Wan Lam


Relevant Degree Programs


Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - Nov 2019)
Alteration of non-coding RNAs as a mechanism of lung cancer gene deregulation (2020)

Lung cancer remains the deadliest form of cancer, and less than half of lung adenocarcinoma (LUAD) patients harbour clinically actionable driver genes, emphasizing the need to explore alternative mechanisms of cancer gene deregulation. The advent of next generation sequencing has begun to reveal the functional importance of long non-coding RNAs (lncRNAs) in human cell biology, which can be exploited by tumours to drive the hallmarks of cancer. Due to their complex tertiary structure and unknown binding motifs there is a growing disparity between number of lncRNAs identified and those that have been functionally characterized. As such, lncRNAs deregulated in cancer may represent critical members of cancer pathways that could hold therapeutic applicability.The goal of this thesis is to identify lncRNAs important to LUAD biology, discover shared features and mechanisms used to regulate cancer driving protein coding genes, and evaluate the clinical relevance of these non-coding genes. We discover and investigate three major mechanisms harnesses by lncRNAs in LUAD: (i) cis-acting regulation of neighbouring genes, (ii) trans-acting regulation through sequence homology and (iii) regulation through shared miRNAs.This work uncovers evidence to suggest that alteration of lncRNAs is a major mechanism of cancer gene regulation in LUAD. Further characterization of these understudied gene regulatory mechanisms could lead to novel therapies that silence oncogenes or reactivate tumour suppressor genes.

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Discovery of oncofetal microRNA-gene networks associated with lung cancer aggressiveness (2019)

Each year about 1.5 million people die from lung cancer worldwide. In order to develop more effective strategies to manage this disease, it is essential to have a better understanding of molecular mechanisms underlying lung tumourigenesis. A recognized fact about cancer in general is that it shares many biological similarities with normal fetal development, such as intense cell proliferation, migration, angiogenesis and immune evasion. These features are gained as a consequence of changes in gene expression, which can result from the disruption in miRNAs. This class of small non-coding RNAs plays a fundamental role in regulating gene expression during normal development and tumourigenesis, nevertheless, comprehensive analyses of miRNAs in the context of lung development and lung cancer had not been previously explored. My hypothesis is that a better understanding of the similarities between human fetal lung development and lung cancer ― in the context of miRNAs ― will provide new insights into biological networks that might be critical to lung cancer pathogenesis.In this thesis, we performed for the first time a comprehensive characterization of miRNA expression in human fetal lung tissue, and identified numerous miRNAs that recapitulate their fetal expression patterns in non-small cell lung cancer (NSCLC). Investigation of genes potentially regulated by these oncofetal miRNAs, led us to discover miRNA-gene networks associated with tumour aggressiveness. Nuclear Factor I/B (NFIB), a transcription factor essential for lung development, was identified as the most prominent target of oncofetal miRNAs. Concordantly, analysis of NFIB expression in multiple NSCLC cohorts revealed its frequent underexpression (in ~40-70% of tumours). We further show that low expression of NFIB was significantly associated with biologically more aggressive subclasses of lung adenocarcinoma, and ultimately, poorer survival in patients with this subtype of NSCLC.Collectively, this work reasserted commonalities between the processes regulating normal development and tumourigenesis, and identified oncofetal miRNA-gene networks biologically relevant to NSCLC. Our study suggests that NFIB may serve as a biomarker for lung adenocarcinomas with poor prognosis, and indicates the possibility that restoration of NFIB expression in these tumours may induce lung cell differentiation, and therefore has the potential to reduce tumour aggressiveness.

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A multidimensional approach to understanding lung tumour aggressiveness (2017)

