Variant frequency distributions reflect pervasive subclonal representation of most somatic coding mutations in FL. (A) Kernel density plots (shaded gray) depict the distribution of somatic nucleotide variant frequencies in each of 8 patients. For each case, peaks can be seen corresponding to clusters of variants with different frequencies. For 6 of 8 cases, the major peak resides less than 20% frequency, indicating that the majority of mutations are present only in a minor subclone within the total tumor cell pool. Red vertical bars represent conservative estimates of tumor purity defined by IGH-BCL2 translocation qPCR assay (supplemental Figure 2). Vertical black bars in LPJ103 and LPJ108 represent homozygous CREBBP mutations that correlate with tumor purity. (B) Variant frequencies in 5 cases were corrected for tumor purity measured by IGH-BCL2 translocation and plotted against depth of sequencing for the given nucleotide. Two clear clusters can be seen, corresponding to mutations that are probably heterozygous in the entire tumor pool (uniformly heterozygous) or within a subclone (subclonally heterozygous). The remaining points probably represent mutations that are homozygous within the entire tumor pool (uniformly homozygous) or within a minor clone (subclonally homozygous). Notably, 3 of 4 MLL2 mutations (pink) fall within uniformly heterozygous cluster and the remaining mutation within the uniformly homozygous cluster. All 4 CREBBP mutations (blue) fall within the uniformly homozygous cluster.

Intratumoral subpopulations are genetically divergent. Variant allele frequencies are compared between the CD20int (y-axis) and CD20hi (x-axis) intratumoral subpopulations. In most cases, only a minority of mutations are represented at approximately equal frequencies in each subpopulation (green dots). Many mutations comparably are enriched in either the CD20int (violet dots) or CD20hi (orange dots) subpopulation, or detected only in the CD20int (blue dots) or CD20hi (red dots) subpopulation. Mutations in selected genes of biologic interest are highlighted by letters A through J. Whereas CREBBP mutations (A) always appear in approximately equal frequencies in tumor cell subpopulations, MLL2 mutations are relatively enriched in 1 subpopulation in 3 of 4 tumors. Diagonal lines represent the threshold for 33% enrichment of variants in 1 subpopulation with reference to the alternate subpopulation (y = 1.33x and x = 1.33y), which allows 99% specificity for true subpopulation bias (supplemental Figure 1).

Subclonal representation of recurrent somatic mutations. Genes containing cSNVs in 2 or more diagnostic specimens within this study exhibit different patterns of representation between CD20int and CD20hi subpopulations. The top panel shows the recurrence frequency of mutations within each gene in our cohort. The bottom panel shows the fraction of those mutations that are represented approximately equally (≤ 33% deviation; green) between each subpopulation, those that show skewed representation (> 33% deviation; purple), and those that are identified exclusively in one subpopulation and not the other (gray). It can be seen that cSNVs in the majority of genes show skewed or unequal representation between tumor subpopulations, and CREBBP mutations are the only variants that are equally represented between subpopulations in all instances.

Relapse emanating from a progressed tumor clone in LPJ041. (A) Molecular cloning of productively rearranged Ig heavy-chain genes show a clonal relationship between the diagnosis and relapse specimen in LPJ041. Fifty-five unique clones were sequenced from the diagnostic specimen (blue), and 43 from the relapse specimen (red). Forty percent of the clones from the diagnostic specimen and 74% of the clones from the relapse specimen shared identical IgHV sequences (indicated by red/blue split). This was the closest sequence to the germ-line identified in the relapse specimen, indicating that the relapse emanated from an evolved tumor clone. Each arrowed line represents a single nucleotide substitution, with numbers representing additional substitutions. (B) Sanger sequencing showed identical t(14;18)(q32;q21) breakpoints in the diagnostic and relapse specimen for LPJ041. Four clones were sequenced from each of the diagnostic and relapse specimens and the breakpoint established by alignment of the sequences with the human genome consensus. (C) Comparison of variant frequencies in diagnostic and relapse specimens for LPJ041 showed the maintenance of the majority of mutations in the relapse that were detected within the diagnostic specimen (green), including 2 mutations in MLL2 that were variably represented in diagnostic subpopulations and a mutation in CREBBP that was uniformly represented at diagnosis. The relapse sample lost a subset of mutations that were identified in the diagnostic specimen (blue), and acquired other mutation that were not detected in the diagnostic specimen (red), including a premature stop codon in TNFAIP3. All highlighted mutations have been validated by Sanger sequencing. (D) Comparison of DNA copy number alterations in diagnostic and relapse specimens of LPJ041 show detectable DNA copy number gains (red) and losses (blue) that are maintained from diagnosis to relapse. The relapse acquires an additional major abnormality, loss of chromosome X.

Relapse emanating from an early precursor clone in LPJ128. (A) Molecular cloning of productively rearranged Ig heavy-chain genes showed identical VDJ breakpoints for the diagnostic and relapse specimens of LPJ128, indicating clonal origin. Seventy-four clones were sequenced from the diagnostic specimen (blue), and 43 from the relapse (red). The sequences obtained from the relapse specimen clones were unique from those obtained from the diagnostic specimen clones, and indicated that the relapse emanated from an early tumor precursor clone. Each arrowed line represents a single nucleotide substitution, with numbers representing additional substitutions. (B) Sanger sequencing showed identical t(14;18)(q32;q21) breakpoints in the diagnostic and relapse specimen for LPJ128, indicating that this is an early event. Four clones were sequenced from each of the diagnostic and relapse specimens and the breakpoint established by alignment of the sequences with the human genome consensus. (C) Comparison of variant frequencies in diagnostic and relapse specimens for LPJ128 showed mutations that the relapse specimen loses the majority of mutations in the relapse that were detected in the diagnostic specimen (blue), including mutations in MLL2 and TNFRSF14 that were variably represented in diagnostic subpopulations. A subset of mutations that were uniformly represented in the diagnostic specimen, including that in CREBBP, were maintained at relapse (green). The relapse also acquired additional mutations (red), including a unique TNFRSF14 mutation after loss of the mutation in the diagnostic specimen. All highlighted mutations were validated by Sanger sequencing. (D) Comparison of DNA copy number alterations in diagnostic and relapse specimens of LPJ128 show detectable DNA copy number gains (red) and losses (blue) that are all lost from diagnosis to relapse.

A model of genetic evolution of FL. A normal B-cell undergoes immunoglobulin recombination, followed by acquisition of the t(14;18)(q32;q21) founder IGH-BCL2 translocation yielding a premalignant tumor cell precursor, detectable in the majority of older adults in the absence of tumors. This precursor acquires 1 or more driver mutations, such as in CREBBP, yielding an early malignant clone. Accelerator mutations are then acquired, such as those in MLL2 and/or TNFRSF14, resulting in a progressed malignant clone with a selective advantage that yields clinical disease. Relapses may originate from either an early malignant clone as in patient LPJ128 (), and therefore possess only founder and driver mutations, or from a progressed malignant clone as in patient LPJ041 (), and therefore possess a full repertoire of founder, driver and accelerator mutations.