Tumor-infiltrating BRAFV600E-specific CD4+ T cells correlated with complete clinical response in melanoma.

T cells specific for neoantigens encoded by mutated genes in cancers are increasingly recognized as mediators of tumor destruction after immune checkpoint inhibitor therapy or adoptive cell transfer. Unfortunately, most neoantigens result from random mutations and are patient specific, and some cancers contain few mutations to serve as potential antigens. We describe a patient with stage IV acral melanoma who achieved a complete response following adoptive transfer of tumor-infiltrating lymphocytes (TILs). Tumor exome sequencing surprisingly revealed fewer than 30 nonsynonymous somatic mutations, including oncogenic BRAFV600E. Analysis of the specificity of TILs identified rare CD4+ T cells specific for BRAFV600E and diverse CD8+ T cells reactive to nonmutated self-antigens. These specificities increased in blood after TIL transfer and persisted long-term, suggesting they contributed to the effective antitumor immune response. Gene transfer of the BRAFV600E-specific T cell receptor (TCR) conferred recognition of class II MHC-positive cells expressing the BRAF mutation. Therapy with TCR-engineered BRAFV600E-specific CD4+ T cells may have direct antitumor effects and augment CD8+ T cell responses to self- and/or mutated tumor antigens in patients with BRAF-mutated cancers.

Identifying Abundant Immunotherapy and Other Targets in Solid Tumors: Integrating RNA-seq and Mass Spectrometry Proteomics Data Sets.

RNA-seq and mass-spectrometry proteomics combined with growing data repositories have greatly increased the capacity to identify candidate proteins or protein sequence variants that share properties of ideal therapy targets, which include being abundant in cancer cells, absent or rare in adult organs (especially vital organs), and shared by many patient tumors. RNA-seq and fixed content arrays can identify genes that are overexpressed or misexpressed in cancer. RNA-seq is uniquely suited to identifying cancer-specific sequence variants. We review factors relevant for determining whether products of genes that are abundant or differentially abundant in RNA-seq are concordant or discordant with proteins that are identified as abundant or differentially abundant in mass-spectrometry proteomics assays.

Neutrophils dominate the immune cell composition in non-small cell lung cancer.

The response rate to immune checkpoint inhibitor therapy for non-small-cell lung cancer (NSCLC) is just 20%. To improve this figure, several early phase clinical trials combining novel immunotherapeutics with immune checkpoint blockade have been initiated. Unfortunately, these trials have been designed without a strong foundational knowledge of the immune landscape present in NSCLC. Here, we use a flow cytometry panel capable of measuring 51 immune cell populations to comprehensively identify the immune cell composition and function in NSCLC. The results show that the immune cell composition is fundamentally different in lung adenocarcinoma as compared with lung squamous cell carcinoma, and that neutrophils are the most prevalent immune cell type. Using T-cell receptor-ß sequencing and tumour reactivity assays, we predict that tumour reactive T cells are frequently present in NSCLC. These results should help to guide the design of clinical trials and the direction of future research in this area.

Tumor-Infiltrating Merkel Cell Polyomavirus-Specific T Cells Are Diverse and Associated with Improved Patient Survival.

Tumor-infiltrating CD8+ T cells are associated with improved survival of patients with Merkel cell carcinoma (MCC), an aggressive skin cancer causally linked to Merkel cell polyomavirus (MCPyV). However, CD8+ T-cell infiltration is robust in only 4% to 18% of MCC tumors. We characterized the T-cell receptor (TCR) repertoire restricted to one prominent epitope of MCPyV (KLLEIAPNC, "KLL") and assessed whether TCR diversity, tumor infiltration, or T-cell avidity correlated with clinical outcome. HLA-A*02:01/KLL tetramer+ CD8+ T cells from MCC patient peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TIL) were isolated via flow cytometry. TCRß (TRB) sequencing was performed on tetramer+ cells from PBMCs or TILs (n = 14) and matched tumors (n = 12). Functional avidity of T-cell clones was determined by IFNγ production. We identified KLL tetramer+ T cells in 14% of PBMC and 21% of TIL from MCC patients. TRB repertoires were strikingly diverse (397 unique TRBs were identified from 12 patients) and mostly private (only one TCRb clonotype shared between two patients). An increased fraction of KLL-specific TIL (>1.9%) was associated with significantly increased MCC-specific survival P = 0.0009). T-cell cloning from four patients identified 42 distinct KLL-specific TCRa/b pairs. T-cell clones from patients with improved MCC-specific outcomes were more avid (P < 0.05) and recognized an HLA-appropriate MCC cell line. T cells specific for a single MCPyV epitope display marked TCR diversity within and between patients. Intratumoral infiltration by MCPyV-specific T cells was associated with significantly improved MCC-specific survival, suggesting that augmenting the number or avidity of virus-specific T cells may have therapeutic benefit. Cancer Immunol Res; 5(2); 137-47. ©2017 AACR.

