Spontaneously regressed CLL tumors are characterized by weak BCR-signaling response and sIgM expression. FACS analysis of the gated CLL fraction of PBMCs. BCR-signaling responses to IgM and IgD stimulation were assessed by phosphoflow using fresh blood samples. Cells were stimulated with anti-human IgM and/or IgD F(ab′)2 antibodies prior to acquisition on a flow cytometer. Combined IgM and IgD stimulation reflects BCR stimulation in vivo, whereas separate IgM and IgD stimulation allows dissection of the relative contribution of IgM and IgD responses to the overall BCR-signaling response in each comparator group. (A) Left panel, The CD19+CD5+ CLL population (shown in red) was gated and analyzed for the phosphorylation of Syk, Erk, and Akt, with the CD19−CD5+ T-cell population (shown in blue) being used as the internal negative control. The positive vs negative gate for p-Syk, p-Erk, and p-Akt was set such that 99% of unstimulated cells would fall within the negative gate. Right panel, Example histograms showing results of a typical indolent M-CLL case and a spontaneous regression case with combined IgM/IgD stimulation. Phosphoprotein response to (B) IgM, (C) IgD, or (D) combined IgM and IgD BCR stimulation, as well as CLL cell-surface (E) IgM (sIgM) and (F) IgD (sIgD) expression. Spontaneous regression cases (REG; n = 14) from the regression time point (T1) were compared against indolent (INDOL) M-CLL (n = 35) and UM-CLL (n = 4) cases. Complete and partial spontaneous regression cases are represented by red and blue dots, respectively, as shown. (G) Expression of sIgM and sIgD was compared between 2 sequential time points in individual spontaneous regression cases. T0 and T1 represent the diagnostic and the regression time point, respectively. Each colored line represents a specific case as indicated. Statistical significance was determined using 1-way ANOVA with Bonferroni post hoc analysis for cohort comparison and the 2-tailed paired Student t test for time point comparison. Statistical significance is indicated by *P < .05, **P < .01, ***P < .001, and ****P < .0001. MFI, mean fluorescence intensity; ns, comparisons that are not statistically significant.