京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12803号 / 論医博第2075号 / 新制||医||1001(附属図書館) / 80847 / (主査)教授 伊達 洋至, 教授 武藤 学, 教授 川上 浩司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Non-small cell lung cancer (NSCLC) makes up the majority of lung cancers, which remains the leading cause of cancer mortality worldwide. NSCLC with mutant epidermal growth factor receptor (EGFR) is currently treated with tyrosine kinase inhibitors (TKIs). TKIs have proven effective in improving survival until resistance is conferred, mostly by way of the exon 20, threonine 790 to methionine (T790M) point mutation in EGFR. The mechanism by which this point mutation arises is poorly understood. Herein we report a possible pathway by which the C to T transition that leads to T790M comes about. We show that activation-induced cytidine deaminase (AID) mRNA expression is induced upon treatment with EGFR TKIs in mutant-EGFR human lung cancer cell lines but not in control cell lines. We also show that stable expression of AID is sufficient to produce resistance to one such TKI, erlotinib, and is sufficient to produce T790M itself. We also report that B-cell lymphoma 6 (BCL6) may precede AID in this pathway. Our results show that BCL6 is upregulated in these cell lines treated with EGFR TKIs but not in normal bronchial cells. We then treated human lung cancer cell lines with EGFR TKIs in combination with BCL6 inhibitors. Our results show that AID is dependent upon BCL6 expression. Finally, we report on results from a transient BCL6 overexpression which lead us to believe that AID mRNA receives input from at least one alternate pathway in addition to BCL6. We also performed these experiments on a family of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, that show they may be involved in this pathway downstream of AID. Taken together, our results suggest a potential pathway involving BCL6, AID, and APOBEC cytidine deaminases that lead to the C to T transition that produces T790M, thereby conferring resistance to EGFR TKIs in mutant-EGFR NSCLC. They also provide potential new targets for treatment should further study confirm our results. / 2021-10-07T00:00:00Z

Tumor lysis syndrome is an oncologic emergency that usually occurs after chemotherapy in patients with hematologic malignancies. Tumor lysis syndrome is rare in cases of solid tumors, especially when it occurs spontaneously. Herein, we present a case of spontaneous tumor lysis syndrome in a 55-year-old woman who presented with dyspnea and was found to have extensive metastatic small cell lung cancer. She developed acute oliguric renal failure and multiple electrolyte abnormalities requiring hemodialysis. The findings of this case suggest that clinicians should maintain a high index of suspicion for patients with malignancies who demonstrate the classic symptom of laboratory abnormalities even in the absence of chemotherapy.

Introduction: Small Cell Lung Cancer (SCLC) is an aggressive malignancy with poor prognosis that accounts for 10% of all clinical lung cancer. SCLC commonly metastasizes to the mediastinum, liver, bone, adrenals, and the brain but secondary endotracheal metastasis is an especially rare occurrence. We discuss the case of a 74-year-old male with principal complaint of cough, wheezing and hemoptysis found to have secondary endotracheal lesions on bronchoscopy. Case report: A 74-year-old male, former smoker with a past medical history of pulmonary embolism, bullous emphysema, and limited stage small cell lung cancer with wedge resection and chemotherapy 3 years ago presented with cough, wheezing, weight loss and intermittent hemoptysis ongoing for few weeks. CT scan of the chest showed multiple polypoid masses arising in the anterior wall of the trachea. He underwent bronchoscopy with biopsy. Pathology was consistent with small-cell lung cancer. Conclusion: Secondary tracheal metastasis from early stage small cell cancer is a rare occurrence. The likelihood of tracheal metastasis of lung cancer is amplified when an endotracheal nodule or eccentric thickening of the tracheal wall is seen on CT of patients with high suspicion. It is important for clinicians to suspect endotracheal lesions when a patient presents with recurrent respiratory complaints despite stable surveillance CT scan of chest in patients with history of lung cancer.

Small Cell Lung Cancer (SCLC) is a highly aggressive neuroendocrine tumor with a 5% survival rate over 5 years. Though SCLC comprises 13% of all cases of lung cancer the median survival time of 14.5 months has seen little improvement over the last four decades. Standard treatment relies on DNA damaging agents such as Cisplatin/Etoposide (EP) which induce a high response rate of 60-70%. Despite this initial response, nearly all patients will relapse rendering first-line therapies ineffective. Furthermore, SCLC has been shown to develop chemotherapy cross-resistance in which resistance to first-line chemotherapies will confer resistance to additional DNA damaging agents thereby reducing treatment efficacy and duration of response. Cross-Resistance constitutes a major clinical issue whose underlying mechanisms remain a mystery. The modest improvements in SCLC patient outcomes over the decades may be partially explained by the existing systems of study. Current methodologies of SCLC study rely on cell lines, patient samples, and Genetically Engineered Mouse Models which have little functional correlation to clinical outcomes. While few sources have proposed Patient Derived Xenograft (PDX) systems as an improved alternative, significant data remains sparse. Without a robust model system which accurately recapitulates patient outcomes, molecular pathways driving resistance cannot be uncovered. Here we present the generation of 34 SCLC PDX models which maintain both genomic and functional fidelity. Furthermore, treatment of a 30-model subset with first-line chemotherapy EP and a novel chemotherapy Olaparib/Temozolomide (OT) allowed for functional and molecular comparison between groups. Our findings demonstrate incomplete independent resistance mechanisms between EP and OT treatment with a small overlap of 31 genes involved in glycolysis and xenobiotic metabolism.

