An example of nested polymerase chain reaction (PCR) analysis for detection of minimal residual disease (MRD) in marrows from 2 follicular lymphoma (FL) patients.
Each sample is subjected to PCR for β-globin housekeeping gene and IgH/BCL2; PCR products undergo gel electrophoresis and (+) bands are detected by ethidium bromide staining (center gels = β-globin and bottom gels = IgH/BCL2 MBR). A band is seen only if an appropriate product is detected; specificity of bands is confirmed by Southern blot using BCL2 probe (top autoradiographs).
(Panel A): Multiple controls are run in parallel with patient samples (see box insert). (+) controls are serial dilutions of an IgH/BCL2 (+) cell line (lanes 4–7). The 106 dilution is run twice; as shown, a (+) result is often detected in only 1of these samples at the lower limit of analytical test sensitivity.
(Panels B andC): Parallel PCR analyses of 5 sequential marrows from 2 different FL patients are shown. For each patient, sample A was at diagnosis, before any treatment, B was 1–2 months after completion of CHOP chemotherapy but before treatment with anti-CD20 monoclonal antibody, and marrows C, D, and E were obtained at 2 months, 6 months, and 12 months after CD20 therapy. PCR of all clinical samples is run in duplicate. The patient in Panel B has a (+) nested PCR for IgH/BCL2 MBR at diagnosis and after CHOP, but the marrow becomes PCR (–) by 2 months after CD20 therapy and remains (–) at 6 and 12 months. The patient in Panel C is also PCR (+) at diagnosis and after CHOP, but also has a (+) band in 1 lane at 2 months after CD20 therapy. The (+) band is confirmed to represent a benign IgH/BCL2-carrying cell, not MRD, as it is not present in the duplicate sample and is clearly a different size from the patient’s FL clone. Note that results are more easily interpreted on the BCL2-probed Southern blots than on the ethidium bromide–stained gels.