Expression and functional characterization of CD33 transcript variants in human acute myeloid leukemia.

With the demonstration of improved survival of some acute myeloid leukemia (AML) patients with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO), CD33 has been validated as a target for antigen-specific immunotherapy. Since previous studies identified a CD33 splice variant missing exon 2 (CD33∆E2) and, consequently, the immune-dominant membrane-distal V-set domain, we investigated the expression and functional characteristics of CD33 transcript variants in AML. In primary AML specimens, we not only found full-length CD33 (CD33FL) and CD33∆E2 but also corresponding variants containing an alternate exon 7 predicted to encode a CD33 protein lacking most of the intracellular domain (CD33E7a and, not previously described, CD33∆E2,E7a) in almost all cases. In acute leukemia cell sublines engineered to express individual CD33 splice variants, all splice variants had endocytic properties. CD33FL and CD33E7a mediated similar degrees of GO cytotoxicity, whereas CD33∆E2 and CD33∆E2,E7a could not serve as target for GO. Co-expression of CD33∆E2 did not interfere with CD33FL endocytosis and did not impact CD33FL-mediated GO cytotoxicity. Together, our findings document a greater-than-previously thought complexity of CD33 expression in human AML. They identify CD33 variants that lack exon 2 and are not recognized by current CD33-directed therapeutics as potential target for future unconjugated or conjugated antibodies.

Proteins associated with pancreatic cancer survival in patients with resectable pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with a dismal prognosis. However, while most patients die within the first year of diagnosis, very rarely, a few patients can survive for >10 years. Better understanding the molecular characteristics of the pancreatic adenocarcinomas from these very-long-term survivors (VLTS) may provide clues for personalized medicine and improve current pancreatic cancer treatment. To extend our previous investigation, we examined the proteomes of individual pancreas tumor tissues from a group of VLTS patients (survival ≥10 years) and short-term survival patients (STS, survival <14 months). With a given analytical sensitivity, the protein profile of each pancreatic tumor tissue was compared to reveal the proteome alterations that may be associated with pancreatic cancer survival. Pathway analysis of the differential proteins identified suggested that MYC, IGF1R and p53 were the top three upstream regulators for the STS-associated proteins, and VEGFA, APOE and TGFß-1 were the top three upstream regulators for the VLTS-associated proteins. Immunohistochemistry analysis using an independent cohort of 145 PDAC confirmed that the higher abundance of ribosomal protein S8 (RPS8) and prolargin (PRELP) were correlated with STS and VLTS, respectively. Multivariate Cox analysis indicated that 'High-RPS8 and Low-PRELP' was significantly associated with shorter survival time (HR=2.69, 95% CI 1.46-4.92, P=0.001). In addition, galectin-1, a previously identified protein with its abundance aversely associated with pancreatic cancer survival, was further evaluated for its significance in cancer-associated fibroblasts. Knockdown of galectin-1 in pancreatic cancer-associated fibroblasts dramatically reduced cell migration and invasion. The results from our study suggested that PRELP, LGALS1 and RPS8 might be significant prognostic factors, and RPS8 and LGALS1 could be potential therapeutic targets to improve pancreatic cancer survival if further validated.

Quantitative glycoproteomics analysis reveals changes in N-glycosylation level associated with pancreatic ductal adenocarcinoma.

