Integrative genomic analysis implicates gain of PIK3CA at 3q26 and MYC at 8q24 in chronic lymphocytic leukemia

Abstract

Purpose: The disease course of chronic lymphocytic leukemia (CLL) varies significantly within cytogenetic groups. We hypothesized that high-resolution genomic analysis of CLL would identify additional recurrent abnormalities associated with short time-to-first therapy (TTFT).

Experimental design: We undertook high-resolution genomic analysis of 161 prospectively enrolled CLLs using Affymetrix 6.0 SNP arrays, and integrated analysis of this data set with gene expression profiles.

Results: Copy number analysis (CNA) of nonprogressive CLL reveals a stable genotype, with a median of only 1 somatic CNA per sample. Progressive CLL with 13q deletion was associated with additional somatic CNAs, and a greater number of CNAs was predictive of TTFT. We identified other recurrent CNAs associated with short TTFT: 8q24 amplification focused on the cancer susceptibility locus near MYC in 3.7%; 3q26 amplifications focused on PIK3CA in 5.6%; and 8p deletions in 5% of patients. Sequencing of MYC further identified somatic mutations in two CLLs. We determined which catalytic subunits of phosphoinositide 3-kinase (PI3K) were in active complex with the p85 regulatory subunit and showed enrichment for the α subunit in three CLLs carrying PIK3CA amplification.

Conclusions: Our findings implicate amplifications of 3q26 focused on PIK3CA and 8q24 focused on MYC in CLL.

Figure 1

A. Overview of CNAs identified in CLL. GISTIC track at bottom shows statistically significant recurrent gains and losses. Gradations of gain (red) to loss (blue) are presented as previously described(9). B. GISTIC plots of amplifications and deletions identified in all CLL tumor samples, with chromosomal locations labeled on the right. The size of each bar shows the G-score which is the frequency times average amplitude of the aberrations. C. GISTIC plots of amplifications and deletions identified in CLL tumor samples from patients who remained untreated, with chromosomal locations labeled on the right. D. TTFT based on number of CNAs in the entire cohort. Median 161.2 months for CNAs < 2 (n = 107), and 49.5 months for CNAs > = 2 (n = 53) (p< 0.0001). E. TTFT based on number of CNAs in patients without deletions of 11q or 17p. Median 161.2 months for CNAs < 2 (n = 105), and 54.8 months for CNAs > = 2 (n = 38) (p = 0.0007).

Figure 2

A. Detailed view of 13q deletion showing GISTIC regions R1, R2 and R3. The red bar on the chromosome indicates the region shown in the expanded figure below. B. TTFT for long vs short 13q deletions, divided by whether patients were treated prior to or after sampling. C. TTFT for biallelic vs monoallelic 13q deletions, divided by whether patients were treated prior to or after sampling. D. TTFT for sole 13q deletion as compared to 13q deletion with any other somatic CNA defined by SNP array, divided by whether patients were treated prior to or after sampling. Previously treated cohort: median not reached for mono- or biallelic 13q deletion (n = 50); 62 months for 13q deletion with other CNAs (n = 27); 84 mos for no 13q deletion (n = 52) (p = 0.0008). Previously untreated cohort: median not reached for mono- or biallelic 13q deletion (n = 52); 40 months for 13q deletion with other CNAs (n = 23); 80 mos for no 13q deletion (n = 49) (p = 0.03). E. Boxplots of DLEU2 and RB1 gene expression, based on 13q deletion status.

Figure 3

A. Amplification of 3q26.32, focused on PIK3CA exon 21. Lower panel shows segmented (top) and raw (bottom) data for tumor and normal as noted. B. TTFT for 3q26 patients with or without deletions of 11q or 17p. Neither, median 87 mos (n = 136); 3q26 amplification alone, 62 mos (n = 7); deletions 11q or 17p alone, 27 mos (n = 15); 3q26 amplification with deletions 11q or 17p, 3.5 mos (n = 2) (p = 0.0001). C. PIK3CA expression determined by gene expression profiling. D. Immunoprecipitation of PIK3CA from two CLLs with broad and two with focal 3q26 amplifications, and 5 controls, with quantitation of bands below. E. Percentage of alpha, beta and delta p110 catalytic subunits in complex with p85, in samples with broad 3q26 amplification (n = 3) and controls without amplification (n = 6), as measured by mass spectrometry (p < 0.05). F. Ratio of alpha:delta p110 catalytic subunit, in complex with p85 regulatory subunit (p = 0.02). N = 3 samples with broad amplifications and 6 control samples, one of which had lower spectral counts(50). G. Heat map showing the CLL cohort ordered by correlation of gene expression with the 3q26 gain samples. Samples with 3q26 gain are labeled in red at right, and samples to the right of the gap show positive correlation with the 3q26 expression pattern. Tick marks at right indicate the locations of genes present on chromosome 3q26 (see Supplementary Table 20 for names).

Figure 4

A. Amplification of 8q24.21, whole chromosome view and focal region. The red bar on the chromosome indicates the region shown in the expanded figure below. Lower panel shows segmented (top) and raw (bottom) data for tumor and normal as noted. B. TTFT for 8q24 patients with or without deletions 11q or 17p. Neither, median 83.1 mos; 8q24 amplification with deletions 11q or 17p, 30 mos; 8q24 alone, 13 mos (p = 0.0001). C. Western blot showing MYC protein levels in 2 samples with 8q24 gain compared to 4 controls, with quantitation of bands below. D. Heat map showing the CLL cohort ordered by correlation of gene expression with the 8q24 gain samples. Samples with 8q24 gain are labeled in red at right, and samples to the right of the gap show positive correlation with the 8q24 expression pattern. Tick marks at right indicate the locations of genes present on chromosome 8q24 (see Supplementary Table 20 for names).

Figure 5

A. Deletion of 8p, whole chromosome view and focal region. The data show segmented data above raw data for tumor or normal as indicated. B. TTFT for deletion 8p patients with or without deletions 11q or 17p. Neither, median 61 mos; 8p deletion alone, 23 mos; deletions 11q or 17p alone, 19 mos; 8p deletion and deletions 11q or 17p, 11 mos (p < 0.0001). C. Heat map showing the CLL cohort ordered by correlation of gene expression with the 8p deletion samples. Samples with 8p deletion are labeled in blue at right, and samples to the right of the gap show positive correlation with the expression pattern of the 8p deletion samples. Tick marks at right indicate the locations of genes present on chromosome 8p (see Supplementary Table 20 for names).