Deep sequencing reveals clonal evolution patterns and mutation events associated with relapse in B-cell lymphomas

Background

Molecular mechanisms associated with frequent relapse of diffuse large B-cell lymphoma (DLBCL) are poorly defined. It is especially unclear how primary tumor clonal heterogeneity contributes to relapse. Here, we explore unique features of B-cell lymphomas - VDJ recombination and somatic hypermutation - to address this question.

Results

We performed high-throughput sequencing of rearranged VDJ junctions in 14 pairs of matched diagnosis-relapse tumors, among which 7 pairs were further characterized by exome sequencing. We identify two distinctive modes of clonal evolution of DLBCL relapse: an early-divergent mode in which clonally related diagnosis and relapse tumors diverged early and developed in parallel; and a late-divergent mode in which relapse tumors developed directly from diagnosis tumors with minor divergence. By examining mutation patterns in the context of phylogenetic information provided by VDJ junctions, we identified mutations in epigenetic modifiers such as KMT2D as potential early driving events in lymphomagenesis and immune escape alterations as relapse-associated events.

Conclusions

Altogether, our study for the first time provides important evidence that DLBCL relapse may result from multiple, distinct tumor evolutionary mechanisms, providing rationale for therapies for each mechanism. Moreover, this study highlights the urgent need to understand the driving roles of epigenetic modifier mutations in lymphomagenesis, and immune surveillance factor genetic lesions in relapse.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0432-0) contains supplementary material, which is available to authorized users.     

The Chlorophyll Biosynthetic Enzyme Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase (Characterization and Partial Purification from Chlamydomonas reinhardtii and Synechocystis sp. PCC 6803)

A universal structural feature of chlorophyll molecules is the isocyclic ring. This ring is formed by the action of the enzyme Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, which catalyzes a complex reaction in which Mg-protoporphyrin IX monomethyl ester is converted to divinyl protochlorophyllide (also called Mg-2,4-divinylpheoporphyrin a5), with the participation of NADPH and O2. Cyclase activity was demonstrated in lysed Chlamydomonas reinhardtii chloroplasts and extracts of Synechocystis sp. PCC 6803. The yield of the reaction product was increased by the addition of catalase and ascorbate or isoascorbate to the incubation mixture. These compounds may act by preventing degradation of the tetrapyrroles by reactive oxygen species. Cyclase activity from C. reinhardtii was not inhibited by the flavoprotein inhibitor quinacrine or by the hemoprotein inhibitors CO, KCN, or NaN3. In contrast, cyclase activity in extracts of C. reinhardtii and Synechocystis sp. PCC 6803 was inhibited by chelators of Fe, suggesting that nonheme Fe is involved in the reaction. Cyclase in lysed C. reinhardtii chloroplasts was associated with the membranes, and attempts to further fractionate or solubilize the activity were unsuccessful. In contrast, cyclase in Synechocystis sp. PCC 6803 extracts could be separated into soluble and membrane components, both of which were required for reconstitution of activity. The membrane component retained activity after it was solubilized by the detergent n-octyl-[beta]-D-glucopyranoside in the presence of glycerol and Mg2+. The solubilized membrane component was purified further by dye-affinity and ion-exchange chromatography.     

Diterpene acids from Helianthus species and their microbiological conversion by Gibberella fujikuroi, mutant B1-41a

The diterpene acid content in 10 species of Helianthus has been investigated. Ent-12,16-cyclokauranoic acid, isolated from H. annuus, is converted into a series of 12,16-cyclogibberellins by cultures of Gibberella fujikuroi, mutant B1-41a, and 12,16-cyclogibberellins A₉, and A₁₂ have been isolated. Ent-12β-acetoxykaurenoic acid and ent-13(S)-angeloxyatisenoic acid have been isolated from H. decapetalus; the metabolism of ent-13(S)-hydroxyatisenoic acid and atisenoic acid by B1-41a is also described.     

14 C incorporation from exogenous compounds into -aminolevulinic acid by greening cucumber cotyledons

Greening cucumber cotyledons accumulate δ-aminolevulinic acid when treated with levulinic acid. A variety of specifically labelled compounds were applied to the tissue and label was measured in the δ-aminolevulinic acid. Glutamate, glutamine and α-ketoglutarate were found to be incorporated into δ-aminolevulinic acid to a much greater extent than were glycine and succinate. A new route of δ-aminolevulinic acid biosynthesis is proposed wherein the carbon skeleton of α-ketoglutarate is incorporated intact.     

Biosynthesis of phycobilins. 15,16-Dihydrobiliverdin IX alpha is a partially reduced intermediate in the formation of phycobilins from biliverdin IX alpha

A partially purified protein fraction from the phycocyanin-containing unicellular rhodophyte, Cyanidium caldarium, reductively transforms biliverdin IX alpha to a violet colored bilin in the presence of NADPH, ferredoxin, and ferredoxin-NADP+ reductase. This bilin has a violin-like absorption spectrum with maxima at 335 and 560 nm in methanolic HCl and at 337, 567, and 603-604 nm in CHCl3. The bilin has been determined to be 15,16-dihydrobiliverdin IX alpha by comparative spectrophotometry and 1H NMR spectroscopy. This product of biliverdin IX alpha reduction is converted enzymatically to phycobilins by further reduction. A general biosynthetic pathway is proposed which accounts for the formation of the phycobilins from biliverdin IX alpha by a two-step reduction process followed by isomerization.     

Some observations on conserved polar side chains in immunoglobulin V-domains

1. The roles of conserved polar residues have been studied in 12 V-domains for which atomic coordinates are available. 2. In most cases a particular residue had a similar side chain conformation in all V-domains examined and the polar group provided the same hydrogen bonds which helped to stabilize the conformations of the domains. 3. In the case of a conserved glutamine/glutamic acid residue the buried side chain could adopt a variety of conformations and the polar group could form different hydrogen bonds from one domain to another. However, they contributed similarly to domain stability. 4. In the case of a conserved threonine/serine residue its side chain showed relative rotations of up to 180 degrees from one domain to another. The hydroxyl group could be buried or exposed at the domain surface. In some domains it formed hydrogen bonds to two other protein atoms but in other domains there was a single hydrogen bond or none at all. The varied roles of this residue are discussed in the text.