Glycosylation plays an important role in epithelial cancers, including pancreatic ductal adenocarcinoma. However, little is known about the glycoproteome of the human pancreas or its alterations associated with pancreatic tumorigenesis. Using quantitative glycoproteomics approach, we investigated protein N-glycosylation in pancreatic tumor tissue in comparison with normal pancreas and chronic pancreatitis tissue. The study lead to the discovery of a roster of glycoproteins with aberrant N-glycosylation level associated with pancreatic cancer, including mucin-5AC (MUC5AC), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), insulin-like growth factor binding protein (IGFBP3), and galectin-3-binding protein (LGALS3BP). Pathway analysis of cancer-associated aberrant glycoproteins revealed an emerging phenomenon that increased activity of N-glycosylation was implicated in several pancreatic cancer pathways, including TGF-ß, TNF, NF-kappa-B, and TFEB-related lysosomal changes. In addition, the study provided evidence that specific N-glycosylation sites within certain individual proteins can have significantly altered glycosylation occupancy in pancreatic cancer, reflecting the complexity of the molecular mechanisms underlying cancer-associated glycosylation events.

Phenotypic and transcriptional fidelity of patient-derived colon cancer xenografts in immune-deficient mice.

Xenografts of human colorectal cancer (CRC) in immune-deficient mice have great potential for accelerating the study of tumor biology and therapy. We evaluated xenografts established in NOD/scid/IL2Rγ-null mice from the primary or metastatic tumors of 27 patients with CRC to estimate their capacity for expanding tumor cells for in vitro studies and to assess how faithfully they recapitulated the transcriptional profile of their parental tumors. RNA-seq analysis of parental human CRC tumors and their derivative xenografts demonstrated that reproducible transcriptional changes characterize the human tumor to murine xenograft transition. In most but not all cases, the human stroma, vasculature, and hematopoietic elements were systematically replaced by murine analogues while the carcinoma component persisted. Once established as xenografts, human CRC cells that could be propagated by serial transplantation remained transcriptionally stable. Three histologically atypical xenografts, established from patients with peritoneal metastases, contained abundant human stromal elements and blood vessels in addition to human tumor cells. The transcriptomes of these mixed tumor/stromal xenografts did not closely resemble those of their parental tumors, and attempts to propagate such xenografts by serial transplantation were unsuccessful. Stable expression of numerous genes previously identified as high priority targets for immunotherapy was observed in most xenograft lineages. Aberrant expression in CRC cells of human genes that are normally only expressed in hematopoietic cells was also observed. Our results suggest that human CRC cells expanded in murine xenografts have great utility for studies of tumor immunobiology and targeted therapies such as immunotherapy but also identify potential limitations.

Significance of expression of ITGA5 and its splice variants in acute myeloid leukemia: a report from the Children's Oncology Group.

Acute myeloid leukemia (AML) encompasses a heterogeneous group of diseases, and novel biomarkers for risk refinement and stratification are needed to optimize patient care. To identify novel risk factors, we performed transcriptome sequencing on 68 diagnostic AML samples and identified 2 transcript variants (-E2 and -E2/3) of the α-subunit (ITGA5) of the very late antigen-5 integrin. We then quantified expression of ITGA5 and these splice variants in specimens from participants of the AAML03P1 trial. We found no association between ITGA5 expression and clinical outcome. In contrast, patients with the highest relative expression (Q4) of the -E2/3 ITGA5 splice variant less likely had low-risk disease than Q1-3 patients (21% vs. 38%, P = 0.027). Q4 patients had worse response to chemotherapy with a higher proportion having persistent minimal residual disease (50% vs. 23%, P = 0.003) and inferior overall survival (at 5 years: 48% vs. 67%, P = 0.015); the latter association was limited to low-risk patients (Q4 vs. Q1-3: 56% vs. 85%, P = 0.043) and was not seen in standard-risk (51% vs. 60%, P = 0.340) or high-risk (33% vs. 38%, P = 0.952) patients. Our exploratory studies indicate that transcriptome sequencing is useful for biomarker discovery, as exemplified by the identification of ITGA5 -E2/3 splice variant as potential novel adverse prognostic marker for low-risk AML that, if confirmed, could serve to further risk-stratify this patient subset.

Autoantibody signatures involving glycolysis and splicesome proteins precede a diagnosis of breast cancer among postmenopausal women.

