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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2022)

Precancer can represent a benign condition in some and a potent precursor to lethal cancer in others. As cancer screening and early detection of cancer improves, precancer diagnoses are becoming quite common, yet our poor understanding of precancer leaves clinicians unclear on how to treat them. Precancerous lesions are often tiny and archived, formalin-fixed, and paraffin-embedded (FFPE), which degrades and damages the small amount of nucleic acid available. This makes the genomic and transcriptomic analysis of precancers quite challenging as many of the lesions, especially the smallest, are often incompatible with standard DNA or RNA sequencing assays. My work reports the optimization of both experimental and computational methods to perform genetic profiling of precancer lesions - including acquired (somatic) and inherited (germline) variation - and the application of these methods to improve our understanding of breast precancer, ductal carcinoma in situ (DCIS).

A low-input FFPE DNA optimized exome sequencing workflow was paired with RNA sequencing, histological analysis, and immune microenvironment spatial profiling to generate a multimodal breast precancer atlas across 85 histological regions microdissected from the lesions of 39 patients diagnosed with pure DCIS. This allowed the measurement of relationships between molecular and phenotypic features, phylogenetic analysis, and identification of immune states revealed by stromal and epithelial-specific profiling. Further, a low-coverage whole-genome sequencing with reference-based imputation method, which provided reliable germline genotyping directly from archival FFPE tissues, was leveraged to evaluate the role of germline genetics in long-term precancer progression. I applied this methodology to samples from 36 patients with DCIS and identified that increased established breast polygenic risk scores were predictive of adverse outcomes.

The improved mapping provided by the breast precancer atlas, and suggestive evidence that germline variants contribute to DCIS outcome represent valuable resources in the study of the natural history of breast precancer. In particular, the findings emphasize the importance of including contextual information - spatial, microenvironmental, and inherited genetic factors - to fully characterize lesions. By only using archival tissue, I set the stage for the large retrospective studies that will be needed to guide the clinical management of precancer.

  • 1 supplemental ZIP
Cover page: Profiling archival samples to examine the molecular underpinnings of early carcinogenesis
  • Article
  • Peer Reviewed
UC San Diego Previously Published Works (2023)
G proteins and G protein-coupled receptors activate a diverse array of signal transduction pathways that promote cell growth and survival. Indeed, hot spot-activating mutations in GNAQ/GNA11, encoding Gαq proteins, are known to be driver oncogenes in uveal melanoma (UM), for which there are limited effective therapies currently available. Focal adhesion kinase (FAK) has been recently shown to be a central mediator of Gαq-driven signaling in UM, and as a result, is being explored clinically as a therapeutic target for UM, both alone and in combination therapies. Despite this, the repertoire of Gαq/FAK-regulated signaling mechanisms have not been fully elucidated. Here, we used a whole-genome CRISPR screen in GNAQ-mutant UM cells to identify mechanisms that, when overactivated, lead to reduced sensitivity to FAK inhibition. In this way, we found that the PI3K/AKT signaling pathway represented a major resistance driver. Our dissection of the underlying mechanisms revealed that Gαq promotes PI3K/AKT activation via a conserved signaling circuitry mediated by FAK. Further analysis demonstrated that FAK activates PI3K through the association and tyrosine phosphorylation of the p85 regulatory subunit of PI3K and that UM cells require PI3K/AKT signaling for survival. These findings establish a novel link between Gαq-driven signaling and the stimulation of PI3K as well as demonstrate aberrant activation of signaling networks underlying the growth and survival of UM and other Gαq-driven malignancies.
    Cover page: Whole-genome CRISPR screening identifies PI3K/AKT as a downstream component of the oncogenic GNAQ–focal adhesion kinase signaling circuitry
    • Article
    UC San Diego Previously Published Works (2020)
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2021)
      Along with early-onset cancers, multiple primary cancers (MPCs) are likely resulting from increased genetic susceptibility; however, the associated predisposition genes or prevalence of the pathogenic variants genes in MPC patients are often unknown. We screened 71 patients with MPC of the stomach, colorectal, and endometrium, sequencing 65 cancer predisposition genes. A subset of 19 patients with early-onset MPC of stomach and colorectum were further evaluated for variants in cancer related genes using both normal and tumor whole exome sequencing. Among 71 patients with MPCs, variants classified to be pathogenic were observed in 15 (21.1%) patients and affected Lynch Syndrome (LS) genes: MLH1 (n = 10), MSH6 (n = 2), PMS2 (n = 2), and MSH2 (n = 1). All carriers had tumors with high microsatellite instability and 13 of them (86.7%) were early-onset, consistent with LS. In 19 patients with early-onset MPCs, loss of function (LoF) variants in RECQL5 were more prevalent in non-LS MPC than in matched sporadic cancer patients (OR = 31.6, 2.73-1700.6, p = 0.001). Additionally, there were high-confidence LoF variants at FANCG and CASP8 in two patients accompanied by somatic loss of heterozygosity in tumor, respectively. The results suggest that genetic screening should be considered for synchronous cancers and metachronous MPCs of the LS tumor spectrum, particularly in early-onset. Susceptibility variants in non-LS genes for MPC patients may exist, but evidence for their role is more elusive than for LS patients.
        Cover page: Prevalence of cancer susceptibility variants in patients with multiple Lynch syndrome related cancers
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2022)

        Purpose

        The contribution of common genetic variants to pre-cancer progression is understudied due to long follow-up time, rarity of poor outcomes and lack of available germline DNA collection. Alternatively, DNA from diagnostic archival tissue is available, but its somatic nature, limited quantity and suboptimal quality would require an accurate cost-effective genome-wide germline genotyping methodology.

