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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23603号 / 医博第4790号 / 新制||医||1055(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 上野 英樹, 教授 金子 新, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The interactions of tumours with normal host tissue are key determinants of cancer growth and progression. The ability or inability of the patient’s immune system to mount a response against the tumour is tightly correlated with prognosis. One of the ways tumours avoid detection and elimination by the immune system is by expressing programmed death ligand 1 (PD-L1). PD-L1 binds to its receptor programmed death 1 (PD1) on T cells, inhibiting T cell responsiveness to antigenic stimuli. Blockade of the PD1/PD-L1 pathway removes this negative signal and restores anti-tumour immunity. While this blockade of PD1/PD-L1 is well established through the use of antibodies, small molecule inhibitors of PD1/PD-L1 are relatively unknown. We employed in silico docking in order to find small molecules capable of binding to either PD1 or PD-L1, and the highest-ranked compounds were tested in biophysical assays for their ability to inhibit PD1/PD-L1 binding. A thermal shift assay identified a pyrazole compound as a possible binding partner for PD-L1, but follow-up assays showed that it had no effect on the PD1/PD-L1 interaction and that its apparent binding was probably due to aggregation. An ELISA assay identified a tryptophan diamine compound as an apparent stabilizer of the PD1/PD-L1 interaction. However this compound, too, was later identified to be inactive in orthogonal assays. We identified a family of salicylic acid derivatives that interfered with TR-FRET measurements – an unusual observation, given that TR-FRET is touted as being insensitive to most mechanisms of compound interference. This discovery should help other fragment- screening groups identify false positives more easily. We also probed the mechanism of inhibition of a recently disclosed family of small molecule PD1/PD-L1 inhibitors from Bristol-Myers Squibb. Concurrently with other groups, we used protein NMR, size exclusion chromatography, and SPR to determine that the compounds were inducing homodimerization through the PD1-binding face of PD-L1. Furthermore, using cellular crosslinking and live cell imaging, we showed that these first generation inhibitors are fairly ineffective at inhibiting this interaction on the cell surface. More potent compounds will be needed to see any cellular effect from this mechanism of action. / Graduate / 2019-02-15

L'apport des anticorps immunomodulateurs ciblant PD-1, PD-L1 et CTLA-4 ont récemment révolutionné la prise en charge thérapeutique du cancer. Cependant, seule une minorité de patients développent des réponses objectives à ces traitements. Par conséquent, de nouvelles innovation thérapeutiques sont nécessaires afin d'augmenter l'immunogénicité des tumeurs et de surmonter la résistance à la thérapie contre les anticorps immunomodulateurs. Les propriétés oncolytiques de certains virus peuvent être exploitées afin de permettre un amorçage de l'immunité anti-tumorale. Différents virus oncolytiques (OVS) sont actuellement en développement clinique intense en combinaison avec des thérapies par anticorps immunomodulateurs. Nous avons trouvé qu'un vaccin viral pédiatrique disponible dans le commerce a des propriétés oncolytiques. Ce virus pédiatrique peut tuer directement les cellules cancéreuses, avec des caractéristiques de mort cellulaire immunogène. De plus, ce virus a des propriétés pro-inflammatoires et peut activer la voie NF-kB indépendamment des voies de danger cellulaire (TLR et IRF). Ces propriétés biologiques in vitro se traduisent in vivo en une activité anti-tumorale. L'Injection intra-tumorale du vaccin a des effets anti-tumoraux directs mais également à médiation immunitaire. De façon intéressante, dans des modèles de souris immunocompétentes porteuses de tumeurs murines, l'injection intra-tumorale de vaccin a un effet synergique avec des anti CTLA-4 permettant la guérison de 100% des souris. Les vaccins sont des produits pédiatriques et adultes de grade clinique. Par conséquent, des stratégies de vaccination in situ par injection intra-tumorale pourraient être rapidement mises en clinique / Immune checkpoint targeted therapies against PD-1, PD-L1 and CTLA-4 are currently revolutionizing cancer care. However, only a minority of patients develop objective responses with these treatments. Therefore, new therapeutic interventions are needed to increase the immunogenicity of tumors in order to overcome resistance to immune checkpoint blockade therapy. Oncolytic properties of common viruses can be exploited for the priming of anti-tumor immunity and such oncolytic