The Role of NELF in Mediating Human Gene Expression

Transcription is the synthesis of RNA from a DNA template. Transcription has been shown to occur in three main steps, initiation, elongation and termination. Additional regulatory steps include a rate limiting step between initiation and elongation of RNA polymerase II (RNA Pol II) termed promoter-proximal pausing of RNA polymerase. Dysregulation at any stage of this process can lead to cancer development. There has been extensive work focusing on how drastic alterations to the transcription process contributes to cancer development; however, there has been less focus on how the fine-tuning steps of transcription regulation contribute to cancer phenotypes. Specifically, my thesis focuses on understanding the contributions of NELF-mediated pausing to cancer development. To begin, I focused on primary cells in order to better understand the function of NELF-mediated pausing in a normal context. My work has identified a global profile of paused genes in human cells. Following the completion of this profile, I wanted to identify pathways that were enriched for paused genes. I found that paused genes are involved in cancer associated pathways such as EGFR and TGFb signaling as well as general transcription and gene expression. After identifying human genes with paused RNA polymerases, I focused on studying NELF. I found that almost all genes with NELF enrichment are paused. A majority of these genes also require NELF to maintain the pause. I then wanted to investigate the biological consequences of NELF-mediated pausing. I found that the expression of more than half of NELF-mediated paused genes are regulated by NELF. These genes include those involved in the cancer-associated pathways EGFR, TGFb and VEGF signaling. I showed that besides localizing to gene promoters, NELF also targets other genomic regions. I found that approximately half of these sites are in enhancers, specifically those enhancers that are actively transcribed. We showed that the abundance of these eRNAs is dependent on NELF. Finally, we found that loss of transcription of these eRNA result in changes in expression of nearby genes. Upon identifying NELF as a key regulator of gene expression in normal cells, I investigated how this process contributed to the gene expression changes found in cancer. To begin, I identified paused genes in HT29 colorectal cancer cells. I found that many of the same pathways show pausing in both normal cells and cancer cells. I also found that most genes utilize pausing to reduce their expression in cancer cells. These genes were enriched for cellular senescence, cellular stress response and WNT signaling. My work provides insight into the role of RNA Pol II pausing in cancer development. I have shown that cancer-associated pathways, such as EGFR, are regulated by RNA Pol II pausing. I have also generated global profiles of pausing in both normal and cancer cells. In comparing these two profiles, I have identified differences in the function of pausing between normal and cancer cells. One of the key differences I identified is the mechanism by which pausing regulates gene expression in normal and cancer cells. Collectively, my work shows that NELF and NELF-mediated pausing are key regulators of gene expression that may be harnessed by cancer cells to promote tumorigenesis.PHDCancer BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138582/1/jmboden_1.pd

PD-L1 Testing for Lung Cancer in 2019: Perspective From the IASLC Pathology Committee

The recent development of immune checkpoint inhibitors (ICIs) has led to promising advances in the treatment of patients with NSCLC and SCLC with advanced or metastatic disease. Most ICIs target programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) axis with the aim of restoring antitumor immunity. Multiple clinical trials for ICIs have evaluated a predictive value of PD-L1 protein expression in tumor cells and tumor-infiltrating immune cells (ICs) by immunohistochemistry (IHC), for which different assays with specific IHC platforms were applied. Of those, some PD-L1 IHC assays have been validated for the prescription of the corresponding agent for first- or second-line treatment. However, not all laboratories are equipped with the dedicated platforms, and many laboratories have set up in-house or laboratory-developed tests that are more affordable than the generally expensive clinical trial–validated assays. Although PD-L1 IHC test is now deployed in most pathology laboratories, its appropriate implementation and interpretation are critical as a predictive biomarker and can be challenging owing to the multiple antibody clones and platforms or assays available and given the typically small size of samples provided. Because many articles have been published since the issue of the IASLC Atlas of PD-L1 Immunohistochemistry Testing in Lung Cancer, this review by the IASLC Pathology Committee provides updates on the indications of ICIs for lung cancer in 2019 and discusses important considerations on preanalytical, analytical, and postanalytical aspects of PD-L1 IHC testing, including specimen type, validation of assays, external quality assurance, and training

The International Association for the Study of Lung Cancer Global Survey on Programmed Death-Ligand 1 Testing for NSCLC