Lung cancer is a deadly disease with a 5-year survival of 18%. Patients with localized disease have improved outcomes, since they are eligible for surgery with curative intent. However, the majority of patients present with metastatic disease, where treatment options are limited. Genetic profiling of lung tumours has lead to the identification of driver mutations and the subsequent development of targeted therapies for some of these mutated genes, which has resulted in improved outcomes for late stage patients. Unfortunately, only subsets of patients harbour these mutations, and patients frequently acquire drug resistance. Novel therapeutics are desperately needed for patients with late stage disease in order to improve patient outcome. To achieve this goal, we require a deeper understanding of the biology that drives aggressive lung tumour biology.In this thesis, we employ two unique approaches to discover genes associated with lung tumour aggressiveness. Firstly, we move beyond the protein-coding landscape and characterize deregulation of small non-coding RNAs in metastatic lung cancer. We further assess the ability of small non-coding RNA expression patterns to classify patients into different outcome groupings. Secondly, we employ an integrative multi-omics approach in order to identify novel oncogenes that have been overlooked by conventional studies within a single dimension (e.g. mutation).Collectively, this work adds to our understanding of aggressive tumour biology. Further characterization of the aggressiveness-associated small non-coding RNAs identified here may inform on novel therapeutic avenues or gene signatures. Importantly, our discovery of a novel lung cancer oncogene may lead to a new therapy for lung cancer.

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Molecular mechanisms underlying tumorigenesis of non-small cell lung cancer subtypes (2014)

Lung cancer is the leading cause of cancer death in Canada and worldwide. A late stage of diagnosis in conjunction with a lack of effective treatment options are largely responsible for the poor survival rates of lung cancer. Histological subtypes of lung cancer are known to respond differently to standard therapies, suggesting they are distinct diseases. A better understanding of the molecular alterations and underlying biology of lung cancer subtypes is therefore necessary for the development of novel detection and treatment strategies in order to improve patient prognosis. We hypothesize that lung adenocarcinoma (AC) and squamous cell carcinoma (SqCC) arise through disparate patterns of molecular alterations and that these differences underlie unique biological mechanisms that contribute to subtype development, phenotypes and response to therapy. In this thesis, I apply multidimensional integrative 'omics approaches to characterize the genomic and epigenomic landscapes of AC and SqCC and elucidate differential patterns of alterations and subtype specific gene disruptions causal to NSCLC and the development of specific subtypes. The integration of DNA copy number, methylation, gene and miRNA expression data on AC and SqCC tumors and patient matched non-malignant tissue identified several subtype specific alterations and revealed unique oncogenic pathways associated with AC and SqCC that can be successfully targeted by existing therapies. By combining genomic analyses with manipulation of candidate genes in vitro and in vivo, we validated the contribution of candidate genes to tumorigenesis and determined the mechanisms through which they contribute to disease pathogenesis. In addition to revealing differentially disrupted genes and pathways we also identified numerous alterations common to both subtypes. Collectively, this work has further characterized the landscape of molecular alterations that define AC and SqCC, and the mechanisms through which these alterations contribute to subtype tumorigenesis. This work has identified novel candidate genes involved in subtype tumorigenesis as well as miRNAs with potential as diagnostic biomarkers for lung cancer. Taken together, these findings underscore the importance of tailoring treatment strategies to the histological subtype based on the underlying biology of that subtype.

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Multi-omics characterization of the molecular effects of smoking and chronic inflammation on the lung (2014)

Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease associated with a 10-fold increased risk of lung cancer (LC), independent of smoking-status. Together these diseases contribute tremendously to morbidity and mortality worldwide. While COPD and lung cancer share common etiologies including genetic susceptibilities and risk factors, the biology driving COPD and LC is largely unknown. No effective treatments exist for either disease, thus a better understanding of the molecular biology underlying these diseases is urgently needed.The overarching hypothesis of this thesis is that specific risk factors, such as smoking and chronic inflammation lead to selective disruption of genes in exposed tissues and that these selectively disrupted genes contribute directly to COPD and lung cancer pathogenesis. Since selection occurs at the DNA level, and tumour and disease systems may be altered at multiple genetic and epigenetic levels; a major hypothesis of this thesis is that loci which sustain high-level concerted genetic, epigenetic and/or transcriptional disruptions in tissues involved in disease pathology are likely indicative of strong selection and may be identified by applying an integrative multi-omics analysis of these tissues. Background pertaining to the rationale, objectives and specific aims of this work are described in Chapter 1. Chapters 2-4 detail the main findings of this thesis, which are that: 1) DNA is altered at the main sites of airflow obstruction in COPD patients (Chapter 2), 2) smoking status impacts miRNA lung tumour biology and patient prognosis (Chapter 3), 3) lung tumours from patients with COPD are molecularly distinct at the genetic and epigenetic levels (Chapter 4) and 4) genes preferentially altered in COPD-related lung tumours are aberrantly methylated in non-malignant airway cells from patients with COPD and lung cancer (Chapter 4). Taken together, this work provides sufficient rationale to explore the clinical application of these findings as potential targets for novel COPD treatments and markers for early lung cancer detection, treatment or targeted chemoprevention. A summary of these key findings, significance, caveats and future directions are discussed in Chapter 5.

