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.

Two types of HERV-K genomes exist which differ in the absence (type 1) or the presence (type 2) of a sequence of 292 nucleotides between the putative pol and env genes. Previously published results from teratocarcinoma cell studies had firmly concluded that the type 1 HERV-K genome was defective in splicing and that only the nondeleted type 2 HERV-K genome containing the 292-nucleotide sequence was capable of being spliced. We now show that in the T47D human breast tumor cell line it is the type 1 HERV-K genome, and not the type 2, which is spliced to subgenomic transcripts.

Detecting clonal T-cell receptor (TCR)-␥ gene rearrangements (GRs) is an important adjunct test fordiagnosing T-cell lymphoma. We compared a recently described assay (BIOMED-2 protocol) , which targets multiple variable (V) gene segments in two polymerase chain reaction (PCR) reactions (multi-V) , with a frequently referenced assay that targets a single V gene segment in four separate PCR reactions (mono-V). A total of 144 consecutive clinical DNA samples were prospectively tested for T-cell clonality by PCR using laboratory-developed mono-V and commercial multi-V primer sets for TCR-␥ GR. The combination of TCR-␤ , mono-V TCR-␥ and multi-V TCR-␥ detected more clonal cases (68/144 , 47%) than any individual PCR assay. We detected clonal TCR-␤ GR in 47/68 (69%) cases. Using either mono-V or multi-V TCR-␥ primers , the sensitivities for detecting clonality were 52/68 (76%) or 51/68 (75%). Using both mono-V and multi-V TCR-␥ primers improved the sensitivity for detecting clonality , 60/68 (88%). Combining either mono-V or multi-V TCR-␥ primers with TCR-␤ primers also improved the sensitivity , 64/68 (94%). Significantly , TCR-␥ V11 GRs could only be detected using the mono-V-PCR primers. We conclude that using more than one T-cell PCR assay can enhance the overall sensitivity for detecting T-cell clonality.