁻ in case 1 and CD180⁺ and CD200⁺ in case 2. In case 5, no clonal B- or T-cell population was identified by FCM.

Histopathological examination of the LN biopsy in case 2 revealed diffuse proliferation of small cells admixed with larger prolymphocytes/paraimmunoblasts. 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 (Figure 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) (Table 1). The tumor cell nuclei of both cases were stained positive by anti-MYC and anti-BCL3 antibodies (Figure 4). The Ki67 index was >90% in case 3 and 40% in case 4.

Fig. 4.

Histopathology of cases 3 (A to C) and 4 (D to I). A, hematoxylin & eosin (H&E) staining (original magnification, 40× objective lens); B, anti-MYC immunostaining (40×); C, anti-BCL3 (40); D, H&E (40×); E, anti-MYC (40×); F, anti-BCL3 (40×); G, anti-CD20 (20×); H, anti-BCL6 (20×); and I, anti-MUM1 (20×). In case 4, there was a vaguely nodular appearance under low-power examination, which was highlighted by CD21-positive follicular dendritic cell networks (not shown). Case 3 was described previously.²¹

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 (Figure 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 (Figure 5).

Fig. 5.

Histopathology of case 5. A, H&E (20×); B, H&E (40×); C, anti-CD3 (20×); D, anti-PD-1 (20×); E, anti-CD20 (40×); F, anti-CD30 (40×); and G, EBER ISH (40×).

Anatomy of IGH::BCL3 fusion gene and other DNA tests

To confirm the IGH::BCL3 fusion gene at the DNA level, we designed oligonucleotide primers that enclosed the proximal or distal breakpoint clusters on BCL3 (Figure 6A),^2,3,5,24 and performed LD-PCR in combination with primers for the intronic Eμ enhancer and IGHM, IGHG, and IGHA constant genes of IGH (Supplementary Table S1).²⁴ As shown in Figure 6B, approximately 2.1- (case 1) and 3.4- (case 2) kb PCR products were generated by the BCL3 intron 1 and Cα/01 primer combination, while in cases 3, 4, and 5, no PCR products were amplified by available primer combinations (Table 3; Figure 6). Nucleotide sequencing of the BCL3 side of the products revealed that breakpoints occurred 788 (case 1) and 941 (case 2) bp upstream of the 5′ end of exon 1 (Figure 6). The sequences of the IGH side matched those of IGHA1 and BCL3-side sequences followed tandem pentametric repeats of GGGCT or related sequences (Figure 6C), indicating that the breakpoints on IGH were localized within the switch region associated with IGHA1 (Sα1). As the result of translocation, IGH and BCL3 were fused in divergent orientation without affecting the protein-coding sequences of BCL3.

Fig. 6.

Anatomy of t(14;19)(q32;q13)/IGH::BCL3 in cases 1 and 2. (A) Schematic diagram of BCL3 at 19q13 (top), IGHEP1 to IGHA1 at 14q32 (middle), and IGH::BCL3 of case 1 on der(14)t(14;19) (bottom). Breakpoints of cases 1 and 2 are indicated by vertical arrows and BCL3 breakpoints described in our previous studies are by open arrowheads; primers for LD-PCR are designed to enclose these breakpoints. cen, centromere; tel, telomere. (B) EtBr-stained agarose gel electrophoresis of LD-PCR encompassing the IGH::BCL3 junctions generated by the BCL3/intron 1 and Cα/01 primer combination. (C) Nucleotide sequences of the IGH::BCL3 junction in cases 1 and 2. Vertical lines indicate nucleotide identity. Tandem pentameric repeats characteristic for IGH switch regions (GGGCT and 4/5-matched sequences) are underscored. In A, the position of the IGH-side breakpoint in case 2 was determined by the position of the primer and the size of the PCR products. The positions of the nucleotides are numbered according to NC_000019.10 and NC_000014.9.

We applied a similar LD-PCR strategy to t(2;14)(p13;q32) in case 1 and found that breakage and union occurred within the upstream sequence of BCL11A at 2p13 and within the switch region associated with IGHG2 (Sγ2) (Table 3; Supplementary Figure S1).^27,28 In case 1, both IGH alleles were involved in the translocations, but because leukemia cells expressed IgMDλ monoclonal immunoglobulins, it is likely that the VDJ-IGHMD complex producing the IgMD heavy chain was translocated to either der(19) or der(2). In case 5, multiplex PCR tests to detect rearrangements of TCRG and TCRB were negative, while AS-PCR designed to detect the single nucleotide substitution of G to T at position 50 (c.G50T) of RHOA was positive, indicating the presence of RHOA^G17V mutation in the DNA material (Table 3).

IGHV status

BIOMED-2 multiplex PCR detected clonal rearrangements of IGK and IGH in all cases. We then amplified the entire lengths of IGHV-D-J sequences using custom IGHV primers in each case, and the nucleotide sequencing of PCR products confirmed a productive VDJ configuration in all cases. The IGHV genes utilized were IGHV1-69 in case 1, IGHV3-7 in case 3, and IGHV4-34 in the remaining 3 cases (Table 3). Sequence identities with germline sequences were 100 and 99.7% in cases 1 and 2, respectively, while they ranged between 84.8 and 96.6% in cases 3, 4, and 5. The lengths of CDR3 in codons ranged between 10 and 21.