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Multi-'omics comparison of lung cancers from current and never smokers (2013)

Lung cancer is the leading cause of cancer death worldwide and a better understanding of the molecular alterations driving tumour biology is required to improve patient prognosis. This is especially true for never smokers (NS), which account for up to 25% of lung cancer cases globally. As the population of current smokers (CS) decreases due to smoking cessation and prevention initiatives, in the coming decades NS and former smokers (FS) will comprise a greater proportion of cases. We used an integrative 'omics approach to study lung cancer genomes of CS and NS to elucidate differential mechanisms and patterns of gene and pathway disruption likely to contribute to lung tumourigenesis. Lung cancers in CS and NS exhibit different clinical features and are known to preferentially select specific mutations (e.g. EGFR, KRAS, EML4-ALK), suggesting they are distinct diseases. Thus, we hypothesize that lung tumours of CS and NS exhibit disparate patterns of molecular alterations on a genome wide scale, reflecting the assumption that they develop through the differential selection of genes and pathways.A large scale, multi-dimensional, genomics study has yet to be performed and holds great potential to reveal novel insights into the mechanisms of lung tumourigenesis in CS and NS. Therefore, we performed DNA copy number, methylation, gene expression and microRNA expression profiling on a panel of lung adenocarcinoma tumours from CS and NS in an attempt to characterize the genomic and epigenomic landscapes of these tumours. In addition to identifying commonly disrupted genes and pathways, our integrative genomic analysis revealed numerous differences between CS and NS lung tumours including: differing extents of copy number and methylation alterations, different patterns of miRNA disruption, and preferential disruption of genes and cellular pathways. Importantly, some of the prominently disrupted genes that drive deregulation of tumour promoting pathways may represent novel therapeutic targets and intervention points.Collectively, this work provides further evidence that lung tumours of CS and NS develop through different molecular alterations which suggests patients will benefit from specific management strategies tailored to their smoking status.

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Concerted genomic and epigenomic alterations in non-small cell lung cancer (2010)

Background: Around the world, lung cancer is the leading cause of cancer-related death and a major public health problem. A diagnosis of lung cancer carries a remarkably poor prognosis, even after years of research into the disease. The advent and availability of tools to survey the genomes and epigenomes of lung cancers is beginning to yield real clues into the molecular nature of the disease. These clues are being turned into new diagnostic and therapeutic tools with increasing regularity. We used modern high-resolution and high-throughput tools to identify novel genes implicated in various lung cancer phenotypes and aspects of lung cancer pathogenesis.Hypotheses: (1) Combined profiling of lung cancer genomes and epigenomes will identify critical lung cancer genes that are simultaneously affected by DNA copy number and DNA methylation aberrations. (2) The susceptibility locus on chromosome 6q, identified through familial linkage studies, contains an unidentified tumor suppressor gene (3) Key genes involved in lung cancer phenotypes can be identified through the elucidation of discriminating genomic and epigenomic alterations.Materials/Methods: Genomic and epigenomic data were analyzed independently and pair-wise to identify genes in NSCLC whose alterations are associated with NSCLC risk, development and phenotype. These genomic and epigenomic data were used in conjunction with a multitude of mRNA and protein-level assays to further refine candidate lists and validate their disruption. Targeted molecular silencing of a candidate TSG was used in conjunction with cellular assays to investigate and confirm the role of this gene in NSCLC.Results: We designed and optimized an experimental/analysis framework for the combined interrogation of epigenomic and genomic data. We used this framework to identify a novel lung cancer tumor suppressor gene, EYA4, that is frequently disrupted in lung cancers, and is associated with NSCLC risk. Following this, we identified subtype-specific genomic and epigenomic alterations with consequent gene expression changes in NSCLC subtypes. Lastly, we identified specific phenotypic characteristics of the subtypes affected by the DNA alterations.Conclusions: Integrated analysis of the genomes and epigenomes of NSCLC tumors provides a unique approach for the discovery of key cancer-related genes.