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Brain metastasis (BM), the most common tumor of the central nervous system, occurs in 20-36% of primary cancers. In particular, 20-40% of patients with non-small cell lung cancer (NSCLC) develop brain metastases, with a dismal survival of approximately 4-11 weeks without treatment, and 16 months with treatment. This highlights a large unmet need to develop novel targeted therapies for the treatment of lung-to-brain metastases (LBM). Genomic interrogation of LBM using CRISPR technology can inform preventative therapies targeting genetic vulnerabilities in both primary and metastatic tumors. Loss-of-function studies present limitations in metastasis research, as knocking out genes essential for survival in the primary tumor cells can thwart the metastatic cascade prematurely. However, transcriptional overexpression of genes using CRISPR activation (CRISPRa) has the potential for overcoming dependencies of gene essentiality. In this thesis, we created and utilized an in vivo genome-wide CRISPRa screening platform to identify novel genes, that when overexpressed, drive LBM. We have developed a patient-derived orthotopic murine xenograft model of LBM using a patient-derived NSCLC cell line (termed CRUK cells) from the Swanton Lab TRACERx study. We introduced a human genome-wide CRISPRa single guide RNA (sgRNA) library into non-metastatic and pro-metastatic lung cancer CRUK cells to achieve 500X representation of each sgRNA in the activation library. We then injected the cells into the lungs of immunocompromised mice and tracked lung tumor development and BM formation. Upon sequencing primary lung tumors and subsequent BM, we will identify enriched sgRNAs which may represent novel drivers of primary lung tumor formation and LBM. To the best of our knowledge, this study is the first in vivo genome-wide CRISPR activation screen using patient-derived NSCLC cells to help elucidate drivers of LBM. This work serves to provide a framework to gain a deeper understanding of the regulators of BM formation which will hopefully lead to targeted drug discovery that will ultimately be used in clinical trials to help eradicate brain metastasis in NSCLC patients. / Thesis / Master of Science (MSc) / Brain metastasis, or the spread of a primary cancer from another organ to the brain, is the most common adult brain tumor. Brain metastases can arise after the treatment of primary tumors and are only detected in the clinic at a highly malignant stage. Current treatments for brain metastasis consist of surgical removal and palliative chemoradiotherapy, which fail to fully eliminate the brain tumor. Over 20% of cancer patients develop brain metastases, with lung, breast, and skin cancers leading as the top three sources of metastasis. In particular, 40% of patients with non-small cell lung cancer develop brain metastasis, with survival of only 4-11 weeks once diagnosed without treatment, and 16 months with treatment. As systemic therapies for the treatment of non-small cell lung cancer are becoming increasingly effective at controlling primary disease, patients are ironically succumbing to their brain tumors. This highlights a large unmet need to develop novel targeted therapies for the treatment of lung-to-brain metastases (LBM). Functional genomic tools provide the opportunity to investigate the genetic underpinnings of LBM. With the advent of gene editing technologies, we are able to overexpress various genes and observe the impact genetic perturbations have on tumor initiation, growth, and metastasis. In this thesis, we devised a pre-clinical animal model of LBM that could be used to study genetic drivers of LBM using a gene overexpression tool such that one gene per tumor cell gets activated. We are then able to model the disease trajectory from a lung tumor to brain metastasis development using patient samples in our animal model and identify genes that, upon overexpression, drive LBM. This platform will lead to potential therapeutic targets to prevent the formation of LBM and prolong the survival of patients with non-small cell lung cancer.

