La diversificazione intraclonale nei geni IGHV nella leucemia linfatica cronica: da un approccio bioinformatico alla clinica.

In dependence of the identity of the variable region of the heavy chain of the immunoglobulin (IGHV) gene respect to the germline, chronic lymphocytic leukemia (CLL) may be subdivided into U-CLL and M-CLL. The evaluation of the IGHV is a hallmark in CLL due to the stability during time and its prognostic and predictive value. Despite this, IGHV intraclonal diversification (ID) has been described in the Sanger era. However, in the Next Generation Sequencing era, no author developed a solid and reliable workflow for ID identification and quantification. It follows that ID characterization is still lacking. Moreover, nobody evaluated the clinical impact of ID in CLL yet. Using the NGS technologies we exploited the immunoglobulin repertoire of 1091 CLL samplesto generate a tailored approach for ID evaluation. Using these data, we developed an innovative methodology to identify systematic sequencing errors (SE) on sequencing data of immunological repertoire (RepSeq), correct them and evaluate ID through the calculation of the inverse Simpson Index (iSI). With focused experiments, we demonstrated the robustness of our approach and the full superimposition of corrected data with the gold standard for RepSeq, namely unique molecular identifiers-based amplification protocol. Moreover, we validate our approach by analyzing other B cell malignancies with documented ID producing a classification coherent with the literature. A validated cutoff of 1.2 of iSI was generated to discriminate CLL samples with ID features (I) and samples without (nI). Among 983 CLL patients with iSI score available, only 15% of samples displayed ID according to 8 the iSI 1.2 cutoff. Both M-CLL and U-CLL have sample with ID, despite a significant ID skewing toward M-CLL was found. No variation in IGHV family or gene usage according to the presence/absence of ID was reported. Analyzing the RepSeq data for the identification of molecular signatures compatible with canonical somatic hypermutation (SHM) processes we observed a significant higher presence of mutations based on Activation induced cytidine deaminase (AICDA) in the context of I-CLL. Indeed, a significant higher AICDA mRNA levels was observed in I-M- CLL. Lastly, taking advantage of 685 CLL patients with time to first treatment (TTFT) available, we observed a significantly longer TTFT of I-M-CLL respect to nI-M-CLL, whereas no differences were observed in U-CLL. In conclusion, we succeeded to quantitative characterize the CLL intraclonal diversification phenomenon and to demonstrate its possible clinical correlation.

In dependence of the identity of the variable region of the heavy chain of the immunoglobulin (IGHV) gene respect to the germline, chronic lymphocytic leukemia (CLL) may be subdivided into U-CLL and M-CLL. The evaluation of the IGHV is a hallmark in CLL due to the stability during time and its prognostic and predictive value. Despite this, IGHV intraclonal diversification (ID) has been described in the Sanger era. However, in the Next Generation Sequencing era, no author developed a solid and reliable workflow for ID identification and quantification. It follows that ID characterization is still lacking. Moreover, nobody evaluated the clinical impact of ID in CLL yet. Using the NGS technologies we exploited the immunoglobulin repertoire of 1091 CLL samplesto generate a tailored approach for ID evaluation. Using these data, we developed an innovative methodology to identify systematic sequencing errors (SE) on sequencing data of immunological repertoire (RepSeq), correct them and evaluate ID through the calculation of the inverse Simpson Index (iSI). With focused experiments, we demonstrated the robustness of our approach and the full superimposition of corrected data with the gold standard for RepSeq, namely unique molecular identifiers-based amplification protocol. Moreover, we validate our approach by analyzing other B cell malignancies with documented ID producing a classification coherent with the literature. A validated cutoff of 1.2 of iSI was generated to discriminate CLL samples with ID features (I) and samples without (nI). Among 983 CLL patients with iSI score available, only 15% of samples displayed ID according to 8 the iSI 1.2 cutoff. Both M-CLL and U-CLL have sample with ID, despite a significant ID skewing toward M-CLL was found. No variation in IGHV family or gene usage according to the presence/absence of ID was reported. Analyzing the RepSeq data for the identification of molecular signatures compatible with canonical somatic hypermutation (SHM) processes we observed a significant higher presence of mutations based on Activation induced cytidine deaminase (AICDA) in the context of I-CLL. Indeed, a significant higher AICDA mRNA levels was observed in I-M- CLL. Lastly, taking advantage of 685 CLL patients with time to first treatment (TTFT) available, we observed a significantly longer TTFT of I-M-CLL respect to nI-M-CLL, whereas no differences were observed in U-CLL. In conclusion, we succeeded to quantitative characterize the CLL intraclonal diversification phenomenon and to demonstrate its possible clinical correlation.