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Development and application of an integrative genomics approach to lung cancer (2010)

Lung cancer has the highest mortality rate amongst all diagnosed malignancies with adenocarcinoma (AC) being the most commonly diagnosed subtype of this disease in North America. The dismal survival statistics of lung cancer patients are largely due to the detection of the disease at an advanced stage and to a lesser extent, the limited efficacy of current front line treatments.Genomic approaches, namely gene expression analysis, have provided tremendous insight intolung cancer. While many gene expression changes have been identified, most changes are likely reactive to changes which have a primary role in cancer development. Moreover, one feature which can discern primary from reactive changes is the presence of concordant DNA level alteration.Many well known genes involved in cancer such as TP53 and CDKN2A have been shown to beaffected by multiple mechanisms of alteration such as somatic mutation in or loss of DNA sequence. For a given gene, one tumor may be affected by one mechanism while another tumor may be affected by a different mechanism. Although this level of multi-dimensional analysis has been performed for specific genes, such analysis has not been done at the genome-wide level.This thesis highlights the development and application of a multi-dimensional genetic andepigenetic approach to identify frequently aberrant genes and pathways in lung AC. I present,first, the design and implementation of the system for integrative genomic multi-dimensional analysis of cancer genomes, epigenomes and transcriptomes (SIGMA²). Next, analyzing a multi-dimensional dataset generated from ten lung AC specimens with non-malignant controls, I identified novel genes and pathways that would have been missed if a non-integrative approach were used. Finally, examining genes involved with EGFR signaling, I identified a gene, signal receptor protein alpha (SIRPA), which had not been previously shown to be associated with lung cancer.Taken together, these findings demonstrate the power of a multi-dimensional approach to identify important genes and pathways in lung cancer. Moreover, identifying key genes using a multi-dimensional approach on a small sample set suggests the need of large datasets may becircumvented by using a more comprehensive approach on a smaller set of samples.

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Global Approaches in Identifying Aberrations in Early Staged Disease in Cancers in Women (2010)

No abstract available.

Understanding the genetic evolution of oral cancer genomes (2010)

Background: Oral cancer is the most common type of head and neck cancer, with a 5-year survival rate of
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Clonal evoluation and lung cancer pharmacogenomics (2009)

Background: Lung cancer is the world's leading cause of cancer death, with a 5 yearsurvival rate of ~16%. Several factors contribute to this poor prognosis: the limited detection of disease at treatable stages, the high metastatic potential of any primary tumour, and the variable effectiveness of chemotherapy. We applied high resolution whole genome profiling technologies to uncover genes associated with specific lung cancer phenotypes and to delineate clonal relationships between tumours.Hypotheses: (i) Shared genetic features in tumours from the same patient are evidenceof a common progenitor. (ii) Continuing clonal evolution facilitates selection forresistance genes during drug exposure. (iii) Drug response can be predicted for pretreatment lung cancer by evaluating specific gene changes.Materials/Methods: DNA alteration data from non-small cell lung cancers (NSCLC) wereintegrated with mRNA/protein expression data to identify genes contributing totumourigenesis. Fine-mapped DNA alteration boundaries were used to evaluate clonality, discriminating multiple primary tumours from intrapulmonary metastasis. Subsequently, this approach was applied to define chemoresistance gene candidates in cells grown under drug selection. Genome alteration data for early stage lung tumourswere also analyzed to define gene changes driving post-treatment recurrence in patients.Results: We optimized collection of and genomic analysis for clinical lung cancer,identifying novel oncogene candidates (including genes contributing to tumourinvasion). In addition, we successfully used DNA alteration boundaries to discriminateclonally-related tumours and define ongoing clonal evolution in both tumours and cancercell lines, providing evidence in support of our first two hypotheses. We also identifieddysregulated genes and gene pathways associated with post-treatment recurrence forclinical lung cancer. These last data suggest that chemoresistance may be an intrinsic process for the majority of cells in a pre-treatment tumour, lending support to our third hypothesis. Significantly, we also detected distinct recurrence-associated gene changes within tumour histology subgroups, indicating that NSCLC may not be treatable as a single disease entity.Conclusions: Global analysis of DNA alterations is an effective means for defining clonal relationships between tumours. Further, tumour phenotypes such as chemoresistance are governed by complex activation of a variety of gene networks.