We assessed the autoantibody repertoire of a mouse model engineered to develop breast cancer and the repertoire of autoantibodies in human plasmas collected at a preclinical time point and at the time of clinical diagnosis of breast cancer. In seeking to identify common pathways, networks, and protein families associated with the humoral response, we elucidated the dynamic nature of tumor antigens and autoantibody interactions. Lysate proteins from an immortalized cell line from a MMTV-neu mouse model and from MCF7 human breast cancers were spotted onto nitrocellulose microarrays and hybridized with mouse and human plasma samples, respectively. Immunoglobulin-based plasma immunoreactivity against glycolysis and spliceosome proteins was a predominant feature observed both in tumor-bearing mice and in prediagnostic human samples. Interestingly, autoantibody reactivity was more pronounced further away than closer to diagnosis. We provide evidence for dynamic changes in autoantibody reactivity with tumor development and progression that may depend, in part, on the extent of antigen-antibody interactions.

Longitudinal screening algorithm that incorporates change over time in CA125 levels identifies ovarian cancer earlier than a single-threshold rule.

PURPOSE: Longitudinal algorithms incorporate change over time in biomarker levels to individualize screening decision rules. Compared with a single-threshold (ST) rule, smaller deviations from baseline biomarker levels are required to signal disease. We demonstrated improvement in ovarian cancer early detection by using a longitudinal algorithm to monitor annual CA125 levels. PATIENTS AND METHODS: We retrospectively evaluated serial preclinical serum CA125 values measured annually in 44 incident ovarian cancer cases identified from participants in the PLCO (Prostate Lung Colorectal and Ovarian) Cancer Screening Trial to determine how frequently and to what extent the parametric empirical Bayes (PEB) longitudinal screening algorithm identifies ovarian cancer earlier than an ST rule. RESULTS: The PEB algorithm detected ovarian cancer earlier than an ST rule in a substantial proportion of cases. At 99% specificity, which corresponded to the ST-rule CA125 cutoff ≥ 35 U/mL that was used in the PLCO trial, 20% of cases were identified earlier by using the PEB algorithm. Among these cases, the PEB signaled abnormal CA125 values, on average, 10 months earlier and at a CA125 concentration 42% lower (20 U/mL) than the ST-rule cutoff. The proportion of cases detected earlier by the PEB algorithm and the earliness of detection increased as the specificity of the screening rule was reduced. CONCLUSION: The PEB longitudinal algorithm identifies ovarian cancer earlier and at lower biomarker concentrations than an ST screening algorithm adjusted to the same specificity. Longitudinal biomarker assessment by using the PEB algorithm may have application for screening other solid tumors in which biomarkers are available.

Interpretation of single and serial measures of HE4 and CA125 in asymptomatic women at high risk for ovarian cancer.

BACKGROUND: Human epididymis protein 4 (HE4) is approved for clinical use with CA125 to predict epithelial ovarian cancer in women with a pelvic mass or in remission after chemotherapy. Previously reported reference ranges for HE4 are inconsistent. METHODS: We report positivity thresholds yielding 90%, 95%, 98%, and 99% specificity for age-defined populations of healthy women for HE4, CA125, and Risk of Ovarian Malignancy Algorithm (ROMA), a weighted average of HE4 and CA125. HE4 and CA125 were measured in 1,780 samples from 778 healthy women aged >25 years with a documented deleterious mutation, or aged >35 years with a significant family history. Effects on marker levels of a woman's age, ethnicity, and epidemiologic characteristics were estimated, as were the population-specific means, variances, and within- and between-woman variances used to generate longitudinal screening algorithms for these markers. RESULTS: CA125 levels were lower with Black ethnicity (P = 0.008). Smoking was associated with higher HE4 (P = 0.007) and ROMA (P < 0.019). Continuous oral contraceptive use decreased levels of CA125 (P = 0.041), and ROMA (P = 0.12). CA125 was lower in women age ≥55, and HE4 increased with age (P < 0.01), particularly among women age ≥55. CONCLUSIONS: Because of the strong effect of age on HE4, thresholds for HE4 are best defined for women of specific ages. Age-specific population thresholds for HE4 for 95% specificity ranged from 41.4 pmol/L for women age 30 to 82.1 pmol/L for women age 80. IMPACT: Incorporation of serial marker values from screening history reduces personalized thresholds for CA125 and HE4 but is inappropriate for ROMA.