DISCUSSION

Here, we characterized 5 B-cell tumors that carried t(14;19)(q32;q13) identified by G-banding and the IGH::BCL3 fusion gene determined by FISH. As described previously,^19,20 the tumors were categorized into two clinically and molecularly different groups: one included B-CLL/SLL cases requiring treatments, and the other was composed of de novo cases of aggressive B-cell lymphoma (Table 1). Although both groups carried IGHV, used by B-CLL cells at higher frequencies than observed in normal B cells,²⁹ the genes were unmutated in the former group and significantly mutated in the latter. Most importantly, the anatomy of t(14;19)(q32;q13)/IGH::BCL3 in cases 1 and 2 was similar to that of previous cases,^2,3,5 while the IGH::BCL3 fusion genes in cases 3, 4, and 5 were not amplified by LD-PCR designed to detect previously cloned BCL3 breakpoints.

The BCL3 BA probe used in this study includes not only BCL3 but also PVR, CEACAM19, CEACAM16, CBLC, and BCAM within the gap between the centromeric and telomeric probes (Supplementary Figure S2). Thus, in BCL3 BA FISH-positive cases, either one of these flanking genes, instead of BCL3, may have been involved. Indeed, a recent study using next-generation sequencing (NGS) found rearrangements of upstream CEACAM16 and downstream CBLC and BCAM with IGH.¹⁹ However, in cases with the latter two rearrangements, the tumors showed marginal zone lymphoma histopathology and lacked expression of BCL3, being clearly different from cases in our study. Bright nuclear staining of BCL3 by IHC observed in cases 3 and 4 suggests that, even though the (14;19)(q32;q13) breakpoints in these two cases were not within the same region as those in cases 1 and 2, they still occurred in the vicinity of BCL3, resulting in the high level expression of the gene product under the influence of juxtaposed IGH.

The BCL3-side breakpoints of t(14;19)(q32;q21)/IGH::BCL3 described in B-CLL/SLL cases were located within a range of the upstream sequences of BCL3,^2,3,19 and those in the majority of cases were clustered immediately 5′ of BCL3 exon 1,^2,3,5 as observed in cases 1 and 2 (Figure 6). The IGH-side breakpoints, on the other hand, preferentially occurred in the Sα1 or Sα2 switch region in our previous studies,^2,3,5 and the Sα1 breakpoints were confirmed by the current study, suggesting that class switch recombination (CSR) from IGHM and IGHA potentially mediates the generation of t(14;19)(q32;q21). However, IGHD and IDHJ breakpoints were identified in a NGS-based study,³⁰ and switch regions associated with IGHG1, IGHG2, and IGHG3 were also affected in another study.¹⁹ Currently, it is unclear whether the preferential Sα involvement observed in our series was due to a bias in case selection or difference in methods between us and others. Nevertheless, the presence of t(14;19)(q32;q13) and t(2;14)(p13;q32), the latter of which also involved Sγ2 switch regions and, therefore, was considered to be mediated by erroneous CSR mechanisms,^27,28 in IGHV-unmutated B-cell tumor cells supports the possibility that CSR can occur outside the germinal center in a T-cell-independent manner and before the initiation of somatic mutations in the IGH gene.^19,20,28

B-cell lymphomas with two or more cytogenetic rearrangements affecting the chromosomal loci of MYC and BCL2 and/or BCL6 are referred to as double-hit (DH) or triple-hit (TH) lymphomas that have become synonymous with high-grade B-cell lymphomas.³¹ The most frequent DH is the combination of a rearrangement of MYC and that of BCL2, and the aggressive clinical behavior of MYC/BCL2 DH tumors has been attributed to a selective and complementary role for MYC and BCL2, i.e., MYC drives cells into active proliferative and metabolic states, while BCL2 is anti-apoptotic without mediating proliferative signals.³¹ The current study showed that MYC/BCL3 DH associated with expression of both the gene products is recurrent. Furthermore, in case 4, although it is unclear whether BCL6 was involved in chromosomal translocation because the telomeric 5′ BCL6 is deleted, it is possible that the regulatory sequences of BCL6 were replaced with those of another gene, leading to deregulated expression of BCL6. In these MYC/BCL3 DH and potential MYC/BCL3/BCL6 TH tumors, BCL3 may have played a role similar to BCL2; in fact, some previous reports suggested the anti-apoptotic and pro-survival functions of BCL3.^6,8,32,33

Although case 5 was initially considered to be angioimmunoblastic T-cell lymphoma (AITL) based on the characteristic histopathology and positive RHOA^G17V mutation, the antigen receptor gene rearrangement test revealed clonal rearrangements of IGK and IGH and t(14;19)(q32;q13) was identified by cytogenetic analysis. As large CD20-positive B cells were scattered or formed clusters within the biopsy specimen and most of the cells were positive for EBER ISH, we concluded that the tumor represented a composite lymphoma of AITL and EBV-positive DLBCL.³⁴ BCL3 rearrangement was reported to be present in Hodgkin lymphoma and T-cell lymphomas including AITL,⁷ but in such cases, B-cell lymphoma components may have been present in the setting of T-cell tumors.