Table 2

Comparison of high-throughput sequencing with real-time PCR MRD monitoring assays. For each patient specimen, IgH rearrangements were amplified from 200 ng of genomic DNA of the indicated specimen types with bar-coded primers adapted for 454 pyrosequencing. The IgH rearrangement libraries were pooled and sequenced. The number of clonal sequences (matching the initial diagnostic specimen clone) and the total number of sequences obtained are listed. Data from pyrosequencing were compared to the results of custom quantitative real-time PCR assays designed to amplify the patient’s malignant clonal sequence. The RT-PCR results were considered positive if >100 copies per microgram of template DNA were detected.

PatientSpecimenClone copies*Total sequences%Clone copiesTotal sequences%RT-PCR (copies/μg)
CLL A sample 1Diagnostic lymph node7,22711,19064.65,7458,93564.3>100,000
CLL A sample 2Blood03410.006700.010
CLL A sample 3Blood381,4772.6603,3501.81,485
CLL A sample 4Blood05880.001,6570.091
CLL A sample 5Blood04300.004910.037
CLL A sample 6Bone marrow01,4710.0212,9910.7314
CLL B sample 1Diagnostic bone marrow2,4614,36356.41,9643,58154.8>100,000
CLL B sample 2Bone marrow1,0801,97454.71,6563,00255.25,496
CLL B sample 3Blood01620.002080.024
CLL B sample 4Blood01140.001170.010
CLL B sample 5Bone marrow18849338.13431,12730.4944
Unrelated CLLBlood05,3260.007,6730.0
Normal controlTonsil014,0070.005,1670.0

*First replicate.

Second replicate.