Alteration of CD20 mRNA expression in B-cell lymphoma cells during the clinical course. (A) RT-PCR (RT) was performed using total RNA from the same tumor cells as in Figure 2B (UPN 3 in Table 2). As positive and negative controls, total RNA from Raji and 293T cells was used (lanes 1 and 2), respectively. I, II, and III (lanes 3-5) correspond to the clinical stages depicted in Figure 2A. (B) Quantitative RT-PCR was performed using the same RNA as in panel A. Arbitrary units of CD20 mRNA expression are indicated in the vertical axis. Note that faint expression of CD20 mRNA could be seen at stage II (column 4) despite a loss of CD20 surface protein expression as shown in Figure 2B. (C) CD20 mRNA expression in the lymphoma cells of UPN 4 (Table 2) was also analyzed. Tumor cells were derived from cerebral fluid at the first relapse after chemotherapy without rituximab (lane 1). Although complete remission was obtained after using rituximab-containing salvage chemotherapy, a second relapse occurred. Tumor cells were once again harvested from this patient's cerebral fluid and analyzed (lane 2). (D) Quantitative RT-PCR was also performed using the same RNA as in (C). Note that CD20 mRNA expression was significantly diminished but could still be observed. In these cells, CD20 protein expression was undetectable using FCM or IHC as indicated in Table 2. Positive and negative controls derived from Raji and 293T cells are indicated by + and −, respectively.