Multiplex PCR for the detection of BCL-1/IGH and BCL-2/IGH gene rearrangements – clinical validation in a prospective study of blood and bone marrow in 258 patients with or suspected of non-Hodgkin's lymphoma.

Figures & data

Figure 1.  Location of the primers on BCL-1, BCL-2 and IGH included in the multiplex PCR.

Figure 2.  Multiplex PCR for detection of t(11;14) and t(14;18). M: molecular marker. Lane 1: cell line DOHH-2, lane 2: cell line WSU-NHL, lane 3: lymph node biopsy from patient no. 261, lane 4: cell line JVM-2, lane 5: blood from patient no. 28, and lane 6: ddH₂O. Multiplex PCR: primers amplifying MBR1, MBR2, and MCR in t(14;18) as well as MTC and mTC1p94 in t(11;14). Split reactions: 5 primer sets detecting t(14;18) MBR1, t(14;18) MBR2, t(14;18) MCR, t(11;14) MTC, and t(11;14) mTC1p94. A control gene (TCF20) is co-amplified in all lanes. White arrows show the translocation positive PCR products in the split reactions, large black arrow exemplifies a PCR product of the control gene, while the thin black arrow exemplifies a weak germline PCR band.

Table I.  PCR positive for t(11;14). Comparison between PCR, flow cytometry, and histology at the same time of sampling.

ID	Age at Diagnosis	Sex	Histological Type	Month from diagnosis	Positivity by PCR* PB/BM	Clonal by FCM PB/BM	Positivity by pathology PB/BM
27	70	F	MCL	46	+/(+)	+/ +	+/ +
216	65	F	MCL	1	+/NA	+/NA	−/ +
28	74	M	MCL	0.0	+/NA	+/NA	+/ +
32	56	M	MCL	6	NA/ +	+/ +	−/ +
41	59	M	MCL	0.0	+/ +	+/ +	+/ +
48	55	M	MCL	10	+/ +	+/ND	−/ +
72	70	M	MCL	0.0	+/NA	+/ +	−/ +
113	59	M	MCL	1	+/ +	+/ +	+/ +
185	49	M	MCL	124	+/(+)	−/ −	−/ +
30	52	M	MCL**	2	+/ +	−/ −	−/ −
183	45	M	MZL	5	NA/ +	NA/ −	−/ +

MCL: mantle cell lymphoma; MZL: marginal zone lymphoma; NA: not available; ND: not done because of too few B-cells; *: PCR positivity is ranked in two categories ((+): weak PCR band; +: medium to strong PCR band); **: the cells showed follicular growth at diagnosis but was later classified as MCL.

Table II.  PCR positive for t(14;18). Comparison between PCR, flow cytometry, and histology at the same time of sampling.

ID	Age at Diagnosis	Sex	Histological Type	Month from diagnosis	Positivity by PCR* PB/BM	Clonal by FCM PB/BM	Positivity by pathology PB/BM
12	65	F	FL	0.0	+/ +	+/ +	−/ −
25	35	F	FL	28	NA/ +	−/ +	−/ +
26	61	F	FL	1	NA/ +	−/ −	−/ −
49	44	F	FL	92	(+)/ +	−/ +	−/ +
64	65	F	FL	27	NA/(+)	−/ −	−/ −
93	51	F	FL	32	+/NA	+/ +	−/ +
99	57	F	FL	11	(+)/ +	−/ +	−/ +
101	75	F	FL	1	(+)/(+)	−/ −	−/ −
115	59	F	FL	32	(+)/(+)	−/ −	−/ +
127	55	F	FL	2	NA/ +	NA/ −	−/ −
152	82	F	FL	0.0	+/ +	−/ −	−/ +
202	81	F	FL	0.0	NA/ +	NA/ND	+/ +
225	62	F	FL	129	(+)/(+)	ND/ND	−/ −
227	54	F	FL	0.0	NA/ +	NA/ −	−/ +
33	51	M	FL	70	+/ +	+/ +	−/ +
44	47	M	FL	13	(+)/ +	−/ −	−/ −
70	78	M	FL	13	(+)/(+)	ND/ND	−/ −
75	57	M	FL	1	(+)/(+)	−/ −	−/ −
97	46	M	FL	6	+/ +	ND/ +	+/ +
103	69	M	FL	1	+/ +	ND/ND	−/ −
112	45	M	FL	131	(+)/(+)	−/ −	−/ −
133	61	M	FL	26	+/ +	−/ −	−/ +
148	40	M	FL	41	(+)/NA	−/ −	−/ +
223	55	M	FL	1	−/(+)	−/ −	−/ +
242	59	M	FL	12	+/ +	+/ +	−/ −
244	40	M	FL	105	+/ +	+/ +	−/ +
261	25	M	FL	30	+/ +	+/ +	−/ +
59	36	F	DLCL	121	NA/ +	NA/ +	−/ +
77	50	F	DLCL	94	(+)/(+)	−/ −	−/ −
85	55	F	DLCL	3	(+)/(+)	−/ −	−/ −
89	78	F	DLCL	6	+/NA	−/NA	−/ +
81	49	M	DLCL	12	(+)/NA	ND/ND	−/ −
86	69	M	DLCL	31	−/ +	−/ −	−/ +
73	63	M	LL	138	(+)/ −	+/ +	+/ +
22	44	F	NHL NOS	1	NA/ +	−/ +	−/ −
172	65	F	NHL NOS	1	+/NA	−/ +	−/ +
102	41	M	Not LM	0.0	(+)/ −	−/ −	−/ −