viruses (OVs) are currently in intense clinical development in combination with immune checkpoint targeted therapies. We have found that commercially available virus vaccines do have oncolytic properties. These pediatric vaccine virus can directly kill cancer cells with features of immunogenic cell death. Moreover, it has pro-inflammatory properties and can activate the NF-Kb pathway in a toll-like receptor and IRF3 independent manner. These in vitro biological properties translate in vivo into anti-tumor activity. Intra-tumoral vaccine therapy has anti-tumor effects which are partly immune mediated. Interestingly, in immunocompetent murine pediatric tumor models, intra-tumoral injection overcome resistance and synergize with immune checkpoint targeted therapy. Vaccines are pediatric and adult clinical grade products. Therefore, in situ intra-tumoral immunization strategies could be implemented quickly in the clinic

Objective: This study aimed to identify the programmed death ligand-1 (PDL1, also termed as CD274) and its positively correlated immune checkpoint genes (ICGs) and to determine the immune subtypes of CD274-centered ICG combinations in oral and squamous cell carcinoma (OSCC). Materials and Methods: Firstly, the 95 ICGs obtained via literature reviews were identified in the Cancer Genome Atlas (TCGA) database in relation to OSCC, and such 88 ICG expression profiles were extracted. ICGs positively correlated with CD274 were utilized for subsequent analysis. The relationship between ICGs positively correlated with CD274 and immunotherapy biomarkers (tumor mutation burden (TMB), and adaptive immune resistance pathway genes) was investigated, and the relationships of these genes with OSCC clinical features were explored. The prognostic values of CD274 and its positively correlated ICGs and also their associated gene pairs were revealed using the survival analysis. Results: Eight ICGs, including CTLA4, ICOS, TNFRSF4, CD27, B- and T-lymphocyte attenuator (BTLA), ADORA2A, CD40LG, and CD28, were found to be positively correlated with CD274. Among the eight ICGs, seven ICGs (CTLA4, ICOS, TNFRSF4, CD27, BTLA, CD40LG, and CD28) were significantly negatively correlated with TMB. The majority of the adaptive immune resistance pathway genes were positively correlated with ICGs positively correlated with CD274. The survival analysis utilizing the TCGA-OSCC data showed that, although CD274 was not significantly associated with overall survival (OS), the majority of ICGs positively correlated with CD274 (BTLA, CD27, CTLA4, CD40LG, CD28, ICOS, and TNFRSF4) were significantly correlated with OS, whereby their low-expression predicted a favorable prognosis. The survival analysis based on the gene pair subtypes showed that the combination subtypes of CD274_low/BTLA_low, CD274_low/CD27_low, CD274_low/CTLA4_low, CD8A_high/BTLA_low, CD8A_high/CD27_low, and CD8A_high/CTLA4_low predicted favorable OS. Conclusion: The results in this study provide a theoretical basis for prognostic immune subtyping of OSCC and highlight the importance of developing future immunotherapeutic strategies for treating oral cancer.

Background: Treatment options for poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinoma are unsatisfactory and prognosis is generally poor. Lenvatinib (LEN), a multi-tyrosine kinase inhibitor targeting fibroblast growth factor receptors (FGFR) 1-4 is approved for advanced radioiodine refractory thyroid carcinoma, but response to single agent is poor in ATC. Recent reports of combining LEN with PD-1 inhibitor pembrolizumab (PEM) are promising. Materials and Methods: Primary ATC (n=93) and PDTC (n=47) tissue samples diagnosed 1997-2019 at five German tertiary care centers were assessed for PD-L1 expression by immunohistochemistry using Tumor Proportion Score (TPS). FGFR 1-4 mRNA was quantified in 31 ATC and 14 PDTC with RNAscope in-situ hybridization. Normal thyroid tissue (NT) and papillary thyroid carcinoma (PTC) served as controls. Disease specific survival (DSS) was the primary outcome variable. Results: PD-L1 TPS≥50% was observed in 42% of ATC and 26% of PDTC specimens. Mean PD-L1 expression was significantly higher in ATC (TPS 30%) than in PDTC (5%; p<0.01) and NT (0%, p<0.001). 53% of PDTC samples had PD-L1 expression ≤5%. FGFR mRNA expression was generally low in all samples but combined FGFR1-4 expression was significantly higher in PDTC and ATC compared to NT (each p<0.001). No impact of PD-L1 and FGFR 1-4 expression was observed on DSS. Conclusion: High tumoral expression of PD-L1 in a large proportion of ATCs and a subgroup of PDTCs provides a rationale for immune checkpoint inhibition. FGFR expression is low thyroid tumor cells. The clinically observed synergism of PEM with LEN may be caused by immune modulation.

