Simultaneous evaluation of T- and B-cell clonality, t(11;14) and t(14;18), in a single reaction by a four-color multiplex polymerase chain reaction assay and automated high-resolution fragment analysis: a method for the rapid molecular diagnosis of lymphoproliferative disorders applicable to fresh frozen and formalin-fixed, paraffin-embedded tissues, blood, and bone marrow aspirates

Abstract

Current polymerase chain reaction (PCR) methods for the molecular diagnosis of B- and T-cell lymphomas by determination of clonality of immunoglobulin heavy chain (IgH) and T-cell receptor-gamma rearrangements and by detection of the chromosomal translocations t(14;18) and t(11;14), require several laborious and costly PCR assays for each of these diagnostic tests. We have developed a multiplex PCR assay for the simultaneous determination of B- and T-cell clonality and the detection of the chromosomal translocations t(14;18) and t(11;14) in a single reaction, using four-color fluorescence and automated high-resolution fragment analysis. The 26 primers combined in the multiplex PCR correspond to the sequences of >90% of the 69 variables and 6 join IgH genes and 100% of the T-cell receptor-gamma variables and join genes that could participate in the respective rearrangements. In addition, they detect the major and the minor breakpoint regions of the t(14;18) and the major breakpoint region of the t(11;14), and amplify the beta-globin gene as an internal control. The specificity of the multiplex PCR was confirmed by analysis of 39 T-cell lymphomas and 58 B-cell lymphomas, including 11 mantle cell lymphomas bearing the t(11;14) and 25 follicular lymphomas bearing the t(14;18), with known rearrangements and/or translocations. Fifteen samples of reactive lymphadenitis remained negative.

Figure 1.

Schematic representation of the fluorescent dye labels and the size rages specific for the PCR fragments derived from each of the diagnostic tests. Panel B: (blue, label 6-FAM); IgH rearrangement fragments VFR3-J appear in a size range between 80 and 140 bp. Fragments of TCR-γ rearrangements appear in a size range between 180 and 280 bp. IgH rearrangement fragments V FR1-J are present in a size range between 300 and 400 bp. Panel G: (green, label 6-JOE); t(11;14) Mbr fragments have sizes between 160 and 300 bp. The control PCR fragment of the β-globin gene has a length of 352 bp. Panel Y: (yellow, label NED); the size of t(14;18) Mbr and mcr fragments is usually between 150 and 300 bp and may, in a rare case, be up to 500 bp. Panel R: (red, label ROX); molecular weight marker. Fragment sizes are indicated below.

Figure 2.

Analysis of control cells by multicolor multiplex PCR, automated HFRA, and GeneScan analysis. Fragments are aligned by size, which is indicated above the panels. A: Polyclonal populations of B and T cells present in a sample of lymphadenopathy. Panel B: Multiple IgH V FR3-J and V FR1-J fragments from polyclonal B cells, and multiple fragments derived from polyclonal TCR-γ rearrangements; panel G, peak of the β-globin fragment at 352 bp. B: The T-cell line Jurkat. Panel B: Fragments from the two clonal TCR-γ rearrangements at 209 bp (V1-J1/J2) and 244 bp (V4P-J1/J2); panel G, β-globin at 352 bp. C: REC-1 cells, derived from a mantle cell lymphoma. Panel B: V FR3-J (103 bp) and V FR3-J (365 bp) fragments of a clonal IgH rearrangement; panel G, peak of the t(11;14) fragment at 229 bp, β-globin at 352 bp. D: DoHH2 cells, derived from a follicular lymphoma. Panel B: V FR3-J (93 bp) and V FR1-J (364 bp) fragments of a clonal IgH rearrangement; panel G, β-globin at 352 bp; panel Y, peak of the t(14;18) Mbr fragment at 242 bp.

Figure 3.

Analysis of clinical samples of B-cell lymphomas by multicolor multiplex PCR, automated HRFA, and GeneScan analysis. Fragments are aligned by size, which is indicated above the panels. A: Sample of a B-cell lymphoma with signals of a clonal IgH rearrangement from both V FR3-J (104 bp) and V FR1-J (369 bp) fragments and low polyclonal background of nonneoplastic B and T cells. B: B-cell lymphoma with signal of a clonal IgH rearrangement from a V FR3-J (125 bp) fragment, and polyclonal V FR1-J and TCR-γ fragments. C: B-cell lymphoma with signal of a clonal IgH rearrangement from a V FR1-J (374 bp) fragment, and polyclonal V FR3-J and TCR-γ fragments.

Figure 4.

Focus on the IgH FR3 region-specific size range. Signals of a clonal IgH rearrangement (133 bp) and nonneoplastic B cells present in the same sample of a B-cell lymphoma. The majority of the IgH V-J fragments differ in length by a multiple of 3 bp.

Figure 5.

Analysis of clinical samples of mantle cell and follicular lymphoma by multicolor multiplex PCR, automated HRFA, and GeneScan analysis. Fragments are aligned by data points, which are indicated above the panels. Fragment lengths are listed below the panel. A: A clonal IgH rearrangement (panel B) with both V FR3-J (96 bp) and V FR1-J (361 bp) fragments, and t(11;14) (panel G, 231 bp) are present in a mantle cell lymphoma. B: A follicular lymphoma bearing a t(14;18)Mbr (panel Y, 242 bp). The IgH FR1-J fragment from a clonal IgH rearrangement (panel B, 363 bp) is amplified. Polyclonal signals from nonneoplastic T cells are present in the same sample (panel B). C: A follicular lymphoma bearing a t(14;18) mcr (panel Y, 225 bp) and polyclonal signals from nonneoplastic B and T cells present in the same sample (panel B).

Figure 6.

Breakpoints in the bcl-2 region and sequences of t(14;18) junctions and breakpoints in the bcl-1 region and sequences of t(11;14) junctions. The breakpoints found in 25 cases of follicular lymphomas are indicated by asterisks. The sequences of the bcl-2-N-IgH J junctions are listed below. The breakpoints on chromosome 11 found in 11 cases of mantle cell lymphoma are indicated by asterisks. The sequences of the bcl-1-N-IgH J junctions are listed below.

Figure 7.

Analysis of clinical samples of T-cell lymphomas by multicolor multiplex PCR, automated HRFA, and GeneScan analysis. A: Two peaks in the TCR-γ-specific size range in panel B demonstrate the clonal TCR-γ rearrangements present in the sample of a T-cell lymphoma. B: TCR-γ rearrangement on one allele and background signals from polyclonal TCR-γ rearrangements of nonneoplastic T cells present in a peripheral T-cell lymphoma. Signals from polyclonal B cells present in the same sample are visible in the IgH-FR3-specific size range, but below detection in the IgH-FR1-specific size range, indicating partial degradation of the DNA sample. C: A T-cell clone with TCR-γ rearrangements on both alleles is present in a high background of nonneoplastic B and T cells in the sample of a peripheral T-cell lymphoma. The clonal TCR-γ rearrangements are visible as two peaks at 212 and 241 bp (panel B). Analysis of the same T-cell lymphoma by the TCR-γ-specific multicolor multiplex PCR is shown in D; a fragment of 149 bp present in panel B shows a clonal TCR-γ rearrangement containing a V3P gene (difference in fragment length, 90 bp). The 245 bp fragment present in panel G shows a second clonal TCR-γ rearrangement with a V1 gene (difference in fragment length, 33 bp).