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R.W.J. Meijers et al.
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Figure 4. Differential expression analysis based on RNA sequencing data. (A) Results of the Spearman correlation of the RNA expression analysis in different chronic lymphocytic leukemia (CLL) samples (n=6 responsive vs. n=6 unresponsive CLL cases). The color scale indicates the degree of correlation varying from blue (low correlation) to red (high correlation). The two panels at the left end indicate the responsiveness (orange = unresponsive, blue = responsive) and the IGHV somatic hypermutation status [red=mutated (M)-CLL, green=unmutated (U)-CLL] of the selected CLL cases. (B) Volcano plot showing differences in transcript levels of genes involved in the B-cell receptor signaling pathway as determined using Qiagen’s Ingenuity Pathway Analysis (IPA). A negative logFC value indicates higher expression of certain genes in unresponsive CLL cases, while a positive logFC value is indicative of higher expression in responsive CLL cases. The logFC value was plotted against the 10log(Pvalue). False discovery rate (FDR) was calculated and transcriptional differences of genes with a logFC value of 1 or -1 in combination with a 10log(Pvalue) above the FDR are indicated in red.
els and autonomous signaling in TKO cells,14 we aimed to gain more insight into possible cell-intrinsic differences, although we cannot formally exclude that Ca2+ levels could also (partly) have been high due to previous anti- genic stimulation in our ex-vivo samples. Using a new cohort, Ca2+ signaling was determined in freshly isolated cells instead of thawed cells, which on average resulted in lower basal Ca2+ levels (data not shown). This might be, in combination with the heterogeneity in basal Ca2+ levels, an underlying explanation for the fact that in this cohort the basal Ca2+ levels were not different between M-CLL and U-CLL cases. Further building on the study of Mockridge et al.,18 who also showed differences in responsiveness to BCR stimulation between CLL cases,
we therefore divided our cohort of patients based on their responsive capacity to BCR stimulation. In both the M- CLL and U-CLL groups, there were cases showing a clear α-IgM response based on Ca2+ influx, while others did not show such a response, indicating that the level of anergy is independent of the IGHV SHM status of the BCR.
The anergic nature of unresponsive CLL was partly confirmed by the marker profile. IgM responders co- express higher levels of surface IgM and IgD, which explains the response to α-IgM as well as α-IgD stimula- tion. The higher expression of the prognostic marker CD38 by the responsive cases is also in line with findings of Mockridge et al.18 suggesting that responsive patients in general have a poor prognosis.2 The strong correlation
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haematologica | 2020; 105(1)