Melanocytic neoplasms have been genetically characterized in detail during the last decade. Recurrent CTNNB1 exon 3 mutations have been recognized in the distinct group of melanocytic tumors showing deep penetrating nevus-like morphology. In addition, they have been identified in 1–2% of advanced melanoma. Performing a detailed genetic analysis of difficult-to-classify nevi and melanomas with CTNNB1 mutations, we found that benign tumors (nevi) show characteristic morphological, genetic and epigenetic traits, which distinguish them from other nevi and melanoma. Malignant CTNNB1-mutant tumors (melanomas) demonstrated a different genetic profile, instead grouping clearly with other non-CTNNB1 melanomas in methylation assays. To further evaluate the role of CTNNB1 mutations in melanoma, we assessed a large cohort of clinically sequenced melanomas, identifying 38 tumors with CTNNB1 exon 3 mutations, including recurrent S45 (n = 13, 34%), G34 (n = 5, 13%), and S27 (n = 5, 13%) mutations. Locations and histological subtype of CTNNB1-mutated melanoma varied; none were reported as showing deep penetrating nevus-like morphology. The most frequent concurrent activating mutations were BRAF V600 (n = 21, 55%) and NRAS Q61 (n = 13, 34%). In our cohort, four of seven (58%) and one of nine (11%) patients treated with targeted therapy (BRAF and MEK Inhibitors) or immune-checkpoint therapy, respectively, showed disease control (partial response or stable disease). In summary, CTNNB1 mutations are associated with a unique melanocytic tumor type in benign tumors (nevi), which can be applied in a diagnostic setting. In advanced disease, no clear characteristics distinguishing CTNNB1-mutant from other melanomas were observed; however, studies of larger, optimally prospective, cohorts are warranted.

Immune checkpoint blockade (ICB) shows remarkable clinical effects in patients with metastatic microsatellite-unstable (MSI) cancer. However, markers identifying potential non-responders are missing. We examined the prevalence of Beta-2-microglobulin (B2M) mutations, a common immune evasion mechanism, in stage IV MSI gastrointestinal cancer and its influence on metastatic pattern and patients’ survival under ICB. Twentyfive patients with metastatic, MSI gastrointestinal adenocarcinoma were included. Eighteen patients received ICB with pembrolizumab and one patient with nivolumab/ ipilimumab. Sequencing was performed to determine B2M mutation status. B2M mutations and loss of B2M expression were detected in 6 out of 25 stage IV MSI cancers. B2M mutations were strongly associated with exclusively peritoneal/peritoneal and lymph node metastases (p=0.0055). However, no significant differences in therapy response (25% vs. 46.6%, p>0.99) and survival (median PFS: 19.5 vs 33.0 months, p=0.74; median OS 39 months vs. not reached, p>0.99) were observed between B2Mmutant and B2M-wild type tumor patients. Among metastatic MSI GI cancers, B2Mmutant tumors represent a biologically distinct disease with distinct metastatic patterns. To assess ICB response in B2M-mutant MSI cancer patients, future studies need to account for the fact that baseline survival of patients with B2M-mutant MSI cancer may be longer than of patients with B2M-wild type MSI cancer.

