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

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 ID_high (iSI ≥ 1.2) vs. ID_low (iSI < 1.2) both in CLL (n = 983) and in other lymphoproliferative disorders (LPD; n = 127). In CLL, ID_high 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), ID_high patients (n = 69) experienced longer time-to-first treatment than ID_low patients (P = 0.015), and multivariate analyses (n = 299) confirmed ID as independent variable. IGHV gene mutations of ID_high cases had molecular signatures indicating ongoing activity of the AID)/Polη-dependent machinery; consistently, ID_high M-CLL expressed higher levels of AID transcripts than ID_low 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.

Fig. 1

Flow-chart of the study with the number of patients analyzed.

Fig. 2. Illustrative phylogenetic trees in dependence of presence of IGHV Intraclonal Diversification (ID).

A ID was calculated from the Hill number-based diversity profiles of diversity indices. Among the different Hill number-derived indices, the inverse Simpson index (iSI, corresponding to a Hill number = 2) was selected. B Phylogenetic tree examples of three different CLL samples with increasing iSI. Nodes in the tree can be either the root node (orange node), leaves (sequences of cells that had no descendants; green nodes), or internal nodes. Internal nodes can be either split nodes, those with more than one child (light blue nodes); or pass-through nodes, those with exactly one child (red nodes). Size of the circle corresponds to the percentage of the specific subclone inside the pathological clone.

Fig. 3. Diversity score in lymphoproliferative disease.

A The scatter plot depicts the iSI calculated for 983 CLL versus the percentage of the Major Clone (clone with the higher percentage) inside the identified pathological clone. B Maximally selected rank statistics graphs for the determination of the best iSI cutoff. C Kaplan-Meier curves comparing TTFT probabilities of 327 M IGHV cases with low intraclonal (iSI < 1.2; ID_low; green line), 69 M IGHV cases with high intraclonal (iSI ≥ 1.2; ID_high) (purple line), 328 U IGHV cases with low intraclonal (light blue line), and 35 U IGHV cases with high intraclonal (red line). The number of patients in each group is reported; P value refers to log-rank test. D Boxplots report the distribution of diversity score calculated by means of Inverse Simpson Index (iSI) in different lymphoproliferative disease. Dotted line refers to iSI cutoff of 1.2.

Fig. 4. Distribution of IGHV families and genes in the CLL cohort among samples with low or high intraclonal diversification (ID).

A The barchart reports the number of CLL cases in dependence of IGHV families according the IGHV mutational status and divided by the presence or not of ID. B The barchart reports the number of CLL cases in dependence of IGHV genes divided according to the IGHV mutational status (mutated: M, unmutated: U) and the presence or not of ID. Light-blue bars represent unmutated IGHV (U-CLL) and ID_low cases, red bars represent U-CLL and ID_high cases, green bars represent mutated IGHV (M-CLL) and ID_low cases, purple bars represent M-CLL and ID_high cases. P value refers to chi-square test.

Fig. 5. Evaluation of mutability rate.

A Activation Induced Cytidine Deaminase (AID) activity. The boxplots on the right report the number of mutations compatible with AID mutational activity in both forward (WRC, W = A/T, R = A/G) and reverse strands (GYW, Y = C/T, W = A/T). The number of mutations occurring in AID coldspots for both the forward and reverse strand are reported on the left (SYC and GRS, S = G/C, Y = C/T, R = A/G). B The boxplots report the expression level of AID samples, 27 ID_high (19 M-, and 8 U-CLL) and 65 ID_low samples (40 M-, and 25 U-CLL). C The graphs report the density of replacement (R) and silent (S) mutations in dependence of the IGHV gene position. Green boxes represent M IGHV and ID_low cases, purple boxes represent M IGHV and ID_high cases. P values refer to student T-test.