Clonal Evolution of Molecular and Cytogenetic Aberrations in 80 CLL Patients

Background: In recent years, the knowledge on the genomic landscape of chronic lymphocytic leukemia (CLL) has dramatically increased. However, the clinical course is highly variable and understanding the clonal evolution is just in the beginnings.

Aim: To characterize mutational and cytogenetic changes during the course of CLL.

Patients and Methods: 80 CLL cases at diagnosis or without prior treatment (time-point A) presenting with persistence of disease at the follow-up evaluation (time-point B) were selected. The time lag was 1-10 years (mean and median: 5 years). All cases were screened at both time-points for following gene mutations by a targeted amplicon sequencing approach: ASXL1, ATM, BIRC3, BRAF, BTK, EGR2, FBXW7, KRAS, MAP2K1, MYD88, NRAS, NFKBIE, NOTCH1, PLCG2, POT1, SAMHD1, SF3B1, SRSF2, TP53, U2AF1, XPO1, and ZRSR2 . The mean overall read depth for all genes was 2,588 (range: 324 - 11,445). Allele frequency of 3% was preset as cut-off for positivity. IGHV mutational status was analyzed in all cases at time-point A. IGHV unmutated status (IGHV-U, sequence identity ≥ 98%) was detected in 55% of cases. Interphase FISH with probes for 17p13, 13q14, 11q22 and centromeric region of chromosome 12 was performed in 65/80 cases at both time-points. Male:female ratio was 1.9:1 and median age was 59 years (range: 38 - 80 years).

Results: 46% (37/80) of the patients presented with mutational alterations at follow-up. Overall, 46 changes of the mutation status in 14 genes were present with following frequencies: TP53 30%, NOTCH1 15%, BIRC3 11%, ATM 9%, SF3B1 9%, NRAS 7%, and POT1, BRAF , EGR2, KRAS, MYD88, NFKBIE, XPO1 and ZRSR2 <4%, each.

Diverse routes of changes were detected. Nine patients (24%) were unmutated at time-point A and showed acquisition of mutations at time-point B, 4 (11%) presented with no changes, and 1 case (3%) lost the known mutation at follow-up. The remaining cases (62%) showed diverse combinations of acquisition, loss and stability of mutations. Out of the 26 cases with acquired mutations during the disease course the mutations were retrospectively detected in 5 patients (19%) 1-9 years (mean: 4 years) prior to follow-up analysis at an allele frequency of 1-2%.

Clonal evolution of gene mutations was associated with the presence of following markers at time-point A: trisomy 12 (77% vs. 40%; p=0.030), IGHV-U (57% vs. 33%, p=0.044), SF3B1 mutations (73% vs. 40%, p=0.024) and begin of therapy before time-point B (60% vs. 25%, p=0.003).

In 65 cases, clonal evolution regarding FISH could be analyzed. At a frequency of 26% (17/65) patients showed cytogenetical evolution over a period of 2-9 years after first analysis (mean and median: 5 years). Overall, 21 changes were detected at following frequencies: del(17p) 47%, del(13q) 35%, del(11q) 29%, and trisomy 12 12%. Clonal evolution regarding FISH was associated with the presence of following markers at time-point A: IGHV-U (42% vs. 7%, p=0.002), NOTCH1 mutations (54% vs. 19%, p=0.029) and begin of therapy before time-point B (37% vs. 11%, p=0.024).

For the three most frequently evolved mutations TP53, NOTCH1 and BIRC3 single associations were analyzed. Gain of TP53 mutations was associated with clonal evolution in FISH (47% vs. 4%. P<0.001), mainly due to the association with the evolution of del(17p) (88% vs. 5%, p<0.001). Moreover, it was more frequent if therapy was applied before time-point B (27% vs. 3%, p=0.006), in IGHV-U cases (27% vs. 6%, p=0.016), and if SF3B1 mutations at time-point A were present (40% vs. 12%, p=0.020). Interestingly, SF3B1 mutated cases seem to be prone to acquire TP53 mutations especially if patients carry IGHV-U status: 50% (5/10). Clonal evolution of NOTCH1 mutations was associated with clonal evolution in FISH in general (24% vs. 2%, p=0.015) and clonal evolution of BIRC3 mutations was associated with del(11q) at time-point A (23% vs. 3%, p=0.028).

Conclusion: 46% of the analyzed cases showed clonal evolution of gene mutations. 62% of these presented with complex pathways suggesting the need for analysis at follow-up not only of already known mutations. Both molecular and cytogenetical evolution were associated with IGHV unmutated status and need for start of therapy. Particularly IGHV unmutated cases with SF3B1 mutations have a high risk to evolve TP53 mutations resulting in therapeutical consequences.