Immunotherapies show promise in the treatment of oncology patients, but complex heterogeneity of the tumor microenvironment makes predicting treatment response challenging. The ability to resolve the relative populations of immune cells present in and around the tumor tissue has been shown to be clinically-relevant to understanding response, but is limited by traditional techniques such as flow cytometry and immunohistochemistry (IHC), due the large amount of tissue required, lack of accurate cell type markers, and many technical and logistical hurdles. One assay (e.g., the ImmunoPrism Immune Profiling Assay) overcomes these challenges by accommodating both small amounts of RNA and highly degraded RNA, common features of RNA extracted from clinically archived solid tumor tissue. The assay is accessed via a reagent kit and cloud-based informatics that provides an end-to-end quantitative, high-throughput immuno-profiling solution for Illumina sequencing platforms. Researchers start with as few as two sections of formalin-fixed paraffin-embedded (FFPE) tissue or 20-40 ng of total RNA (depending on sample quality), and the protocol generates an immune profile report quantifying eight immune cell types and ten immune escape genes, capturing a complete view of the tumor microenvironment. No additional bioinformatic analysis is required to make use of the resulting data. With the appropriate sample cohorts, the protocol may also be used to identify statistically significant biomarkers within a patient population of interest.

Background: Transposable elements (TEs) are major components of plant genomes. Despite being regarded as junk DNA at first, TEs play important roles for the organisms they are found in. The most obvious and easily recognizable effects caused by TEs result from their mobility, which can disrupt coding sequences or promoter regions. However, with the recent advances in transcriptomics, it is becoming increasingly evident that TEs can act as an additional layer of gene expression regulation through a number of processes, which can involve production of non-coding RNAs. Here, we describe how Tnt1, a stress-responsive LTR-retrotransposon, interferes with gene expression and modulate a number of developmental aspects in tobacco. Results: Through an RNAi approach, we generated tobacco (HP) lines knocked-down for Tnt1 expression. Quantitative RT-PCR experiments confirm that Tnt1 is downregulated in HP lines after ethylene exposure. A RNA-seq experiment was performed and through two independent bioinformatic approaches (with different stringencies) we found 932 and 97 differentially expressed genes in HP lines. A number of phenotypes were observed in such lines, namely lesion mimicry in leaves, underdevelopment of the root system, overproduction of root hairs and early loss of seed viability. Folding prediction of part of the Tnt1 mRNA reveals putative stem-loop secondary structures containing transcriptional regulation sequences, suggesting it could be a source of small RNAs. We also propose a model to explain the Tnt1 expression in both homeostatic and stress conditions, and how it could interact with stress-responsive genes. Conclusions: Our results are consistent that interferences with Tnt1 transcript levels correlate with transcriptomic and phenotypic changes, suggesting a functional role for this element during plant development and stress response.

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Significance: Non-Small Cell Lung Cancer (NSCLC) is responsible for the largest number of cancer-related fatalities, accounting for 22.8% of all cancer deaths in the United States in 2015. Targeted therapy precisely treats mutated cancer pathways and has significantly higher response rates compared to chemotherapy. However, tumor heterogeneity is prevalent in NSCLC patients, and the common practice of detecting mutations using tissue biopsy from a fraction of a single tumor can be less than satisfactory. Failure to detect molecular resistance emerging from heterogenic tumor cell population is a major reason that, even though highly effective, targeted therapy has not contributed greatly to prolong patient survival. We aim to develop a cost-effective, highly sensitive and specific liquid biopsy for advanced stage NSCLC patients that is able to capture a comprehensive tumor profile and monitor tumor response to therapy. Innovation: BEST (Blocker-based Enrichment System for Tumor DNA) blocks primer extension of over 90% of wild type DNA and allows preferential amplification of the mutant allele. BEST NSCLC liquid biopsy is highly sensitive (limit of detection of > = 0.005%), comprehensive (detects 56 mutations in the EGFR, KRAS, BRAF, ALK, and ROS1 oncogenes), and rapid (estimated 4-day turnaround time). BEST is comprised of a multiplex of circulating tumor DNA enrichment reaction and a multiplex plasma cDNA enrichment reaction. Our BEST NSCLC liquid biopsy panel enriches >100 fold for the above driver mutations. The ability to detect a myriad of mutations in heterogenic tumor cell populations allows dynamic and personalized treatment during the course of the disease. Summary: Our primary focus is to offer a test to detect actionable mutations and subsequently monitor tumor dynamics and the rise of molecular resistance. Our liquid biopsy will be also ideal for patients in follow-up visits in conjunction with the typical X-ray or CT image scans. Our test is also suitable for vulnerable patients from whom a solid biopsy cannot be obtained due to illness or inaccessibility. Citation Format: Christopher Kasbek, Yang Song, Danielle Quintanilha, Si Chen, QingXuan Song, Tianjiao Wang, Jun T. Huang. Developing a next-generation noninvasive clinical test for non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1393.