Chimeric RNAs reveal putative neoantigen peptides for developing tumor vaccines for breast cancer.

Introduction: We present here a strategy to identify immunogenic neoantigen candidates from unique amino acid sequences at the junctions of fusion proteins which can serve as targets in the development of tumor vaccines for the treatment of breastcancer. Method: We mined the sequence reads of breast tumor tissue that are usually discarded as discordant paired-end reads and discovered cancer specific fusion transcripts using tissue from cancer free controls as reference. Binding affinity predictions of novel peptide sequences crossing the fusion junction were analyzed by the MHC Class I binding predictor, MHCnuggets. CD8+ T cell responses against the 15 peptides were assessed through in vitro Enzyme Linked Immunospot (ELISpot). Results: We uncovered 20 novel fusion transcripts from 75 breast tumors of 3 subtypes: TNBC, HER2+, and HR+. Of these, the NSFP1-LRRC37A2 fusion transcript was selected for further study. The 3833 bp chimeric RNA predicted by the consensus fusion junction sequence is consistent with a read-through transcription of the 5'-gene NSFP1-Pseudo gene NSFP1 (NSFtruncation at exon 12/13) followed by trans-splicing to connect withLRRC37A2 located immediately 3' through exon 1/2. A total of 15 different 8-mer neoantigen peptides discovered from the NSFP1 and LRRC37A2 truncations were predicted to bind to a total of 35 unique MHC class I alleles with a binding affinity of IC50<500nM.); 1 of which elicited a robust immune response. Conclusion: Our data provides a framework to identify immunogenic neoantigen candidates from fusion transcripts and suggests a potential vaccine strategy to target the immunogenic neopeptides in patients with tumors carrying the NSFP1-LRRC37A2 fusion.

A potential diagnostic serum immunological marker panel to differentiate between primary and secondary knee osteoarthritis.

Inflammation contributes to knee osteoarthritis (KOA) where many immunological mediators participate in its initiation and progression. Most clinicians manage primary (pKOA) and secondary osteoarthritis (sKOA) alike. Whether immunological profiles of pKOA and sKOA differ remains obscure. Hence, we aimed to differentially identify potential serum immunologic diagnostic markers of pKOA and of sKOA. This case control study used 46 KOA patients (pKOA, n = 30; sKOA, n = 16), and 60 age, gender matched controls (normal healthy, n = 30; systemic lupus erythematosus [SLE] disease controls, n = 30) where serum was assayed for cytokines (TNF-α, IL-1ß, IL-6, IL-10) and nitric oxide derivatives (NOx). Sandwich ELISA assessed cytokine levels, while the 'Griess assay' quantified NOx levels. The diagnostic accuracy of optimal marker combinations was evaluated by the CombiROC web tool. Compared with pKOA, sKOA serum displayed significantly elevated levels of pro inflammatory cytokines (TNF-α, IL-1ß, IL-6) with a concurrent decrease in the anti-inflammatory cytokine, IL-10 (P<0.05). This was reiterated by significantly higher Th1:Th2 (TNF-α: IL-10) serum cytokine ratio observed in sKOA compared to that of pKOA. The CombiROC curves identified TNF-α, IL-1ß, IL-6 and NOx as the best performing panel of potential diagnostic markers to discriminate pKOA from control groups (~97% accuracy, 90% Sensitivity [SE] and 98% specificity [SP]), while TNF-α, IL-1ß and IL-6 discriminated sKOA from control groups (~100% accuracy, 100% SE, and 98% SP). The study identified discrete serum immune biomarker panels to differentiate between pKOA (TNF-α, IL-1ß, IL-6 and NOx) and sKOA (TNF-α, IL-1ß and IL-6). These findings may assist in developing distinct therapeutic agents for the two types of KOA.