Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffinembedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.cancer diagnostics | high-content cellular analysis | image analysis | mTOR | multiplexing

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BackgroundThymic epithelial tumors are PD-L1–expressing tumors of thymic epithelial origin characterized by varying degrees of lymphocytic infiltration and a predisposition towards development of paraneoplastic autoimmunity. PD-1–targeting antibodies have been evaluated, largely in patients with thymic carcinoma. We sought to evaluate the efficacy and safety of the anti-PD-L1 antibody, avelumab (MSB0010718C), in patients with relapsed, advanced thymic epithelial tumors and conduct correlative immunological studies.MethodsSeven patients with thymoma and one patient with thymic carcinoma were enrolled in a phase I, dose-escalation trial of avelumab (MSB0010718C), and treated with avelumab at doses of 10 mg/kg to 20 mg/kg every 2 weeks until disease progression or development of intolerable side effects. Tissue and blood immunological analyses were conducted.ResultsTwo of seven (29%) patients with thymoma had a confirmed Response Evaluation Criteria in Solid Tumors–defined partial response, two (29%) had an unconfirmed partial response and three patients (two thymoma; one thymic carcinoma) had stable disease (43%). Three of four responses were observed after a single dose of avelumab. All responders developed immune-related adverse events that resolved with immunosuppressive therapy. Only one of four patients without a clinical response developed immune-related adverse events. Responders had a higher absolute lymphocyte count, lower frequencies of B cells, regulatory T cells, conventional dendritic cells, and natural killer cells prior to therapy.ConclusionThese results demonstrate anti-tumor activity of PD-L1 inhibition in patients with relapsed thymoma accompanied by a high frequency of immune-related adverse events. Pre-treatment immune cell subset populations differ between responders and non-responders.Trial registrationClinicalTrials.gov - NCT01772004. Date of registration – January 21, 2013.Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0723-9) contains supplementary material, which is available to authorized users.

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Cancer is a multifaceted disease characterized by heterogeneous genetic alterations and cellular metabolism, at the organ, tissue, and cellular level. Key features of cancer heterogeneity are summarized by 10 acquired capabilities, which govern malignant transformation and progression of invasive tumors. The relative contribution of these hallmark features to the disease process varies between cancers. At the DNA and cellular level, germ-line and somatic gene mutations are found across all cancer types, causing abnormal protein production, cell behavior, and growth. The tumor microenvironment and its individual components (immune cells, fibroblasts, collagen, and blood vessels) can also facilitate or restrict tumor growth and metastasis. Oncology research is currently in the midst of a tremendous surge of comprehension of these disease mechanisms. This will lead not only to novel drug targets but also to new challenges in drug discovery. Integrated, multi-omic, multiplexed technologies are essential tools in the quest to understand all of the various cellular changes involved in tumorigenesis. This review examines features of cancer heterogeneity and discusses how multiplexed technologies can facilitate a more comprehensive understanding of these features.

Nucleotides with a dye attached to the terminal phosphate with four or more phosphates (tetra- or pentaphosphates) are superior substrates than the corresponding triphosphates for DNA and RNA polymerases. When fluorogenic dyes are directly attached to the terminal phosphate, they can be released by the action of polymerase and alkaline phosphatase. The released dye changes color and fluorescence properties. The fluorescent signal can also be amplified by using multiple labeled nucleotides to detect small amounts of template. We have explored the utility of these nucleotides in a variety of applications including homogeneous SNP detection methods, DNA sequencing, and quantitation of PCR and RCA.

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A number of terminal phosphate-labeled nucleotides with three or more phosphates and with varied length linkers attached between the terminal phosphate and the dye have been synthesized. These nucleotides have been tested as substrates for different DNA and RNA polymerases. We have also explored their utility in DNA sequencing, SNP analysis, nucleic acid amplification, quantitative PCR, and other biochemical assays.