Discovery and preliminary confirmation of novel early detection biomarkers for triple-negative breast cancer using preclinical plasma samples from the Women's Health Initiative observational study.

Triple-negative breast cancer is a particularly aggressive and lethal breast cancer subtype that is more likely to be interval-detected rather than screen-detected. The purpose of this study is to discover and initially validate novel early detection biomarkers for triple-negative breast cancer using preclinical samples. Plasma samples collected up to 17 months before diagnosis from 28 triple-negative cases and 28 matched controls from the Women's Health Initiative Observational Study were equally divided into a training set and a test set and interrogated by a customized antibody array. Data were available on 889 antibodies; in the training set, statistically significant differences in case versus control signals were observed for 93 (10.5 %) antibodies at p < 0.05. Of these 93 candidates, 29 were confirmed in the test set at p < 0.05. Areas under the curve for these candidates ranged from 0.58 to 0.79. With specificity set at 98 %, sensitivity ranged from 4 to 68 % with 20 candidates having a sensitivity ≥ 20 % and 6 having a sensitivity ≥ 40 %. In an analysis of KEGG gene sets, the pyrimidine metabolism gene set was upregulated in cases compared to controls (p = 0.004 in the testing set) and the JAK/Stat signaling pathway gene set was downregulated (p = 0.003 in the testing set). Numerous potential early detection biomarkers specific to triple-negative breast cancer in multiple pathways were identified. Further research is required to followup on promising candidates in larger sample sizes and to better understand their potential biologic importance as our understanding of the etiology of triple-negative breast cancer continues to grow.

Impact of screening test performance and cost on mortality reduction and cost-effectiveness of multimodal ovarian cancer screening.

Ongoing ovarian cancer screening trials are investigating the efficacy of a two-step screening strategy using currently available blood and imaging tests [CA125 and transvaginal sonography (TVS)]. Concurrently, efforts to develop new biomarkers and imaging tests seek to improve screening performance beyond its current limits. This study estimates the mortality reduction, years of life saved, and cost-effectiveness achievable by annual multimodal screening using increasing CA125 to select women for TVS, and predicts improvements achievable by replacing currently available screening tests with hypothetical counterparts with better performance characteristics. An existing stochastic microsimulation model is refined and used to screen a virtual cohort of 1 million women from ages 45 to 85 years. Each woman is assigned a detailed disease course and screening results timeline. The preclinical behavior of CA125 and TVS is simulated using empirical data derived from clinical trials. Simulations in which the disease incidence and performance characteristics of the screening tests are independently varied are conducted to evaluate the impact of these factors on overall screening performance and costs. Our results show that when applied to women at average risk, annual screening using increasing CA125 to select women for TVS achieves modest mortality reduction (~13%) and meets currently accepted cost-effectiveness guidelines. Screening outcomes are relatively insensitive to second-line test performance and costs. Identification of a first-line test that does substantially better than CA125 and has similar costs is required for screening to reduce ovarian mortality by at least 25% and be reasonably cost-effective.

Extensive gene-specific translational reprogramming in a model of B cell differentiation and Abl-dependent transformation.

To what extent might the regulation of translation contribute to differentiation programs, or to the molecular pathogenesis of cancer? Pre-B cells transformed with the viral oncogene v-Abl are suspended in an immortalized, cycling state that mimics leukemias with a BCR-ABL1 translocation, such as Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). Inhibition of the oncogenic Abl kinase with imatinib reverses transformation, allowing progression to the next stage of B cell development. We employed a genome-wide polysome profiling assay called Gradient Encoding to investigate the extent and potential contribution of translational regulation to transformation and differentiation in v-Abl-transformed pre-B cells. Over half of the significantly translationally regulated genes did not change significantly at the level of mRNA abundance, revealing biology that might have been missed by measuring changes in transcript abundance alone. We found extensive, gene-specific changes in translation affecting genes with known roles in B cell signaling and differentiation, cancerous transformation, and cytoskeletal reorganization potentially affecting adhesion. These results highlight a major role for gene-specific translational regulation in remodeling the gene expression program in differentiation and malignant transformation.

Integrative analysis of N-linked human glycoproteomic data sets reveals PTPRF ectodomain as a novel plasma biomarker candidate for prostate cancer.