FL: follicular lymphoma, DLCL: diffuse large cell lymphoma; LL: lymphoplasmacytic lymphoma; NHL NOS: NHL not otherwise specified; LM: lymphoid malignancy; NA: not available; ND: not done because of too few B-cells; *: PCR positivity is ranked in two categories ((+): weak PCR band; +: medium to strong PCR band).

Figure 3.  Breakpoints from the fusion DNA found in patients included in this study. Arrows indicate breakpoints and numbers refer to patient IDs. A. Sequence of BCL-1 MTC region. Sequence start at position 431 in the Genebank sequence with accession no. s77049. B. Sequence of BCL-1 mTC1p94 region. Reverse complement of accession no. ap001824 in the Genebank database. Sequence start at position 41535. C. Sequence of BCL-2 MBR region. Sequence start at position 2981 in the Genebank sequence with accession no. m14745 [8]. D. Sequence of BCL-2 MCR region. Sequence from Ngan, 1989 [27]. Sequence starts at position 905 and ends at position 1432.

Table III.  PCR negative for t(11;14) and t(14;18). Patients and samples.

					n FCM + *	n Pathology + *	FCM+ and Pathology +	FCM+ or Pathology +
Histological Type	n	Median age	Age range	F/M	PB+ or BM +	PB+ or BM +	PB+ or BM +	PB+ or BM +
MCL	13	64	43–81	4/9	9	9	7	11
FL	23	54	34–79	14/9	9	4	3	10
DLCL	57	66	22–89	25/32	11	7	4	14
Other NHL's	31	–	–	–	–	–	–	–
Other LM's	40	–	–	–	–	–	–	–
Not LM	46	–	–	–	–	–	–	–

n: number; FCM+: monoclonal in FCM; MCL: mantle cell lymphoma; FL: follicular lymphoma, DLCL: diffuse large cell lymphoma; LM: lymphoid malignancy. Other NHL's includes NHL not otherwise specified, Burkitt's lymphoma, marginal zone lymphoma, precursor lymphoma/leukaemia, periphery T cell lymphoma, anaplastic large cell lymphoma, and lymphoplasmacytic lymphoma. Other LM's includes Hodgkin's disease, multiple myeloma, and chronic lymphatic leukaemia. *: Number of cases that were not available or not done because of too few B-cells are not included.

Cleary ML, Smith SD, Sklar J. Cloning and structural analysis of cDNAs for BCL-2 and a hybrid BCL-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell 1986; 47: 19–28

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Ngan BY, Nourse J, Cleary ML. Detection of chromosomal translocation t(14;18) within the minor cluster region of BCL-2 by polymerase chain reaction and direct genomic sequencing of the enzymatically amplified DNA in follicular lymphomas. Blood 1989; 73: 1759–62

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