Introduction: Lung cancer is the leading cause of cancer death worldwide. In Canada, in 2021 alone, an estimated 21,000 patients have died from this disease. Non-small cell lung cancer (NSCLC) constitutes 85% of all lung cancer cases diagnosed. Over the past 30 years, treatment of unresected locally advanced (LA)-NSCLC evolved from treatment with chest radiotherapy (RT) alone to the current standard of care (SOC) of concurrent chemo-radiation (cCRT), followed by consolidative immunotherapy. Modern RT has influenced the survival of LA-NSCLC patients. In this work we analyzed data from provincial and local institutional databases to evaluate whether, i) the use of modern imaging with 18F-deoxyglucose (FDG)-positron emission tomography (PET), ii) dose of chest RT to tumors and iii) unintentional irradiation of normal tissues during treatment for lung cancer, influence outcomes of patients managed with RT. Methodology: Ontario provincial databases were searched through the Institute of Clinical Evaluative Sciences (IC/ES) for stage III NSCLC patients diagnosed between 2007 and 2017. Surgical patients were excluded, and all patients that received RT with or without chemotherapy were selected. Patients were divided into groups of different RT doses (<40Gy, 40-55.9Gy, and ≥56Gy) and whether they underwent diagnostic FDG-PET. For the next study phase (the institutional level), we retrospectively identified and reviewed LA-NSCLC patients treated at local health integration network area 4 (LHIN4) cancer centres (Juravinski and Walker Family Cancer Centres) from 2009 to 2019. We selected patients treated in that period with chest RT > 40Gy with or without chemotherapy. Patients’ data were reviewed individually for disease characteristics, staging investigations, RT treatment parameters and survival outcomes. Dosimetric analysis was performed on both groups of patients (RT alone group and cCRT group). Results: The provincial analysis included 5,577 stage III patients who had received chest RT without surgery between January 2007 and March 2017. Within this group, 39.8% (2,225) received RT alone, 47.4% (2,645) received concurrent chemo-radiotherapy (cCRT), and 12.6% (707) received sequential chemo-radiotherapy (sCRT). Median overall survival (OS) with RT alone in three dose groups <40Gy, 40-55.9Gy, ≥ 56Gy was 7.2, 8.5 and 13.3 months compared to 16.5, 15.8 and 22 months for cCRT patients. Higher RT dose and PET utilization were independently associated with improved survival in multivariate analysis. At the institutional analysis, 84 patients were treated with RT alone, 184 with cCRT and patients with sequential CRT were excluded. In the RT alone group, the median, 1- and 3-year overall survival were 18.1 months, 64.4% and 24.3%, respectively. In comparison, the median, 1- and 3-year survival outcomes in the cCRT group were 36.3 months, 82.5%, and 50.4%, respectively. Additionally, 79.8% of patients in the radiation alone group and 95.1% in cCRT group had PET staging. In univariate analysis, the RT dose prescribed to the tumor and RT dose delivered to the heart were significantly associated with survival, while multivariate analysis only showed the significant association between RT dose to heart and overall survival. Conclusions: Our population-based analysis confirmed that radiation monotherapy remains a widely used treatment modality in LA-NSCLC. Higher RT doses and utilization of FDG-PET imaging are associated with improved survival in patients with unresected LA-NSCLC managed with RT. The institutional analysis suggests that in well-staged patients with LA-NSCLC, chest RT of ≥40Gy is associated with improved survival outcomes that compare favorably with historical results of definitive RT alone treatment. Further, survival of patients staged well with FDG-PET and treated with SOC cCRT was higher than historical reports. Importantly, in this study we found that RT dose delivered to the heart associates negatively with patient survival. These findings can help improve clinical decision-making in the management of unresected LA-NSCLC and can serve as basis for future clinical trials. / Thesis / Master of Science (MSc) / Lung cancer is the leading cause of cancer death in Canada and worldwide. These tumors are present as two main histological types, small cell and non-small cell lung cancer, the latter of which consists the majority of the cases diagnosed. Although treatments with surgery or radiotherapy provide reasonable outcomes in lung cancer cases detected early, a high proportion of patients present with localized but advanced disease that is inoperable. Over the last three decades, treatment of locally advanced non-small cell lung cancer has evolved from radiation alone to chemoradiation and immunotherapy. These developments have increased the survival of these patients. In this thesis, we tried to dissect the elements that play roles in the survival of locally advanced non-small cell lung cancer patients. To do this, we evaluated such patients at two levels. First, at the provincial level, we evaluated the type of treatments, and we explored the association of metabolic imaging with positron emission tomography (PET) and the use of high-dose chest radiotherapy with patient survival. Second, at the institutional level, we assessed patients’ outcomes with a more detailed approach. We analyzed the type of treatment along with a detailed dosimetric analysis. The results of our analysis suggest that the use of PET scans and curative radiotherapy is associated with improved survival. On the other hand, the unintentional treatment of the heart with increasing doses of radiotherapy, taking place during chest radiation for lung cancer, is associated with poor outcomes. These results provide a basis for further investigation to improve outcomes of radiotherapy in this disease.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24288号 / 医博第4904号 / 新制||医||1061(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山本 洋介, 教授 中本 裕士, 教授 森田 智視 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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