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Genetic alterations and lineage specificity in lung cancer (2009)

Background: Lung cancer is the world's leading cause of cancer mortality. The main factors contributing to this are the late stage of disease at the time of diagnosis and a lack of effective chemotherapeutic strategies. A better understanding of the molecular origins and basic biology of lung cancer will lead to the development of early detection techniques and novel therapies to address these issues. I applied an integrative genomics approach utilizing high-resolution whole genome profiling technologies to uncover causal gene disruption in lung cancer and identify candidates associated with the development of specific lung cancer subtypes. Hypotheses: (i) Genes key to lung tumorigenesis will be identified in recurrently altered genomic regions. (ii) Lung cancer subtypes require distinct genetic alterations for neoplastic development.Materials/ Methods: DNA copy number data from lung cancer specimens were integrated with expression data to identify genes contributing to tumorigenesis. Subsequently, this approach was applied to compare lung cancer subtypes to discover genes and pathways specifically disrupted in each. Quantitative RT-PCR and immunohistochemistry were performed to validate results from microarray experiments and cell models were utilized to confirm the functional significance of identified genes.Results: I identified novel gene candidates frequently deregulated in lung cancer which contribute to tumorigenesis, supporting the first hypothesis. In addition, the comparison of lung cancer subtypes identified subtype-specific genetic events and delineated genes and pathways important in their differential development, supporting the second hypothesis. Significantly, I discovered a novel squamous cell lineage specific oncogene, BRF2, which affects polymerase III transcribed genes. Conclusions: Integrative genomic analysis is an effective means for identifying key gene disruptions in lung cancer. Furthermore, these findings suggest that lung cancer subtypes require distinct genetic alterations for tumorigenesis, uncovering the specific targets disrupted by these alterations for the first time. Most importantly, activation of BRF2 represents a novel mechanism of tumorigenesis through the increase of polymerase III mediated transcription and the targeted activation of this gene in SqCC suggests that it may be an excellent candidate for new treatment strategies tailored to this subtype. Together, this work highlights the need for tailoring therapies to the specific cancer subtypes.

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Mantle cell lymphoma pathogenesis (2009)

Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma with a median patient survival time of 3 years. Although the characteristic t(11;14)(q13;q32) is found in virtually all cases, experimental evidence suggests that this event alone is insufficient to result in lymphoma and secondary genomic alterations are required. Therefore, secondary genetic alterations have been proposed as essential in MCL pathogenesis. Within this thesis I describe the creation of a novel assay to determine segmental copy number alterations at a previously unprecedented resolution. This new assay necessitated the development of new analytical software to visualize and analyze the high density data sets created. The creation of this software is described in detail. With these tools in place we assayed model genomes of MCL for recurrent segmental copy number alterations. These recurrent regions were defined; however, among these were copy number variations that appeared in both cases and controls. Investigation of these natural copy number variations in this thesis revealed that the human genome has a higher plasticity than previously appreciated. In fact, thousands of loci within the genome were found to be variable in copy number that may influence sensory perception and possibly disease susceptibility. I next investigated the genomes of MCL tumor samples to determine which somatic copy number alterations are related to a poor clinical course. Among the numerous loci that showed frequent copy number alterations in MCL genomes, many were associated with poor patient outcome. Among these, the loss of 9p21 was a strong factor in determining the clinical course of patients with MCL (P=0.0004). Three additional loci (4q13, 8q24, and 13q14) were combined with 9p21 to create a survival model that was very predictive of patient outcome (P=5.87 x 10-6). Interestingly, a previously uncharacterized locus (4q13) was within this survival model. Investigating this locus further revealed that the expression of two genes (CCNG2 and CCNI) influences the overall survival of patients with MCL (P=0.0292 and 0.0201, respectively).

