Rapid and accurate detection of monoclonal immunoglobulin heavy chain gene rearrangement by DNA melting curve analysis in the LightCycler System

Abstract

The detection of immunoglobulin heavy chain gene rearrangement (IgH-R) is a standard tool for distinguishing polyclonal from monoclonal B-cell populations. Current DNA-based polymerase chain reactions (PCR) strategies can diagnose monoclonal IgH-R either by measuring the length of the amplicon or by detecting gel mobility variations owing to sequence-dependent conformational changes. However, amplification and analysis remain sequential operations usually requiring manual transfer. We have developed a novel PCR strategy for detecting monoclonal IgH-R that monitors fluorescence of the specific double-stranded DNA binding dye SYBR Green I during melting curve analysis using the LightCycler System. We compared polyacrylamide gel electrophoresis (PAGE) versus melting curve analysis in 130 clinical DNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues (mostly skin biopsies) of 128 patients. The identical FR3 primers were used to amplify the IgH variable region for both analytic techniques. We detected IgH-R in 24 DNA samples from FFPE tissue of 22 patients. Melting curve analysis, compared to PAGE, revealed no false negative and no false positive results, yielding both sensitivity and specificity equal to 100%. We also compared Southern blot analysis versus melting curve analysis in 23 clinical DNA samples from fresh-frozen lymph nodes of 23 patients. We detected IgH-R by melting curve analysis in 7 DNA samples from fresh-frozen lymph nodes. Melting curve analysis, compared to Southern blot analysis, revealed sensitivity equal to 58.3% (7 of 12) and specificity equal to 100% (11 of 11). We conclude that continuous fluorescence monitoring of PCR products with DNA melting curve analysis can rapidly and reproducibly distinguish polyclonal from monoclonal B-cell populations.

Figure 1.

Optimization of PCR cycles. DNA from 100% Namalwa B-cell line (N, top curve), a mixture of 50% Namalwa and 50% tonsil (0.5 N + 0.5 T, middle curve), and 100% tonsil (T, bottom curve) was amplified for 20 (A), 30 (B), 40 (C), 50 (D), and 60 (E) cycles. Namalwa started to produce a sharp −dF/dT peak after 30 PCR cycles (B). The 100% Namalwa/100% tonsil −dF/dT peak height ratio reached 3.88 at 40 cycles (C and Table 1 ). Additional cycles did not significantly improve this ratio. Therefore, we set the criteria for diagnosing a monoclonal B-cell DNA sample as having a −dF/dT peak height at least twice as high, and less than one-half as wide, as a polyclonal tonsil DNA sample after 40 cycles. x axis = temperature (°C); y axis = −dF/dT, where F = fluorescence and T = temperature.

Figure 2.

Minimal detection of B-cell clonality. Namalwa B-cell line DNA (50%, 25%, 12.5%, 6.25%, 3.13%, and 1.56%) were serially diluted into tonsil DNA. After 40 cycles of amplification in the LightCycler System, a distinct peak with the expected Tm of Namalwa was still detectable at the 12.5% level by DNA melting curve analysis. Axes definitions are the same as in Figure 1 .

Figure 3.

Detecting B-cell clonality in representative clinical DNA samples using melting curve analysis. Sharp peaks above dashed line (−dF/dT = 0.35) indicate clonally rearranged IgH: Namalwa B-cell line and four-patient DNA samples from three patients (see Table 2 ). DNA samples 1 and 2, obtained from a single individual, share the same Tm value indicating clonal identity. Broad peaks below dashed line (−dF/dT = 0.35) indicate polyclonal IgH rearrangements: tonsil and five unnumbered patient DNA samples. Axes definitions are the same as in Figure 1 .