In an attempt to identify prostate cancer biomarkers with greater diagnostic and prognostic capabilities, we have developed an integrative proteomic discovery workflow focused on N-linked glycoproteins that refines the target selection process. In this work, hydrazide-based chemistry was used to identify N-linked glycopeptides from 22Rv1 prostate cancer cells cultured in vitro, which were compared with glycopeptides identified from explanted 22Rv1 murine tumor xenografts. One hundred and four human glycoproteins were identified in the former analysis and 75 in the latter, with 40 proteins overlapping between data sets. Of the 40 overlapping proteins, 80% have multiple literature references to the neoplastic process and ∼40% to prostatic neoplasms. These include a number of well-known prostate cancer-associated biomarkers, such as prostate-specific membrane antigen (PSMA). By integrating gene expression data and available literature, we identified members of the overlap data set that deserve consideration as potential prostate cancer biomarkers. Specifically, the identification of the extracellular domain of protein tyrosine phosphatase receptor type F (PTPRF) was of particular interest due to the direct involvement of PTPRF in the control of ß-catenin signaling, as well as dramatically elevated gene expression levels in the prostate compared to other tissues. In this investigation, we demonstrate that the PTPRF E-subunit is more abundant in human prostate tumor tissue compared to normal control and also detectable in murine plasma by immunoblot and ELISA. Specifically, PTPRF distinguishes between animals xenografted with the 22Rv1 cells and control animals as early as 14 days after implantation. This result suggests that the ectodomain of PTPRF has the potential to function as a novel plasma or tissue-based biomarker for prostate cancer. The workflow described adds to the literature of potential biomarker candidates for prostate cancer and demonstrates a pathway to developing new diagnostic assays.

Quantitative proteomic profiling identifies protein correlates to EGFR kinase inhibition.

Clinical oncology is hampered by lack of tools to accurately assess a patient's response to pathway-targeted therapies. Serum and tumor cell surface proteins whose abundance, or change in abundance in response to therapy, differentiates patients responding to a therapy from patients not responding to a therapy could be usefully incorporated into tools for monitoring response. Here, we posit and then verify that proteomic discovery in in vitro tissue culture models can identify proteins with concordant in vivo behavior and further, can be a valuable approach for identifying tumor-derived serum proteins. In this study, we use stable isotope labeling of amino acids in culture (SILAC) with proteomic technologies to quantitatively analyze the gefitinib-related protein changes in a model system for sensitivity to EGF receptor (EGFR)-targeted tyrosine kinase inhibitors. We identified 3,707 intracellular proteins, 1,276 cell surface proteins, and 879 shed proteins. More than 75% of the proteins identified had quantitative information, and a subset consisting of 400 proteins showed a statistically significant change in abundance following gefitinib treatment. We validated the change in expression profile in vitro and screened our panel of response markers in an in vivo isogenic resistant model and showed that these were markers of gefitinib response and not simply markers of phospho-EGFR downregulation. In doing so, we also were able to identify which proteins might be useful as markers for monitoring response and which proteins might be useful as markers for a priori prediction of response.

Concordant release of glycolysis proteins into the plasma preceding a diagnosis of ER+ breast cancer.

Although the identification of peripheral blood biomarkers would enhance early detection strategies for breast cancer, the discovery of protein markers has been challenging. In this study, we sought to identify coordinated changes in plasma proteins associated with breast cancer based on large-scale quantitative mass spectrometry. We analyzed plasma samples collected up to 74 weeks before diagnosis from 420 estrogen receptor (ER)(+) cases and matched controls enrolled in the Women's Health Initiative cohort. A gene set enrichment analysis was applied to 467 quantified proteins, linking their corresponding genes to particular biologic pathways. On the basis of differences in the concentration of individual proteins, glycolysis pathway proteins exhibited a statistically significant difference between cases and controls. In particular, the enrichment was observed among cases in which blood was drawn closer to diagnosis (effect size for the 0-38 weeks prediagnostic group, 1.91; P, 8.3E-05). Analysis of plasmas collected at the time of diagnosis from an independent set of cases and controls confirmed upregulated levels of glycolysis proteins among cases relative to controls. Together, our findings indicate that the concomitant release of glycolysis proteins into the plasma is a pathophysiologic event that precedes a diagnosis of ER(+) breast cancer.

Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries.

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.