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were similar and significantly shorter than those of cases lacking all these mutations (P≤0.0001 in all pairwise com- parisons with WT) (Figure 1C), in keeping with previously reported studies,11,16,19,32 irrespective of IGHV status (Online Supplementary Figure S1A) or concomitant SF3B1/NOTCH1 mutations (Online Supplementary Figure S1B).
SF3B1-mutated chronic lymphocytic leukemia displays lower expression of CD20 than wild-type cases and similar to NOTCH1-mutated cases
Low CD20 expression represents a hallmark of CLL compared to other lymphoproliferative disorders or nor- mal B cells (Online Supplementary Figure 2A).7 We previous- ly showed that a reduction of CD20 could be due to NOTCH1 mutation-driven epigenetic dysregulation involving histone deacetylases.5,13 To expand this observa- tion, here we investigated whether low CD20 expression was also associated with SF3B1 mutations in CLL, possi- bly via a non-mutational activation of the NOTCH1 path- way.15 In keeping with previous observations,5,33 in the whole cohort of 537 cases, CLL bearing trisomy 12 (121/537 cases, 22.5%) had significantly higher expression of CD20 (P<0.0001) (Online Supplementary Figure S2A) and a strong association with NOTCH1 mutations (36/121, 29.8%, c2 P<0.0001); conversely, SF3B1 mutations were infrequent (6/121, 5.0%, c2 P=0.0815) (Online Supplementary Figure S2B, Online Supplementary Table S2). Therefore, to avoid confounding effects, for the main sub- sequent analyses we considered only cases without evi- dence of trisomy 12 (416/537 cases), irrespectively of other concomitant chromosomal abnormalities.
CD20 expression, determined by mean fluorescence intensity, was lower in SF3B1-mutated cases than in WT cases (SF3B1-mutated, 42 cases, median 7396; WT, 327 cases, median 9538; P=0.0024) (Figure 2A), without differ- ences from NOTCH1-mutated cases 47 cases, median 8438; P=0.3748) (Figure 2A), whose CD20 expression was lower than that of WT cases (P=0.0248), as reported pre- viously.5,13
CD20 expression is known to be heterogeneous and dis- persed in CLL, often spanning a 2- to 3-log range of fluo- rescence intensity within the CD19+/CD5+ pathological population.34,35 We, therefore, implemented a complemen- tary strategy of analysis, evaluating the percentage of the “CD20dim fraction”, defined as the differential population fraction between the two sides of the fluorescence distri- bution with respect to the mode (Online Supplementary Figure S2C).28 As a statistically robust measure of skew- ness, this method allowed us to discriminate cases with homogeneous or heterogeneous CD20 expression (Online Supplementary Figure S2D), with an inverse correlation with mean fluorescence intensity (rho= -0.417, P<0.001) (Figure 2B) and was consistent over time in sequential blood samples of treatment-naïve CLL cases (Online Supplementary Figure S2E). As shown in Figure 2C, the magnitude of the expansion of the CD20dim fraction was clearly increased in CLL cases with mutations of SF3B1 (median 15.5) or NOTCH1 (median 13.4) compared to WT cases (median 8.2; P<0.001 for both comparisons) while there were no evident differences between the SF3B1 mutational hotspots (Figure 2C, right panel). Again, no difference was found between NOTCH1-mutated and SF3B1-mutated cases (P=0.6365). Of note, when analyzed separately, the six cases with concomitant mutations of
SF3B1 and NOTCH1 showed a trend for a progressive expansion of the CD20dim fraction (median 19.3; P=0.0466 vs. WT) (Online Supplementary Figure S2F). Although excluded upfront from the analyses, the lower expression of CD20 was also confirmed in the trisomy 12 subset in NOTCH1-mutated CLL5,13 and with a trend in the context of the few SF3B1-mutated cases (Online Supplementary Figure S2G).
SF3B1-mutated cases show evidence of activation of the NOTCH1 pathway
To evaluate whether activation of the NOTCH1 path- way also occurs in primary SF3B1-mutated CLL, we per- formed GEP on 28 cases, 13 WT versus nine SF3B1-mutat- ed (VAF: range 21-48%, mean 37%) or six NOTCH1-mutated (VAF: range 17-51%, mean 30%) cases, all with unmutated IGHV and devoid of trisomy 12.
In the SF3B1-mutated category, 585 array probes (502 upregulated and 83 downregulated) corresponding to 443 known genes (402 upregulated and 41 downregulated) were differentially expressed (Online Supplementary Table S3) compared to WT samples. On the other hand, when comparing the NOTCH1-mutated and the WT categories, we identified 2,097 differentially expressed array probes (1,147 upregulated and 950 downregulated) (Online Supplementary Table S4), corresponding to 1,274 known genes (840 upregulated and 434 downregulated). When this NOTCH1 gene signature was applied to all samples, SF3B1-mutated cases clustered with the NOTCH1-mutat- ed cases (Online Supplementary Figure S3A), suggesting the presence of a common underlying signature. In fact, by merging the 443 SF3B1-mutated differentially expressed genes with the 1,274 NOTCH1-mutated differentially expressed genes, 419/443 (94.6%) were shared and con- cordantly regulated in the two signatures (390 upregulated and 29 downregulated) (Figure 3A, B). Consistently, when the SF3B1 signature was applied to all samples, NOTCH1-mutated cases clustered together with SF3B1-mutated cases (Online Supplementary Figure S3B). GEP data were externally validated by quantitative reverse transcriptase polymerase reaction (RT-qPCR), selecting four genes related to the NOTCH1 pathway (CD300A, IL1R2, HEY1, HES4) (Online Supplementary Figure S3C). Although in these signatures the differential expression of the single probe dedicated to MS4A1 (the gene encoding CD20) in our GEP platform (see Online Supplementary Methods) was not statistically significant upon correction for the false discovery rate (Online Supplementary Tables S3 and S4), a re-evaluation of MS4A1 transcript expression by RT-qPCR confirmed its significantly lower levels in NOTCH1-mutated versus WT cases (P=0.001) and in SF3B1-mutated versus WT cases (P<0.001) (Online Supplementary Figure S3D).
Gene set enrichment analysis of NOTCH1-mutated versus WT cases revealed a significant enrichment in 11 out of 17 NOTCH1-related datasets (Online Supplementary Table S5) linked to NOTCH1 transcriptional activity and overexpression (Figure 3C, Online Supplementary Figure S4A). Interestingly, one of the most significantly enriched datasets was a recently published CLL-specific NOTCH1 signature.15 The gene set enrichment analysis, using the same 17 NOTCH1-related datasets, when repeated on SF3B1-mutated versus WT cases, identified ten datasets as significantly enriched (Figure 3D, Online Supplementary Figure S4B, Online Supplementary Table S5), including the
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