Alterações no genoma de células tumorais são eventos comuns durante a carcinogênese. Tais aberrações genômicas - como mutações e variações estruturais - são diretamente relacionadas a detecção e morte de células neoplásicas pelo sistema imune. Além da contribuição da perda de função de genes supressores tumorais (GSTs) no desenvolvimento neoplásico, GSTs também influenciam a resposta imune no câncer. Por exemplo, o GST PTEN afeta diretamente a via interferon através da desfosforilação do fator regulador de interferon 3 (IRF3). No entanto, se mantém inconclusivo se a inativação de PTEN diretamente influencia a resposta imune através de IRF3 ou por provocar altos níveis de instabilidade genômica. Para responder essa questão, conduzimos uma análise PanCancer de 33 tipos tumorais da coorte The Cancer Genome Atlas para identificar se existem associações entre a inativação do gene PTEN e alterações genômicas específicas que podem suprimir ou ativar a resposta imune antitumoral. O status de inativação de PTEN foi determinado a partir de dados de variação no número de cópias e presença de mutações de ponto. Nesse estudo, investigamos o efeito da inativação de PTEN nas alterações genômicas de tumores e na abundância de 22 células do sistema imune derivadas do algoritmo CIBERSORT. Observamos que a inativação de PTEN foi significantemente associada com níveis elevados de aneuploidia, mutações, e heterogeneidade intratumoral. Além disso, nossos achados mostraram que a inativação de PTEN é altamente específica para cada tipo tumoral. Da mesma maneira, observamos que pacientes com tumores PTEN-inativos podem apresentar variações na resposta a imunoterapia. Tal observação deriva da correlação significativa entre a inativação de PTEN e a expressão dos alvos terapêuticos proteína programada 1 da morte celular (PD1), seu ligante (PDL1), e indoleamina 2,3 dioxigenase (IDO1). Na análise PanCancer, a inativação de PTEN também foi significativamente associada à composição de células do sistema imune no microambiente tumoral, incluindo células T regulatórias (Treg) e células CD8+. A partir desses achados, conduzimos uma análise aprofundada de tumores de próstata primários e metastáticos com a perda de PTEN. Através de uma análise in silico, nós observamos que tumores primários e metastáticos apresentam maiores densidades de Treg quando há perda da proteína PTEN. Nós também observamos que, dependendo do local de metástase prostática, a deficiência de PTEN é associada a um perfil de células imunes supressivas no microambiente tumoral. A partir da análise de uma coorte brasileira composta por 94 tumores primários de próstata, observamos que a perda de PTEN se associa a uma maior densidade de Tregs e uma maior expressão da proteína imunossupressora IDO1. Além disso, tumores PTEN-deficientes com altas densidades de Tregs apresentaram o pior prognóstico entre pacientes. Coletivamente, nós demonstramos que a inativação de PTEN é associada a um estado imunossuprimido no microambiente tumoral. Ademais, a perda de PTEN possivelmente se associa a resposta imune antitumoral através da combinação de duas diferentes vias - uma dependente de IRF3 e outra relacionada ao efeito no genoma de células cancerosas. Ensaios funcionais são necessários para validar os achados desse estudo; porém, sugerimos que a avaliação do status de inativação de PTEN pode ter alto potencial para discernir pacientes que responderão à imunoterapia. / Cancer-cell genomes undergo several abnormalities during carcinogenesis. Indeed, many of the tumor-specific genomic changes, such as mutations and chromosomal aberrations, are related to how the host immune system responds to detect and kill tumor cells. In addition to these general effects, loss of function of specific tumor suppressor genes (TSG) contributes to tumor development and progression and at the same time also regulates several facets of the immune response in cancer. For instance, the TSG phosphatase and tensin homolog (PTEN) was shown to directly regulate the anti-viral interferon response by licensing the interferon regulatory factor 3 (IRF3). However, it is still unclear whether PTEN directly influences the immune response through the interferon network or by provoking higher levels of genomic instability. To address this question, we conducted a PanCancer analysis of 33 tumor types from The Cancer Genome Atlas to determine whether there were associations between PTEN inactivation and specific genomic features that are linked to immunosuppressive states in cancer. PTEN inactivation status was determined by combining copy number and point mutation data. Then, we performed a parallel analysis of genomic instability and immune-cell abundances derived from the CIBERSORT algorithm comparing PTEN deficient to intact tumors. We found that PTEN inactivation was strongly associated with enhanced levels of aneuploidy, mutation load, immunogenic mutations, and tumor heterogeneity. Furthermore, we found that the outcome of PTEN inactivation status was highly specific to each tumor type and the induced changes appeared to lead to variation in immune responses in different cancers. Response to current immunotherapeutic approaches depends on the expression of targeted immune checkpoints, and we found that tumors with PTEN deficiency had altered expression of programmed death protein 1 (PD1), its ligand (PDL1), and the immunosuppressive protein indoleamine 2,3-dioxygenase (IDO1). We also found that PTEN inactivation led to a distinct immune-cell composition in the tumor microenvironment, including regulatory T cells and CD8+ T cells. Lastly, we performed an in-depth analysis of the immune-cell content of prostate tumors that harbored PTEN protein loss. Through an in silico analysis of 622 tumors, we found that both primary and metastatic lesions had higher densities of regulatory T cells when PTEN was lost. Then, the analysis of 94 primary prostate tumors from Brazil demonstrated that PTEN protein loss was significantly associated with high Treg density and IDO1 protein expression. Moreover, PTEN-null tumors with high Treg density exhibited the worse outcome among patients. We also found that, depending on the prostate cancer metastatic site, PTEN deficiency was linked to variation in the immunosuppressive immune cell landscape. Collectively, we show that PTEN inactivation associates with the anti-tumor immune response likely through direct avenues (via licensing of IRF3) and indirectly by influencing the genome of cancer cells. Functional studies are required to validate our in silico findings; however, we speculate that determining PTEN inactivation status may allow clinicians to distinguish patients that are more likely to respond to current immunotherapies.

