Plasma cell-free DNA hydroxymethylation profiling reveals anti-PD-1 treatment response and resistance biology in non-small cell lung cancer.

BACKGROUND: Treatment with immune checkpoint inhibitors (ICIs) targeting programmed death-1 (PD-1) can yield durable antitumor responses, yet not all patients respond to ICIs. Current approaches to select patients who may benefit from anti-PD-1 treatment are insufficient. 5-hydroxymethylation (5hmC) analysis of plasma-derived cell-free DNA (cfDNA) presents a novel non-invasive approach for identification of therapy response biomarkers which can tackle challenges associated with tumor biopsies such as tumor heterogeneity and serial sample collection. METHODS: 151 blood samples were collected from 31 patients with non-small cell lung cancer (NSCLC) before therapy started and at multiple time points while on therapy. Blood samples were processed to obtain plasma-derived cfDNA, followed by enrichment of 5hmC-containing cfDNA fragments through biotinylation via a two-step chemistry and binding to streptavidin coated beads. 5hmC-enriched cfDNA and whole genome libraries were prepared in parallel and sequenced to obtain whole hydroxymethylome and whole genome plasma profiles, respectively. RESULTS: Comparison of on-treatment time point to matched pretreatment samples from same patients revealed that anti-PD-1 treatment induced distinct changes in plasma cfDNA 5hmC profiles of responding patients, as judged by Response evaluation criteria in solid tumors, relative to non-responders. In responders, 5hmC accumulated over genes involved in immune activation such as inteferon (IFN)-γ and IFN-α response, inflammatory response and tumor necrosis factor (TNF)-α signaling, whereas in non-responders 5hmC increased over epithelial to mesenchymal transition genes. Molecular response to anti-PD-1 treatment, as measured by 5hmC changes in plasma cfDNA profiles were observed early on, starting with the first cycle of treatment. Comparison of pretreatment plasma samples revealed that anti-PD-1 treatment response and resistance associated genes can be captured by 5hmC profiling of plasma-derived cfDNA. Furthermore, 5hmC profiling of pretreatment plasma samples was able to distinguish responders from non-responders using T cell-inflamed gene expression profile, which was previously identified by tissue RNA analysis. CONCLUSIONS: These results demonstrate that 5hmC profiling can identify response and resistance associated biological pathways in plasma-derived cfDNA, offering a novel approach for non-invasive prediction and monitoring of immunotherapy response in NSCLC.

Epigenomic Blood-Based Early Detection of Pancreatic Cancer Employing Cell-Free DNA.

BACKGROUND & AIMS: Early detection of pancreatic cancer (PaC) can drastically improve survival rates. Approximately 25% of subjects with PaC have type 2 diabetes diagnosed within 3 years prior to the PaC diagnosis, suggesting that subjects with type 2 diabetes are at high risk of occult PaC. We have developed an early-detection PaC test, based on changes in 5-hydroxymethylcytosine (5hmC) signals in cell-free DNA from plasma. METHODS: Blood was collected from 132 subjects with PaC and 528 noncancer subjects to generate epigenomic and genomic feature sets yielding a predictive PaC signal algorithm. The algorithm was validated in a blinded cohort composed of 102 subjects with PaC, 2048 noncancer subjects, and 1524 subjects with non-PaCs. RESULTS: 5hmC differential profiling and additional genomic features enabled the development of a machine learning algorithm capable of distinguishing subjects with PaC from noncancer subjects with high specificity and sensitivity. The algorithm was validated with a sensitivity for early-stage (stage I/II) PaC of 68.3% (95% confidence interval [CI], 51.9%-81.9%) and an overall specificity of 96.9% (95% CI, 96.1%-97.7%). CONCLUSIONS: The PaC detection test showed robust early-stage detection of PaC signal in the studied cohorts with varying type 2 diabetes status. This assay merits further clinical validation for the early detection of PaC in high-risk individuals.

Structural and biophysical correlation of anti-NANP antibodies with in vivo protection against P. falciparum.

The most advanced P. falciparum circumsporozoite protein-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective antibodies and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. In this study, antibody apparent affinity correlates best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I ß- and Asn pseudo 310 turns; such insights can be incorporated into design of more effective immunogens and antibodies for passive immunization.

Remote Activation of a Latent Epitope in an Autoantigen Decoded With Simulated B-Factors.

Mutants of a catalytically inactive variant of Proteinase 3 (PR3)-iPR3-Val103 possessing a Ser195Ala mutation relative to wild-type PR3-Val103-offer insights into how autoantigen PR3 interacts with antineutrophil cytoplasmic antibodies (ANCAs) in granulomatosis with polyangiitis (GPA) and whether such interactions can be interrupted. Here we report that iHm5-Val103, a triple mutant of iPR3-Val103, bound a monoclonal antibody (moANCA518) from a GPA patient on an epitope remote from the mutation sites, whereas the corresponding epitope of iPR3-Val103 was latent to moANCA518. Simulated B-factor analysis revealed that the binding of moANCA518 to iHm5-Val103 was due to increased main-chain flexibility of the latent epitope caused by remote mutations, suggesting rigidification of epitopes with therapeutics to alter pathogenic PR3·ANCA interactions as new GPA treatments.

Non-progressing cancer patients have persistent B cell responses expressing shared antibody paratopes that target public tumor antigens.

There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility.

Antibodies to influenza nucleoprotein cross-react with human hypocretin receptor 2.

The sleep disorder narcolepsy is linked to the HLA-DQB1*0602 haplotype and dysregulation of the hypocretin ligand-hypocretin receptor pathway. Narcolepsy was associated with Pandemrix vaccination (an adjuvanted, influenza pandemic vaccine) and also with infection by influenza virus during the 2009 A(H1N1) influenza pandemic. In contrast, very few cases were reported after Focetria vaccination (a differently manufactured adjuvanted influenza pandemic vaccine). We hypothesized that differences between these vaccines (which are derived from inactivated influenza viral proteins) explain the association of narcolepsy with Pandemrix-vaccinated subjects. A mimic peptide was identified from a surface-exposed region of influenza nucleoprotein A that shared protein residues in common with a fragment of the first extracellular domain of hypocretin receptor 2. A significant proportion of sera from HLA-DQB1*0602 haplotype-positive narcoleptic Finnish patients with a history of Pandemrix vaccination (vaccine-associated narcolepsy) contained antibodies to hypocretin receptor 2 compared to sera from nonnarcoleptic individuals with either 2009 A(H1N1) pandemic influenza infection or history of Focetria vaccination. Antibodies from vaccine-associated narcolepsy sera cross-reacted with both influenza nucleoprotein and hypocretin receptor 2, which was demonstrated by competitive binding using 21-mer peptide (containing the identified nucleoprotein mimic) and 55-mer recombinant peptide (first extracellular domain of hypocretin receptor 2) on cell lines expressing human hypocretin receptor 2. Mass spectrometry indicated that relative to Pandemrix, Focetria contained 72.7% less influenza nucleoprotein. In accord, no durable antibody responses to nucleoprotein were detected in sera from Focetria-vaccinated nonnarcoleptic subjects. Thus, differences in vaccine nucleoprotein content and respective immune response may explain the narcolepsy association with Pandemrix.