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Additional file 1: Supplementary Table 1. Antibodies used for BCR signaling activity assessment. Supplementary Table 2. Results of relative telomere length (RTL) quantification in the basic CLL cohort (n = 198). Supplementary Table 3. TP53 mutation evolution cohort ��� overview of clinico-biological features and results in baseline and follow-up samples, PART A Abbreviations: ALZ ��� alemtuzumab; BR - bendamustine and rituximab; CLB ��� chlorambucil; CR - cyclophosphamide and rituximab; del - deletion; F ��� female; FCR - fludarabine, cyclophosphamide, and rituximab; FISH - fluorescence in situ hybridization; hyper CVAD - hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone; IGHV ��� immunoglobulin heavy chain variable region; M ��� male; M ��� mutated; mo ��� months; U ��� unmutated; yrs ��� years. Supplementary Table 3. TP53 mutation evolution cohort ��� overview of clinico-biological features and results in baseline and follow-up samples, PART B Abbreviations: LTD ��� lymphocyte doubling time; mo ��� months; mut ��� mutated; p ��� phosphorylated; RTL ��� relative telomere length; VAF ��� variant allele frequency; wt ��� wildtype. Supplementary Figure 1. RTL of the entire CLL cohort was compared to the telomere length of a control DNA sample pooled from healthy individuals, arbitrarily set to RTL = 1 (green line). Median RTL of CLL cases (black line) was 0.81, range 0.46 ��� 1.25. Supplementary Figure 2. A) Rai (n = 130) and B) Binet (n = 133) stages at diagnosis in untreated CLL samples and their associations with RTL. The significant associations were P 0 vs III-IV = 0.0018; P I-II vs III-IV = 0.0108; P A vs C = 0.0043. Supplementary Figure 3. Overall survival (OS) of A) the entire CLL cohort (n = 198) divided by RTL above (���RTL long���) and below (���RTL short���) median RTL value (median RTL = 0.84; OS long RTL = 119; OS short RTL = 91; OS P = 0.0004); and B) of CLL patients with mutated TP53 status (n = 40) divided by RTL above (���RTL long���) and below (���RTL short���) median RTL value in this subgroup (median RTL = 0.78; OS long RTL = 86; OS short RTL = 55; OS P = 0.046). Supplementary Figure 4. hTERT expression in A) the baseline samples (P shortening vs. stable = 0.66; P stable vs. prolonging = 0.08; P shortening vs. prolonging = 0.45) and in B) the follow-up samples (P shortening vs. stable = 0.77; P stable vs. prolonging = 0.52; P shortening vs. prolonging = 0.82), (RTL shortening n = 10; stable n = 10; prolonging n = 6). C) Comparison of hTERT values in serial samples. hTERT expression did not associate with RTL prolongation. Supplementary Figure 5. Lymphocyte doubling time did not correlate with a change of RTL time during the disease course (Pearson r = -0.175; P = 0.45). Supplementary Figure 6. p-ZAP70/SYK significantly correlated with p-ERK1/2 in A) baseline samples (Pearson R = 0.82; P = 0.002) and B) follow-up samples (Pearson R = 0.92; P < 0.001).

Genome methylation profiles define na��ve-like (n-CLL), memory-like (m-CLL), and intermediate (i-CLL) subsets of chronic lymphocytic leukaemia (CLL). The profiles can be easily determined by the analysis of the five-CpG signature. m-CLL, i-CLL, and n-CLL with the good, intermediate, and poor prognoses, respectively, differ by the somatic hypermutation status of the immunoglobulin heavy chain variable gene (IGHV), a widely used prognostic predictor in CLL. We have previously shown that the expression of WNT5A, encoding a ROR1 ligand, distinguishes patients with the worse outcome within the prognostically favourable IGHV-mutated subgroup. To analyse the mechanisms controlling WNT5A expression, we investigated the methylation status of 54 CpG sites within the WNT5A promoter and its relation to the WNT5A gene expression. In a cohort of 59 CLL patients balanced for combinations of IGHV and WNT5A statuses, we identified three promoter CpG sites whose methylation level correlated with the WNT5A expression within the IGHV-mutated subgroup. Further, we complemented our data with the methylation status of the five-CpG signature. IGHV-mutated/WNT5A-negative and IGHV-mutated/WNT5A-positive cases overlapped with m���CLL and i���CLL methylation subgroups, respectively, while most IGHV���unmutated samples were assigned to n-CLL. Median methylation levels of all the three CpG sites in the WNT5A promoter were lowest in i-CLL. Finally, a detailed analysis of m-CLL and i-CLL showed that undetectable WNT5A expression predicts longer treatment-free survival with higher statistical significance than the classification according to the five-CpG signature. To conclude, a favourable m-CLL subgroup is associated with mutated IGHV and undetectable WNT5A expression due to its promoter methylation.