Keloid disease (KD) is a cutaneous fibroproliferative tumour characterised by heterogeneity, locally aggressive invasion and therapeutic resistance. Clinical, histological and molecular differences between the keloid scar centre and margin as well as recent evidence of the importance of epithelial-mesenchymal interactions (EMI) in KD pathobiology contribute to the complexity and diversity of KD, which coupled with the lack of a validated animal model have hindered research and effective management. Despite significant progress in the field of KD research, reliance on conventional monolayer cell culture and whole tissue analysis methods have failed to fully reflect the natural architecture, pathology and complexity of KD in vivo. In order to address these challenges, a site-specific in situ approach was therefore employed here for the first time in KD research. The first aim of this work was to compare the value of this contemporary approach with traditional methods of tissue dissection. The second aim was to compare the genomic expression between well-defined, distinct keloid sites and normal skin (NS). The third aim was to develop and explore hypotheses arising from this site-specific gene expression profiling approach, so as to enhance understanding of KD pathobiology as a basis for improved diagnostic and therapeutic strategies in future KD management. The fourth aim was to probe these hypotheses with relevant functional in vitro studies. The current site-specific in situ approach was achieved through a combination of laser capture microdissection and whole genome microarray, allowing separation of epidermis from dermis for keloid centre, margin and extralesional sites compared with NS. This in situ approach yielded selective, accurate and sensitive data, exposing genes that were overlooked with alternative methods of dissection. Identification of significant upregulation of the aldo-keto reductase enzyme AKR1B10 in all three sites of the keloid epidermis (KE) in situ, implicated dysregulation of the retinoic acid (RA) pathway in KD pathogenesis. This hypothesis was supported by showing that induced AKR1B10 overexpression in NS keratinocytes reproduced the keloid RA pathway expression pattern. Moreover, co-transfection with a luciferase reporter plasmid revealed reduced RA response element activity. Paracrine signals released by AKR1B10-overexpressing keratinocytes into conditioned medium resulted in TGFβ1 and collagen upregulation in keloid fibroblasts, suggesting the disturbed RA metabolism exerts a pro-fibrotic effect through pathological EMI, thus further supporting the hypothesis of RA deficiency in KE. Gene expression profiling further revealed an upregulation of NRG1 and ErbB2 in keloid margin dermis. Exogenous NRG1 led to enhanced keloid fibroblast migration with increased Src and PTK2 expression, which were attenuated with ErbB2 siRNA studies. Together with the observed failure to recover this expression with NRG1 treatment, suggested the novel KD pathobiology hypothesis that NRG1/ErbB2/Src/PTK2 signaling plays a role in migration at the keloid margin. In addition to these hypotheses, LCM methodology with comprehensive analysis of the data permitted the development of additional novel working hypotheses that will inform future KD research, including inflammatory gene dysregulation and cancer-like stem cells that may contribute to the therapeutic resistance characteristic of KD.

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.

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