        Experimental design

        Blood and tissue DNA from 10 individuals were used to benchmark the accuracy of Single Nucleotide Polymorphisms (SNP) genotypes, Polygenic Risk Scores (PRS) or HLA haplotypes using low-coverage whole-genome sequencing (lc-WGS) and genotype imputation. Tissue-derived PRS were further evaluated for 36 breast cancer patients (11.7 years median follow-up time) diagnosed with DCIS and used to model the risk of Breast Cancer Subsequent Events (BCSE).

        Results

        Tissue-derived germline DNA profiling resulted in accurate genotypes at common SNPs (blood correlation r2 > 0.94) and across 22 disease-related polygenic risk scores (PRS, mean correlation r = 0.93). Imputed Class I and II HLA haplotypes were 96.7% and 82.5% concordant with clinical-grade blood HLA haplotypes, respectively. In DCIS patients, tissue-derived PRS was significantly associated with BCSE (HR = 2, 95% CI 1.2-3.8). The top and bottom decile patients had an estimated 28% and 5% chance of BCSE at 10 years, respectively.

        Conclusions

        Archival tissue DNA germline profiling using lc-WGS and imputation, represents a cost and resource-effective alternative in the retrospective design of long-term disease genetic studies. Initial results in breast cancer suggest that common risk variants contribute to pre-cancer progression.
          Cover page: Accurate genome-wide genotyping from archival tissue to explore the contribution of common genetic variants to pre-cancer outcomes
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2021)
          BACKGROUNDThe aberrant activation of the PI3K/mTOR signaling circuitry is one of the most frequently dysregulated signaling events in head and neck squamous cell carcinoma (HNSCC). Here, we conducted a single-arm, open-label phase IIa clinical trial in individuals with oral premalignant lesions (OPLs) to explore the potential of metformin to target PI3K/mTOR signaling for HNSCC prevention.METHODSIndividuals with OPLs, but who were otherwise healthy and without diabetes, underwent pretreatment and posttreatment clinical exam and biopsy. Participants received metformin for 12 weeks (week 1, 500 mg; week 2, 1000 mg; weeks 3-12, 2000 mg daily). Pretreatment and posttreatment biopsies, saliva, and blood were obtained for biomarker analysis, including IHC assessment of mTOR signaling and exome sequencing.RESULTSTwenty-three participants were evaluable for response. The clinical response rate (defined as a ≥50% reduction in lesion size) was 17%. Although lower than the proposed threshold for favorable clinical response, the histological response rate (improvement in histological grade) was 60%, including 17% complete responses and 43% partial responses. Logistic regression analysis revealed that when compared with never smokers, current and former smokers had statistically significantly increased histological responses (P = 0.016). Remarkably, a significant correlation existed between decreased mTOR activity (pS6 IHC staining) in the basal epithelial layers of OPLs and the histological (P = 0.04) and clinical (P = 0.01) responses.CONCLUSIONTo our knowledge this is the first phase II trial of metformin in individuals with OPLs, providing evidence that metformin administration results in encouraging histological responses and mTOR pathway modulation, thus supporting its further investigation as a chemopreventive agent.TRIAL REGISTRATIONNCT02581137FUNDINGNIH contract HHSN261201200031I, grants R01DE026644 and R01DE026870.
            Cover page: Inhibition of mTOR signaling and clinical activity of metformin in oral premalignant lesions
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2022)
            Microenvironmental and molecular factors mediating the progression of Breast Ductal Carcinoma In Situ (DCIS) are not well understood, impeding the development of prevention strategies and the safe testing of treatment de-escalation. We addressed methodological barriers and characterized the mutational, transcriptional, histological, and microenvironmental landscape across 85 multiple microdissected regions from 39 cases. Most somatic alterations, including whole-genome duplications, were clonal, but genetic divergence increased with physical distance. Phenotypic and subtype heterogeneity was frequently associated with underlying genetic heterogeneity and regions with low-risk features preceded those with high-risk features according to the inferred phylogeny. B- and T-lymphocytes spatial analysis identified three immune states, including an epithelial excluded state located preferentially at DCIS regions, and characterized by histological and molecular features of immune escape, independently from molecular subtypes. Such breast pre-cancer atlas with uniquely integrated observations will help scope future expansion studies and build finer models of outcomes and progression risk.
              Cover page: The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ
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