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.