Introduction: Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) is required to determine the eligibility for pembrolizumab monotherapy in advanced NSCLC worldwide and for several other indications depending on the country. Four assays have been approved/ Communauté Européene–In vitro Diagnostic (CV-IVD)–marked, but PD-L1 IHC seems diversely implemented across regions and laboratories with the application of laboratory-developed tests (LDTs). Method: To assess the practice of PD-L1 IHC and identify issues and disparities, the International Association for the Study of Lung Cancer Pathology Committee conducted a global survey for pathologists from January to May 2019, comprising multiple questions on preanalytical, analytical, and postanalytical conditions. Result: A total of 344 pathologists from 64 countries participated with 41% from Europe, 24% from North America, and 18% from Asia. Besides biopsies and resections, cellblocks were used by 75% of the participants and smears by 11%. The clone 22C3 was most often used (69%) followed by SP263 (51%). They were applied as an LDT by 40% and 30% of the users, respectively, and 76% of the participants developed at least one LDT. Half of the participants reported a turnaround time of less than or equal to 2 days, whereas 13% reported that of greater than or equal to 5 days. In addition, quality assurance (QA), formal training for scoring, and standardized reporting were not implemented by 18%, 16%, and 14% of the participants, respectively. Conclusions: Heterogeneity in PD-L1 testing is marked across regions and laboratories in terms of antibody clones, IHC assays, samples, turnaround times, and QA measures. The lack of QA, formal training, and standardized reporting stated by a considerable minority identifies a need for additional QA measures and training opportunities

A Grading System for Invasive Pulmonary Adenocarcinoma: A Proposal From the International Association for the Study of Lung Cancer Pathology Committee

Introduction: A grading system for pulmonary adenocarcinoma has not been established. The International Association for the Study of Lung Cancer pathology panel evaluated a set of histologic criteria associated with prognosis aimed at establishing a grading system for invasive pulmonary adenocarcinoma. Methods: A multi-institutional study involving multiple cohorts of invasive pulmonary adenocarcinomas was conducted. A cohort of 284 stage I pulmonary adenocarcinomas was used as a training set to identify histologic features associated with patient outcomes (recurrence-free survival [RFS] and overall survival [OS]). Receiver operating characteristic curve analysis was used to select the best model, which was validated (n = 212) and tested (n = 300, including stage I–III) in independent cohorts. Reproducibility of the model was assessed using kappa statistics. Results: The best model (area under the receiver operating characteristic curve [AUC] = 0.749 for RFS and 0.787 for OS) was composed of a combination of predominant plus high-grade histologic pattern with a cutoff of 20% for the latter. The model consists of the following: grade 1, lepidic predominant tumor; grade 2, acinar or papillary predominant tumor, both with no or less than 20% of high-grade patterns; and grade 3, any tumor with 20% or more of high-grade patterns (solid, micropapillary, or complex gland). Similar results were seen in the validation (AUC = 0.732 for RFS and 0.787 for OS) and test cohorts (AUC = 0.690 for RFS and 0.743 for OS), confirming the predictive value of the model. Interobserver reproducibility revealed good agreement (k = 0.617). Conclusions: A grading system based on the predominant and high-grade patterns is practical and prognostic for invasive pulmonary adenocarcinoma

New Approaches to SCLC Therapy: From the Laboratory to the Clinic

The outcomes of patients with SCLC have not yet been substantially impacted by the revolution in precision oncology, primarily owing to a paucity of genetic alterations in actionable driver oncogenes. Nevertheless, systemic therapies that include immunotherapy are beginning to show promise in the clinic. Although, these results are encouraging, many patients do not respond to, or rapidly recur after, current regimens, necessitating alternative or complementary therapeutic strategies. In this review, we discuss ongoing investigations into the pathobiology of this recalcitrant cancer and the therapeutic vulnerabilities that are exposed by the disease state. Included within this discussion, is a snapshot of the current biomarker and clinical trial landscapes for SCLC. Finally, we identify key knowledge gaps that should be addressed to advance the field in pursuit of reduced SCLC mortality. This review largely summarizes work presented at the Third Biennial International Association for the Study of Lung Cancer SCLC Meeting. (C) 2020 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved

Towards standards for human fecal sample processing in metagenomic studies

International audienceTechnical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses