Lung cancer is the leading cause of cancer deaths worldwide due to the late stage of diagnosis as most patients do not present symptoms until late-stage disease. The two most common forms of non-small cell lung cancer are adenocarcinoma followed by squamous cell carcinoma, which differ in location, appearance and molecular phenotypes and require different approaches for treatment. Prior to the development of invasive lung cancer, premalignant lesions, or regions of abnormal cellular architecture, are often present in the lung. Understanding the pathologic heterogeneity and cellular and molecular alterations in premalignant lesions that precede or are adjacent to invasive carcinomas may help identify the earliest changes in lung carcinogenesis that could be used to identify biomarkers of progression and targets for intervention. Previous work by our group profiling endobronchial biopsies by bulk mRNA sequencing identified gene expression alterations associated with high grade squamous premalignant lesion histology and progression towards advanced histology. Molecular profiling combined with careful pathologic assessment of premalignant lesions are important steps in uncovering the biological processes that are dysregulated in premalignant lesions that progress; however, lung tissue is often limited and fixed. Biopsies of lung tissue, obtained using forceps or needles, are an important diagnostic and prognostic clinical tool although, only tiny amounts of tissue may be left for research. As a result, it is important to work closely with pathologists to review clinical pathology samples and optimize methodologies to use the limited biospecimens available to study these early changes associated with the development of lung cancer. My thesis work focused on the detailed pathologic annotation of lung premalignant lesion biopsies and whole tumor samples and the development of methodologies to profile these small biospecimens to advance lung cancer interception research. Lung squamous cell carcinoma is thought to originate from bronchial premalignant lesions that progress through a series of histological grades to dysplasia (mild, moderate, and severe), carcinoma in situ, and invasive carcinoma. Endobronchial biopsies of these premalignant lesions have heterogeneous pathologic features, and it is not known which features are associated with progression to cancer. In Aim 1 of my thesis, in collaboration with thoracic pathologists, whole slide biopsy images were annotated to identify the histologic grades of surface epithelium, including the presence of angiogenic squamous dysplasia, and stromal features such as the presence of a fibrotic basal membrane under the epithelium. Analyzing these annotations across 284 biopsies identified a significant association between former smokers and the presence of a fibrotic basal membrane. Additionally, we found that when exposed to asbestos, approximately two thirds of patients developed angiogenic dysplasia, while those who were never exposed had a 50% chance of developing it. We also observed a reserve cell dysplasia-like pattern characterized by its nuclear uniformity through all layers, increased nucleus to cytoplasm ratio, hyperchromia, and presence of cilia. This type of dysplasia is not described in the lung to date but is well described in the cervix as a transitional subtype between reserve cell hyperplasia and squamous dysplasia. We quantified the percentage of each histologic grade present in the epithelium of each biopsy and found that there was a weak, but significant, negative correlation between the percentage of normal epithelium and reserve cell dysplasia-like epithelium. The results suggest that reserve cell dysplasia in the lung may be worthy of more study, as it is found to be only inversely correlated with normal epithelium. More studies must be done to elucidate its true role in the premalignant to malignant process. To complement the pathologic analysis of endobronchial biopsies in Aim 2, we examined 31 lung cancer resection cases where premalignant lesions were present in the tumor margins. Our goal was to profile the RNA and DNA of multiple regions within each case to identify molecular alterations associated with the transition from premalignant to tumor tissue. The 31 cases were stained with H&E and mIHC panels using markers corresponding to epithelial, immune, and stromal cell types. We annotated the H&E stains alone and together with the mIHC stains and found that we annotated significantly more unique regions when considering both data modalities. We captured the annotated regions using an optimized laser capture microdissection (LCM) protocol. The goal of Aim 3 was to first optimize the LCM and isolation protocols for limited FFPE lung samples. We accomplished this using a smaller group of eight samples, of which six were LCM’d, by comparing different DNA and RNA isolation kits to select the kit that offered the highest quality and greatest amount of isolated DNA and RNA. The LCM tissue from the 31 cases is currently undergoing RNA and DNA sequencing and we hope the data analysis will identify unique tissue microenvironments associated with premalignant lesion progression. These findings contribute to existing lung cancer and premalignancy research, which, as a field, aims to identify progressing lesions and treat patients at earlier stages to decrease mortality. My thesis work has focused on characterizing the pathologic features of lung squamous premalignant lung lesions and their associations with premalignant progression to cancer and other clinical covariates such as smoking status and carcinogenic exposures. To fully understand these pathologic features, I have developed methods to isolate high quality RNA and DNA from these limited biospecimens to allow for the identification of the molecular alterations underpinning the pathological changes.