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The role of specific genomic alterations in small cell lung cancer aggressiveness (2008)

Small Cell Lung Cancer (SCLC) is a very aggressive neuroendocrine tumour of the lung, which demonstrates a 5 year survival of only 10% for extensive stage disease (20-30% for limited stage), with only modest improvement over the last few decades. Identification of new molecular diagnostic and therapeutic targets is thus imperative. Previous efforts in identifying molecular changes in SCLC by gene expression profiling using microarrays have facilitated disease classification but yielded very limited information on SCLC biology. Previous DNA studies have been successful in identifying several loci important to SCLC. However the low resolution of conventional chromosomal Comparative Genomic Hybridization (CGH) has limited the findings to large chromosomal regions with only a few specific candidate genes discovered to date. Thus, to further understand the biological behaviour of SCLC, better methods for studying the genomic alterations in SCLC are necessary.This thesis highlights the development of array CGH technology for the high resolution dissection of aneuploidy in cancer genomes and the application of this new technology to the study of SCLC. I present the development of the first whole genome CGH array which offered unprecedented resolution in the profiling of cancer genomes allowing fine mapping of genes in a single experiment. Through application of DNA based analysis in conjunction with integrated expression analysis and comparison of SCLC to less aggressive non-small cell lung tumours I have identified novel patterns of pathway disruption specific to SCLC. This included alteration to Wnt pathway members and striking patterns of cell cycle activation through predominantly downstream disruption of signalling pathways including direct activation of the E2F transcription factors, which are normally repressed by the Rb gene.Analysis of targets of the E2F/Rb pathway identified EZH2 as being specifically hyper-activated in SCLC, compared to NSCLC. EZH2 is a polycomb group gene involved in the control of many cellular functions including targeted DNA methylation and escape from senescence in hematopoietic stem cells.Taken together these results suggest that in SCLC, downstream disruption may replace multiple upstream alterations leading to activation independent of a specific mitogenic pathway, and that EZH2 represents a potentially important therapeutic target.

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Master's Student Supervision (2010 - 2018)
Assessing a hyperspectral image analysis system to study tumor and immune cell spatial organizations within lung tumor microenvironments (2018)

Lung cancer is the leading cause of cancer-associated death worldwide. To improve clinical management, biological discovery underlying disease pathogenesis and improved sensitivity of early detection imaging modalities are required. Presently, therapeutic targeting immune components of the tumor microenvironment led to immunotherapies, potent therapeutic agents revolutionizing clinical practice. Increasingly recognized, the native spatial organization of tumor microenvironment immune cells could potentially serve as a surrogate for predicting prognosis and stratifying tumor immunogenicity. However, conventional methods, including histopathology and genomic profiling, are limited in measuring such parameters. Image analysis systems are being tested for these applications, but further development and optimization is required before clinical adoption. Dr. Calum MacAulay and Dr. Martial Guillaud’s research team at the British Columbia Cancer Research Centre (BCCRC) developed a novel hyperspectral image analysis system capable of spatially profiling tumor microenvironments in situ. Herein, this thesis reports the workflow optimization of this system prototyped with the lung adenocarcinoma (AC) microenvironment. This study was conducted with 21 primary lung AC. Adjacent tumor sections were stained for nuclei with hematoxylin and markers of tumor-infiltrating lymphocytes (CD3, CD79a, CD8) or adaptive immune resistance (PD1, PDL1, CD8) by multiplex immunohistochemistry. After a lung pathologist identified areas of interest on tissue sections, five representative regions within each area were imaged. Image stacks of sixteen different illumination wavelengths (from 420 to 720 nm) were then processed by spectral unmixing, segmented, and immunohistochemical marker positivity thresholds applied. Processed two-dimensional images were quantified for cell types and neighboring cell type spatial correlations. Additionally, three lung AC within this cohort were selected to test workflow of image registration software to interpolate cell spatial distributions between sections. The system was able to determine immune cell-to-cell spatial correlations and distribution within and across sections. Furthermore, technical factors were identified that affected the workflow of this system, including staining, image acquisition, segmentation, specialized equipment, and sampling strategy. Here we describe a valuable platform to quantitatively and spatially profile tumor heterogeneity that could then be used to correlate with lung cancer prognosis and treatment outcomes, although further optimization of this method is required.