L’adénocarcinome canalaire du pancréas (ACP) est un problème majeur de santé publique. L’ACP se développe principalement à partir de deux lésions précurseurs : les néoplasies intra-épithéliales pancréatiques et les tumeurs intracanalaires papillaires et mucineuses du pancréas (TIPMP). Les mécanismes moléculaires sous-tendant l’oncogenèse pancréatique sont nombreux. Nous avons étudié le mécanisme de cancérogenèse MSI (MicroSatellite Instability) où il existe une déficience dans le système de réparation des erreurs de réplication de l’ADN ou système MMR (Mismatch Repair). Ce mécanisme de cancérogenèse original est caractérisé par une instabilité génétique de l’ADN affectant les séquences répétées microsatellites du génome. Le phénotype MSI a été décrit dans le syndrome de Lynch (SL), dans lequel il existe une mutation germinale d’un des gènes du système MMR (MLH1, MSH2, MSH6 et PMS2). L’intérêt de l’étude des cancers MSI s’est accru de façon considérable avec le développement des immunothérapies dirigées contre les checkpoints immunitaires (ICK), en particulier PD-1/PD-L1. Nous avons confirmé que la fréquence du phénotype MSI se situe entre 1-2%. Nous avons montré que l’immunohistochimie est la méthode de screening plus adaptée pour l’identification de l’ACP MSI en comparaison avec les techniques de biologie moléculaire. Le phénotype MSI a été plus fréquemment observé dans un contexte de TIPMP. Les cas MSI identifiés présentaient des caractéristiques biologiques évocatrices du SL. Egalement, nos résultats confirment la présence d’un processus de carcinogenèse MSI immunogénique, mais suggèrent des évènements somatiques spécifiques à l’organe d’origine du cancer. Par ailleurs, les ACP MSI étaient caractérisés par un infiltrat inflammatoire riche en lymphocytes cytotoxiques T CD8+ et surexprimaient l’ICK PD-L1 permettant de supposer une probable réponse clinique de l’ACP MSI à l’immunothérapie anti-PD1/PD-L1. / Pancreatic ductal adenocarcinoma (PDAC) is a major health problem in France and around the world. PDAC is developed mainly from two precursor lesions: pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasm (IPMN). There are several molecular mechanisms underlying pancreatic oncogenesis. Particularly, we were interested in the MSI (MicroSatellite Instability) which is due to a defective DNA Mismatch Repair (MMR) system, which normally functions to recognize and repair erroneous insertions, deletions, and mis-incorporation of bases that can arise during DNA replication and recombination. The MSI phenotype was first described in the familial cancer condition known as Lynch syndrome (LS), where the MMR genes MLH1, MSH2, MSH6 or PMS2 harbor germline mutations. Interest in MSI tumors has recently increased after studies have highlighted the concomitant expression of multiple active immune checkpoint (ICK) markers including PD-1 and PD-L1 and the role of the MSI status to predict clinical benefit from immune checkpoint blockade. A Our results indicate that the MSI phenotype occurs in PDAC with a frequency of 1-2%. Our data showed that IHC using antibodies against the four MMR proteins was more sensitive for the assessment of MSI status than PCR-based methods. In addition, we demonstrate for the first time a statistically significant positive association between MSI and IPMNs in PDAC. MSI PDAC, including IPMN, are unlikely to be sporadic since they display molecular features that are usually observed in LS-related neoplasms. Also, our results highlight that an MSI-driven immunogenic pathway to cancer is active in MSI PDACs but suggest that MSI-driven somatic events may be tissue-specific. We observed a stronger lymphocytic tumor infiltration by activated TCD8 cells in MSI PDAC compared to MSS PDAC and found a positive association between PD-L1 expression and MSI status, suggesting that MSI PDAC could be responsive to ICK blockade therapy.