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XLSX file - 48KB, IPA biological functions analysis.

XLSX file - 343KB, Genes associated with SCC carcinogenesis.

XLS file - 32KB, MYC activity prediction and its target genes.

PDF file - 85KB

PDF file - 690KB, Supplementary Figure S1. Laser capture microdissection. Supplementary Figure S2. Experimental validation of PTBP3 expression. Supplementary Table S1. Patient demographics. List of SCC patients with their age, gender, grade of premalignant lesion, and assays performed. Supplementary Table S2. Alignment statistics. The number and percentage of reads aligning uniquely to the nuclear (autosomal and sex) or mitochondrial chromosomes, or not uniquely aligned, are shown for each sample.

XLS file - 35KB, TP53 activity prediction and its target genes.

A 26-year-old woman with end-stage lung disease secondary to cystic fibrosis, cirrhosis secondary to hepatitis C, and insulin-dependent diabetes mellitus presented with acute pneumonia. She was treated with intravenous piperacillin/tazobactam and tobramycin. Other medications included dexamethasone, bumetanide, pantoprazole, dornase, and insulin. At admission, the patient's hematocrit was 30.8% (Table 1). On day 8 of hospitalization, she developed severe anemia with a hematocrit of 11.5%. Her blood type was group A, Rh D-positive, and the results of her antibody screen (performed in solid-phase, low-ionic-strength solution), which had previously been positive for only anti-E antibody, were now positive with a panreactive pattern. The result of the direct antiglobulin test (DAT)3 was 2+ (moderately positive) for IgG, and the eluate was nonreactive. Six units of red blood cells (RBCs) were requested for transfusion. Her plasma was weakly to moderately cross-match incompatible with all group A–positive, E antigen–negative units tested. Given the severity of her anemia, 6 of these units were emergently released for transfusion to stabilize the patient while the investigation of her anemia and the new serologic findings continued. View this table: Table 1. Laboratory results for day 1 and day 8 of hospitalization. Only 2 mechanisms were likely responsible for such an abrupt and severe decrease in the hematocrit: bleeding and hemolysis. Bleeding was initially suspected; however, no source of hemorrhage was found. On the other hand, laboratory findings revealed high lactate dehydrogenase (LDH) activity, hemoglobinuria, and unconjugated hyperbilirubinemia, all of which were consistent with hemolysis. The positive DAT result indicated an immune-mediated etiology. The panreactive pattern of the antibody panel results suggested warm autoantibodies, but the nonreactive eluate eliminated this possibility because panreactivity should also be observed in the eluate. The anti-E antibody first detected at admission indicated that the patient had received a transfusion or had become pregnant at some point in her life. There was no history of recent transfusion, however, and …

Lung squamous cell carcinoma (SCC) is thought to arise from premalignant lesions in the airway epithelium; therefore, studying these lesions is critical for understanding lung carcinogenesis. Previous microarray and sequencing studies designed to discover early biomarkers and therapeutic targets for lung SCC had limited success identifying key driver events in lung carcinogenesis, mostly due to the cellular heterogeneity of patient samples examined and the interindividual variability associated with difficult to obtain airway premalignant lesions and appropriate normal control samples within the same patient. We performed RNA sequencing on laser-microdissected representative cell populations along the SCC pathologic continuum of patient-matched normal basal cells, premalignant lesions, and tumor cells. We discovered transcriptomic changes and identified genomic pathways altered with initiation and progression of SCC within individual patients. We used immunofluorescent staining to confirm gene expression changes in premalignant lesions and tumor cells, including increased expression of SLC2A1, CEACAM5, and PTBP3 at the protein level and increased activation of MYC via nuclear translocation. Cytoband enrichment analysis revealed coordinated loss and gain of expression in chromosome 3p and 3q regions, respectively, during carcinogenesis. This is the first gene expression profiling study of airway premalignant lesions with patient-matched SCC tumor samples. Our results provide much needed information about the biology of premalignant lesions and the molecular changes that occur during stepwise carcinogenesis of SCC, and it highlights a novel approach for identifying some of the earliest molecular changes associated with initiation and progression of lung carcinogenesis within individual patients. Cancer Prev Res; 7(5); 487–95. ©2014 AACR.

Little is known about the mechanisms involved in the initiation and progression of lung cancer, even though lung cancer contributes to the most cancer-related deaths in the world. Lung squamous cell carcinoma (SCC) is the second most common histological subtype, and often arises centrally from a large airway, usually a bronchus. It is believed that squamous lung cancer develops through a series of genetic and epigenetic changes that alter the normal epithelium from squamous metaplasia, to dysplasia, then carcinoma in situ, and finally to invasive SCC. Although there have been studies devoted to discovering the genetic and molecular changes observed in the tumor, few studies have contributed to the investigation of squamous metaplasia or dysplasia. To better understand carcinogenesis in the lung, especially those steps involved in the early and precancerous stages, a precise study of the biology of premalignant lesions is needed. To achieve this, we used laser capture microdissection to specifically isolate normal airway basal cells, premalignant cells, and tumor regions within individual patients with squamous lung cancer. The RNAs obtained were successfully sequenced using Next Generation Sequencing. Using two different statistical models of linear mixed effects and edgeR (empirical analysis of digital gene expression data in R), we identified a list of genes that were differentially expressed in the premalignant lesions and in tumors when compared to the normal basal cells. We were able to validate at RNA and protein levels several of the genes identified to have higher expression in premalignant lesions and tumors than in normal basal cells. We performed a gene network analysis and found that some of these genes are known to be involved in the development of other squamous tumors, cell cycle progression, cell death and survival, and cellular assembly, while some genes are uncharacterized or novel to cancer development. We also discovered that, even though TP53 RNA expression was not significantly different among normal, premalignant, and tumor cells, several proteins that were upregulated in the premalignant lesions were known to target TP53 protein for degradation, cytoplasmic localization, or disruption of its function. This suggests that the disruption of TP53 pathway began at the premalignant stage of carcinogenesis, and persisted through the formation of invasive carcinoma. This is the first comprehensive gene expression profiling of airway premalignant lesions and allows us to study the biology behind these lesions in the setting of carcinogenesis. Citation Format: Aik T. Ooi, Adam Gower, Kelvin Zhang, Jessica Vick, Longsheng Hong, Michael Fishbein, Brian Nagao, W Dean Wallace, David Elashoff, Marc Lenburg, Steven Dubinett, Avrum Spira, Brigitte Gomperts. Examining early events in carcinogenesis by profiling premalignant lesions in the airways of patients with lung squamous cell carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1063. doi:10.1158/1538-7445.AM2013-1063