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Cisplatin partially impedes lung adenocarcinoma-mediated M2 macrophage polarization (2016)

Lung cancer is the leading cause of cancer mortality and at 18%, has one of the lowest five-year survival rates of all malignancies. The majority of patients (>80%) are diagnosed with locally advanced or metastatic disease for which the standard of care is platinum-based doublet chemotherapy. However, chemotherapy has modest effects on overall survival, highlighting the need for novel and more effective treatments. Within the past decade, the role of the immune system in tumorigenesis has become increasingly appreciated. Targeting the immune cells within the tumor microenvironment is a growing field of study that holds exciting therapeutic potential. Macrophages are a prominent immune cell type in the lung and lung tumors. It is widely accepted that a spectrum of macrophage activation states exists, with the exact phenotype dependent upon the precise composition of signals within the microenvironment. At opposite ends of this spectrum there exist M1 macrophages which are pro-inflammatory and have antitumor functions, and M2 macrophages which are anti-inflammatory and act in wound healing and thus promote tumorigenesis. I hypothesized that macrophage differentiation is skewed by lung adenocarcinoma cells to an M2 phenotype and that cisplatin, a commonly prescribed chemotherapeutic, affects macrophage polarity. I co-cultured human monocytes with human lung adenocarcinoma cells in the absence and presence of physiologically relevant concentrations of cisplatin. Co-cultured macrophages displayed increased differentiation and an M2 polarity, in part potentially through IL-6 secretion by tumor cells. Cisplatin impeded macrophage differentiation, with treated macrophages displaying decreased size, granularity, and surface marker expression; however, CD206 expression, an M2 marker, remained elevated, suggesting a role for CD206 in response to treatment. Additionally, I optimized single cell analysis of clinical specimens in preparation for future projects, specifically ex vivo analysis of the effect of standard first line chemotherapy on macrophage polarity and other immune cells in advanced non-small cell lung cancer. Collectively, this work has demonstrated that macrophage polarity is affected by lung adenocarcinoma cells and by cisplatin. Moreover, the optimization of single cell analysis has prepared for the study of the effect of chemotherapy on macrophage polarity over the course of treatment using more physiologically representative specimens.

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Molecular genetic characterization of retinoblastoma tumors lacking RB1 mutations (2013)

Retinoblastoma is a rare childhood cancer of the retina and is the most common intraocular tumor in children. Classically, retinoblastoma results from biallelic loss of the RB1 tumor suppressor gene. As with other cancer types, dysregulation of a single gene alone is not considered sufficient for complete transformation to malignancy. Frequent regions of genetic alteration harbouring additional genes, implicated in retinoblastoma oncogenesis and progression, include chromosomes 1q, 2p, 6p, 13q and 16q.Sensitive molecular genetic screening techniques are capable of identifying RB1 mutations in 98% of unilateral retinoblastoma tumors. The remaining 2% harbour no identifiable RB1 inactivating alterations, and therefore molecular interrogation of these cases would likely reveal alternative genetic events driving retinoblastoma tumorigenesis in the absence of RB1 inactivation. Towards this objective, in this thesis work, I describe genetic alterations identified by tiling path array comparative genomic hybridization in a rare sample set composed of 23 RB1⁺/⁺ tumors. In addition to gene disruption by copy number alteration, mechanisms of gene disruption resulting in no overall change in copy number or change in copy number with allelic imbalance were also investigated utilizing genome-wide SNP array analysis on five of the RB1⁺/⁺ tumors. The most striking recurrent genetic alteration identified in retinoblastoma tumors lacking RB1 inactivating mutations, was focal high-level MYCN amplification, which occurred at a frequency of approximately 48%. The MYCN amplified RB1⁺/⁺ tumors also exhibited a statistically significant lower proportion of their genome affected by genomic instability when compared with the RB1 ⁻/⁻ tumors. In a subset of five matched tumor and blood normal samples the occurrence of copy neutral LOH was explored, although none was observed. Allele specific copy number analysis identified instances of allelic imbalance, including all five MYCN amplifications. Amplification of MYCN may represent a rare and novel alternate mechanism of retinoblastoma tumorigenesis. This work provides insight into the role of mutational events driving tumorigenesis in retinoblastoma in the absence of RB1 inactivation.

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