Tumor mutational burden and immune infiltration as independent predictors of response to neoadjuvant immune checkpoint inhibition in early TNBC in GeparNuevo.

BACKGROUND: The predictive value of tumor mutational burden (TMB), alone or in combination with an immune gene expression profile (GEP), for response to neoadjuvant therapy in early triple negative breast cancer (TNBC) is currently not known, either for immune checkpoint blockade (ICB) or conventional chemotherapy. PATIENTS AND METHODS: We obtained both whole exome sequencing and RNA-Seq data from pretreatment samples of 149 TNBC of the recent neoadjuvant ICB trial, GeparNuevo. In a predefined analysis, we assessed the predictive value of TMB and a previously developed immune GEP for pathological complete remission (pCR). RESULTS: Median TMB was 1.52 mut/Mb (range 0.02-7.65) and was significantly higher in patients with pCR (median 1.87 versus 1.39; P = 0.005). In multivariate analysis, odds ratios for pCR per mut/Mb were 2.06 [95% confidence intervals (CI) 1.33-3.20, P = 0.001] among all patients, 1.77 (95% CI 1.00-3.13, P = 0.049) in the durvalumab treatment arm, and 2.82 (95% CI 1.21-6.54, P = 0.016) in the placebo treatment arm, respectively. We also found that both continuous TMB and immune GEP (or tumor infiltrating lymphocytes) independently predicted pCR. When we stratified patients in groups based on the upper tertile of TMB and median GEP, we observed a pCR rate of 82% (95% CI 60% to 95%) in the group with both high TMB and GEP in contrast to only 28% (95% CI 16% to 43%) in the group with both low TMB and GEP. CONCLUSIONS: TMB and immune GEP add independent value for pCR prediction. Our results recommend further analysis of TMB in combination with immune parameters to individually tailor therapies in breast cancer.

Type I interferon gene signature test-low and -high patients with systemic lupus erythematosus have distinct gene expression signatures.

OBJECTIVES: Type I interferon (IFN) is implicated in systemic lupus erythematosus (SLE) pathogenesis. We aimed to identify type I IFN signaling-dependent and -independent molecular pathways in a large population of patients with SLE. METHODS: Baseline blood samples from adult patients with moderate to severe SLE from two Phase IIb studies (NCT01438489, n = 265; NCT01283139, n = 416) were profiled using whole transcriptome array analyses. Type I IFN gene signature (IFNGS) test status (high or low) was determined using a validated qualitative polymerase chain reaction-based test. IFN-type-specific signatures were developed by stimulating healthy blood with IFN-ß, IFN-γ, IFN-λ, IFN-ω, or pooled IFN-α. These, and multiple literature-derived cell type and cytokine pathway signatures, were evaluated in individual and pooled study populations. A Fisher's exact test was used for associations, adjusted for false discovery rate. RESULTS: Whole blood samples from IFNGS test-high patients were enriched versus IFNGS test-low patients for CD40L signaling (Q < 0.001), CXC cytokine (Q < 0.001), TLR8-mediated monocyte activation (Q < 0.001), IgG (Q < 0.001), major histocompatibility complex class I (Q < 0.001), and plasma cell (Q < 0.001) gene expression signatures. IFNGS test-low patients had significant enrichment of eosinophil (Q < 0.001), IFN-γ-specific (Q = 0.005), and T-cell or B-cell (Q < 0.001) signatures. Similar enrichment profiles were demonstrated in patients with primary Sjögren's syndrome, systemic sclerosis, and dermatomyositis. CONCLUSIONS: IFNGS test-high patients overexpressed many gene signatures associated with SLE pathogenesis compared with IFNGS test-low patients, reflecting broad immune activation. These results provide new insights into the molecular heterogeneity underlying SLE pathogenesis, highlighting shared mechanisms beyond type I IFN, across several autoimmune diseases. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01438489 and NCT01283139.

Enhanced B Cell Alloantigen Presentation and Its Epigenetic Dysregulation in Liver Transplant Rejection.

T cell suppression prevents acute cellular rejection but causes life-threatening infections and malignancies. Previously, liver transplant (LTx) rejection in children was associated with the single-nucleotide polymorphism (SNP) rs9296068 upstream of the HLA-DOA gene. HLA-DOA inhibits B cell presentation of antigen, a potentially novel antirejection drug target. Using archived samples from 122 white pediatric LTx patients (including 77 described previously), we confirmed the association between rs9296068 and LTx rejection (p = 0.001, odds ratio [OR] 2.55). Next-generation sequencing revealed that the putative transcription factor (CCCTC binding factor [CTCF]) binding SNP locus rs2395304, in linkage disequilibrium with rs9296068 (D' 0.578, r(2) = 0.4), is also associated with LTx rejection (p = 0.008, OR 2.34). Furthermore, LTx rejection is associated with enhanced B cell presentation of donor antigen relative to HLA-nonidentical antigen in a novel cell-based assay and with a downregulated HLA-DOA gene in a subset of these children. In lymphoblastoid B (Raji) cells, rs2395304 coimmunoprecipitates with CTCF, and CTCF knockdown with morpholino antisense oligonucleotides enhances alloantigen presentation and downregulates the HLA-DOA gene, reproducing observations made with HLA-DOA knockdown and clinical rejection. Alloantigen presentation is suppressed by inhibitors of methylation and histone deacetylation, reproducing observations made during resolution of rejection. Enhanced donor antigen presentation by B cells and its epigenetic dysregulation via the HLA-DOA gene represent novel opportunities for surveillance and treatment of transplant rejection.

Relationship between disease activity and type 1 interferon- and other cytokine-inducible gene expression in blood in dermatomyositis and polymyositis.

The objective of this study was to evaluate the relationship between blood mRNA, disease activity and treatment effects in a longitudinal study of patients with dermatomyositis (DM) or polymyositis (PM). In all, 24 patients with DM or PM were followed for up to 6 years (mean of 1.9 years) at 2-7 follow-up visits while receiving standard clinical care. Clinical data and blood samples collected at 80 patient visits were used for the analysis of cytokine-induced gene expression for the signaling pathways of type 1 interferon (IFN), tumor necrosis factor-α, IL-1ß, granulocyte-monocyte colony-stimulating factor, IL-10 and IL-13. A type 1 IFN signature score, but not other cytokine signature scores in the blood of patients with DM or PM, correlated highly with disease activity, decreased significantly with immunomodulatory therapies and showed concordant changes with major changes in disease activity. Type 1 IFN signature score in the blood correlates with disease activity in longitudinal follow-up of individual patients with DM or PM. The type 1 IFN-inducible gene transcripts in the blood have potential utility for monitoring disease activity in patients with DM or PM.

Allospecific CD154+ T cells associate with rejection risk after pediatric liver transplantation.

Antigen-specific T cells, which express CD154 rapidly, but remain untested in alloimmunity, were measured with flow cytometry in 16-h MLR of 58 identically-immunosuppressed children with liver transplantation (LTx), to identify Rejectors (who had experienced biopsy-proven rejection within 60 days posttransplantation). Thirty-one children were sampled once, cross-sectionally. Twenty-seven children were sampled longitudinally, pre-LTx, and at 1-60 and 61-200 days after LTx. Results were correlated with proliferative alloresponses measured by CFSE-dye dilution (n = 23), and CTLA4, a negative T-cell costimulator, which antagonizes CD154-mediated effects (n = 31). In cross-sectional observations, logistic regression and leave-one-out cross-validation identified donor-specific, CD154 + T-cytotoxic (Tc)-memory cells as best associated with rejection outcomes. In the longitudinal cohort, (1) the association between CD154 + Tc-memory cells and rejection outcomes was replicated with sensitivity/specificity 92.3%/84.6% for observations at 1-60 days, and (2) elevated pre-LTx CD154 + Tc-memory cell responses were associated with significantly increased incidence (p = 0.02) and hazard (HR = 7.355) of rejection in survival/proportional hazard analysis. CD154 expression correlated with proliferative alloresponses (r = 0.835, p = 7.1e-07), and inversely with CTLA4 expression of allospecific CD154 + Tc-memory cells (r =-0.706, p = 3.0e-05). Allospecific CD154 + T-helper-memory cells, not CD154 + Tc-memory, were inhibited by increasing Tacrolimus concentrations (p = 0.026). Collectively, allospecific CD154 + T cells provide an estimate of rejection risk in children with LTx.

Pharmacogenomics and translational simulations to bridge indications for an anti-interferon-α receptor antibody.

A type I interferon (IFN) gene signature shared by systemic lupus erythematous (SLE) and systemic sclerosis (SSc) was used to evaluate an anti-type I IFN-α receptor (IFN-αR) monoclonal antibody, MEDI-546, in a phase I trial in SSc. MEDI-546 suppressed IFN signature in blood and skin of SSc patients in a dose-dependent manner. To bridge clinical indications to SLE, we developed a model incorporating (i) pharmacokinetics (PK) and pharmacodynamics (PD) in SSc patients, (ii) internalization kinetics of MEDI-546/IFN-αR complex, and (iii) the different IFN signatures between SSc and SLE. Simulations predicted that i.v. administration of MEDI-546 at 300- or 1,000-mg monthly doses could suppress IFN signature in blood to levels of healthy subjects in 53 and 68% of SLE patients, respectively. An innovative approach utilizing a novel biomarker characterized the PD of MEDI-546 by modeling and simulation and allowed rapid progression of MEDI-546 from a phase I study in SSc to a randomized, multiple-dose phase II trial.

A novel oncogenic role for the miRNA-506-514 cluster in initiating melanocyte transformation and promoting melanoma growth.

Malignant melanoma is the most aggressive form of skin cancer and its incidence has doubled in the last two decades. It represents only 4% of skin cancer cases per year, but causes as many as 74% of skin cancer deaths. Early detection of malignant melanoma is associated with survival rates of up to 90%, but later detection (stage III to stage IV) is associated with survival rates of only 10%. Dysregulation of microRNA (miRNA) expression has been linked to tumor development and progression by functioning either as a tumor suppressor, an oncogene or a metastasis regulator in multiple cancer types. To understand the role of miRNA in the pathogenesis of malignant melanoma and identify biomarkers of metastasis, miRNA expression profiles in skin punches from 33 metastatic melanoma patients and 14 normal healthy donors were compared. We identified a cluster of 14 miRNAs on the X chromosome, termed the miR-506-514 cluster, which was consistently overexpressed in nearly all melanomas tested (30-60 fold, P<0.001), regardless of mutations in N-ras or B-raf. Inhibition of the expression of this cluster as a whole, or one of its sub-clusters (Sub-cluster A) consisting of six mature miRNAs, led to significant inhibition of cell growth, induction of apoptosis, decreased invasiveness and decreased colony formation in soft agar across multiple melanoma cell lines. Sub-cluster A of the miR-506-514 cluster was critical for maintaining the cancer phenotype, but the overexpression of the full cluster was necessary for melanocyte transformation. Our results provide new insights into the functional role of this miRNA cluster in melanoma, and suggest new approaches to treat or diagnose this disease.