A Retrospective Analysis of 450 TP53 Mutations in a Real Life Cohort of CLL from the French Innovative Leukemia Organization (FILO) Group

In Chronic Lymphocytic Leukemia (CLL), it is well established that 17p deletions are associated with adverse prognosis and chemotherapy resistance. 17p deletions are most often associated with TP53 mutations, but TP53 mutations can occur in the absence of 17p deletion in about half of the cases with a similar unfavorable prognostic influence. Some patients harbor several subclones with different TP53 mutations. Nonetheless, little is known on the functional effect of the various alterations and of their associations.

We have retrospectively collected 450 TP53 variants from a real life cohort of 337 patients treated in centers of the FILO group (French Innovative Leukemia Organization). Among those, 218 were evidenced by Sanger sequencing (exons 4 to 9) with a sensitivity around 15% and 232 by next generation sequencing (exons 2 to 11) with a VAF (variant allelic frequency) >1%. All TP53 missense variants were previously described in the UMD TP53 mutation database strengthening the specificity of the data of this cohort.

Full clinical data were available for 245 patients. FISH analysis was performed in 230 cases, 17p deletion was absent in 100/230 cases (43%) confirming the importance of looking for TP53 mutation in these cases. Only 76 patients (31%) had an initial TP53 mutation assessment. The other 169 patients received a median of 2 treatments (0-11) before TP53 mutation was searched. Treatments included Fludarabine or Bendamustine based in 144 cases, Chlorambucil based in 47 cases and miscellaneous other treatments in 57 cases. After detection of TP53 mutation, 113 patients received either Ibrutinib or Idelalisib whereas 89 did not because there were not yet available. Overall survival of patients who received a BCR inhibitor is significantly better than those who did not (p<0,0001).

We analysed the mutation profile of TP53 . Mutations distribution along the TP53 gene was in agreement with the 2500 chronic lymphoproliferative disorders included in the UMD_TP53 database. There were 340 missense mutations (76%) and 110 null TP53 variants (24%), either nonsense mutations (n= 23), or frameshift insertions or deletions (n=51) or splice site mutations (n=36), leading to the potential loss of TP53 expression. Eighty patients (35%) had only a single TP53 alteration while 150 (65%) carried more than one TP53 alteration. The type of mutation was unrelated to the presence of several clones, of 17p deletion or the IGHV mutational status. But among the 80 patients with a single TP53 alteration, 70 had a TP53 missense mutant (versus only 10 null TP53 mutations, p<10-6) suggesting that the dominant negative effect of this mutant alleviates the need of a second event such as loss of 17p. Interestingly, the 10 patients who harboured only one null TP53 mutation appeared to have a milder CLL course and no strong chemorefractoriness.

We then studied the association between IGHV mutational status, presence of 17p deletion or presence of multiclonal TP53 mutations. IGHV status was mutated in 35/ 157 (22%) patients in which it was available. As expected, more multiclonal mutations cases were detected by NGS rather than by Sanger. Thirty cases harbored only small clones (VAF<15%), that would have been missed by Sanger sequencing. Two or more mutations were identified in 71 patients (21%), and 7 of them had 4 to 8 detectable clones. Presence of several clones also appeared independent of IGHV status and of presence or absence of 17p deletion. Richter's transformation occured in 29 patients experienced. We did not identify any specific pattern of TP53 mutation among those (hotspot, variant type or presence of subclones). Only 2 of them harbored mutated IGHV . Survival after Richter's transformation was very poor.

In conclusion, detection of 17p deletion by FISH analysis is currently included in the workout before therapy, whereas TP53 mutations are still not explored enough in routine practice. This retrospective real life cohort highlights the need for early detection of TP53 mutation in order to elude inefficient treatment and further development of resistant clones. Moreover, this cohort evidences that P53 alteration is a very heterogeneous process with multiple combinations depending on the penetrance of the various alterations, with disparity between missense and null variants. Whether or not this is linked to the clinical course of the disease is currently under study.