IGHV3-21–associated gene expression profiles in CLL. (A) Heat map generated with 122 probes found to be differentially expressed between IGHV3-21 patients (red bar under the tree) and non–IGHV3-21 patients (blue bar) by supervised analyses (Supplemental Materials). The color scale identifies relative gene expression changes normalized by the SD of 1 with 0 representing the mean expression level of a given gene. Expression for the 2 probes for the IGLV3-21(V_λ2-14) gene is reported below the heat map (location of these probes in the heat map is indicated by dashes); results of IGV light chain rearrangements, when available (ie, for all IGHV3-21 cases and 1 non–IGHV3-21 CLL), are reported below the corresponding hybridization spot. Location in the heat map of the VIPR1 and TGFβ2 genes are indicated by 1 and 2 asterisks, respectively. (B) Heat map generated with 20 genes found to be differentially expressed between homHCDR3–IGHV3-21 cases (yellow bar under the tree) and nonhomHCDR3–IGHV3-21 cases (orange bar) by supervised analyses (see “Materials and methods”). Location in the heat map of the WNT-16 gene is indicated by 3 asterisks. (C) Validation of microarray results by QRT-PCR. Three representative genes were selected to validate microarray results by QRT-PCR: VIPR1 and TGFβ2 were chosen to characterize non–IGHV3-21 versus IGHV3-21 CLLs (13 total cases), while WNT-16 was chosen among the genes differentially expressed by homHCDR3–IGHV3-21 and nonhomHCDR3–IGHV3-21 cases (6 cases). The incorporation of the SYBR Green dye into the PCR products was monitored in real time, and the resulting threshold cycles (Ct) (ie, the PCR cycle number corresponding to the beginning of the exponential growth of PCR products) were computed. Ct values were converted into attomoles by means of QRT-PCR experiments carried out with serial dilution of known concentrations of VIPR1-, TGFβ2-, WNT-16–, and β₂M-specific amplicons. Results for each CLL case represent the values of the relative expression levels of VIPR1/β₂M and TGFβ2/β₂M in IGHV3-21 cases (red bars) and non–IGHV3-21 cases (blue bars) and WNT-16/β₂M in homHCDR3–IGHV3-21 cases (yellow bars) and nonhomHCDR3–IGHV3-21 VH3-21 cases (orange bars). The panel on the bottom right represents ethidium bromide–stained agarose gels showing the expression of VIPR1, TGFβ2, and WNT-16 by conventional RT-PCR in 8 cell lines and in 3 representative CLL cases (B338, B80, and B419). Asterisks refer to molecular weight markers.