# Measurement of Immunoglobulin Intraclonal diversification refines the clinical impact of IGHV mutational status in chronic lymphocytic leukemia

## TL;DR

This study introduces a new method to measure intraclonal diversification in chronic lymphocytic leukemia, showing it can refine the clinical significance of IGHV mutations.

## Contribution

A novel NGS-based approach to quantify intraclonal diversification and its clinical relevance in mutated CLL.

## Key findings

- IDhigh cases are more common in mutated CLL and are associated with better clinical outcomes.
- IDhigh M-CLL patients have longer time-to-first treatment compared to IDlow patients.
- IDhigh cases show molecular signatures of active AID/Polη machinery and higher AID transcript levels.

## Abstract

Chronic lymphocytic leukemia (CLL) cells may bear mutations in IGHV genes, the 2%-cutoff allowing to discriminate two subsets, unmutated (U)- or mutated (M)-CLL, with different clinical course. IGHV genes may also incorporate additional ongoing mutations, a phenomenon known as intraclonal diversification (ID). Here, through an original bioinformatic workflow for NGS data, we used the inverse Simpson Index (iSI) as diversity measure among IGHV sequences to dichotomize cases with different ID levels into IDhigh (iSI ≥ 1.2) vs. IDlow (iSI < 1.2) both in CLL (n = 983) and in other lymphoproliferative disorders (LPD; n = 127). In CLL, IDhigh cases accounted for 14.6%, overrepresented in M-CLL (P = 0.0028), while higher percentages were documented in GC-derived LPD. In M-CLL (n = 396), IDhigh patients (n = 69) experienced longer time-to-first treatment than IDlow patients (P = 0.015), and multivariate analyses (n = 299) confirmed ID as independent variable. IGHV gene mutations of IDhigh cases had molecular signatures indicating ongoing activity of the AID)/Polη-dependent machinery; consistently, IDhigh M-CLL expressed higher levels of AID transcripts than IDlow M-CLL (P = 0.012). In conclusion, we propose a robust NGS protocol to quantitatively evaluate ID in CLL, demonstrating that: i) all CLL patients presented ID although at various degree; ii) high degree of ID has clinical relevance identifying a M-CLL subset with better outcome.