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.