Bigdye sequencing system

Tumor biopsy specimens prior to treatment were obtained from 302 patients with de novo DLBCL, which have previously been classified by gene expression profiling, and 116 patients with follicular lymphoma (FL); 75 patients with chronic lymphocytic leukemia (CLL); 68 patients with Hodgkin lymphoma (HL). All samples were studied according to a protocol approved by the National Cancer Institute Institutional Review Board. Genomic DNA from patient samples was extracted with the DNeasy Tissue kit (Qiagen) according to the manufacturer’s instructions. The primers used to amplify RNF31 exon 10 are: RNF31_E10_F, 5′-CTGGGCTGGGTGCCTTTTCCTGTCAGG-3′ and RNF31_E10_R, 5′- GAGTAATTCTTGGACCAGGTATCG-3′. The PCR products were purified using the MinElute 96 UF PCR Purification Kit (Qiagen) and subsequently sequenced using BigDye sequencing system (Applied Biosystems) from both strands.

Genomic DNA from patient samples was extracted with the AllPrep DNA/RNA Kit (Qiagen) according to the manufacturer’s instructions. PCR was performed with a GeneAmp XL PCR kit (Applied Biosystems) as previously described (23 (link), 24 (link)). The sequences for primers applied to amplify MYD88, CD79A, CD79B, CARD11, and TNFAIP3 are summarized in Supplementary Table S2. The PCR products were visualized by electrophoresis on a 1% agarose gel and ethidium bromide staining. The templates were purified using the QuickStep2 96-well PCR purification Kit (Edge BioSystems) and subsequently sequenced (BigDye sequencing system, Applied Biosystems). Mutations were confirmed on independent PCR products and sequenced from both strands.
Genetic mutations in the B-cell receptor (BCR) pathway have been shown to result in constitutive activity of NFκB, leading to deregulated proliferation and survival signals (23 (link), 25 (link)). In addition, mutations in this pathway are predicted to result in intrinsic resistance to targeted agents such as ibrutinib. Indeed, mutations in CARD11 or TNFAIP3 have been shown to inhibit clinical response to ibrutinib in R/R DLBCL (26 (link)). We therefore performed targeted sequencing of genes in the BCR pathway (MYD88, CD79A, CD79B, CARD11, and TNFAIP3) to understand if mutations abrogated lenalidomide activity.